CN114555795A - 酮还原酶多肽及多核苷酸 - Google Patents

酮还原酶多肽及多核苷酸 Download PDF

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CN114555795A
CN114555795A CN202080067803.2A CN202080067803A CN114555795A CN 114555795 A CN114555795 A CN 114555795A CN 202080067803 A CN202080067803 A CN 202080067803A CN 114555795 A CN114555795 A CN 114555795A
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杰克·梁
南希塔·苏布兰马尼安
沙琳·奇恩
大卫·威廉·霍曼
卡蒂·惠伦
马修·布莱克·琼斯
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Abstract

本发明提供了与天然存在的野生型酮还原酶相比具有改进的特性的工程化酮还原酶，以及编码工程化酮还原酶的多核苷酸，能够表达工程化酮还原酶的宿主细胞，以及使用工程化酮还原酶合成手性醇的方法。本发明还提供了使用工程化酶的方法。

Description

酮还原酶多肽及多核苷酸
本申请要求2019年9月26日提交的美国临时专利申请序列第62/906,268号的优先权，该美国临时专利申请出于所有目的通过引用以其整体并入。
对序列表、表格或计算机程序的引用
根据37C.F.R.§1.821，以计算机可读形式(CRF)通过EFS-Web以文件名CX8-195WO2_ST25.txt与本申请同时提交的序列表通过引用并入本文。序列表的电子拷贝于2020年9月23日生成，其文件大小为664千字节。
发明领域
本发明提供了与天然存在的野生型酮还原酶相比具有改进的特性的工程化酮还原酶，以及编码工程化酮还原酶的多核苷酸，能够表达工程化酮还原酶的宿主细胞，以及使用工程化酮还原酶的方法。
背景
属于酮还原酶(KRED)或羰基还原酶类(EC1.1.1.184)的酶可用于从对应的前手性酮底物并通过对应的外消旋醛底物的立体选择性还原来合成光学活性醇。KRED通常将酮底物和醛底物转化为对应的醇产物，但是也可以催化逆反应，将醇底物氧化为对应的酮/醛产物。酶诸如KRED对酮和醛的还原以及对醇的氧化需要辅因子，最常见的是还原型烟酰胺腺嘌呤二核苷酸(NADH)或还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)，以及用于氧化反应的烟酰胺腺嘌呤二核苷酸(NAD+)或烟酰胺腺嘌呤二核苷酸磷酸(NADP+)。NADH和NADPH充当电子供体，而NAD+和NADP+充当电子受体。经常观察到酮还原酶和醇脱氢酶接受磷酸化或非磷酸化的辅因子(处于其氧化态和还原态)，但最通常不是对两者都接受。
为了绕过用于产生关键化合物的许多化学合成程序，酮还原酶越来越多地被用于将不同的酮底物和醛底物酶促转化为手性醇产物。这些应用可以利用表达酮还原酶的全细胞来生物催化酮和醛的还原或生物催化醇的氧化，或者在全细胞中多种酮还原酶的存在将不利地影响期望产物的立体纯度(stereopurity)和产率的那些情况下使用纯化的酶。
“苦味”是啤酒的一个重要的品尝属性，通常来自于啤酒花(植物啤酒花(Humuluslupulus L.)的花)的添加。异-α-酸是在酿造过程期间，由啤酒花植物的蛇麻素腺体(lupulin glands)中天然存在的化合物葎草酮的异构化而形成的。具体来说，六种主要的异-α-酸对苦味负有责任：顺式-异葎草酮、反式-异葎草酮、顺式-异辅葎草酮(cis-isocohumulone)、反式-异辅葎草酮、顺式-异近葎草酮(cis-isoadhumulone)和反式-异近葎草酮。
然而，异-α-酸不是光稳定的，并且光诱导形成3-甲基-2-丁烯-1-硫醇(3-MBT)使啤酒具有明显的光击或臭鼬味和香气。这要求用棕色的瓶子或罐包装啤酒。另一个解决办法是通过还原异-α-酸的羰基基团生成相应的二氢-(ρ)-异-α-酸(dihydro-(rho)-iso-α-acids)来产生完全光稳定的啤酒。这些还原的二氢-(ρ)-异-α-酸是稳定的，并且可以装在透明瓶或绿色瓶中。
然而，目前只能使用有毒、危险和非食品级化学品(例如硼氢化钠)将异-α-酸转化为二氢-(ρ)-异-α-酸。因此，将异-α-酸安全和食品级地转化为二氢-(ρ)-异-α-酸具有相当大的商业价值。
发明概述
本发明提供了与天然存在的野生型酮还原酶相比具有改进的特性的工程化酮还原酶，以及编码工程化酮还原酶的多核苷酸，能够表达工程化酮还原酶的宿主细胞，以及使用工程化酮还原酶的方法。
本发明提供了与来自开菲尔乳杆菌(Lactobacillus kefir)的天然存在的野生型酮还原酶(SEQ ID NO:2)或当与其他工程化酮还原酶(包括SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330的工程化酮还原酶多肽)相比时，在将异-α-酸转化为相应的二氢-(ρ)-异-α-酸方面具有改进的酶活性的工程化酮还原酶(“KRED”)。
在一些另外的实施方案中，工程化酶除了具有改进的酶活性以外还具有一种或更多种改进的特性。酶特性的改进包括但不限于改进的跨越一系列底物的活性、改进的在高底物浓度的活性和改进的在低辅因子浓度的活性。
本发明提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:4具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：12、21、87、93、97、110、145、148、152、153、194、196、197、200、206、212和226，其中所述位置参考SEQID NO:4编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：12I、21R、87L、93D、93M、93T、93V、97G、110I、145C、145G、145M、145S、148I、152G、152S、153C、153R、153V、194H、194N、194R、196H、196K、196R、197G、197R、200L、200Q、200R、206V、212S和226L，其中所述位置参考SEQ ID NO:4编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：V12I、L21R、V87L、I93D、I93M、I93T、I93V、K97G、L110I、L145C、L145G、L145M、L145S、V148I、T152G、T152S、L153C、L153R、L153V、P194H、P194N、P194R、L196H、L196K、L196R、D197G、D197R、E200L、E200Q、E200R、M206V、T212S和I226L，其中所述位置参考SEQ ID NO:4编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:6具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：12/110/145/152、12/145、87/110/145、87/110/145/194、87/145/194、110、110/145/152/197、110/145/194、145、145/152、145/197/226和152，其中所述位置参考SEQ ID NO:6编号。在一些实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：12I/110I/145M/152G、12I/145M、87L/110I/145M、87L/110I/145M/194H、87L/110I/145M/194N、87L/145M/194H、110I、110I/145M/152G/197G、110I/145M/194H、145M、145M/152G、145M/197G/226L和152S，其中所述位置参考SEQ ID NO:6编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：V12I/L110I/L145M/T152G、V12I/L145M、V87L/L110I/L145M、V87L/L110I/L145M/P194H、V87L/L110I/L145M/P194N、V87L/L145M/P194H、L110I、L110I/L145M/T152G/D197G、L110I/L145M/P194H、L145M、L145M/T152G、L145M/D197G/I226L和T152S，其中所述位置参考SEQ ID NO:6编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:80具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：17、21、46、56、72、79、95、101、110、152、162、190、198、210、211和227，其中所述位置参考SEQ IDNO:80编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：17Q、17S、21A、46V、56C、72A、79L、95I、101C、101L、101T、110V、152K、152L、162G、190A、198A、198Q、210F、210W、211R和227V，其中所述位置参考SEQ ID NO:80编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：L17Q、L17S、L21A、K46V、V56C、K72A、E79L、V95I、D101C、D101L、D101T、I110V、T152K、T152L、A162G、P190A、D198A、D198Q、T210F、T210W、L211R和C227V，其中所述位置参考SEQ ID NO:80编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:80具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：17、79、157、159、190/191/194、190/194、191/194、194、198和211，其中所述位置参考SEQ ID NO:80编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：17M、17Q、17S、79L、157C、159T、190A/191T/194E、190A/194E、191T/194E、194E、198A、198Q和211R，其中所述位置参考SEQ ID NO:80编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：L17M、L17Q、L17S、E79L、N157C、S159T、P190A/I191T/P194E、P190A/P194E、I191T/P194E、P194E、D198A、D198Q和L211R，其中所述位置参考SEQ ID NO:80编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:104具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：17/46/190、17/46/198/211、17/96/194/198、17/190/198、46/190/194/198和46/194/198，其中所述位置参考SEQ ID NO:104编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：17M/46V/190A、17M/46V/198A/211R、17M/96V/194E/198Q、17M/190A/198A、17M/190A/198Q、46V/190A/194E/198Q和46V/194E/198Q，其中所述位置参考SEQ ID NO:104编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：Q17M/K46V/P190A、Q17M/K46V/D198A/L211R、Q17M/I96V/P194E/D198Q、Q17M/P190A/D198A、Q17M/P190A/D198Q、K46V/P190A/P194E/D198Q和K46V/P194E/D198Q，其中所述位置参考SEQ ID NO:104编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:172具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：45、101、179、194、204、226和231，其中所述位置参考SEQ ID NO:172编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：45L、101R、101Y、179M、194E、204Q、226V和231G，其中所述位置参考SEQ ID NO:172编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：E45L、D101R、D101Y、Y179M、P194E、E204Q、I226V和A231G，其中所述位置参考SEQ ID NO:172编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:186具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：95/96/97/150/153/205、95/96/150/153/205/206/211/249、95/97/143/145/150/153/202/205、95/97/143/145/150/153/249、95/97/150/153、95/97/150/153/202/205/206、95/150/153/205/206/211、95/150/153/205/211、95/150/153/206/249、96/150/153、96/150/153/206、97/150/153、97/150/153/205、97/150/153/205/211、97/150/153/206、143/144/145/150/153/202/205/249、143/145/150/153、144/145/150/153/205/206、144/150/153、144/150/153/202/205/206、145/150/153/206/249、145/153/211、150/153/202/206/249、150/153/205/211、150/153/206/211、150/153/211和150/153/249，其中所述位置参考SEQ IDNO:186编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：95A/96A/97A/150A/153A/205A、95A/96A/150A/153A/205A/206A/211A/249A、95A/97A/143A/145A/150A/153A/202A/205A、95A/97A/143A/145A/150A/153A/249A、95A/97A/150A/153A、95A/97A/150A/153A/202A/205A/206A、95A/150A/153A/205A/206A/211A、95A/150A/153A/205A/211A、95A/150A/153A/206A/249A、96A/150A/153A、96A/150A/153A/206A、97A/150A/153A、97A/150A/153A/205A、97A/150A/153A/205A/211A、97A/150A/153A/206A、143A/144A/145A/150A/153A/202A/205A/249A、143A/145A/150A/153A、144A/145A/150A/153A/205A/206A、144A/150A/153A、144A/150A/153A/202A/205A/206A、145A/150A/153A/206A/249A、145A/153A/211A、150A/153A/202A/206A/249A、150A/153A/205A/211A、150A/153A/206A/211A、150A/153A/211A和150A/153A/249A，其中所述位置参考SEQ ID NO:186编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：V95A/I96A/K97A/D150A/L153A/M205A、V95A/I96A/D150A/L153A/M205A/M206A/L211A/W249A、V95A/K97A/S143A/M145A/D150A/L153A/W202A/M205A、V95A/K97A/S143A/M145A/D150A/L153A/W249A、V95A/K97A/D150A/L153A、V95A/K97A/D150A/L153A/W202A/M205A/M206A、V95A/D150A/L153A/M205A/M206A/L211A、V95A/D150A/L153A/M205A/L211A、V95A/D150A/L153A/M206A/W249A、I96A/D150A/L153A、I96A/D150A/L153A/M206A、K97A/D150A/L153A、K97A/D150A/L153A/M205A、K97A/D150A/L153A/M205A/L211A、K97A/D150A/L153A/M206A、S143A/I144A/M145A/D150A/L153A/W202A/M205A/W249A、S143A/M145A/D150A/L153A、I144A/M145A/D150A/L153A/M205A/M206A、I144A/D150A/L153A、I144A/D150A/L153A/W202A/M205A/M206A、M145A/D150A/L153A/M206A/W249A、M145A/L153A/L211A、D150A/L153A/W202A/M206A/W249A、D150A/L153A/M205A/L211A、D150A/L153A/M206A/L211A、D150A/L153A/L211A和D150A/L153A/W249A，其中所述位置参考SEQ ID NO:186编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:186具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：7/147、103/147、110、110/179/194、147和249，其中所述位置参考SEQ ID NO:186编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：7Q/147I、103R/147I、110V、110V/179M/194E、147I和249Y，其中所述位置参考SEQ ID NO:186编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：H7Q/L147I、T103R/L147I、I110V、I110V/Y179M/P194E、L147I和W249Y，其中所述位置参考SEQ ID NO:186编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:194具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：7/12/54/110/150/153/194/205/211/249、12/54/72/110/150/152/153/194/205/211/249、12/72/101/103/110/152/249、12/72/110/147/152/204、45/54/72/110/152/194/204、72/110/147/150/152/153/194/205/211/249和110/150/153/179/194/205/211/249，其中所述位置参考SEQ ID NO:194编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：7Q/12I/54S/110V/150D/153L/194E/205M/211L/249Y、12I/54S/72T/110V/150D/152M/153L/194E/205M/211L/249Y、12I/72S/101Y/103Q/110V/152M/249Y、12I/72S/110V/147I/152M/204Q、45L/54S/72S/110V/152M/194E/204Q、72S/110V/147M/150D/152M/153L/194E/205M/211L/249Y和110V/150D/153L/179M/194E/205M/211L/249Y，其中所述位置参考SEQ ID NO:194编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：H7Q/V12I/T54S/I110V/A150D/A153L/P194E/A205M/A211L/W249Y、V12I/T54S/K72T/I110V/A150D/T152M/A153L/P194E/A205M/A211L/W249Y、V12I/K72S/R101Y/T103Q/I110V/T152M/W249Y、V12I/K72S/I110V/L147I/T152M/E204Q、E45L/T54S/K72S/I110V/T152M/P194E/E204Q、K72S/I110V/L147M/A150D/T152M/A153L/P194E/A205M/A211L/W249Y和I110V/A150D/A153L/Y179M/P194E/A205M/A211L/W249Y，其中所述位置参考SEQ ID NO:194编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:252具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：7/12/54/179/249、7/152、12/54/72/152/179/249、40、54/72、72/147/152/179/249和249，其中所述位置参考SEQ ID NO:252编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：7Q/12I/54S/179Y/249Y、7Q/152M、12I/54S/72T/152M/179Y/249Y、40E、54S/72S、72S/147M/152M/179Y/249Y和249Y，其中所述位置参考SEQ IDNO:252编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：H7Q/V12I/T54S/M179Y/W249Y、H7Q/T152M、V12I/T54S/K72T/T152M/M179Y/W249Y、H40E、T54S/K72S、K72S/L147M/T152M/M179Y/W249Y和W249Y，其中所述位置参考SEQID NO:252编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:270具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：92/93、150/152、150/152/153和194/195，其中所述位置参考SEQ ID NO:270编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：92A/93E、150D/152A/153L、150Y/152A、150Y/152S和194S/195A，其中所述位置参考SEQ ID NO:270编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：G92A/I93E、A150D/M152A/A153L、A150Y/M152A、A150Y/M152S和E194S/R195A，其中所述位置参考SEQ ID NO:270编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:272具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：92/93/95、93、93/95、93/95/109、93/95/109/114、93/95/114、93/109/114和114，其中所述位置参考SEQ ID NO:272编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：92A/93D/95R、93A/95K/109R、93A/95R/109D/114T、93D/95R、93E/109R/114A、93M、93R/95A/114T和114A，其中所述位置参考SEQ ID NO:272编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：G92A/I93D/V95R、I93A/V95K/K109R、I93A/V95R/K109D/N114T、I93D/V95R、I93E/K109R/N114A、I93M、I93R/V95A/N114T和N114A，其中所述位置参考SEQ ID NO:272编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:286具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：12/45/72/109/249、12/45/93/249、12/45/249、12/109/249、45/72/249、45/109/249、45/249、96和145/150，其中所述位置参考SEQ ID NO:286编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：12I/45E/72T/109D/249Y、12I/45E/93A/249Y、12I/45E/249Y、12I/109D/249Y、45E/72T/249Y、45E/109D/249Y、45E/249Y、96A和145A/150A，其中所述位置参考SEQ ID NO:286编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：V12I/L45E/S72T/K109D/W249Y、V12I/L45E/I93A/W249Y、V12I/L45E/W249Y、V12I/K109D/W249Y、L45E/S72T/W249Y、L45E/K109D/W249Y、L45E/W249Y、I96A和M145A/Y150A，其中所述位置参考SEQ ID NO:286编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:328具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：150、150/151、150/195和195，其中所述位置参考SEQ ID NO:328编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：150A、150A/151A、150A/195S、195A和195S，其中所述位置参考SEQ ID NO:328编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：Y150A、Y150A/P151A、Y150A/R195S、R195A和R195S，其中所述位置参考SEQ ID NO:328编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:330具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：12/72/109/195、17/73/200、17/115、68/72/101/152/205、68/72/124、68/72/124/152、68/101/124/152/205、68/124/205、72/109/152/195、72/109/195、72/152、72/152/195、72/195、73、73/147、79、93、93/95/145/195、93/109/114/145/195、93/195、95/195、96/108/147/200、96/194/200、101/205、145/195、147、147/200、192、194、194/200、195、198和200，其中所述位置参考SEQ ID NO:330编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：12V/72S/109K/195R、17A/73V/200P、17A/115Q、68E/72D/101K/152Q/205L、68R/72D/101Q/152Q/205L、68R/72R/124E、68R/72R/124E/152Q、68R/101Q/124E/152Q/205L、68R/124E/205L、72D/152Q、72K/152M/195R、72K/195R、72S/109K/152M/195R、72S/109K/195R、73V、73V/147I、79A、93A、93A/95R/145A/195R、93A/109K/114T/145A/195R、93A/195R、95R/195R、96P/108S/147I/200P、96P/194N/200P、101M/205L、145A/195A、147I、147I/200P、192R、194N、194N/200P、195R、198G、198R和200P，其中所述位置参考SEQ ID NO:330编号。在一些另外的实施方案中，工程化酮还原酶变体包含选自以下的至少一个取代或取代集：I12V/T72S/D109K/S195R、M17A/L73V/E200P、M17A/L115Q、A68E/T72D/R101K/A152Q/A205L、A68R/T72D/R101Q/A152Q/A205L、A68R/T72R/L124E、A68R/T72R/L124E/A152Q、A68R/R101Q/L124E/A152Q/A205L、A68R/L124E/A205L、T72D/A152Q、T72K/A152M/S195R、T72K/S195R、T72S/D109K/A152M/S195R、T72S/D109K/S195R、L73V、L73V/L147I、E79A、I93A、I93A/V95R/M145A/S195R、I93A/D109K/N114T/M145A/S195R、I93A/S195R、V95R/S195R、I96P/R108S/L147I/E200P、I96P/E194N/E200P、R101M/A205L、M145A/S195A、L147I、L147I/E200P、K192R、E194N、E194N/E200P、S195R、Q198G、Q198R和E200P，其中所述位置参考SEQ ID NO:330编号。
本发明还提供了工程化酮还原酶变体，所述工程化酮还原酶变体包含含有与SEQID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330具有至少90％序列同一性的序列的多肽序列。在一些实施方案中，工程化酮还原酶变体包含含有与SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330具有至少95％序列同一性的序列的多肽序列。在一些另外的实施方案中，工程化酮还原酶变体包含SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330中列出的多肽序列。在一些另外的实施方案中，工程化酮还原酶变体包含编码表5-1、表6-1、表7-1、表8-1、表17-2、表18-1、表19-1、表19-2、表20-1、表20-2、表21-1、表22-1和/或表24-1中提供的变体的多肽序列。在一些另外的实施方案中，工程化酮还原酶变体包含选自SEQ ID NO:6至SEQ ID NO:412中列出的偶数编号的序列的多肽序列。
本发明还提供了编码本文提供的工程化酮还原酶变体的工程化多核苷酸序列。在一些实施方案中，工程化多核苷酸序列包含与选自SEQ ID NO:5至SEQ ID NO:411中列出的奇数编号的序列的序列至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多相同的多核苷酸序列。本发明还提供了包含编码本文提供的工程化酮还原酶变体的工程化多核苷酸序列的载体。在一些实施方案中，载体还包含至少一种控制序列。在一些实施方案中，载体包括SEQ ID NO:413和/或414。
本发明还提供了包含载体的宿主细胞，所述载体包含编码本文提供的工程化酮还原酶变体的多核苷酸。
本发明还提供了产生本文提供的工程化酮还原酶变体的方法，所述方法包括在本文提供的宿主细胞产生工程化酮还原酶变体的条件下培养所述宿主细胞。在一些实施方案中，方法还包括回收由宿主细胞产生的工程化酮还原酶变体的步骤。在一些另外的实施方案中，产生工程化酮还原酶变体的方法包括培养包含SEQ ID NO:413和/或414的载体的宿主细胞。
本发明还提供了固定化的工程化酮还原酶变体。
本发明还提供了包含至少一种本文提供的工程化酮还原酶变体的组合物。在一些实施方案中，组合物包含至少一种本文提供的固定化的工程化酮还原酶变体。
附图简述
图1提供了比较SEQ ID NO:6和SEQ ID NO:80的多肽在高底物浓度的KRED活性的典型HPLC反应谱(reaction profile)。
图2提供了在实施例11中描述的实验结果，所选变体在高底物和低NADP浓度的KRED活性(％转化)。
图3提供了描绘由SEQ ID NO:194的多肽产生的Rho物质(species)的典型HPLC反应谱。
图4提供了比较SEQ ID NO:328和SEQ ID NO:330的多肽在高底物和低NADP浓度的KRED活性的典型HPLC反应谱。对于图4，实线＝4g/L酶；虚线＝1g/L酶；菱形＝SEQ ID NO:328；空心正方形＝SEQ ID NO:330。
图5提供了比较SEQ ID NO:270、SEQ ID NO:328、SEQ ID NO:330、SEQ ID NO:348、SEQ ID NO:346和SEQ ID NO:356的多肽在高底物和低NADP浓度的KRED活性的典型HPLC反应谱。对于图5，空心三角形＝SEQ ID NO:270；实心三角形＝SEQ ID NO:328；空心圆形＝SEQ ID NO:348；实心圆形＝SEQ ID NO:356；空心正方形＝SEQ ID NO:330；实心正方形＝SEQ ID NO:346。
发明描述
本发明提供了与天然存在的野生型酮还原酶相比具有改进的特性的工程化酮还原酶，以及编码工程化酮还原酶的多核苷酸，能够表达工程化酮还原酶的宿主细胞，以及使用工程化酮还原酶的方法。
定义
参考本发明，本文的描述中使用的技术和科学术语将具有本领域普通技术人员通常理解的含义，除非另有具体定义。因此，以下术语意在具有以下含义。本文提及的所有专利和出版物，包括这些专利和出版物中公开的所有序列，都通过引用明确并入。除非另外指示，本发明的实践包括分子生物学、发酵、微生物学和相关领域中常用的常规技术，这些技术是本领域技术人员已知的。除非本文另外定义，本文使用的所有技术和科学术语具有与本发明所属领域内的普通技术人员通常理解的相同含义。尽管与本文描述的那些相似或等效的任何方法和材料都可以用于实践或测试本发明，但描述了优选的方法和材料。实际上，不意图本发明局限于本文描述的特定方法学、方案和试剂，因为这些可以取决于使用它们的环境而变化。本文提供的标题不是对本发明的各方面或实施方案的限制。
尽管如此，为了便于理解本发明，下文定义了许多术语。数值范围包括限定该范围的数字。因此，本文公开的每个数值范围意图包括落在这样的较宽数值范围内的每一较窄数值范围，如同这样的较窄数值范围在本文被全部清楚地写出。还意图本文公开的每个最大的(或最小的)数值限制包含每个较低(或较高)的数值限制，如同这样的较低(或较高)数值限制在本文被清楚地写出。
如本文使用的，术语“包含(comprising)”及其同源词以其包括性意义使用(即，等同于术语“包括(including)”及其相应的同源词)。
如在本文和在所附权利要求书中使用的，单数的“一(a)”、“一(an)”和“该(the)”包括复数指代物，除非上下文另外明确规定。因此，例如对“宿主细胞”的提及包括多于一个这样的宿主细胞。
除非另外指示，分别地，核酸以5’至3’的方向从左向右书写，而氨基酸序列以氨基至羧基的方向从左向右书写。
本文提供的标题不是对可以通过整体参考说明书而获得的本发明的各个方面或实施方案的限制。因此，下文定义的术语通过整体参考说明书被更充分地定义。
“酮还原酶”和“KRED”在本文中可互换使用，以指具有将羰基基团还原为其对应醇的酶促能力的多肽。更具体地，本发明的酮还原酶多肽能够将异-α-酸的混合物还原成相应的二氢-(ρ)-异-α-酸，如方案1所示。本发明的酮还原酶来源于开菲尔乳杆菌(L.kefir)的天然存在的KRED(SEQ ID NO:2)。然而，术语KRED和酮还原酶不限于此，并且可以指天然存在的酶或来源于各种细菌、植物、藻类和/或动物物种的酶。酶可以是合成的、人造的或通过本领域技术人员已知的各种方法产生。
“异-α-酸”或“iso”在本文可互换地用于指异葎草酮的异构体、差向异构体、非对映异构体、互变异构体和对映异构体，异葎草酮是一种来源于啤酒花(啤酒花植物Humuluslupulus L.的花)的化合物。“异-α-酸”或“iso”包括顺式-异葎草酮、反式-异葎草酮、顺式-异辅葎草酮、反式-异辅葎草酮、顺式-异近葎草酮和反式-异近葎草酮，但不限于此。“异-α-酸”或“iso”还包括任何天然存在或合成的异构体、差向异构体、非对映异构体、互变异构体、对映异构体或具有类似化学特性(特别地，使啤酒或其他酒精饮料或类似饮料具有苦的味道或苦味)的其他衍生物或类似化合物。这包括异葎草酮特窗酸(tetronic acid)核心的任何异构体、差向异构体、非对映异构体、对映异构体或互变异构体。
“二氢-(ρ)-异-α-酸”或“rho”在本文中可互换地用于指通过还原由本文定义的“异-α-酸”或“iso”的羰基基团而生成的化合物。“二氢-(ρ)-异-α-酸”或“rho”可通过一种或更多种KRED多肽的转化从“异-α-酸”或“iso”产生，如本文所述。
如本文使用的，术语“蛋白”、“多肽”和“肽”在本文中可互换使用，以指通过酰胺键共价连接的至少两个氨基酸的聚合物，而不论长度或翻译后修饰(例如，糖基化、磷酸化、脂质化、豆蔻酰化(myristilation)、泛素化等)如何。此定义内包括D-氨基酸和L-氨基酸以及D-氨基酸和L-氨基酸的混合物。
如本文使用的，“多核苷酸”和“核酸”指的是共价连接在一起的两个或更多个核苷。多核苷酸可以完全由核糖核苷(即RNA)组成、完全由2’脱氧核糖核苷(即DNA)或核糖核苷和2’脱氧核糖核苷的混合物组成。虽然核苷典型地将经由标准磷酸二酯连接连接在一起，但多核苷酸可以包括一个或更多个非标准连接。多核苷酸可以是单链或双链的，或者可以包括单链区域和双链区域二者。此外，虽然多核苷酸将通常包含天然存在的编码核碱基(即腺嘌呤、鸟嘌呤、尿嘧啶、胸腺嘧啶和胞嘧啶)，但它可以包含一种或更多种修饰的和/或合成的核碱基(例如肌酐、黄嘌呤、次黄嘌呤等)。优选地，这样的修饰的或合成的核碱基将是编码核碱基。
如本文使用的，“编码序列”是指核酸(例如基因)编码蛋白质的氨基酸序列的部分。
如本文使用的，“天然存在的”或“野生型”是指在自然界中发现的形式。例如，天然存在的或野生型多肽或多核苷酸序列是存在于可以从自然界的来源分离的生物体中并且没有被人工操作有意修饰的序列。
如本文使用的，当在本发明中关于(例如，细胞、核酸或多肽)使用时，“非天然存在的”或“工程化”或“重组”是指如下材料或与该材料的天然或自然形式对应的材料：已经以自然界本来不存在的方式被修饰或与其相同但由合成材料产生或衍生和/或通过使用重组技术操作产生。非限制性实例包括，除其他以外，表达自然(非重组)形式的细胞中未发现的基因或表达原本以不同水平表达的自然基因的重组细胞。
如本文使用的，“序列同一性百分比”、“同一性百分比”和“相同百分比”指的是多核苷酸序列或多肽序列之间的比较，并且通过在比较窗上比较两个最佳比对序列来确定，其中与参考序列相比，比较窗中的多核苷酸或多肽序列的部分可以包含添加或缺失(即空位)，用于这两个序列的最佳比对。百分比如下计算：通过确定两个序列中出现相同的核酸碱基或氨基酸残基，或者核酸碱基或氨基酸残基与空位比对的位置的数目，以产生匹配位置的数目，将匹配位置的数目除以比较窗中位置的总数，并将结果乘以100以产生序列同一性百分比。对最佳比对和序列同一性百分比的确定使用BLAST和BLAST 2.0算法来进行(例如参见，Altschul等人，J.Mol.Biol.215:403-410[1990]；和Altschul等人，Nucleic AcidsRes.3389-3402[1977])。公众可通过美国国家生物技术信息中心网站获得用于进行BLAST分析的软件。
简言之，BLAST分析包括首先通过识别问询序列中具有长度W的短字(word)来识别高评分序列对(HSP)，该高评分序列对当与数据库序列中的相同长度的字对齐时匹配或满足某个正值阈值评分T。T被称为相邻字评分阈值(Altschul等人，同上)。这些最初的邻近字击中(word hit)充当启动搜索的种子以找到包含它们的更长HSP。然后字击中沿着每个序列的两个方向延伸直到累积比对评分不能增加的程度。对于核苷酸序列，累积评分使用参数M(用于匹配残基对的奖励评分；总是＞0)和N(用于错配残基的惩罚评分；总是＜0)计算。对于氨基酸序列，评分矩阵用于计算累积评分。在以下情况时，停止字击中在每一个方向的延伸：累积比对评分从其最大达到值下降了量X；由于累积了一个或更多个负评分残基比对，累积评分达到0或小于0；或到达任一序列末端。BLAST算法参数W、T和X决定比对的灵敏度和速度。BLASTN程序(对于核苷酸序列)使用以下作为默认值：字长(W)为11、期望值(E)为10、M＝5、N＝-4、以及两条链的比较。对于氨基酸序列，BLASTP程序使用以下作为默认值：字长(W)为3、期望值(E)为10、和BLOSUM62评分矩阵(参见例如，Henikoff和Henikoff,Proc.Natl.Acad.Sci.USA 89:10915[1989])。
许多其他算法是可获得的和本领域已知的，这些算法在提供两个序列的同一性百分比方面与BLAST起相似作用。用于比较的序列的最佳比对可以使用本领域已知的任何合适的方法进行(例如，通过Smith和Waterman,Adv.Appl.Math.2:482[1981]的局部同源性算法；通过Needleman和Wunsch,J.Mol.Biol.48:443[1970]的同源性比对算法；通过Pearson和Lipman,Proc.Natl.Acad.Sci.USA 85:2444[1988]的搜索相似性的方法；和/或通过这些算法的计算机化实现[GCG Wisconsin软件包中的GAP、BESTFIT、FASTA和TFASTA])，或通过使用本领域通常已知的方法进行目视检查。此外，序列比对和序列同一性百分比的确定可以使用所提供的默认参数，采用GCG Wisconsin软件包(Accelrys,Madison WI)中的BESTFIT或GAP程序。
如本文使用的，“参考序列”指的是另一序列被与之比较的确定序列。参考序列可以是更大序列的子集，例如，全长基因或多肽序列的区段(segment)。通常，参考序列为至少20个核苷酸或氨基酸残基的长度、至少25个残基的长度、至少50个残基的长度，或核酸或多肽的全长。由于两个多核苷酸或多肽可以各自(1)包含两个序列之间相似的序列(即完整序列的一部分)，并且(2)还可以包含两个序列之间趋异的(divergent)序列，因此两个(或更多个)多核苷酸或多肽之间的序列比较通常通过在比较窗上比较两个多核苷酸的序列来鉴定和比较序列局部区域的相似性来进行。术语“参考序列”不意图局限于野生型序列，并且可以包括工程化序列或改变的序列。例如，在一些实施方案中，“参考序列”可以是先前工程化或改变的氨基酸序列。
如本文使用的，“比较窗”指的是至少约20个连续核苷酸位置或氨基酸残基的概念性区段(conceptual segment)，其中序列可以与至少20个连续核苷酸或氨基酸的参考序列比较，并且其中与参考序列(其不包含添加或缺失)相比，序列在比较窗中的部分可以包含20％或更少的添加或缺失(即空位)，以用于两个序列的最佳比对。比较窗可以比20个连续残基更长，并任选地包括30、40、50、100或更长的窗。
当如本文使用的，当在给定氨基酸或多核苷酸序列的编号的上下文中使用时，“对应于”、“关于”或“相对于”是指，当将给定氨基酸或多核苷酸序列与指定参考序列相比时，该参考序列的残基的编号。换言之，给定聚合物的残基编号或残基位置关于参考序列被指定，而不是通过给定氨基酸或多核苷酸序列内残基的实际数字位置被指定。例如，可以通过引入空位将给定氨基酸序列诸如工程化酮还原酶的氨基酸序列与参考序列比对，以优化两个序列之间的残基匹配。在这些情况中，尽管存在空位，对给定氨基酸或多核苷酸序列中的残基关于与其比对的参考序列进行编号。如本文使用的，如下文进一步描述的对残基位置的提及，诸如“Xn”，应被理解为指“对应于......的残基”，除非另外明确说明。因此，例如“X94”指的是多肽序列中在位置94处的任何氨基酸。
如本文使用的，“立体选择性”指一种立体异构体相对于另一种立体异构体或另一组立体异构体在化学或酶促反应中的优先形成。立体选择性可以是部分的，此时一种立体异构体的形成优于另一种，或者立体选择性可以是完全的，此时仅形成一种立体异构体。当立体异构体是对映异构体时，立体选择性被称为对映选择性，即两种对映异构体的总和中一种对映异构体的分数(通常报道为百分比)。本领域中通常可选择地将其报道(通常为百分比)为根据下式从中计算的对映异构体过量(e.e.)：[主要对映异构体-次要对映异构体]/[主要对映异构体+次要对映异构体]。当立体异构体是非对映异构体时，立体选择性被称为非对映选择性，即两种非对映异构体的混合物中一种非对映异构体的分数(通常报道为百分比)，通常可选择地报告为非对映异构体过量(d.e.)。对映异构体过量和非对映异构体过量是立体异构体过量的类型。还应理解，立体选择性不限于单一立体异构体，并且可以描述立体异构体的组。
如本文使用的，“高立体选择性”是指能够以至少约75％的立体异构体过量将底物转化为其对应的手性醇产物的化学或酶促反应。
如本文使用的，“增加的酶促活性”和“增加的活性”是指工程化酶的改进的特性，其可以通过与参考酶相比，比活性(例如，产生的产物/时间/重量蛋白)的增加或底物向产物的转化百分比(例如，使用指定量的酮还原酶，在指定时间段内，起始量的底物向产物的转化百分比)的增加来表示。确定酶活性的示例性方法在实施例中提供。与酶活性相关的任何特性都可以被影响，包括经典的酶特性Km、Vmax或kcat，其改变可以导致酶促活性的增加。酮还原酶活性可以通过用于测量酮还原酶的标准测定中的任何一种来测量，诸如底物或产物浓度的变化，或辅因子浓度的变化(在不存在辅因子再生系统的情况下)。酶活性的比较使用确定的酶制品、设定条件下的确定测定和一种或更多种确定底物来进行，如本文进一步详细描述的。通常，当比较细胞裂解物中的酶时，确定细胞的数目和测定的蛋白的量，并且使用相同的表达系统和相同的宿主细胞以使由宿主细胞产生并存在于裂解物中的酶的量的变化最小化。
如本文使用的，“转化”指的是将底物酶促转化成对应的产物。
如本文使用的，“转化百分比”指的是在指定条件下在一定时间段内转化成产物的底物的百分比。因此，例如，酮还原酶多肽的“酶促活性”或“活性”可以表示为底物转化为产物的“转化百分比”。
如本文使用的，“热稳定的(thermostable)”或“热稳定的(thermal stable)”可互换使用以指这样的多肽，其与未处理的酶相比，当暴露于一组温度条件(例如，40℃-80℃)一定时间段(例如，0.5hr-24hr)时耐受失活，因此在暴露于升高的温度后，保持一定水平的残余活性(诸如例如，多于60％至80％)。
如本文使用的，“溶剂稳定的”是指与未处理的酶相比，多肽在暴露于不同浓度(例如，5％-99％)的溶剂后，维持相似活性(例如，多于例如60％至80％)的能力。
如本文使用的，“氨基酸差异”或“残基差异”是指多肽序列的一个位置处的氨基酸残基相对于参考序列中对应位置处的氨基酸残基的差异。本文中氨基酸差异的位置通常被称为“Xn”，其中n指的是残基差异所基于的参考序列中的对应位置。例如，“与SEQ ID NO:2相比在位置X40处的残基差异”是指在对应于SEQ ID NO:2的位置40的多肽位置处的氨基酸残基的差异。因此，如果SEQ ID NO:2的参考多肽在位置40处具有组氨酸，那么“与SEQ IDNO:2相比在位置X40处的残基差异”是指在对应于SEQ ID NO:2的位置40的多肽位置处除组氨酸以外的任何残基的氨基酸取代。在本文的大多数情况下，在一个位置处的特定氨基酸残基差异指示为“XnY”，其中“Xn”指定如上文描述的对应位置，并且“Y”是在工程化多肽中发现的氨基酸(即，与参考多肽中的不同的残基)的单字母标识符。在一些情况下，本发明还提供由常规符号“AnB”表示的特定氨基酸差异，其中A是参考序列中的残基的单字母标识符，“n”是参考序列中的残基位置的编号，并且B是工程化多肽序列中残基取代的单字母标识符。在一些实例中，本发明的多肽可以相对于参考序列包含一个或更多个氨基酸残基差异，其由相对于参考序列存在残基差异的一列指定位置指示。在一些实施方案中，当多于一种氨基酸可以在多肽的指定残基位置中使用时，可以使用的各种氨基酸残基由“/”隔开(例如X192A/G)。本发明包括含有一个或更多个氨基酸差异的工程化多肽序列，所述一个或更多个氨基酸差异包括保守氨基酸取代和非保守氨基酸取代的任一种/或两者。本发明的序列表中包括的特定重组酮还原酶多肽的氨基酸序列包括起始甲硫氨酸(M)残基(即，M代表残基位置1)。然而，技术人员理解，该起始甲硫氨酸残基可以通过诸如宿主细胞中或体外翻译系统中的生物加工机制去除，以产生缺乏起始甲硫氨酸残基但在其他方面保留酶的特性的成熟蛋白。因此，如本文使用的术语“相对于SEQ ID NO:2在位置Xn处的氨基酸残基差异”可以指位置“Xn”或已经被加工以缺少起始甲硫氨酸的参考序列的对应位置(例如，位置(X-1)n)。
如本文使用的，措辞“保守氨基酸取代”指的是具有相似侧链的残基的可互换性，并且因此通常包括用相同或相似的氨基酸定义类别内的氨基酸取代多肽中的氨基酸。例如但不限于，在一些实施方案中，具有脂肪族侧链的氨基酸被另一种脂肪族氨基酸(例如，丙氨酸、缬氨酸、亮氨酸和异亮氨酸)取代；具有羟基侧链的氨基酸被另一种具有羟基侧链的氨基酸(例如，丝氨酸和苏氨酸)取代；具有芳族侧链的氨基酸被另一种具有芳族侧链的氨基酸(例如，苯丙氨酸、酪氨酸、色氨酸和组氨酸)取代；具有碱性侧链的氨基酸被另一种具有碱性侧链的氨基酸(例如，赖氨酸和精氨酸)取代；具有酸性侧链的氨基酸被另一种具有酸性侧链的氨基酸(例如，天冬氨酸或谷氨酸)取代；和/或疏水氨基酸或亲水氨基酸分别被另一种疏水氨基酸或亲水氨基酸取代。示例性保守取代在表1中提供。
Figure BDA0003565647960000211
如本文使用的，措辞“非保守取代”指的是用具有显著不同的侧链特性的氨基酸取代多肽中的氨基酸。非保守取代可以使用定义的组之间而不是之内的氨基酸，并且影响：(a)取代区域中的肽骨架的结构(例如，脯氨酸取代甘氨酸)，(b)电荷或疏水性，或(c)侧链体积。例如但不限于，示例性非保守取代可以是用碱性或脂肪族氨基酸取代酸性氨基酸；用小氨基酸取代芳香族氨基酸；和用疏水氨基酸取代亲水氨基酸。
如本领域技术人员将理解的，除非另外说明，否则以上定义的类别中的一些并不相互排斥。因此，具有显示两种或更多种物理化学特性的侧链的氨基酸可以被包括在多个类别中。对任何氨基酸或残基的适当分类对本领域技术人员来说将是明显的，特别是根据本文提供的详细发明。
如本文使用的，“缺失”指的是通过从参考多肽去除一个或更多个氨基酸来修饰多肽。缺失可以包括去除1个或更多个氨基酸、2个或更多个氨基酸、5个或更多个氨基酸、10个或更多个氨基酸、15个或更多个氨基酸、或者20个或更多个氨基酸、多达构成多肽的氨基酸总数的10％或多达构成多肽的氨基酸总数的20％，同时保留酶促活性和/或保留工程化酶的改进的特性。缺失可以涉及多肽的内部部分和/或末端部分。在各种实施方案中，缺失可以包括连续的区段或可以是不连续的。
如本文使用的，“插入”指的是通过向参考多肽添加一个或更多个氨基酸来修饰多肽。在一些实施方案中，改进的工程化酮还原酶包括向天然存在的酮还原酶多肽插入一个或更多个氨基酸，以及向工程化酮还原酶多肽插入一个或更多个氨基酸。插入可以处于多肽的内部部分或者可以是插入到羧基或氨基末端。如本文使用的插入包括如本领域已知的融合蛋白。插入可以是氨基酸的连续区段或由天然存在的多肽中的一个或更多个氨基酸分开。
术语“氨基酸取代集”或“取代集”是指与参考序列相比，多肽序列中的一组氨基酸取代。取代集可以具有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个或更多个氨基酸取代。在一些实施方案中，取代集是指存在于实施例中提供的表格中所列的变体KRED的任一个中的氨基酸取代集。
如本文使用的，“片段”指的是具有氨基末端和/或羧基末端缺失，但其中剩余的氨基酸序列与序列中的对应位置相同的多肽。片段通常可以具有全长酮还原酶多肽例如SEQID NO:4的多肽的约80％、约90％、约95％、约98％或约99％。在一些实施方案中，片段是“有生物活性的”(即，它表现出与全长序列相同的酶促活性)。
如本文使用的，“分离的多肽”是指与天然伴随多肽的其他污染物，例如，蛋白、脂质和多核苷酸基本上分离的多肽。该术语包括已经从其天然存在的环境或表达系统(例如，宿主细胞或体外合成)中取出或纯化的多肽。改进的酮还原酶可以存在于细胞内、存在于细胞培养基中，或以各种形式制备，诸如裂解物或分离的制品。如此，在一些实施方案中，本发明的工程化酮还原酶多肽可以是分离的多肽。
如本文使用的，“大体上纯的多肽”指的是其中多肽物质是存在的主要物质(即，以摩尔或重量计，其比组合物中的任何其他单独的大分子物质更丰富)的组合物，并且当目标物质以摩尔或％重量计构成存在的大分子物质的至少约50％时，该组合物通常为大体上纯的组合物。通常，以存在于组合物中的所有大分子物质的摩尔或％重量计，基本上纯的工程化酮还原酶多肽组合物将占约60％或更多、约70％或更多、约80％或更多、约90％或更多、约91％或更多、约92％或更多、约93％或更多、约94％或更多、约95％或更多、约96％或更多、约97％或更多、约98％或更多、或约99％。溶剂物质、小分子(<500道尔顿)和元素离子物质不被认为是大分子物质。在一些实施方案中，分离的改进的酮还原酶多肽是基本上纯的多肽组合物。
如本文使用的，当关于核酸或多肽使用时，术语“异源的”是指正常情况下生物体(例如，野生型生物体)不表达及分泌的序列。在一些实施方案中，该术语涵盖包含两个或更多个子序列的序列，发现所述子序列彼此之间关系与在自然界中正常存在的关系不同，或所述序列被重组工程化，使得其表达水平或与细胞中的其他核酸或其他分子的物理关系或结构不是正常存在于自然界中的。例如，异源的核酸通常被重组地产生，具有以自然界中未发现的方式排列的来自不相关的基因的两个或更多个序列(例如，本发明的核酸开放阅读框(ORF)可操作地连接至被插入到表达盒诸如载体中的启动子序列)。在一些实施方案中，“异源多核苷酸”指的是通过实验室技术被引入到宿主细胞中的任何多核苷酸，并且包括从宿主细胞中取出、经受实验室操作、并且然后重新引入到宿主细胞的多核苷酸。
如本文使用的，“密码子优化的”是指编码蛋白的多核苷酸的密码子改变为在特定生物体中优先使用的那些密码子，使得编码的蛋白在感兴趣的生物体中有效地表达。在一些实施方案中，编码酮还原酶的多核苷酸可以被密码子优化以用于从所选择的用于表达的宿主生物体优化产生。
如本文使用的，“控制序列”在本文中被定义为包括对本发明的多核苷酸和/或多肽的表达是必需或有利的所有组分。每个控制序列对于感兴趣的多核苷酸可以是天然的或外源的。这样的控制序列包括但不限于前导序列、多腺苷酸化序列、前肽序列、启动子、信号肽序列和转录终止子。
如本文使用的，“可操作地连接”在本文中被定义为控制序列被合适地放置(即，以功能性关系)在相对于感兴趣的多核苷酸的某一位置处的配置，使得控制序列指导或调控感兴趣的多核苷酸和/或多肽的表达。
如本文使用的，措辞“辅因子再生系统”或“辅因子再循环系统”是指参与使辅因子的氧化形式还原的反应(例如，NADP+至NADPH)的一组反应物。通过酮还原酶催化的酮底物的还原而被氧化的辅因子通过辅因子再生系统以还原形式再生。辅因子再生系统包括化学计量还原剂，所述化学计量还原剂是还原氢等同物的来源，并且能够使辅因子的氧化形式还原。辅因子再生系统还可以包含催化剂，例如催化使辅因子的氧化形式被还原剂还原的酶催化剂。分别由NAD+或NADP+再生NADH或NADPH的辅因子再生系统是本领域已知的，并且可以用于本文描述的方法中。
如本文使用的，“合适的反应条件”是指生物催化反应溶液中的那些条件(例如，酶载量、底物载量、辅因子载量、温度、pH、缓冲剂、共溶剂等的范围)，在这些条件下本发明的酮还原酶多肽能够立体选择性地使底物化合物还原为产物化合物。在本发明中提供并通过实施例说明了示例性的“合适的反应条件”。
如本文使用的，诸如“化合物载量”、“酶载量”或“辅因子载量”中的“载量”指的是在反应开始时反应混合物中组分的浓度或量。
如本文使用的，在生物催化剂介导的方法的上下文中，“底物”指的是由生物催化剂作用的化合物或分子。例如，在本文公开的方法中，酮还原酶生物催化剂的示例性底物是异-α-酸。
如本文使用的，在生物催化剂介导的方法的上下文中，“产物”指的是由生物催化剂的作用产生的化合物或分子。
如本文使用的，本文使用的“平衡”是指如由化学或酶促反应的正向速率常数和反向速率常数确定的在化学或酶促反应中产生化学物质的稳定状态浓度的过程(例如，两种物质A和B的相互转化)，包括立体异构体的相互转化。
如本文使用的，“氧代”是指＝O。
如本文使用的，“氧基”是指二价基团-O-，其可以具有各种取代基以形成不同的氧基基团，包括醚和酯。
如本文使用的，“羧基”是指-COOH。
如本文使用的，“羰基”是指-C(O)-，其可以具有各种取代基以形成不同的羰基基团，包括酸、酰基卤、醛、酰胺、酯和酮。
如本文使用的，“羟基”是指-OH。
如本文使用的，“任选的”和“任选地”意指随后描述的事件或情形可以发生或可以不发生，并且该描述包括其中事件或情形发生的情况和其中事件或情形不发生的情况。本领域普通技术人员将理解，对于被描述为包含一个或更多个任选的取代基的任何分子，仅意在包括空间上可实现的和/或合成上可行的化合物。
如本文使用的，“任选地被取代的”是指一种或一系列化学基团中的所有后续修饰对象(modifier)。例如，在术语“任选地被取代的芳基烷基”中，分子的“烷基(alkyl)”部分和“芳基”部分可以被取代或可以不被取代，并且对于一系列“任选地被取代的烷基、环烷基、芳基和杂芳基”，烷基基团、环烷基基团、芳基基团和杂芳基基团彼此独立地可以被取代或可以不被取代。
工程化酶多肽
酮还原酶(KRED)或羰基还原酶生物催化剂(EC 1.1.1.184)可用于从醛和酮合成醇，以及从对应的前立体异构体酮底物合成光学活性仲醇。KRED也可以催化逆反应(即，醇底物向对应的醛/酮产物的氧化)。KRED对酮和醛的还原以及对醇的氧化使用辅因子，最常见的是还原型烟酰胺腺嘌呤二核苷酸(NADH)或还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)，以及用于氧化反应的烟酰胺腺嘌呤二核苷酸(NAD)或烟酰胺腺嘌呤二核苷酸磷酸(NADP+)。NADH和NADPH充当电子供体，而NAD+和NADP+充当电子受体。
如本领域已知的，KRED酶可见于广泛范围的细菌和酵母中(参见例如，Hummel和Kula Eur.J.Biochem.,184:1-13[1989])。已经报道了许多KRED基因和酶序列，包括木兰假丝酵母(Candida magnoliae)(Genbank登录号JC7338；GI:11360538)；近平滑假丝酵母(Candida parapsilosis)(Genbank登录号BAA24528.1；GI:2815409)，赭色掷孢酵母(Sporobolomyces salmonicolor)(Genbank登录号AF160799；GI:6539734)，开菲尔乳杆菌(Genbank登录号AAP94029.1；GI:33112056)，短乳杆菌(Lactobacillus brevis)(Genbank登录号1NXQ_A；GI:30749782)和布氏嗜热厌氧菌(Thermoanaerobium brockii)(Genbank登录号P14941；GI:1771790)的那些KRED基因和酶序列。
酮还原酶的立体选择性已经被应用于制备重要的药物构建单元(pharmaceuticalbuilding block)(参见例如，Broussy等人，Org.Lett.,11:305-308[2009])。天然存在或工程化的KRED在产生有用的化学化合物的生物催化过程中的具体应用已针对以下被证明：4-氯乙酰乙酸酯的还原(参见例如，Zhou,J.Am.Chem.Soc.,105:5925-5926[1983]；Santaniello,J.Chem.Res.,(S)132-133[1984]；美国专利第5,559,030号；美国专利第5,700,670号；和美国专利第5,891,685号)、二氧代羧酸的还原(参见例如，美国专利第6,399,339号)、叔丁基(S)-氯-5-羟基-3-氧代己酸酯的还原(参见例如，美国专利第6,645,746号；和WO 01/40450)、基于吡咯并三嗪的化合物的还原(参见例如，美国申请公布第2006/0286646号)；取代的苯乙酮的还原(参见例如，美国专利第6,800,477号和第8,748,143号)、以及酮噻茂烷(ketothiolane)的还原(WO 2005/054491)。
异-α-酸(“iso”)以顺式和反式差向异构体的复杂混合物存在。存在总共三种不同的侧链(iso上的“R”)，通常称为n-、ad-和co-(分别是iBu、sBu和iPr)。此外，预计ad-iso(R＝s-Bu)作为一对对映体存在。n-、ad-和co-iso中的每一个也作为对应的顺式/反式-异构体对，以及潜在的由邻近待还原的酮的C4叔醇衍生的对映异构体对存在。如果不考虑特窗酸核心的互变异构体，总共存在异-α-酸的多达16种异构体(4个R的，顺式/反式和对映异构体)。
酶对其作用的底物通常有很好的选择性。因此，单种酶或甚至两种酶的简单混合物可以将异-α-酸的所有16种异构体完全转化为相应的二氢-(ρ)-异α-酸是出乎意料的。令人惊讶的是，本发明包括使用一种或更多种酶和辅因子的简单混合物将异-α-酸转化为二氢-(ρ)-异-α-酸的方法。此外，利用酶(KRED)进行生物转化允许将从异-α-酸制造二氢-(ρ)异-α-酸标记/认证为“天然”。
本发明提供了工程化酮还原酶，这些酶能够通过区域选择性地仅还原叔醇附近异戊烯基侧链上的酮，将异-α-酸的16种主要异构体不加选择地还原为相应的二氢-(ρ)-异-α-酸，如方案1所描绘的。
方案1
Figure BDA0003565647960000271
选择SEQ ID NO:4的酮还原酶多肽作为用于开发本发明提供的改进的酶的初始骨架。SEQ ID NO:4的酶来源于来自开菲尔乳杆菌的野生型酮还原酶(SEQ ID NO:2)。选择SEQID NO:4的多肽作为初始骨架，是由于其将异-α-酸转化为相应的二氢-(ρ)-异α-酸的高活性，以及其相对的底物混杂性和将一系列异葎草酮异构体和差向异构体转化为相应的二氢-(ρ)-异-α-酸的能力。此外，SEQ ID NO:4的多肽在各种反应条件下均表现出活性。发现SEQ ID NO:2的野生型序列在将异-α-酸转化为相应的二氢-(ρ)-异-α-酸方面不具有可检测到的活性。
本发明的工程化酮还原酶多肽是与SEQ ID NO:4的工程化酮还原酶相比具有改进的特性的工程化酮还原酶。在一些实施方案中，与SEQ ID NO:4的工程化多肽相比，本发明的工程化酮还原酶多肽具有改进的将异-α-酸转化为相应的二氢-(ρ)-异-α-酸的活性。在一些其他实施方案中，与SEQ ID NO:4的工程化多肽相比，本发明的工程化酮还原酶多肽具有改进的对一系列异-α-酸底物的活性。在一些其他实施方案中，与SEQ ID NO:4的工程化多肽相比，本发明的工程化酮还原酶多肽具有改进的对一系列底物和辅因子浓度的活性。在一些其他实施方案中，与SEQ ID NO:4的工程化多肽相比，本发明的工程化酮还原酶多肽具有改进的在高底物浓度的活性。在一些其他实施方案中，与SEQ ID NO:4的工程化多肽相比，本发明的工程化酮还原酶多肽具有改进的在低辅因子浓度的活性。
在一些实施方案中，工程化酮还原酶多肽具有改进的对一种或更多种底物的活性。在一些实施方案中，底物包括异-α-酸的混合物。在一些实施方案中，底物包括顺式-异葎草酮。在一些实施方案中，底物包括反式-异葎草酮。在一些实施方案中，底物包括顺式-异辅葎草酮。在一些实施方案中，底物包括反式-异辅葎草酮。在一些实施方案中，底物包括顺式-异近葎草酮。在一些实施方案中，底物包括反式-异近葎草酮。
在一些实施方案中，工程化酮还原酶多肽具有改进的对一种或更多种底物的活性。在一些实施方案中，工程化酮还原酶多肽具有改进的对异-α-酸的混合物的活性。在一些实施方案中，工程化酮还原酶多肽具有改进的对顺式-异葎草酮的活性。在一些实施方案中，工程化酮还原酶多肽具有改进的对反式-异葎草酮的活性。在一些实施方案中，工程化酮还原酶多肽具有改进的对顺式-异辅葎草酮的活性。在一些实施方案中，工程化酮还原酶多肽具有改进的对反式-异辅葎草酮的活性。在一些实施方案中，工程化酮还原酶多肽具有改进的对顺式-异近葎草酮的活性。在一些实施方案中，工程化酮还原酶多肽具有改进的对反式-异近葎草酮的活性。
在一些实施方案中，工程化酮还原酶多肽在存在辅因子再循环系统的情况下将底物化合物转化为产物化合物。在一些实施方案中，辅因子再循环系统包含第二酶，例如葡萄糖脱氢酶。在一些实施方案中，辅因子再循环系统包括异丙醇。
在一些实施方案中，工程化酮还原酶多肽能够在合适的反应条件下以相对于参考多肽SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330的活性增加至少约1.2倍、1.5倍、2倍、3倍、4倍、5倍、10倍、20倍、30倍、40倍、50倍或100倍的活性将底物化合物转化为产物化合物。在一些实施方案中，工程化酮还原酶多肽能够在合适的反应条件下，以约48h、约36h、约24h的反应时间或甚至更短的时间长度，以至少约40％、至少约50％、至少约60％、至少约70％、至少约80％、或至少约90％、至少约95％、至少约98％、至少约99％的转化率百分比将底物化合物转化为产物化合物。
合适的反应条件可包括提供将底物化合物生物催化转化成相应的产物化合物的反应参数的组合。因此，在方法的一些实施方案中，反应参数的组合包括：(a)约0.1g/L至220g/L底物化合物；(b)约0.5g/L至50g/L工程化多肽；(c)约10％-60％异丙醇中约0.01g/L至10g/L NADP+；(d)约5mM至200mM三乙醇胺*H2SO4；(e)约0mM至5mM MgSO4或MgCl2；(f)温度为约25℃至60℃；和(g)6-10的pH。因此，在方法的一些实施方案中，反应参数的组合包括：(a)约10g/L至220g/L底物化合物；(b)约0.5g/L至50g/L工程化多肽；(c)约10％-60％异丙醇中约0.01g/L至10g/L NADP+；(d)约5mM-200mM磷酸钾或磷酸钠；(e)约0mM至5mM MgSO4或MgCl2；(f)温度为约25℃至60℃；和(g)约6至10的pH。
在一些实施方案中，反应参数的组合包括：(a)约80g/L底物化合物；(b)约20g/L工程化多肽；(c)40％异丙醇中约0.01g/L NADP+；(d)约100mM三乙醇胺*H2SO4；(e)约2mMMgSO4；(f)约40℃；和(g)约8的pH。在一些实施方案中，反应参数的组合包括：(a)约160g/L底物化合物；(b)约20g/L工程化多肽；(c)40％异丙醇中约0.01g/L NADP+；(d)约100mM磷酸钾；(e)约2mM MgSO4；(f)约40℃；和(g)约8的pH。
另外的示例性反应条件包括实施例中提供的测定条件。
在一些实施方案中，改进的工程化酮还原酶包含天然存在的酮还原酶多肽中的氨基酸残基缺失，或其他工程化酮还原酶多肽中的氨基酸残基缺失。因此，在本发明的一些实施方案中，缺失包括酮还原酶多肽的1个或更多个氨基酸、2个或更多个氨基酸、3个或更多个氨基酸、4个或更多个氨基酸、5个或更多个氨基酸、6个或更多个氨基酸、8个或更多个氨基酸、10个或更多个氨基酸、15个或更多个氨基酸，或20个或更多个氨基酸、多达氨基酸总数的10％、多达氨基酸总数的10％、多达氨基酸总数的20％，或多达氨基酸总数的30％的缺失，只要酮还原酶的功能活性被保持。在一些实施方案中，缺失可以包括1-2个、1-3个、1-4个、1-5个、1-6个、1-7个、1-8个、1-9个、1-10个、1-11个、1-12个、1-14个、1-15个、1-16个、1-18个、1-20个、1-22个、1-24个、1-25个、1-30个、1-35个或约1-40个氨基酸残基的缺失。在一些实施方案中，缺失的数目可以为1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、14个、15个、16个、18个、20个、22个、24个、26个、30个、35个或约40个氨基酸。在一些实施方案中，缺失可以包括1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、18个或20个氨基酸残基的缺失。
如本文描述的，本发明的酮还原酶多肽可以呈融合多肽的形式，其中酮还原酶多肽与其他多肽诸如抗体标签(例如，myc表位)或纯化序列(例如，His标签)融合。因此，在一些实施方案中，可使用与其他多肽融合或不与其他多肽融合的酮还原酶多肽。
在一些实施方案中，本文描述的多肽不限于遗传编码氨基酸。除了遗传编码氨基酸之外，本文描述的多肽可以完全或部分地包含天然存在的和/或合成的非编码氨基酸。本文描述的多肽可以包含的某些常见的非编码氨基酸包括但不限于：遗传编码氨基酸的D-立体异构体；2,3-二氨基丙酸(Dpr)；α-氨基异丁酸(Aib)；ε-氨基己酸(Aha)；δ-氨基戊酸(Ava)；N-甲基甘氨酸或肌氨酸(MeGly或Sar)；鸟氨酸(Orn)；瓜氨酸(Cit)；叔丁基丙氨酸(Bua)；叔丁基甘氨酸(Bug)；N-甲基异亮氨酸(MeIle)；苯基甘氨酸(Phg)；环己基丙氨酸(Cha)；正亮氨酸(Nle)；萘基丙氨酸(Nal)；2-氯苯丙氨酸(Ocf)；3-氯苯丙氨酸(Mcf)；4-氯苯丙氨酸(Pcf)；2-氟苯丙氨酸(Off)；3-氟苯丙氨酸(Mff)；4-氟苯丙氨酸(Pff)；2-溴苯丙氨酸(Obf)；3-溴苯丙氨酸(Mbf)；4-溴苯丙氨酸(Pbf)；2-甲基苯丙氨酸(Omf)；3-甲基苯丙氨酸(Mmf)；4-甲基苯丙氨酸(Pmf)；2-硝基苯丙氨酸(Onf)；3-硝基苯丙氨酸(Mnf)；4-硝基苯丙氨酸(Pnf)；2-氰基苯丙氨酸(Ocf)；3-氰基苯丙氨酸(Mcf)；4-氰基苯丙氨酸(Pcf)；2-三氟甲基苯丙氨酸(Otf)；3-三氟甲基苯丙氨酸(Mtf)；4-三氟甲基苯丙氨酸(Ptf)；4-氨基苯丙氨酸(Paf)；4-碘苯丙氨酸(Pif)；4-氨基甲基苯丙氨酸(Pamf)；2,4-二氯苯丙氨酸(Opef)；3,4-二氯苯丙氨酸(Mpcf)；2,4-二氟苯丙氨酸(Opff)；3,4-二氟苯丙氨酸(Mpff)；吡啶-2-基丙氨酸(2pAla)；吡啶-3-基丙氨酸(3pAla)；吡啶-4-基丙氨酸(4pAla)；萘-1-基丙氨酸(1nAla)；萘-2-基丙氨酸(2nAla)；噻唑基丙氨酸(taAla)；苯并噻吩基丙氨酸(bAla)；噻吩基丙氨酸(tAla)；呋喃基丙氨酸(fAla)；高苯丙氨酸(hPhe)；高酪氨酸(hTyr)；高色氨酸(hTrp)；五氟苯丙氨酸(5ff)；苯乙烯基丙氨酸(sAla)；蒽基丙氨酸(aAla)；3,3-二苯基丙氨酸(Dfa)；3-氨基-5-苯基戊酸(Afp)；青霉胺(Pen)；1,2,3,4-四氢异喹啉-3-羧酸(Tic)；β-2-噻吩基丙氨酸(Thi)；甲硫氨酸亚砜(Mso)；N(w)-硝基精氨酸(nArg)；高赖氨酸(hLys)；膦酰基甲基苯丙氨酸(pmPhe)；磷酸丝氨酸(pSer)；磷酸苏氨酸(pThr)；高天冬氨酸(hAsp)；高谷氨酸(hGlu)；1-氨基环戊-(2或3)-烯-4-羧酸；哌啶酸(PA)；氮杂环丁烷-3-羧酸(ACA)；1-氨基环戊烷-3-羧酸；烯丙基甘氨酸(aOly)；炔丙基甘氨酸(pgGly)；高丙氨酸(hAla)；正缬氨酸(nVal)；高亮氨酸(hLeu)、高缬氨酸(hVal)；高异亮氨酸(hIle)；高精氨酸(hArg)；N-乙酰赖氨酸(AcLys)；2,4-二氨基丁酸(Dbu)；2,3-二氨基丁酸(Dab)；N-甲基缬氨酸(MeVal)；高半胱氨酸(hCys)；高丝氨酸(hSer)；羟基脯氨酸(Hyp)和高脯氨酸(hPro)。本文描述的多肽可以包含的另外的非编码氨基酸对本领域技术人员是明显的。这些氨基酸可以处于L-构型或D-构型。
本领域技术人员将认识到，带有侧链保护基团的氨基酸或残基也可以构成本文描述的多肽。在这种情况下属于芳族类别的这样的受保护的氨基酸的非限制性实例包括(保护基团在圆括号中列出)但不限于：Arg(tos)、Cys(甲苄基)、Cys(硝基吡啶亚磺酰基)、Glu(δ-苄基酯)、Gln(呫吨基)、Asn(N-δ-呫吨基)、His(bom)、His(苄基)、His(tos)、Lys(fmoc)、Lys(tos)、Ser(O-苄基)、Thr(O-苄基)和Tyr(O-苄基)。
可以构成本文描述的多肽的构象上受限制的非编码氨基酸包括但不限于N-甲基氨基酸(L-构型)；1-氨基环戊-(2或3)-烯-4-羧酸；哌可酸(pipecolic acid)；氮杂环丁烷-3-羧酸；高脯氨酸(hPro)；以及1-氨基环戊烷-3-羧酸。
如以上描述的，被引入天然存在的多肽以产生工程化酮还原酶的各种修饰可以靶向酶的特定特性。
编码工程化酶的多核苷酸
在另一方面，本发明提供了编码工程化酮还原酶的多核苷酸。多核苷酸可以可操作地连接至控制基因表达的一个或更多个异源调控序列，以创建能够表达多肽的重组多核苷酸。包含编码工程化酮还原酶的异源多核苷酸的表达构建体可被引入合适的宿主细胞以表达相应的酮还原酶多肽。
因为知晓对应于各种氨基酸的密码子，蛋白序列的可得性提供了对能够编码目标的所有多核苷酸的描述。其中相同氨基酸由可选择的或同义密码子编码的遗传密码的简并性允许产生极大数目的核酸，所有这些核酸编码本发明的改善的酮还原酶。因此，鉴定了特定的氨基酸序列后，本领域技术人员可以通过以不改变蛋白的氨基酸序列的方式简单修改序列的一个或更多个密码子来制备任何数目的不同核酸。在此方面，本发明特别设想了通过选择基于可能的密码子选择的组合可以进行的每个和每一个可能的多核苷酸变异，并且所有这样的变异应被认为针对本文公开的任何多肽(包括实施例中的表格中呈现的氨基酸序列)具体公开。在各种实施方案中，优选地选择适应在其中产生蛋白的宿主细胞的密码子。例如，在细菌中使用的优选的密码子被用于在细菌中表达基因；在酵母中使用的优选的密码子被用于酵母中的表达；并且在哺乳动物中使用的优选的密码子被用于在哺乳动物细胞中表达。
在一些实施方案中，多核苷酸包括编码天然存在的酮还原酶多肽氨基酸序列的核苷酸序列，如SEQ ID NO:1所呈现的。在一些实施方案中，多核苷酸具有包含与SEQ ID NO:3、5、79、103、171、185、193、251、269、271、285、327和/或329的核酸序列(各自分别编码SEQID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330的相同多肽序列)至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的同一性的核酸序列。
在一些实施方案中，酶多核苷酸编码具有酶活性与本文公开的特性的工程化多肽，其中所述多肽包含与选自本文提供的SEQ ID NO的参考序列或任何变体(例如实施例中提供的那些)的氨基酸序列具有至少60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列，和与一种或更多种参考多核苷酸或如实施例中公开的任何变体的氨基酸序列相比的一个或更多个残基差异(例如1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个氨基酸残基位置)。在一些实施方案中，参考多肽序列选自SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330。
在一些实施方案中，编码工程化酮还原酶的多核苷酸包括与选自SEQ ID NO:3、5、79、103、171、185、193、251、269、271、285、327和/或329的序列具有至少60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的多核苷酸序列。在一些实施方案中，编码工程化酮还原酶的多核苷酸包括SEQ ID NO:5、79、103、171、185、193、251、269、271、285、327和/或329。在一些实施方案中，编码工程化酮还原酶的多核苷酸包括与选自SEQ ID NO:5至411的序列具有至少60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的多核苷酸序列。在一些实施方案中，编码工程化酮还原酶的多核苷酸包括选自SEQ ID NO:5至411的多核苷酸序列。
在一些实施方案中，工程化酮还原酶序列包括包含被鉴定为有益的位置的序列，如实施例中描述的。
在一些实施方案中，将编码改进的酮还原酶的分离的多核苷酸以各种方式操作，以提供多肽的改进的表达和/或产生。取决于使用的表达载体，在将分离的多核苷酸插入载体前对分离的多核苷酸的操作可能是期望的或必要的。用于利用重组DNA方法修饰多核苷酸和核酸序列的技术是本领域熟知的。
对于细菌宿主细胞，用于指导本发明的核酸构建体的转录的合适启动子包括从以下获得的启动子：大肠杆菌(E.coli)lac操纵子、天蓝色链霉菌(Streptomycescoelicolor)琼脂糖酶基因(dagA)、枯草芽孢杆菌(Bacillus subtilis)果聚糖蔗糖酶基因(sacB)、地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因(amyL)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)麦芽糖淀粉酶基因(amyM)、解淀粉芽孢杆菌(Bacillusamyloliquefaciens)α-淀粉酶基因(amyQ)、地衣芽孢杆菌青霉素酶基因(penP)、枯草芽孢杆菌xylA和xylB基因、及原核β-内酰胺酶基因(参见例如，Villa-Kamaroff等人，Proc.NatlAcad.Sci.USA 75:3727-3731[1978])、以及tac启动子(参见例如，DeBoer等人，Proc.NatlAcad.Sci.USA 80:21-25[1983])。另外的合适的启动子对于本领域技术人员是已知的。
对于丝状真菌宿主细胞，用于指导本发明的核酸构建体的转录的合适启动子包括从以下的基因获得的启动子：米曲霉(Aspergillus oryzae)TAKA淀粉酶、米黑根毛霉(Rhizomucor miehei)天冬氨酸蛋白酶、黑曲霉(Aspergillus niger)中性α-淀粉酶、黑曲霉酸稳定型α-淀粉酶、黑曲霉或泡盛曲霉(Aspergillus awamori)葡糖淀粉酶(glaA)、米黑根毛霉脂肪酶、米曲霉碱性蛋白酶、米曲霉磷酸丙糖异构酶、构巢曲霉(Aspergillusnidulans)乙酰胺酶和尖孢镰刀菌(Fusarium oxysporum)胰蛋白酶样蛋白酶(WO 96/00787)、以及NA2-tpi启动子(来自黑曲霉中性α-淀粉酶基因和米曲霉磷酸丙糖异构酶基因的启动子的杂合体)及其突变启动子、截短启动子和杂合启动子。
在酵母宿主中，可用的启动子包括但不限于来自以下的基因的启动子：酿酒酵母(Saccharomyces cerevisiae)烯醇化酶(ENO-1)、酿酒酵母半乳糖激酶(GAL1)、酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)和酿酒酵母3-磷酸甘油酸激酶，以及用于酵母宿主细胞的其他可用启动子(参见例如，Romanos等人，Yeast 8:423-488[1992])。
控制序列还可以是合适的转录终止子序列，转录终止子序列是由宿主细胞识别以终止转录的序列。终止子序列被可操作地连接至编码多肽的核酸序列的3’末端。在本发明中可以使用在所选择的宿主细胞中起作用的任何终止子。
例如，用于丝状真菌宿主细胞的示例性转录终止子可以从以下的基因获得：米曲霉TAKA淀粉酶、黑曲霉葡糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、黑曲霉α-葡萄糖苷酶和尖孢镰刀菌胰蛋白酶样蛋白酶。
用于酵母宿主细胞的示例性终止子可以从以下的基因获得：酿酒酵母烯醇化酶、酿酒酵母细胞色素C(CYC1)、和酿酒酵母甘油醛-3-磷酸脱氢酶，以及本领域已知的用于酵母宿主细胞的其他可用的终止子(参见例如，Romanos等人，同上)。
控制序列还可以是合适的前导序列，前导序列是对宿主细胞的翻译重要的mRNA的非翻译区。前导序列被可操作地连接至编码多肽的核酸序列的5’末端。可以使用在所选择的宿主细胞中有功能的任何前导序列。用于丝状真菌宿主细胞的示例性前导序列从以下的基因获得：米曲霉TAKA淀粉酶和构巢曲霉磷酸丙糖异构酶。用于酵母宿主细胞的合适的前导序列从以下的基因获得：酿酒酵母烯醇化酶(ENO-1)、酿酒酵母3-磷酸甘油酸激酶、酿酒酵母α-因子和酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)。
控制序列还可以是多腺苷酸化序列，多腺苷酸化序列是被可操作地连接至核酸序列的3’末端的序列，并且在转录时，其被宿主细胞识别为将多腺苷残基添加至转录的mRNA的信号。在本发明中可以使用在所选择的宿主细胞中起作用的任何多腺苷酸化序列。用于丝状真菌宿主细胞的示例性多腺苷酸化序列可以来自以下的基因：米曲霉TAKA淀粉酶、黑曲霉葡糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、尖孢镰刀菌胰蛋白酶样蛋白酶和黑曲霉α-葡糖苷酶，以及本领域已知的用于酵母宿主细胞的其他可用的多腺苷酸化序列(参见例如，Guo等人，Mol.Cell.Biol.,15:5983-5990[1995])。
控制序列还可以是信号肽编码区域，其编码与多肽的氨基末端连接并指导所编码的多肽进入细胞的分泌途径的氨基酸序列。核酸序列的编码序列的5’末端可以固有地包含信号肽编码区，所述信号肽编码区符合翻译阅读框地(in translation reading frame)与编码分泌多肽的编码区的区段天然地连接。可选地，编码序列的5’末端可以包含对编码序列是外源的信号肽编码区。当编码序列不天然包含信号肽编码区时可能需要外源信号肽编码区。
可选择地，外源信号肽编码区可以简单替换天然信号肽编码区以增加多肽的分泌。然而，指导所表达的多肽进入所选择的宿主细胞的分泌途径的任何信号肽编码区可以在本发明中使用。
用于细菌宿主细胞的有效的信号肽编码区是从以下的基因获得的信号肽编码区：芽孢杆菌NClB 11837麦芽糖淀粉酶、嗜热脂肪芽孢杆菌α-淀粉酶、地衣芽孢杆菌枯草杆菌蛋白酶、地衣芽孢杆菌β-内酰胺酶、嗜热脂肪芽孢杆菌中性蛋白酶(nprT、nprS、nprM)和枯草芽孢杆菌prsA、以及本领域已知的另外的信号肽(参见例如，Simonen等人，Microbiol.Rev.,57:109-137[1993])。
用于丝状真菌宿主细胞的有效的信号肽编码区包括但不限于从以下的基因获得的信号肽编码区：米曲霉TAKA淀粉酶、黑曲霉中性淀粉酶、黑曲霉葡糖淀粉酶、米黑根毛霉天冬氨酸蛋白酶、特异腐质霉(Humicola insolens)纤维素酶和绵毛状腐质霉(Humicolalanuginosa)脂肪酶。用于酵母宿主细胞的可用的信号肽可以来自以下的基因：酿酒酵母α因子和酿酒酵母转化酶，以及另外的可用的信号肽编码区(参见例如，Romanos等人，1992，同上)。
控制序列还可以是编码定位于多肽的氨基末端的氨基酸序列的前肽编码区。所得多肽被称为酶原(proenzyme)或多肽原(或在某些情况下为酶原(zymogen))。多肽原通常是无活性的并且可以通过前肽从多肽原的催化裂解或自动催化裂解转化为成熟的有活性的多肽。前肽编码区可以从以下的基因获得：枯草芽孢杆菌碱性蛋白酶(aprE)、枯草芽孢杆菌中性蛋白酶(nprT)、酿酒酵母α-因子、米黑根毛霉天冬氨酸蛋白酶和嗜热毁丝霉(Myceliophthora thermophila)乳糖酶(WO 95/33836)。
在信号肽和前肽区域两者均存在于多肽的氨基末端时，前肽区域紧邻多肽的氨基末端定位并且信号肽区域紧邻前肽区域的氨基末端定位。
添加允许相对于宿主细胞的生长来调控多肽的表达的调控序列也可能是期望的。调控系统的实例是引起基因表达响应于化学或物理刺激(包括调控化合物的存在)而开启或关闭的那些。在原核宿主细胞中，合适的调控序列包括lac、tac和trp操纵子系统。在酵母宿主细胞中，合适的调控系统包括例如ADH2系统或GAL1系统。在丝状真菌中，合适的调控序列包括TAKAα-淀粉酶启动子、黑曲霉葡糖淀粉酶启动子和米曲霉葡糖淀粉酶启动子。
调控序列的其他实例是允许基因扩增的那些。在真核系统中，这些包括在氨甲蝶呤存在下扩增的二氢叶酸还原酶基因以及用重金属扩增的金属硫蛋白基因。在这些情况下，编码本发明的KRED多肽的核酸序列与调控序列可操作地连接。
因此，在一些实施方案中，本发明也涉及重组的表达载体，该重组的表达载体包含编码工程化酮还原酶多肽或其变体的多核苷酸，以及取决于其将要引入的宿主的类型，一个或多个表达调控区域，诸如启动子和终止子，复制起点等。以上描述的各种核酸和控制序列可以被连接在一起以产生重组表达载体，所述重组表达载体可以包括一个或更多个方便的限制性位点，以允许在这样的位点处插入或取代编码多肽的核酸序列。可选择地，本发明的核酸序列可以通过将包含该序列的核酸序列或核酸构建体插入到用于表达的适当的载体中来表达。在产生表达载体时，编码序列位于载体中，使得编码序列与用于表达的适当的控制序列可操作地连接。
重组表达载体可以是任何载体(例如，质粒或病毒)，其可以方便地经受重组DNA程序并且可以引起多核苷酸序列的表达。载体的选择通常将取决于载体与待引入载体的宿主细胞的相容性。载体可以是线性质粒或闭合的环状质粒。
表达载体可以是自主复制载体(即，作为染色体外实体存在的载体)，其复制独立于染色体复制(例如，质粒、染色体外元件、微型染色体或人工染色体)。载体可以包含用于确保自我复制的任何工具(means)。可选择地，载体可以是当被引入宿主细胞中时被整合到基因组中并与其被整合进的染色体一起复制的载体。此外，可以使用单一载体或质粒或者一起包含待引入到宿主细胞基因组中的总DNA的两种或更多种载体或质粒、或转座子。
本发明的表达载体优选地含有一个或更多个选择性标志物(selectablemarkers)，其允许容易地选择转化的细胞。选择性标志物可以是基因，其产物提供杀生物剂或病毒抗性、重金属抗性、针对营养缺陷型的原养型等。细菌的选择性标志物的实例是来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因或赋予抗生素抗性诸如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的标志物。用于酵母宿主细胞的合适的标志物为ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。
用于在丝状真菌宿主细胞中使用的选择性标志物包括但不限于amdS(乙酰胺酶)、argB(鸟氨酸氨甲酰基转移酶)、bar(膦丝菌素乙酰基转移酶)、hph(潮霉素磷酸转移酶)、niaD(硝酸盐还原酶)、pyrG(乳清酸核苷-5’-磷酸脱羧酶)、sC(硫酸腺苷酰转移酶)和trpC(邻氨基苯甲酸合酶)及其等同物。用于在曲霉属真菌细胞中使用的实施方案包括构巢曲霉或米曲霉的amdS和pyrG基因以及吸水链霉菌(Streptomyces hygroscopicus)的bar基因。
本发明的表达载体可以包含允许载体整合到宿主细胞的基因组中或允许载体在细胞中独立于基因组自主复制的一个或更多个元件。为了整合到宿主细胞基因组中，载体可以依赖于核酸序列，所述核酸序列编码用于通过同源或非同源重组将载体整合到基因组中的多肽或载体的任何其他元件。
可选择地，表达载体可以包含用于指导通过同源重组整合到宿主细胞的基因组中的另外的核酸序列。另外的核酸序列使载体能够在一条或更多条染色体中的一个或更多个精确位置处整合到宿主细胞基因组中。为了增加在准确位置整合的可能性，整合元件应当优选地包含与对应的靶序列高度同源的足够数目的核酸，诸如100个至10,000个碱基对，优选地400个至10,000个碱基对，并且最优选地800个至10,000个碱基对，以增强同源重组的可能性。整合元件可以是与宿主细胞的基因组中的靶序列同源的任何序列。此外，整合元件可以是非编码或编码核酸序列。在另一方面，载体可以通过非同源重组整合到宿主细胞的基因组中。
对于自主复制，载体还可以包含复制起点，使得载体能够在所讨论的宿主细胞中自主复制。细菌复制起点的实例是允许在大肠杆菌中复制的P15A ori或质粒pBR322、pUC19、pACYC177(该质粒具有P15A ori)、或pACYC184的复制起点，和允许在芽孢杆菌中复制的pUB110、pE194、pTA1060或pAMβ1的复制起点。用于在酵母宿主细胞中使用的复制起点的实例是2μm复制起点、ARS1、ARS4、ARS1和CEN3的组合以及ARS4和CEN6的组合。复制起点可以是具有使其在宿主细胞中的功能对温度敏感的突变的复制起点(参见例如，Ehrlich,Proc.Natl.Acad.Sci.USA 75:1433[1978])。
可以将多于一个拷贝的本发明的核酸序列插入宿主细胞中以增加基因产物的产量。核酸序列拷贝数的增加可以通过将该序列的至少一个另外的拷贝整合到宿主细胞基因组中，或者通过将可扩增的选择性标志物基因包含在核酸序列中来获得，其中可以通过在合适的选择剂的存在下培养细胞来选择含有选择标志物基因的扩增的拷贝并从而含有核酸序列的另外的拷贝的细胞。
本发明并不意图局限于本文公开的表达载体。本领域技术人员将认识到，任何合适的表达载体可以用于本发明。用于本发明的许多表达载体是商购可得的。合适的商业表达载体包括但不限于p3xFLAGTMTM表达载体(Sigma-Aldrich)，其包括用于在哺乳动物宿主细胞中表达的CMV启动子和hGH多腺苷酸化位点以及用于在大肠杆菌中扩增的pBR322复制起点和氨苄青霉素抗性标志物。其他商购可得的合适的表达载体包括但不限于pBluescriptII SK(-)和pBK-CMV载体(Stratagene)，以及源自pBR322(Gibco BRL)、pUC(Gibco BRL)、pREP4、pCEP4(Invitrogen)或pPoly的质粒(参见，Lathe等人，Gene 57:193-201[1987])。
用于表达工程化多肽的宿主细胞
本发明还提供了包含编码本发明的改进的酮还原酶多肽的多核苷酸的宿主细胞，所述多核苷酸被可操作地连接至一个或更多个控制序列以在宿主细胞中表达酮还原酶。用于表达由本发明的表达载体编码的KRED多肽的宿主细胞是本领域熟知的，并且包括但不限于细菌细胞，诸如大肠杆菌、开菲尔乳杆菌、短乳杆菌、微小乳杆菌(Lactobacillusminor)、链霉菌属和鼠伤寒沙门氏菌(Salmonella typhimurium)细胞；真菌细胞，诸如酵母细胞(例如，酿酒酵母或巴斯德毕赤酵母(Pichia pastoris)(ATCC登录号201178))；昆虫细胞，诸如果蝇属(Drosophila)S2和夜蛾属(Spodoptera)Sf9细胞；动物细胞，诸如CHO、COS、BHK、293和Bowes黑素瘤细胞；以及植物细胞。用于上文描述的宿主细胞的适当的培养基和生长条件是本领域熟知的。
用于表达酮还原酶的多核苷酸可以通过本领域已知的各种方法引入细胞中。技术包括，除其他以外，电穿孔、生物弹射粒子轰击(biolistic particle bombardment)、脂质体介导的转染、氯化钙转染和原生质体融合。用于将多核苷酸引入细胞中的各种方法对技术人员将是明显的。
大肠杆菌W3110是可用于本发明的宿主菌株，尽管并不意图本发明局限于这种特定的宿主菌株。表达载体通过将编码改进的酶的多核苷酸可操作地连接至质粒pCK110900中来创建，所述编码改进的酶的多核苷酸可操作地连接至在lacI阻遏物的控制下的lac启动子。表达载体还包含P15a复制起点和氯霉素抗性基因。大肠杆菌W3110中包含主题多核苷酸的细胞可以通过使细胞经受氯霉素选择来分离。
产生工程化酮还原酶多肽的方法
在一些实施方案中，为了制备本发明的改进的KRED多核苷酸和多肽，催化还原反应的天然存在的酮还原酶从开菲尔乳杆菌获得(或来源)。在一些实施方案中，对亲本多核苷酸序列密码子优化以增强酮还原酶在指定宿主细胞中的表达。作为说明，编码开菲尔乳杆菌的野生型KRED多肽的亲本多核苷酸序列由寡核苷酸构建，所述寡核苷酸基于可从Genbank数据库获得的开菲尔乳杆菌KRED序列的已知多肽序列来制备。对亲本多核苷酸序列进行密码子优化以用于在大肠杆菌中表达，并将密码子优化的多核苷酸克隆到表达载体中，将酮还原酶基因的表达置于lac启动子和lacI阻遏物基因的控制下。鉴定在大肠杆菌中表达活性酮还原酶的克隆，并对基因测序以确认其身份。
在一些实施方案中，如以上讨论的，工程化酮还原酶可以通过使编码天然存在的酮还原酶的多核苷酸经受诱变和/或定向演化方法来获得。诱变可以根据本领域已知的任何技术来进行，包括随机诱变和定点诱变。定向演化可以用本领域已知的任何技术包括重排来进行，以筛选改进的启动子变体。诱变和定向演化方法是本领域熟知的(参见例如，美国专利号5,605,793、5,811,238、5,830,721、5,834,252、5,837,458、5,928,905、6,096,548、6,117,679、6,132,970、6,165,793、6,180,406、6,251,674、6,265,201、6,277,638、6,287,861、6,287,862、6,291,242、6,297,053、6,303,344、6,309,883、6,319,713、6,319,714、6,323,030、6,326,204、6,335,160、6,335,198、6,344,356、6,352,859、6,355,484、6,358,740、6,358,742、6,365,377、6,365,408、6,368,861、6,372,497、6,337,186、6,376,246、6,379,964、6,387,702、6,391,552、6,391,640、6,395,547、6,406,855、6,406,910、6,413,745、6,413,774、6,420,175、6,423,542、6,426,224、6,436,675、6,444,468、6,455,253、6,479,652、6,482,647、6,483,011、6,484,105、6,489,146、6,500,617、6,500,639、6,506,602、6,506,603、6,518,065、6,519,065、6,521,453、6,528,311、6,537,746、6,573,098、6,576,467、6,579,678、6,586,182、6,602,986、6,605,430、6,613,514、6,653,072、6,686,515、6,703,240、6,716,631、6,825,001、6,902,922、6,917,882、6,946,296、6,961,664、6,995,017、7,024,312、7,058,515、7,105,297、7,148,054、7,220,566、7,288,375、7,384,387、7,421,347、7,430,477、7,462,469、7,534,564、7,620,500、7,620,502、7,629,170、7,702,464、7,747,391、7,747,393、7,751,986、7,776,598、7,783,428、7,795,030、7,853,410、7,868,138、7,783,428、7,873,477、7,873,499、7,904,249、7,957,912、7,981,614、8,014,961、8,029,988、8,048,674、8,058,001、8,076,138、8,108,150、8,170,806、8,224,580、8,377,681、8,383,346、8,457,903、8,504,498、8,589,085、8,762,066、8,768,871、9,593,326和所有相关的非美国的对应专利；Ling等人，Anal.Biochem.,254(2):157-78[1997]；Dale等人，Meth.Mol.Biol.,57:369-74[1996]；Smith,Ann.Rev.Genet.,19:423-462[1985]；Botstein等人，Science,229:1193-1201[1985]；Carter,Biochem.J.,237:1-7[1986]；Kramer等人，Cell,38:879-887[1984]；Wells等人，Gene,34:315-323[1985]；Minshull等人，Curr.Op.Chem.Biol.,3:284-290[1999]；Christians等人，Nat.Biotechnol.,17:259-264[1999]；Crameri等人，Nature,391:288-291[1998]；Crameri,等人，Nat.Biotechnol.,15:436-438[1997]；Zhang等人，Proc.Nat.Acad.Sci.U.S.A.,94:4504-4509[1997]；Crameri等人，Nat.Biotechnol.,14:315-319[1996]；Stemmer,Nature,370:389-391[1994]；Stemmer,Proc.Nat.Acad.Sci.USA,91:10747-10751[1994]；WO 95/22625；WO 97/0078；WO 97/35966；WO 98/27230；WO 00/42651；WO 01/75767和WO 2009/152336；所有这些通过引用并入本文)。
对诱变处理后获得的克隆筛选具有期望的改进的酶特性的工程化酮还原酶。测量来自表达文库的酶活性可以使用标准生物化学技术来进行，所述标准生物化学技术监测随着NADH或NADPH被转化为NAD+或NADP+，NADH或NADPH浓度的降低速率(通过吸光度或荧光的降低)。在这个反应中，当酮还原酶将酮底物还原为对应的羟基基团时，NADH或NADPH被酮还原酶消耗(氧化)。如通过每单位时间吸光度或荧光的降低测量的NADH或NADPH浓度的降低速率指示固定量的裂解物(或由其制成的冻干粉末)中的KRED多肽的相对(酶促)活性。产物的立体化学可以通过各种已知技术来确定，并如实施例中所提供的。在期望的改进的酶特性为热稳定性时，酶活性可在将酶制品经受确定的温度和测量热处理后余下的酶促活性的量之后测量到。然后对包含编码酮还原酶的多核苷酸的克隆进行分离、测序以鉴定核苷酸序列的改变(如果有的话)、并且用于在宿主细胞中表达酶。
当已知工程化多肽的序列时，编码酶的多核苷酸可以根据已知的合成方法通过标准固相方法来制备。在一些实施方案中，多达约100个碱基的片段可以被单独地合成、然后连接(例如，通过酶促连接方法或化学连接方法或聚合酶介导的方法)以形成任何期望的连续序列。例如，本发明的多核苷酸和寡核苷酸可以通过化学合成来制备(例如，使用由Beaucage等人，Tet.Lett.,22:1859-69[1981]描述的经典亚磷酰胺法，或由Matthes等人，EMBO J.,3:801-05[1984]描述的方法，如自动化合成方法中通常实践的)。根据亚磷酰胺方法，寡核苷酸被合成(例如，在自动DNA合成仪中)、纯化、退火、连接并克隆入适当的载体。此外，基本上任何核酸可以从各种商业来源中的任一种获得(例如，The Midland CertifiedReagent Company,Midland,TX，The Great American Gene Company,Ramona,CA，ExpressGen Inc.Chicago,IL，Operon Technologies Inc.,Alameda,CA，以及许多其他商业来源)。
在宿主细胞中表达的工程化酮还原酶可以使用用于蛋白纯化的熟知的技术中的任一种或更多种从细胞和/或培养基回收，所述技术尤其包括溶菌酶处理、声处理(sonication)、过滤、盐析、超速离心和色谱。用于裂解和从细菌诸如大肠杆菌高效提取蛋白的合适的溶液是以商标名CelLyticTM(Sigma-Aldrich)商购可得的。
分离酮还原酶多肽的色谱技术，包括但不限于反相色谱、高效液相色谱、离子交换色谱、凝胶电泳和亲和色谱。用于纯化特定酶的条件将部分地取决于诸如净电荷、疏水性、亲水性、分子量、分子形状等因素，并且对于本领域技术人员将是明显的。
在一些实施方案中，亲和技术用于分离改进的酮还原酶。对于亲和色谱纯化，可使用特异性结合酮还原酶多肽的任何抗体。为了产生抗体，可以通过注射酮还原酶免疫接种各种宿主动物，包括但不限于兔、小鼠、大鼠等。酮还原酶多肽可以通过侧链官能基团或附接至侧链官能基团的接头的手段被附接至合适的载体诸如BSA。根据宿主物种，可以使用各种佐剂增强免疫应答，佐剂包括但不限于弗氏(完全和不完全)、矿物凝胶诸如氢氧化铝、表面活性物质诸如溶血卵磷脂、普朗尼克多元醇、聚阴离子、肽、油乳剂、钥孔
Figure BDA0003565647960000431
血蓝蛋白(keyhole limpet hemocyanin)、二硝基苯酚，以及潜在有用的人类佐剂诸如BCG(卡介苗)和短棒状杆菌(Corynebacterium parvum)。
酮还原酶可以以表达酶的细胞的形式、作为粗提取物、或作为分离的或纯化的制品来制备及使用。酮还原酶可以被制备为冻干物、呈粉末形式(例如，丙酮粉末)或被制备为酶溶液。在一些实施方案中，酮还原酶可以是基本上纯的制品形式。
在一些实施方案中，酮还原酶多肽可以被附接至固体基底。基底可以是固相、表面和/或膜。固体支持物可以包括有机聚合物，诸如聚苯乙烯、聚乙烯、聚丙烯、聚氟乙烯、聚氧乙烯(polyethyleneoxy)和聚丙烯酰胺以及它们的共聚物和接枝物。固体支持物还可以是无机的，诸如玻璃、二氧化硅、可控孔隙玻璃(CPG)、反相二氧化硅或金属诸如金或铂。基底的构型可以呈珠、球、颗粒(particle)、小粒(granule)、凝胶、膜或表面的形式。表面可以是平坦的、基本上平坦的或非平坦的。固体支持物可以是多孔的或无孔的，并且可以具有溶胀或非溶胀特征。固体支持物可以被配置为孔、凹陷(depression)或其他容器(container)、器皿(vessel)、特征或位置的形式。多于一个支持物可以被配置在阵列上于多个位置处，所述位置可用试剂的自动递送或通过检测方法和/或仪器寻址。
如本领域技术人员已知的，酮还原酶催化的还原反应通常需要辅因子。由本文描述的工程化酮还原酶催化的还原反应通常也需要辅因子，尽管工程化酮还原酶的许多实施方案比用野生型酮还原酶催化的反应需要少得多的辅因子。如本文使用的，术语“辅因子”是指与酮还原酶联合运作的非蛋白化合物。适合于与本文描述的工程化酮还原酶一起使用的辅因子包括但不限于NADP+(烟酰胺腺嘌呤二核苷酸磷酸)、NADPH(NADP+的还原形式)、NAD+(烟酰胺腺嘌呤二核苷酸)和NADH(NAD+的还原形式)。通常，将辅因子的还原形式添加到反应混合物中。还原的NAD(P)H形式可以使用辅因子再生系统任选地由氧化的NAD(P)+形式再生。术语“辅因子再生系统”是指参与使辅因子的氧化形式还原的反应(例如，NADP+到NADPH)的一组反应物。通过酮还原酶催化的酮底物的还原而被氧化的辅因子通过辅因子再生系统以还原形式再生。辅因子再生系统包括化学计量还原剂，所述化学计量还原剂是还原氢等同物的来源，并且能够使辅因子的氧化形式还原。辅因子再生系统还可以包含催化剂，例如催化使辅因子的氧化形式被还原剂还原的酶催化剂。辅助因子再生系统还可以包括辅底物，诸如异丙醇。分别由NAD+或NADP+再生NADH或NADPH的辅因子再生系统是本领域已知的，并且可以用于本文描述的方法中。
实验
本发明的各种特征和实施方案在以下代表性实施例中进行了说明，这些实施例旨在说明而非限制。
在以下实验公开内容中，以下缩写适用：ppm(百万分率)；M(摩尔/升)；mM(毫摩尔/升)，uM和μM(微摩尔/升)；nM(纳摩尔/升)；mol(摩尔)；gm和g(克)；mg(毫克)；ug和μg(微克)；L和l(升)；ml和mL(毫升)；cm(厘米)；mm(毫米)；um和μm(微米)；sec.(秒)；min(分钟)；h和hr(小时)；U(单位)；MW(分子量)；rpm(转/分)；℃(摄氏度)；RT(室温)；CDS(编码序列)；DNA(脱氧核糖核酸)；RNA(核糖核酸)；HPLC(高效液相色谱)；FIOPC(相对于阳性对照的倍数改进)；HTP(高通量)；LB(Luria肉汤)；KPO4(磷酸钾)；KPO3(亚磷酸钾)；TEoA(三乙醇胺)；PMBS(硫酸多粘菌素B)；Sigma-Aldrich(Sigma-Aldrich,St.Louis,MO)；Millipore(Millipore,Corp.,Billerica MA)；Difco(Difco Laboratories,BD DiagnosticSystems,Detroit,MI)；Daicel(Daicel,West Chester,PA)；Genetix(Genetix USA,Inc.,Beaverton,OR)；Molecular Devices(Molecular Devices,LLC,Sunnyvale,CA)；AppliedBiosystems(Applied Biosystems,Life Technologies,Corp.的一部分,Grand Island,NY),Agilent(Agilent Technologies,Inc.,Santa Clara,CA)；Thermo Scientific(Thermo Fisher Scientific的一部分,Waltham,MA)；Corning(Corning,Inc.,Palo Alto,CA)；和Bio-Rad(Bio-Rad Laboratories,Hercules,CA)。
在本发明的开发中使用了以下序列。
SEQ ID NO:413：
Figure BDA0003565647960000461
Figure BDA0003565647960000471
Figure BDA0003565647960000481
SEQ ID NO:414：
Figure BDA0003565647960000482
Figure BDA0003565647960000491
实施例1
包含重组KRED基因的大肠杆菌表达宿主
用于产生本发明的变体的初始KRED酶从Codexis收集的商购可得KRED酶小组获得。在初始筛选期间，变体SEQ ID NO:4产生最多的产物，如通过LC/MS确定的。将KRED编码基因克隆到表达载体系统中，包括pCK110900(参见美国专利申请公布第2006/0195947号的图3)、SEQ ID NO:413或SEQ ID NO:414中，可操作地连接到在lacl阻遏物的控制下的lac启动子。表达载体系统还包含P15a复制起点和氯霉素抗性基因。本发明并不意图局限于本文公开的表达载体。本领域技术人员将认识到，任何合适的表达载体可以用于本发明，包括但不限于p3xFLAGTMTM表达载体(Sigma-Aldrich)、pBluescriptII SK(-)和pBK-CMV载体(Stratagene)，以及来源于pBR322(Gibco BRL)、pUC(Gibco BRL)、pREP4、pCEP4(Invitrogen)或pPoly(参见，Lathe等人，Gene 57:193-201[1987])的质粒。
使用本领域已知的标准方法将所得质粒转化到大肠杆菌W3110中。如本领域已知的，通过使细胞经受氯霉素选择来分离转化体(参见例如，美国专利第8,383,346号和WO2010/144103)。
实施例2
HTP含KRED的湿细胞沉淀的制备
将来自单克隆菌落的含有重组KRED编码基因的大肠杆菌细胞接种到96孔浅孔微量滴定板的孔中的含有1％葡萄糖和30μg/mL氯霉素的190μl LB中。将板用O2可透过的密封件(seal)密封，并使培养物在20℃、200rpm和85％湿度生长过夜。然后，将20μl的每种细胞培养物转移到含有380μL TB和30μg/mL CAM的96孔深孔板的孔中。将深孔板用O2可透过的密封件密封，并且在30℃、250rpm和85％湿度孵育，直到达到0.6-0.8的OD600。然后将细胞培养物用达到1mM的最终浓度的IPTG诱导，并且在与最初使用的相同条件下孵育过夜。然后用在4℃、4000rpm持续10分钟的离心将细胞沉淀。弃去上清液，并且在裂解前将沉淀冷冻在-80℃。
实施例3
HTP含KRED的细胞裂解物的制备
首先，通过加入150μL含有100mM pH 8三乙醇胺*H2SO4与2mM MgSO4或100mM pH 8磷酸钾与2mM MgSO4、1g/L溶菌酶和0.5g/L PMBS的裂解缓冲液裂解如实施例2所述产生的细胞沉淀。然后，在桌上型摇动器(bench top shaker)上在室温摇动细胞沉淀2小时。将板在4℃以4000rpm离心15分钟，以去除细胞碎片。然后将上清液用于生物催化反应以确定它们的活性水平。
实施例4
从摇瓶(SF)培养物制备冻干裂解物
摇瓶程序可用于生成工程化KRED多肽摇瓶粉末(SFP)，其可用于二次筛选测定和/或用于本文所述的生物催化过程。与HTP测定中使用的细胞裂解物相比，酶的摇瓶粉末(SFP)制品提供了更纯化的工程化酶的制品(例如，多达总蛋白的30％)，并且还允许使用更浓缩的酶溶液。首先，将如上文描述地生长的选定HTP培养物铺板在具有1％葡萄糖和30μg/ml氯霉素(CAM)的LB琼脂板上，并在37℃生长过夜。将来自每个培养物的单个菌落转移到6ml具有1％葡萄糖和30μg/ml CAM的LB中。使培养物在30℃在250rpm生长18h，并以约1:50传代培养至250ml含30μg/ml CAM的TB中，至0.05的最终OD600。使培养物在30℃在250rpm生长约3小时，达到0.8-1.0之间的OD600，并用1mM IPTG诱导。然后使培养物在30℃在250rpm生长20h。将培养物离心(4000rpm，在4℃持续20分钟)。弃去上清液，并将沉淀重悬在35ml的50mM pH 8磷酸钾与2mM MgSO4中。将重悬的细胞离心(4000rpm，在4℃持续20分钟)。弃去上清液，并将沉淀重悬在6ml的50mM pH 8磷酸钾与2mM MgSO4中，并使用来自ConstantSystems(One Shot)的细胞破坏仪裂解细胞。使裂解物沉淀(10,000rpm，在4℃持续60min)并且将上清液冷冻并冻干以产生摇瓶(SF)酶。
实施例5
来源于SEQ ID NO:4的工程化多肽的演化与针对改进的KRED活性的筛选
基于筛选变体还原异-α-酸底物的结果，选择SEQ ID NO:4作为亲本酶。使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码SEQ ID NO:4的具有KRED活性的多肽的工程化多核苷酸(即，SEQ ID NO:3)用于生成表5-1的另外的工程化多肽。与起始多肽相比，这些多肽显示出从异-α-酸形成二氢-(ρ)-异-α-酸的改进。使用上文描述的定向演化方法与以下表5-2所述的HTP测定和分析方法一起，从SEQ ID NO:4的“骨架”氨基酸序列产生工程化多肽。
定向演化始于SEQ ID NO:3中所列的多核苷酸。然后选择工程化多肽作为起始的“骨架”基因序列。工程化多肽的文库使用各种熟知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生并使用HTP测定和分析方法进行筛选，该HTP测定和分析方法测量多肽将异-α-酸底物转化为期望的二氢-(ρ)-异-α-酸产物的能力。
酶测定以96孔形式在200μL总体积/孔中进行，其包括在100mM pH 8三乙醇胺*H2SO4与2mM MgSO4中的50％v/v HTP酶裂解物、8g/L异-α-酸底物(
Figure BDA0003565647960000522
异构化啤酒花提取物溶液,Kalsec)以及40体积％异丙醇(IPA)中的0.1g/L NADP。将板密封并在40℃以600rpm摇动孵育20-24小时。
20-24小时后，加入1000μL的具有0.1％乙酸的乙腈。将板密封，并在4℃以4000rpm离心10分钟。将猝灭的样品在50:50乙腈:水混合物中进一步稀释4-5x，然后进行HPLC分析。HPLC运行参数在下表5-2中描述。
Figure BDA0003565647960000521
Figure BDA0003565647960000531
Figure BDA0003565647960000532
Figure BDA0003565647960000541
实施例6
来源于SEQ ID NO:6的工程化多肽的演化与针对改进的KRED活性的筛选
使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码SEQ ID NO:6的具有KRED活性的多肽的工程化多核苷酸(即，SEQ ID NO:5)用于生成表6-1的另外的工程化多肽。与起始多肽相比，这些多肽显示出从异-α-酸形成二氢-(ρ)-异-α-酸的改进。使用上文描述的定向演化方法与表5-2中所述的HTP测定和分析方法一起，从SEQ ID NO:6的“骨架”氨基酸序列产生工程化多肽。
定向演化始于SEQ ID NO:5中所列的多核苷酸。然后选择工程化多肽作为起始的“骨架”基因序列。工程化多肽的文库使用各种熟知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生并使用HTP测定和分析方法进行筛选，该HTP测定和分析方法测量多肽将异-α-酸底物转化为期望的二氢-(ρ)-异-α-酸产物的能力。
酶测定以96孔形式在200μL总体积/孔中进行，其包括在100mM pH 8三乙醇胺*H2SO4与2mM MgSO4中的50％v/v HTP酶裂解物、16g/L或40g/L异-α-酸底物(
Figure BDA0003565647960000542
异构化啤酒花提取物溶液,Kalsec)以及40体积％异丙醇(IPA)中的0.1g/L NADP。将板密封并在40℃以600rpm摇动孵育20-24小时。
20-24小时后，加入1000μL的具有0.1％乙酸的乙腈。将板密封，并在4℃以4000rpm离心10分钟。将猝灭的样品在50:50乙腈:水混合物中进一步稀释10-20x，然后进行HPLC分析。HPLC运行参数在表5-2中描述。
Figure BDA0003565647960000551
实施例7
来源于SEQ ID NO:80的工程化多肽的演化与针对改进的KRED活性的筛选
使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码SEQ ID NO:80的具有KRED活性的多肽的工程化多核苷酸(即，SEQ ID NO:79)用于生成表7-1的另外的工程化多肽。与起始多肽相比，这些多肽显示出从异-α-酸形成二氢-(ρ)-异-α-酸的改进。使用上文描述的定向演化方法与表5-2中所述的HTP测定和分析方法一起，从SEQ ID NO:80的“骨架”氨基酸序列产生工程化多肽。
定向演化始于SEQ ID NO:79中所列的多核苷酸。然后选择工程化多肽作为起始的“骨架”基因序列。工程化多肽的文库使用各种熟知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生并使用HTP测定和分析方法进行筛选，该HTP测定和分析方法测量多肽将异-α-酸底物转化为期望的二氢-(ρ)-异-α-酸产物的能力。
酶测定以96孔形式在200μL总体积/孔中进行，其在100mM pH 8磷酸钾与2mMMgSO4中的包括25％v/v HTP酶裂解物、60g/L或80g/L异-α-酸底物(
Figure BDA0003565647960000562
异构化啤酒花提取物溶液,Kalsec)以及40体积％异丙醇(IPA)中的0.02g/L NADP。将板密封并在45℃以600rpm摇动孵育20-24小时。
20-24小时后，加入1000μL的具有0.1％乙酸的乙腈。将板密封，并在4℃以4000rpm离心10分钟。将猝灭的样品在50:50乙腈:水混合物中进一步稀释20-40x，然后进行HPLC分析。HPLC运行参数在表5-2中描述。
Figure BDA0003565647960000561
Figure BDA0003565647960000571
实施例8
来源于SEQ ID NO:80的工程化多肽的演化与针对改进的KRED活性的筛选
使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码具有SEQ ID NO:80的KRED活性的多肽的工程化多核苷酸(即，SEQ ID NO:79)用于生成表8-1的另外的工程化多肽。与起始多肽相比，这些多肽显示出从异-α-酸形成二氢-(ρ)-异-α-酸的改进。使用上文描述的定向演化方法与表5-2中所述的HTP测定和分析方法一起，从SEQ ID NO:80的“骨架”氨基酸序列产生工程化多肽。
定向演化始于SEQ ID NO:79中所列的多核苷酸。然后选择工程化多肽作为起始的“骨架”基因序列。工程化多肽的文库使用各种熟知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生并使用HTP测定和分析方法进行筛选，该HTP测定和分析方法测量多肽将异-α-酸底物转化为期望的二氢-(ρ)-异-α-酸产物的能力。
酶测定以96孔形式在200μL总体积/孔中进行，其包括在100mM pH 8磷酸钾与2mMMgSO4中的10％-20％v/v HTP酶裂解物、80g/L或160g/L异-α-酸底物(
Figure BDA0003565647960000572
异构化啤酒花提取物溶液,Kalsec)以及40体积％异丙醇(IPA)中的0.02g/L NADP。将板密封并在45℃以600rpm摇动孵育20-24小时。
20-24小时后，加入1000μL的具有0.1％乙酸的乙腈。将板密封，并在4℃以4000rpm离心10分钟。将猝灭的样品在50:50乙腈:水混合物中进一步稀释20-40x，然后进行HPLC分析。HPLC运行参数在表5-2中描述。
Figure BDA0003565647960000581
实施例9
来源于SEQ ID NO:6和SEQ ID NO:80的工程化多肽的演化与针对改进的在高底物浓度的KRED活性的筛选
通过将200mg酶粉末溶于5mL的100mM pH 8三乙醇胺*H2SO4与2mM MgSO4中，制备40g/L酶储备溶液。取2mL等分试样，并进行连续两次1:1v/v稀释至各自20g/L和10g/L。在空气中用搅拌棒将500μL酶储备溶液(10g/L、20g/L或40g/L)加入小瓶中。向搅拌的酶储备溶液加入100μL的在缓冲液中的1g/L NADP和在400μL异丙醇(IPA)中的25g/L异-α-酸的等分试样。最终反应组成为40％的IPA中的5g/L、10g/L或20g/L酶、10g/L异-α-酸和0.1g/LNADP。将小瓶置于25℃或40℃的加热块中，并于1h、2h、4h、8h和24h后取样，并通过HPLC-UV进行分析。比较SEQ ID NO:6和SEQ ID NO:80的典型反应谱示于图1。
实施例10
来源于SEQ ID NO:80、104、100、136、116、132、162、150、152、144和146的工程化多肽的演化与针对改进的在高底物和低NADP浓度的KRED活性的筛选
通过将100mg酶粉末溶于500μL的100mM pH 8磷酸钾缓冲液与2mM MgSO4和0.1g/LNADP，制备200g/L酶储备溶液。向96深孔板的孔加入40μL酶/NADP储备溶液、80μL异丙醇和80μL的40wt％异-α-酸水溶液。最终反应组成为40％的IPA中的40g/L酶、160g/L异-α-酸和0.02g/L NADP。将板密封并在40℃孵育24小时，并且然后猝灭，并通过HPLC-UV分析。数据在表11-1示出和在图2中描绘。
Figure BDA0003565647960000591
实施例11
来源于SEQ ID NO:104的工程化多肽的演化与针对改进的KRED活性的筛选
如实施例8所述，使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码SEQ ID NO:104的具有KRED活性的多肽的工程化多核苷酸(即SEQ ID NO:103)用于生成表11-1的另外的改进的工程化多肽。
Figure BDA0003565647960000601
实施例12
包含重组KRED基因的大肠杆菌表达宿主
用于产生本发明的变体的初始KRED酶从Codexis收集的商购可得的KRED酶小组获得。在初始筛选期间，SEQ ID NO:172的多肽或SEQ ID NO:270的多肽产生最多的产物，如通过LC/MS确定的。将KRED编码基因克隆到SEQ ID NO:413或SEQ ID NO:414的表达载体，可操作地连接到在lacl阻遏物的控制下的lac启动子。表达载体还包含P15a复制起点和氯霉素抗性基因。使用本领域已知的标准方法将所得质粒转化到大肠杆菌W3110中。如本领域已知的，通过使细胞经受三氯生选择来分离转化体(参见例如，美国专利第8,383,346号和WO2010/144103)。
实施例13
HTP含KRED的湿细胞沉淀的制备
将来自单克隆菌落的含有重组KRED编码基因的大肠杆菌细胞接种到96孔浅孔微量滴定板的孔中的含有1％葡萄糖和0.12μg/mL三氯生的190μl LB中。将板用O2可透过的密封件密封，并使培养物在20℃、200rpm和85％湿度生长过夜。然后，将20μl的每种细胞培养物转移到含有380μL TB和30μg/mL CAM的96孔深孔板的孔中。将深孔板用O2可透过的密封件密封，并且在30℃、250rpm和85％湿度孵育，直到达到0.6-0.8的OD600。然后将细胞培养物用达到1mM的最终浓度的IPTG诱导，并且在与最初使用的相同条件下孵育过夜。然后用在4℃、4000rpm持续10分钟的离心将细胞沉淀。弃去上清液，并且在裂解前将沉淀冷冻在-80℃。
实施例14
HTP含KRED的细胞裂解物的制备
首先，通过加入150μL含有100mM pH 8磷酸钾与2mM MgSO4或者100mM pH 8磷酸钾与2mM MgSO4、1g/L溶菌酶和0.5g/L PMBS的裂解缓冲液裂解如实施例2所述产生的细胞沉淀。然后，在桌上型摇动器上在室温摇动细胞沉淀2小时。将板在4℃以4000rpm离心15分钟，以去除细胞碎片。然后将上清液用于生物催化反应以确定它们的活性水平。
实施例15
从摇瓶(SF)培养物制备冻干裂解物
摇瓶程序可用于生成工程化KRED多肽摇瓶粉末(SFP)，其可用于二次筛选测定和/或用于本文所述的生物催化过程。与HTP测定中使用的细胞裂解物相比，酶的摇瓶粉末(SFP)制品提供了更纯化的工程化酶的制品(例如，多达总蛋白的30％)，并且还允许使用更浓缩的酶溶液。首先，将如上文描述地生长的选定HTP培养物铺板在具有1％葡萄糖和0.12μg/ml三氯生的LB琼脂板上，并在37℃生长过夜。将来自每个培养物的单个菌落转移到6ml的具有1％葡萄糖和30μg/ml CAM的LB中。使培养物在30℃在250rpm生长18h，并以约1:50传代培养至250ml含0.12μg/ml三氯生的TB中，至0.05的最终OD600。使培养物在30℃在250rpm生长约3小时，达到0.8-1.0之间的OD600，并用1mM IPTG诱导。然后使培养物在30℃在250rpm生长20h。将培养物离心(4000rpm，在4℃持续20分钟)。弃去上清液，并将沉淀重悬在35ml的50mM pH 8磷酸钾与2mM MgSO4中。将重悬的细胞离心(4000rpm，在4℃持续20分钟)。弃去上清液，并将沉淀重悬在6ml的50mM pH 8磷酸钾与2mM MgSO4中，并使用来自ConstantSystems(One Shot)的细胞破坏仪裂解细胞。使裂解物沉淀(10,000rpm，在4℃持续60min)并且将上清液冷冻并冻干以产生摇瓶(SF)酶。
实施例16
来源于SEQ ID NO:172的工程化多肽的演化与针对改进的KRED活性的筛选
基于筛选变体还原异-α-酸底物的结果，选择SEQ ID NO:172的多肽作为亲本酶。使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码SEQ ID NO:172的具有KRED活性的多肽的工程化多核苷酸用于产生表16-1的另外的工程化多肽。与起始多肽相比，这些多肽显示出从异-α-酸形成二氢-(ρ)-异-α-酸的改进。使用上文描述的定向演化方法与以下表16-1中描述的HTP测定和分析方法一起，从SEQ ID NO:172的“骨架”氨基酸序列生成工程化多肽。
定向演化始于SEQ ID NO:171中所列的多核苷酸。然后选择工程化多肽作为起始的“骨架”基因序列。工程化多肽的文库使用各种熟知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生并使用HTP测定和分析方法进行筛选，该HTP测定和分析方法测量多肽将异-α-酸底物转化为期望的二氢-(ρ)-异-α-酸产物的能力。
酶测定以96深孔板形式在100μL总体积/孔中进行，其包括100mM pH 8磷酸钾与2mM MgSO4中的20％v/v HTP酶裂解物、40％v/v的40wt％异-α-酸底物水溶液(
Figure BDA0003565647960000621
异构化啤酒花提取物溶液,Kalsec)以及40vol％异丙醇(IPA)中的0.02g/L NADP。将板密封并在45℃以600rpm摇动孵育20-24小时。
20-24小时后，加入1000μL的具有0.1％乙酸的乙腈。将板密封，并在4℃以4000rpm离心10分钟。将猝灭的样品在50:50乙腈:水混合物中进一步稀释4-5x，然后进行HPLC分析。HPLC运行参数在表5-2中描述。
Figure BDA0003565647960000631
实施例17
来源于SEQ ID NO:186的工程化多肽的演化与针对改进的对反式-ISO的KRED活性的筛选
基于筛选变体还原异-α-酸底物的结果，选择SEQ ID NO:186作为亲本酶。使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
SEQ ID NO:185的编码具有KRED活性的多肽的工程化多核苷酸用于生成表17-2的另外的工程化多肽。与起始多肽相比，这些多肽显示出从异-α-酸形成二氢-(ρ)-异-α-酸的改进。使用上文描述的定向演化方法与表5-2中所述的HTP测定和分析方法一起，从SEQ IDNO:186的“骨架”氨基酸序列产生工程化多肽。
定向演化始于SEQ ID NO:185中所列的多核苷酸。然后选择工程化多肽作为起始的“骨架”基因序列。工程化多肽文库使用各种熟知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生并使用HTP测定和分析方法进行筛选，该HTP测定和分析方法测量多肽将异-α-酸底物转化为期望的二氢-(ρ)-异-α-酸产物的能力。
酶测定以96深孔板形式在100μL总体积/孔中进行，其包括100mM pH 8磷酸钾与2mM MgSO4中的50％v/v HTP酶裂解物、10％v/v的10g/L SEQ ID NO:186、1g/L异-α-酸底物(
Figure BDA0003565647960000643
异构化啤酒花提取物溶液,Kalsec)以及40vol％异丙醇(IPA)中的0.01g/LNADP。将板密封并在30℃以600rpm摇动孵育44-48小时。
20-24小时后，加入1000μL的具有0.1％乙酸的乙腈。将板密封，并在4℃以4000rpm离心10分钟。将猝灭的样品在50:50乙腈:水混合物中进一步稀释4-5x，然后进行HPLC分析。HPLC运行参数在下表17-1中描述。参见图3。对反式ISO显示改进的活性的变体显示在表17-2中。
Figure BDA0003565647960000641
Figure BDA0003565647960000642
Figure BDA0003565647960000651
实施例18
来源于SEQ ID NO:186的工程化多肽的演化与针对改进的KRED活性的筛选
如实施例16中所述，使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码SEQ ID NO:186的具有KRED活性的多肽的工程化多核苷酸(即SEQ ID NO:185)用于生成表18-1的另外的改进的工程化多肽。
Figure BDA0003565647960000661
实施例19
来源于SEQ ID NO:194、SEQ ID NO:252的工程化多肽的演化与针对改进的在高底物和低NADP浓度的KRED活性的筛选
通过将100mg酶粉末溶于500μL的100mM pH 8磷酸钾缓冲液与2mM MgSO4和0.1g/LNADP，制备200g/L酶储备溶液。向96深孔板中的孔加入40μL的酶/NADP储备溶液的等分试样、80μL异丙醇和80μL的40wt％异-α-酸水溶液。最终反应组成为40％IPA中的40g/L酶、160g/L异-α-酸和0.02g/L NADP。将板密封并在40℃孵育24小时，并且然后猝灭，并通过HPLC-UV分析。数据在表19-1、表19-2和表19-3中示出。
Figure BDA0003565647960000662
Figure BDA0003565647960000671
Figure BDA0003565647960000672
Figure BDA0003565647960000673
实施例20
来源于SEQ ID NO:270和SEQ ID NO:272的工程化多肽的演化与针对改进的在高底物和低NADP浓度的KRED活性的筛选
如实施例18中所述，使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码SEQ ID NO:270的具有KRED活性的多肽的工程化多核苷酸(即SEQ ID NO:269)和编码SEQ ID NO:272的具有KRED活性的多肽的工程化多核苷酸(即SEQ ID NO:271)用于生成表20-1和表20-2的另外的改进的工程化多肽。
Figure BDA0003565647960000681
Figure BDA0003565647960000682
实施例21
来源于SEQ ID NO:286的工程化多肽的演化与针对改进的在高底物和低NADP浓度的KRED活性的筛选
如实施例18中所述，使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
编码SEQ ID NO:286的具有KRED活性的多肽的工程化多核苷酸(即SEQ ID NO:285)用于生成表21-1的另外的改进的工程化多肽。
Figure BDA0003565647960000691
实施例22
来源于SEQ ID NO:328的工程化多肽的演化与针对改进的KRED活性的筛选
基于筛选变体还原异-α-酸底物的结果，选择SEQ ID NO:328作为亲本酶。使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
SEQ ID NO:327的编码具有KRED活性的多肽的工程化多核苷酸用于生成表22-1的另外的工程化多肽。与起始多肽相比，这些多肽显示出从异-α-酸形成二氢-(ρ)-异-α-酸的改进。使用上文描述的定向演化方法与表5-2中所述的HTP测定和分析方法一起，从SEQ IDNO:328的“骨架”氨基酸序列产生工程化多肽。
定向演化始于SEQ ID NO:327中所列的多核苷酸。然后选择工程化多肽作为起始的“骨架”基因序列。工程化多肽的文库使用各种熟知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生并使用HTP测定和分析方法进行筛选，该HTP测定和分析方法测量多肽将异-α-酸底物转化为期望的二氢-(ρ)-异-α-酸产物的能力。
酶测定以96孔圆底板在200μL总体积/孔中进行，其包括100mM pH 8磷酸钾与2mMMgSO4中的20％v/v HTP酶裂解物、40％v/v的40wt％异-α-酸底物水溶液(
Figure BDA0003565647960000702
异构化啤酒花提取物溶液,Kalsec)以及40vol％异丙醇(IPA)中的0.02g/L NADP。将板密封并在45℃以600rpm摇动孵育20-24小时。
20-24小时后，加入1000μL的具有0.1％乙酸的乙腈。将板密封，并在4℃以4000rpm离心10分钟。将猝灭的样品在50:50乙腈:水混合物中进一步稀释4-5x，然后进行HPLC分析。HPLC运行参数在表5-2和表17-1中描述。改进的变体在表22-1中显示。
Figure BDA0003565647960000701
实施例23
SEQ ID NO:328和SEQ ID NO:330在高底物和低NADP载量的性能
将8g的40wt％ISO水溶液的样品溶解在8mL异丙醇(IPA)和2mL 100mM pH 8磷酸钾缓冲液中。用4N NaOH和/或10％H3PO4调节pH至8。用搅拌棒并在加热块中在40℃于氮气罩下将4.5mL的经调整pH的ISO/IPA/缓冲溶液加入到隔板-加盖的小瓶(septum-capped vials)中。向溶液加入具有10mM MgSO4和0.2g/L NADP的100mM pH 8磷酸钾缓冲液中0.5mL的10g/L或40g/L KRED(最终KRED和NADP载量分别为1g/L或4g/L KRED和0.02g/L NADP)。在各个时间间隔，通过注射器从反应中取出20μL等分试样，用1000μL的具有0.1％乙酸的1:1乙腈/水猝灭。在20℃以4,000rpm离心5分钟后，将20μL上清液用180μL的具有0.1％乙酸的1:1乙腈/水稀释，进行HPLC分析(表5-2和表17-1)。见图4。
实施例24
来源于SEQ ID NO:330的工程化多肽的演化与针对改进的KRED活性的筛选
基于筛选变体还原异-α-酸底物的结果，选择SEQ ID NO:330作为亲本酶。使用良好建立的技术(例如饱和诱变，和先前鉴定的有益突变的重组)产生工程化基因的文库。每种基因编码的多肽如实施例2中描述地以HTP产生，并且可溶性裂解物如实施例3中描述地产生。
SEQ ID NO:329的编码具有KRED活性的多肽的工程化多核苷酸用于生成表24-1的另外的工程化多肽。与起始多肽相比，这些多肽显示出从异-α-酸形成二氢-(ρ)-异-α-酸的改进。使用上文描述的定向演化方法与以下表24-1所述的HTP测定和分析方法一起，从SEQID NO:330的“骨架”氨基酸序列产生工程化多肽。
定向演化始于SEQ ID NO:329中所列的多核苷酸。然后选择工程化多肽作为起始的“骨架”基因序列。工程化多肽的文库使用各种熟知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生并使用HTP测定和分析方法进行筛选，该HTP测定和分析方法测量多肽将异-α-酸底物转化为期望的二氢-(ρ)-异-α-酸产物的能力。
酶测定以96圆底板形式在200μL总体积/孔中进行，其包括100mM pH 8磷酸钾与2mM MgSO4中的50％v/v HTP酶裂解物、10％v/v的40wt％异-α-酸底物水溶液(
Figure BDA0003565647960000711
异构化啤酒花提取物溶液,Kalsec)以及40vol％异丙醇(IPA)中的0.02g/L NADP。将板密封并在45℃以600rpm摇动孵育20-24小时。
Figure BDA0003565647960000721
实施例25
SEQ ID NO:270、SEQ ID NO:328、SEQ ID NO:330、SEQ ID NO:348、SEQ ID NO:346和SEQ ID NO:356在高底物和低NADP载量的性能
将80mg的每种变体的样品溶解在1mL NADP储备溶液中，该储备溶液由100mL的100mM pH 9磷酸钾和10mM MgSO4中的10mg NADP组成。将储备溶液进行5次连续1:1稀释，以得到NADP储备溶液中80g/L、40g/L、20g/L、10g/L和5g/L酶的储备溶液。在圆底板中，将储备溶液的20uL等分试样加入40uL的40wt％ISO溶液和40uL的IPA中，以得到20mM pH 8磷酸钾和2mM MgSO4与0.02g/L NADP中40vol％IPA中的160g/L ISO的最终组成。最终酶浓度分别为16g/L、8g/L、4g/L、2g/L和1g/L。将这些板密封并置于摇动器中在40℃和600rpm持续24小时。24小时后，将板在20℃以4,000rpm离心10分钟，并将100ul上清液转移到深孔板中1mL的具有0.1％乙酸的1:1乙腈/水中。将具有猝灭的反应混合物的深孔板在20℃在4,000rpm离心10分钟，并将5uL上清液转移到200uL的具有0.1％乙酸的1:1乙腈/水中，按照表5-2和表17-1进行HPLC分析。见图5。
出于所有目的，本申请中引用的所有出版物、专利、专利申请和其他文件在此通过引用以其整体并入，其程度如同每个单独的出版物、专利、专利申请或其他文件被单独地指出出于所有目的通过引用并入一样。
虽然已经示出和描述了各种具体的实施方案，但是将理解，可以做出各种改变而不偏离本发明的精神和范围。
序列表
<110> 科德克希思公司
<120> 酮还原酶多肽及多核苷酸
<130> CX8-195WO1
<140>
<141> 2020-09-24
<150> US 62/906,268
<151> 2019-09-26
<160> 414
<210> 1
<211> 759
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的野生型Kred
<400> 1
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
<210> 2
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的野生型Kred
<400> 2
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 3
<211> 759
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 3
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacagtga 759
<210> 4
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 4
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 5
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 5
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 6
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 6
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 7
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 7
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaggga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 8
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 8
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Arg Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 9
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 9
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgaggg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 10
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 10
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Arg Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 11
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 11
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tttctgggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 12
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 12
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 13
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 13
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgggtctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 14
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 14
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Gly Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 15
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 15
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggcatga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 16
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 16
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg His Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 17
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 17
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcaggggttg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 18
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 18
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Val Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 19
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 19
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 20
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 20
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 21
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 21
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttggtgggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 22
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 22
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Gly Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 23
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 23
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tttgtgggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 24
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 24
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Cys Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 25
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 25
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacggttg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 26
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 26
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Val Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 27
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 27
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcag ggatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 28
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 28
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Arg Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 29
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 29
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
agggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 30
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 30
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Arg Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 31
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 31
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcaggggatg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 32
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 32
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Asp Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 33
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 33
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cattggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 34
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 34
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Ile Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 35
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 35
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgtgtg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 36
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 36
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Cys Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 37
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 37
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctgcag 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 38
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 38
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Gln Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 39
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 39
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagacca ggcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 40
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 40
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Arg Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 41
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 41
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctgcgg 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 42
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 42
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Arg Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 43
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 43
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatggtgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 44
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 44
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Val Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 45
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 45
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatgg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 46
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 46
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Met Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 47
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 47
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcgg tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 48
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 48
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Gly Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 49
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 49
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagacca atcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 50
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 50
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Asn Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 51
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 51
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctgttg 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 52
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 52
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Leu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 53
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 53
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 54
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 54
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 55
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 55
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggctgtg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 56
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 56
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Leu Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 57
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 57
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggacgg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 58
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 58
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Thr Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 59
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 59
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaagtccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 60
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 60
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Ser Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 61
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 61
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc atcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 62
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 62
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr His Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 63
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 63
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgtctctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 64
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 64
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Ser Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 65
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 65
atgaccgatc gtctgaagca taaagtagcc atcattaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 66
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 66
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 67
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 67
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattgg gagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 68
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 68
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Gly Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 69
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 69
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgacctt ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggctcga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 70
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 70
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Leu Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 71
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 71
atgaccgatc gtctgaagca taaagtagcc atcattaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 72
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 72
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 73
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 73
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 74
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 74
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 75
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 75
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc ctcggaaaga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 76
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 76
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 77
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 77
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgggcctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 78
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 78
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Gly Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 79
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 79
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 80
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 80
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 81
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 81
atgaccgatc gtctgaagca taaagtagcc atcattaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgggcctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 82
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 82
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Gly Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 83
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 83
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc atcggaaaga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 84
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 84
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr His Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 85
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 85
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcataca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgggcctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc ctcggaaagg tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 86
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 86
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Gly Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Gly Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 87
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 87
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagacca accggaaaga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 88
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 88
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Asn Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 89
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 89
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc atcggaaaga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaacgccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 90
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 90
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr His Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 91
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 91
atgaccgatc gtctgaagca taaagtagcc atcgtaaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgagcctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 92
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 92
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Ser Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 93
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 93
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc ctcggaaagg tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggctgtg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 94
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 94
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Gly Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Leu Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 95
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 95
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc atcggaaaga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 96
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 96
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr His Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 97
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 97
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttctggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc ctcggaaaga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 98
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 98
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Leu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 99
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 99
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg cgtaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 100
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 100
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Arg Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 101
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 101
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaag tggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 102
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 102
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Ser Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 103
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 103
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaca gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 104
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 104
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Gln Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 105
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 105
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgctctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 106
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 106
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Ala Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 107
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 107
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgaagctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 108
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 108
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Lys Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 109
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 109
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
acgactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 110
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 110
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Thr Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 111
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 111
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaagtg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 112
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 112
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 113
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 113
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
tgtactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 114
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 114
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Cys Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 115
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 115
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 116
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 116
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 117
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 117
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgattgtat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 118
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 118
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Cys Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 119
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 119
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
gggggtgtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 120
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 120
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Gly Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 121
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 121
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg ggattattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 122
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 122
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ile Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 123
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 123
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgttgg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 124
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 124
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Trp Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 125
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 125
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtttt ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 126
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 126
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Phe Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 127
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 127
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
gctgcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 128
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 128
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Ala Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 129
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 129
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatcgt tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 130
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 130
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Val Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 131
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 131
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaagtggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 132
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 132
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Val Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 133
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 133
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
ctgactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 134
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 134
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Leu Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 135
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 135
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 136
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 136
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 137
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 137
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgttgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 138
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 138
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Leu Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 139
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 139
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgagttggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 140
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 140
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Leu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 141
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 141
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acggcgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 142
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 142
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ala Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 143
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 143
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgct atcaagaccg agcgcaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 144
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 144
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 145
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 145
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggccct atcaagaccg agcgcaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 146
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 146
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Glu Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 147
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 147
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatactg cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 148
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 148
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Cys Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 149
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 149
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 150
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 150
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 151
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 151
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaag tggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 152
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 152
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Ser Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 153
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 153
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggccct accaagaccg agcgcaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 154
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 154
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Thr Lys
180 185 190
Thr Glu Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 155
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 155
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgct accaagaccg agcgcaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 156
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 156
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Thr Lys
180 185 190
Thr Glu Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 157
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 157
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgctactaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 158
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 158
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Thr Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 159
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 159
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 160
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 160
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 161
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 161
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgcgctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 162
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 162
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Ala Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 163
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 163
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tgctctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 164
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 164
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Ala Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 165
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 165
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaagtagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccc atcaagaccc cgcggaagga tgctctggaa 600
ggttgggagg aaatgatgtc acagcgtacg cgtaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 166
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 166
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Val Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Pro Arg Lys Asp Ala Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Arg Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 167
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 167
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaca gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaagtagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 168
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 168
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Gln Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Val Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 169
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 169
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaagtagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tgatctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 170
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 170
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Val Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Asp Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 171
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 171
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 172
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 172
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 173
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 173
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaca gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaagtagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 174
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 174
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Gln Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Val Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 175
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 175
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttgttaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcccg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ttaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 176
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 176
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Val
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 177
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 177
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagc agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 178
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 178
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 179
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 179
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catgggtgtg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 180
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 180
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Val Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 181
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 181
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
tatactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 182
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 182
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Tyr Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 183
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 183
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggacatggat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 184
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 184
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Met Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 185
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 185
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 186
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 186
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 187
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 187
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gggtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 188
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 188
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Gly Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 189
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 189
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 190
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 190
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 191
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 191
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtttgaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 192
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 192
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 193
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 193
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 194
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 194
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 195
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 195
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 196
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 196
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 197
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 197
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcaattgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 198
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 198
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ile
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 199
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 199
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 200
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 200
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 201
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 201
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 202
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 202
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 203
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 203
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcga tcgcggggct cgtaggcgat ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 204
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 204
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ala Gly Leu Val Gly Asp Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 205
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 205
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtagccaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 206
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 206
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ala
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 207
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 207
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 208
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 208
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 209
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 209
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcagcttc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 210
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 210
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Ala Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 211
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 211
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcga tcatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcaatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 212
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 212
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 213
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 213
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcga tcatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatggcttc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 214
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 214
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Ala Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 215
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 215
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatggcttc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 216
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 216
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Ala Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 217
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 217
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcagccgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcaatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 218
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 218
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ala
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 219
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 219
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcaattgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcgcga tcgcggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggtgctgagg aagcaatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 220
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 220
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ile
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ala Ile
130 135 140
Ala Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Ala Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 221
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 221
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatggcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 222
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 222
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 223
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 223
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcaattgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggtgctgagg aagcagcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 224
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 224
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ile
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Ala Glu Glu Ala Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 225
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 225
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcgcgg cggcggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggtgctgagg aagcaatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cggggcaacc gcacag 756
<210> 226
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 226
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ala Ala
130 135 140
Ala Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Ala Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Ala Thr Ala Gln
245 250
<210> 227
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 227
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcgcga tcgcggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 228
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 228
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ala Ile
130 135 140
Ala Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 229
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 229
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcaattgc gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcgcga tcgcggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cggggcaacc gcacag 756
<210> 230
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 230
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ile
85 90 95
Ala Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ala Ile
130 135 140
Ala Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Ala Thr Ala Gln
245 250
<210> 231
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 231
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtagccaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcga tcatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatggcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 232
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 232
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ala
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 233
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 233
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcgg cgatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggtgctgagg aagcagcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 234
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 234
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ala
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Ala Glu Glu Ala Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 235
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 235
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcagccaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcagcttc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cggggcaacc gcacag 756
<210> 236
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 236
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ala
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Ala Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Ala Thr Ala Gln
245 250
<210> 237
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 237
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcga tcatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatggcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cggggcaacc gcacag 756
<210> 238
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 238
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Ala Thr Ala Gln
245 250
<210> 239
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 239
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcgg cggcggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcagcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 240
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 240
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ala
130 135 140
Ala Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 241
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 241
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcga tcgcggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatggcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cggggcaacc gcacag 756
<210> 242
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 242
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ala Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Ala Thr Ala Gln
245 250
<210> 243
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 243
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cggggcaacc gcacag 756
<210> 244
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 244
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Ala Thr Ala Gln
245 250
<210> 245
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 245
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcgg cgatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 246
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 246
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ala
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 247
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 247
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggtgctgagg aaatggcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cggggcaacc gcacag 756
<210> 248
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 248
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Ala Glu Glu Met Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Ala Thr Ala Gln
245 250
<210> 249
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 249
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggcaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgct ccgactgcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatggcttc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cggggcaacc gcacag 756
<210> 250
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 250
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Ala Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Thr Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Ala Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Ala Thr Ala Gln
245 250
<210> 251
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 251
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggacatggat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 252
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 252
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Met Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 253
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 253
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 254
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 254
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 255
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 255
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaagctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cgtactcgca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat tgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 256
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 256
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Arg Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Ile Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 257
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 257
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 258
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 258
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 259
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 259
atgaccgatc gtctgaagca gaaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaagctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat tgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 260
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 260
Met Thr Asp Arg Leu Lys Gln Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Ile Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 261
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 261
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 262
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 262
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 263
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 263
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaaatc ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat tgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 264
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 264
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Ile Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Ile Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 265
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 265
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgagcgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat tgtaggcgct ccgatggcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 266
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 266
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Ile Val Gly Ala Pro Met Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 267
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 267
atgaccgacc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgagcgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
tatactcaga cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgatggcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccc cacggaagga tcagctggaa 600
ggttgggagg aagcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 268
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 268
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Tyr Thr Gln Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Met Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Pro Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 269
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 269
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgatggcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 270
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 270
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Met Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 271
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 271
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 272
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 272
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 273
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 273
atgaccgacc gtctgaagca caaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggacatggat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 274
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 274
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Met Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 275
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 275
atgaccgacc gtctgaagca gaaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 276
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 276
Met Thr Asp Arg Leu Lys Gln Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 277
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 277
atgaccgatc gtctgaagca caaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgtcgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat ggtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 278
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 278
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Met Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 279
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 279
atgaccgatc gtctgaagca taaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtgag 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggacatggat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 280
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 280
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg Glu Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Met Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 281
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 281
atgaccgacc gtctgaagca caaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgtcgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacga cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgat ccgactctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggacatggat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 282
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 282
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Met Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 283
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 283
atgaccgacc gtctgaagca gaaagtagcc atcgttaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggacatggat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg aaatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 284
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 284
Met Thr Asp Arg Leu Lys Gln Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Met Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 285
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 285
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggcggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 286
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 286
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 287
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 287
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccgtcggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 288
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 288
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ser Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 289
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 289
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgatggcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagacct cggctaaaga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 290
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 290
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Met Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Ser Ala Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 291
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 291
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagccgagg gcgtcattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgatggcag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 292
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 292
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Ala Glu Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Met Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 293
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 293
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgat ccggcgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 294
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 294
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Ala Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 295
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 295
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggtgatg gccgtattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 296
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 296
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Asp Gly Arg Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 297
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 297
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggtgctg gcaaaattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtcgtgtt ctgtccgtga atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 298
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 298
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ala Gly Lys Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Arg Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 299
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 299
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggtgctg gccgtattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtga cgctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 300
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 300
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ala Gly Arg Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Thr Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 301
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 301
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggtcgtg gcgccattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtga cgctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 302
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 302
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Arg Gly Ala Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Thr Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 303
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 303
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggatgg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 304
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 304
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Met Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 305
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 305
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggtgagg gcgtcattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtcgtgtt ctgtccgtgg cgctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 306
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 306
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Glu Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Arg Val Leu Ser
100 105 110
Val Ala Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 307
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 307
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtgg cgctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 308
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 308
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Ala Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 309
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 309
atgaccgacc gtctgaagca caaagtagcc atcattaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtcggaaga agcaggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagctgatg gccgtattaa aagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct cgtaggcgat ccgatgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aacggaagga tcagctggaa 600
ggttgggagg agatgatgtc acagcgtacg ctgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcgtctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 310
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 310
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Ala Asp Gly Arg Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Asp Pro Met Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Leu Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 311
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 311
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtggccaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggcggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 312
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 312
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ala
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 313
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 313
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtgatcaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagctcga tcgcagggct ggtaggcgcc ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtggacc gcacag 756
<210> 314
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 314
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ala Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Trp Thr Ala Gln
245 250
<210> 315
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 315
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagcctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 316
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 316
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 317
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 317
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 318
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 318
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 319
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 319
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aagcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 320
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 320
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 321
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 321
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 322
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 322
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 323
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 323
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagcctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggggctg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 324
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 324
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ala Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 325
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 325
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtctgaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgagtctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 326
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 326
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Leu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 327
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 327
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 328
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 328
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 329
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 329
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 330
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 330
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 331
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 331
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcggaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 332
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 332
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 333
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 333
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 334
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 334
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 335
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 335
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aggcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 336
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 336
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ala Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 337
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 337
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggctat ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 338
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 338
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Tyr Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 339
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 339
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct gccgccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 340
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 340
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Ala Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 341
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 341
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 342
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 342
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 343
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 343
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggatcg ggcgtattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 344
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 344
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Arg Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 345
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 345
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggggcag gggtcattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 346
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 346
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ala Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 347
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 347
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggggcag ggcgtattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgctgggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 348
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 348
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ala Gly Arg Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ala Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 349
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 349
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggggcag gggtcattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaagtt ctgtccgtca ctctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgctgggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 350
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 350
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ala Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Thr Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ala Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 351
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 351
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgctgggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aggcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 352
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 352
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ala Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ala Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 353
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 353
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggatcg gggtcattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 354
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 354
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 355
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 355
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaagctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgatggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 356
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 356
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Met Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 357
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 357
atgaccgatc gtctgaagca caaagtagcc atcgtcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgtcgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaggtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 358
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 358
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 359
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 359
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgtcgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaggtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgatggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 360
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 360
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Met Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 361
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 361
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgtcgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtaaggtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 362
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 362
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Ser Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Lys Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 363
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 363
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaagctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agcgcaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 364
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 364
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Arg Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 365
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 365
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcaggggccg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 366
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 366
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ala Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 367
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 367
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gttcgtgtca acacagtaca tccgggcgcg atcaagacca actcgaagga tcagctggag 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 368
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 368
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Asn Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 369
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 369
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gttcgtgtca acacagtaca tccgggcgcg atcaagacca actcgaagga tcagctgcca 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 370
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 370
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Asn Ser Lys Asp Gln Leu Pro Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 371
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 371
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aatcgaagga tcagctgcca 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 372
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 372
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Pro Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 373
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 373
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggcggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 374
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 374
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Ala Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 375
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 375
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttccgaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagacca actcgaagga tcagctgcca 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 376
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 376
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Pro
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Asn Ser Lys Asp Gln Leu Pro Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 377
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 377
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttccgaa gagcgttgaa 300
cggactacca cggaggaatg gagtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat cgtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aatcgaagga tcagctgcca 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 378
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 378
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Pro
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Ser Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Ile Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Pro Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 379
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 379
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atccgcaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 380
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 380
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Arg
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 381
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 381
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat tgtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 382
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 382
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Ile Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 383
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 383
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cgcactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat cgtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aatcgaagga tcagctgcca 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 384
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 384
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Ile Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Pro Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 385
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 385
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccgtgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aatcgaagga tcagctggag 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 386
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 386
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Val Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 387
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 387
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccgtgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggttatcaa gagcgttgaa 300
cgcactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggat cgtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aatcgaagga tcagctggag 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 388
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 388
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Val Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Ile Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 389
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 389
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacagc cggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atcaggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 390
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 390
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Ala Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Gln Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 391
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 391
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacagc cggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccgtgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg aatcgaagga tcagctgcca 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 392
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 392
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Ala Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Val Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Pro Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 393
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 393
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tggcctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 394
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 394
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gly Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 395
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 395
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcaggctgg acgaccctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcgcctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 396
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 396
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Arg Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 397
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 397
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga acgtggctgg acggacctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
caaactacta cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgcaggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc agctgatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 398
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 398
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Arg Gly Trp Thr Asp Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Gln Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Gln Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Leu Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 399
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 399
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agagggctgg acggacctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
aagactacta cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgcaggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc agctgatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 400
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 400
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Glu Gly Trp Thr Asp Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Lys Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Gln Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Leu Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 401
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 401
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcgggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
atgactacta cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc agctgatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 402
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 402
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Met Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Leu Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 403
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 403
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga acgtggctgg acgcgcctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtg agggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 404
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 404
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Arg Gly Trp Thr Arg Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Glu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 405
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 405
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga acgtggctgg acgcgcctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cgcactacta cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtg agggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgcaggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 406
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 406
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Arg Gly Trp Thr Arg Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Glu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Gln Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 407
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 407
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga agcgggctgg acggacctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgcaggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc aggcaatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 408
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 408
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Ala Gly Trp Thr Asp Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Gln Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Ala Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 409
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 409
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga acgtggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
cggactacca cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtg agggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccggccgccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc agctgatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 410
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 410
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Arg Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Arg Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Glu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Ala Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Leu Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 411
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 411
atgaccgatc gtctgaagca caaagtagcc atcatcaccg gcgggacaat gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgagaaagc cgccaaatca atcggtggca gtgatgttat tcgctttgtc 180
cagcacgatg cgtccgaaga acgtggctgg acgacgctgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccct ggtgaacaat gcagggattg gggtaattaa gagcgttgaa 300
caaactacta cggaggaatg gcgtgatgtt ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtg agggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttatggggct ggtaggcgct ccgcaggccg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggcgcg atcaagaccg agtcgaagga tcagctggaa 600
ggttgggagc agctgatgtc acagcgtacg gcgaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtacacc gcacag 756
<210> 412
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自开菲尔乳杆菌(Lactobacillus kefir)的Kred的工程化变体
<400> 412
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Ser Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Glu Glu Arg Gly Trp Thr Thr Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Leu Val Asn Asn Ala Gly Ile Gly Val Ile
85 90 95
Lys Ser Val Glu Gln Thr Thr Thr Glu Glu Trp Arg Asp Val Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Glu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Met Gly Leu Val Gly Ala Pro Gln Ala Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Ala Ile Lys
180 185 190
Thr Glu Ser Lys Asp Gln Leu Glu Gly Trp Glu Gln Leu Met Ser Gln
195 200 205
Arg Thr Ala Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 413
<211> 4205
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pJVFabI#2-Bla质粒序列
<400> 413
aacggctttg ccgcggccct ctcacttccc tgttaagtat cttcctggca tcttccagga 60
aatctccgcc ccgttcgtaa gccatttccg ctcgccgcag tcgaacgacc gagcgtagcg 120
agtcagtgag cgaggaagcg gaatatatcc tgtatcacat attctgctga cgcaccggtg 180
cagccttttt tctcctgcca catgaagcac ttcactgaca ccctcatcag tgaaccaccg 240
ctggtagcgg tggttttttt aggcctatgg cctttttttt ttgtgggaaa cctttcgcgg 300
tatggtatta aagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac 360
gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa 420
ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct 480
gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg 540
cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc 600
tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga 660
agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa 720
ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc 780
gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga 840
cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt 900
agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct 960
cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg 1020
ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt tccactgcga tgctggttgc 1080
caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg 1140
tgcggacatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc 1200
gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct 1260
gcaactctct cagggccagg cggttaaggg caatcagctg ttgcccgtct cactggtgaa 1320
aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 1380
attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cggtacccga 1440
taaaagcggc ttcctgacag gaggccgttt tgtttctcga gttaattaag gcagtgagcg 1500
caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 1560
tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacggcta 1620
tgaccatgat tacggattca ctggccgtcg ttttacaatc tagaggccag cctggccata 1680
aggagatata catatgagta ttcaacattt ccgtgtcgcc cttattccct tttctgcggc 1740
attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 1800
tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 1860
gagttttcgc cccgaagagc gttttccaat gatgagcact tttaaagttc tgctatgtgg 1920
cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc 1980
tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 2040
agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 2100
tctgacaacg atcggaggac cgaaggagct aaccgttttt ttgcacacca tgggggatca 2160
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 2220
tgacaccacg atgcctacag caatggcaac aacgttgcgc aaactattaa ctggcgaact 2280
acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 2340
accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 2400
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 2460
cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgtaata gacagatcgc 2520
tgagataggt gcctcactga ttaagcattg gggccaaact ggccaccatc accatcacca 2580
ttagggaaga gcagatgggc aagcttgacc tgtgaagtga aaaatggcgc acattgtgcg 2640
acattttttt ttgaattcta cgtaaaaagc agccgataca tcggctgctt tttttttctg 2700
cagggtgaaa caaaacggtt gaccacatga agtaaacacg gtacggtgcg gtagtttatc 2760
acagttaaat tgctaacgca gtcaggcaaa gtccatgggt tttctttccg gtaagcgcat 2820
tctggtaacc ggtgttgcca gcaaactatc catcgcctac ggtatcgctc aggcgatgca 2880
ccgcgaagga gctgaactgg cattcaccta ccagaacgac aaactgaaag gccgcgtaga 2940
agaatttgcc gctcaattgg gttctgacat cgttctgcag tgcgatgttg cagaagatgc 3000
cagcatcgac accatgttcg ctgaactggg gaaagtttgg ccgaaatttg acggttttgt 3060
acactctatt gtttttgcac ctggcgatca gctggatggt gactatgtta acgccgttac 3120
ccgtgaaggc ttcaaaattg cccacgacat cagctcctac agcttcgttg caatggcaaa 3180
agcttgccgc tccatgctga atccgggttc tgccctgctg accctttcct accttggcgc 3240
tgagcgcgct atcccgaact acaacgttat gggtctggca aaagcgtctc tggaagcgaa 3300
cgtgcgctat atggcgaacg cgatgggtcc ggaaggtgtg cgtgttaacg ccatctctgc 3360
tggtccgatc cgtactctgg cggcctccgg tatcaaagac ttccgcaaaa tgctggctca 3420
ttgcgaagcc gttaccccga ttcgccgtac cgttactatt gaagatgtgg gtaactctgc 3480
ggcattcctg tgctccgatc tctctgccgg tatctccggt gaagtggtcc acgttgacgg 3540
cggtttcagc attgctgcaa tgaacgaact cgaactgaaa taactgcagg agctcaaaca 3600
gcagcctgta ttcaggctgc tttttagaaa tattttatct gattaataag atgatcttct 3660
tgagatcgtt ttggtctgcg cgtattctct tgctctgaaa acgaaaaaac cgccttgcag 3720
ggcggttttt cgaaggttct ctgagctacc aactctttga accgaggtaa ctggcttgga 3780
ggagcgcagt caccaaaact tgtcctttca gtttagcctt aaccggcgca tgacttcaag 3840
actaactcct ctaaatcaat taccagtggc tgctgccagt ggtgcttttg catgtctttc 3900
cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcggact gaacgggggg 3960
ttcgtgcata cagtccagct tggagcgaac tgcctacccg gaactgagtg tcaggcgtgg 4020
aatgagacaa acgcggccat aacagcggaa tgacaccggt aaaccgaaag gcaggaacag 4080
gagagcgcac gagggagccg ccagggggaa acgcctggta tctttatagt cctgtcgggt 4140
ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg cggagcctat 4200
ggaaa 4205
<210> 414
<211> 4205
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pJVFabI#5-Bla质粒序列
<400> 414
aacggctttg ccgcggccct ctcacttccc tgttaagtat cttcctggca tcttccagga 60
aatctccgcc ccgttcgtaa gccatttccg ctcgccgcag tcgaacgacc gagcgtagcg 120
agtcagtgag cgaggaagcg gaatatatcc tgtatcacat attctgctga cgcaccggtg 180
cagccttttt tctcctgcca catgaagcac ttcactgaca ccctcatcag tgaaccaccg 240
ctggtagcgg tggttttttt aggcctatgg cctttttttt ttgtgggaaa cctttcgcgg 300
tatggtatta aagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac 360
gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa 420
ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct 480
gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg 540
cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc 600
tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga 660
agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa 720
ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc 780
gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga 840
cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt 900
agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct 960
cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg 1020
ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt tccactgcga tgctggttgc 1080
caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg 1140
tgcggacatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc 1200
gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct 1260
gcaactctct cagggccagg cggttaaggg caatcagctg ttgcccgtct cactggtgaa 1320
aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 1380
attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cggtacccga 1440
taaaagcggc ttcctgacag gaggccgttt tgtttctcga gttaattaag gcagtgagcg 1500
caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 1560
tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacggcta 1620
tgaccatgat tacggattca ctggccgtcg ttttacaatc tagaggccag cctggccata 1680
aggagatata catatgagta ttcaacattt ccgtgtcgcc cttattccct tttctgcggc 1740
attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 1800
tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 1860
gagttttcgc cccgaagagc gttttccaat gatgagcact tttaaagttc tgctatgtgg 1920
cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc 1980
tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 2040
agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 2100
tctgacaacg atcggaggac cgaaggagct aaccgttttt ttgcacacca tgggggatca 2160
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 2220
tgacaccacg atgcctacag caatggcaac aacgttgcgc aaactattaa ctggcgaact 2280
acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 2340
accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 2400
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 2460
cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgtaata gacagatcgc 2520
tgagataggt gcctcactga ttaagcattg gggccaaact ggccaccatc accatcacca 2580
ttagggaaga gcagatgggc aagcttgacc tgtgaagtga aaaatggcgc acattgtgcg 2640
acattttttt ttgaattcta cgtaaaaagc agccgataca tcggctgctt tttttttctg 2700
cagggtgaaa caaaacggtt gactacatga agtaaacacg gtacggtgcg gtagtttatc 2760
acagttaaat tgctaacgca gtcaggcaaa gtccatgggt tttctttccg gtaagcgcat 2820
tctggtaacc ggtgttgcca gcaaactatc catcgcctac ggtatcgctc aggcgatgca 2880
ccgcgaagga gctgaactgg cattcaccta ccagaacgac aaactgaaag gccgcgtaga 2940
agaatttgcc gctcaattgg gttctgacat cgttctgcag tgcgatgttg cagaagatgc 3000
cagcatcgac accatgttcg ctgaactggg gaaagtttgg ccgaaatttg acggttttgt 3060
acactctatt gtttttgcac ctggcgatca gctggatggt gactatgtta acgccgttac 3120
ccgtgaaggc ttcaaaattg cccacgacat cagctcctac agcttcgttg caatggcaaa 3180
agcttgccgc tccatgctga atccgggttc tgccctgctg accctttcct accttggcgc 3240
tgagcgcgct atcccgaact acaacgttat gggtctggca aaagcgtctc tggaagcgaa 3300
cgtgcgctat atggcgaacg cgatgggtcc ggaaggtgtg cgtgttaacg ccatctctgc 3360
tggtccgatc cgtactctgg cggcctccgg tatcaaagac ttccgcaaaa tgctggctca 3420
ttgcgaagcc gttaccccga ttcgccgtac cgttactatt gaagatgtgg gtaactctgc 3480
ggcattcctg tgctccgatc tctctgccgg tatctccggt gaagtggtcc acgttgacgg 3540
cggtttcagc attgctgcaa tgaacgaact cgaactgaaa taactgcagg agctcaaaca 3600
gcagcctgta ttcaggctgc tttttagaaa tattttatct gattaataag atgatcttct 3660
tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa acgaaaaaac cgccttgcag 3720
ggcggttttt cgaaggttct ctgagctacc aactctttga accgaggtaa ctggcttgga 3780
ggagcgcagt caccaaaact tgtcctttca gtttagcctt aaccggcgca tgacttcaag 3840
actaactcct ctaaatcaat taccagtggc tgctgccagt ggtgcttttg catgtctttc 3900
cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcggact gaacgggggg 3960
ttcgtgcata cagtccagct tggagcgaac tgcctacccg gaactgagtg tcaggcgtgg 4020
aatgagacaa acgcggccat aacagcggaa tgacaccggt aaaccgaaag gcaggaacag 4080
gagagcgcac gagggagccg ccagggggaa acgcctggta tctttatagt cctgtcgggt 4140
ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg cggagcctat 4200
ggaaa 4205

Claims (37)

1.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性。
2.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:4具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且在选自以下位置中的一个或更多个位置处具有至少一个取代或取代集：12、21、87、93、97、110、145、148、152、153、194、196、197、200、206、212和226，其中所述位置参考SEQ ID NO:4编号。
3.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:6具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：12/110/145/152、12/145、87/110/145、87/110/145/194、87/145/194、110、110/145/152/197、110/145/194、145、145/152、145/197/226和152，其中所述位置参考SEQ ID NO:6编号。
4.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:80具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：17、21、46、56、72、79、95、101、110、152、162、190、198、210、211和227，其中所述位置参考SEQ ID NO:80编号。
5.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:80具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：17、79、157、159、190/191/194、190/194、191/194、194、198和211，其中所述位置参考SEQ ID NO:80编号。
6.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:104具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：17/46/190、17/46/198/211、17/96/194/198、17/190/198、46/190/194/198和46/194/198，其中所述位置参考SEQ ID NO:104编号。
7.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:172具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：45、101、179、194、204、226和231，其中所述位置参考SEQ ID NO:172编号。
8.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:186具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：95/96/97/150/153/205、95/96/150/153/205/206/211/249、95/97/143/145/150/153/202/205、95/97/143/145/150/153/249、95/97/150/153、95/97/150/153/202/205/206、95/150/153/205/206/211、95/150/153/205/211、95/150/153/206/249、96/150/153、96/150/153/206、97/150/153、97/150/153/205、97/150/153/205/211、97/150/153/206、143/144/145/150/153/202/205/249、143/145/150/153、144/145/150/153/205/206、144/150/153、144/150/153/202/205/206、145/150/153/206/249、145/153/211、150/153/202/206/249、150/153/205/211、150/153/206/211、150/153/211和150/153/249，其中所述位置参考SEQ ID NO:186编号。
9.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:186具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：7/147、103/147、110、110/179/194、147和249，其中所述位置参考SEQ ID NO:186编号。
10.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:194具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：7/12/54/110/150/153/194/205/211/249、12/54/72/110/150/152/153/194/205/211/249、12/72/101/103/110/152/249、12/72/110/147/152/204、45/54/72/110/152/194/204、72/110/147/150/152/153/194/205/211/249和110/150/153/179/194/205/211/249，其中所述位置参考SEQ ID NO:194编号。
11.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:252具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：7/12/54/179/249、7/152、12/54/72/152/179/249、40、54/72、72/147/152/179/249和249，其中所述位置参考SEQ ID NO:252编号。
12.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:270具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：92/93、150/152、150/152/153和194/195，其中所述位置参考SEQ ID NO:270编号。
13.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:272具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：92/93/95、93、93/95、93/95/109、93/95/109/114、93/95/114、93/109/114和114，其中所述位置参考SEQ ID NO:272编号。
14.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:286具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：12/45/72/109/249、12/45/93/249、12/45/249、12/109/249、45/72/249、45/109/249、45/249、96和145/150，其中所述位置参考SEQ ID NO:286编号。
15.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:328具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：150、150/151、150/195和195，其中所述位置参考SEQ ID NO:328编号。
16.一种工程化酮还原酶变体，所述工程化酮还原酶变体与SEQ ID NO:330具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性，并且具有选自以下的至少一个取代或取代集：12/72/109/195、17/73/200、17/115、68/72/101/152/205、68/72/124、68/72/124/152、68/101/124/152/205、68/124/205、72/109/152/195、72/109/195、72/152、72/152/195、72/195、73、73/147、79、93、93/95/145/195、93/109/114/145/195、93/195、95/195、96/108/147/200、96/194/200、101/205、145/195、147、147/200、192、194、194/200、195、198和200，其中所述位置参考SEQ ID NO:330编号。
17.根据权利要求1-16中任一项所述的工程化酮还原酶变体，所述工程化酮还原酶变体包含与SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330具有至少90％序列同一性的多肽序列。
18.根据权利要求1-17中任一项所述的工程化酮还原酶变体，所述工程化酮还原酶变体包含与SEQ ID NO:4、6、80、104、172、186、194、252、270、272、286、328和/或330具有至少95％序列同一性的多肽序列。
19.根据权利要求1-18中任一项所述的工程化酮还原酶变体，所述工程化酮还原酶变体包含SEQ ID NO:6、80、104、172、186、194、252、270、272、286、328和/或330中列出的多肽序列。
20.根据权利要求1-19中任一项所述的工程化酮还原酶变体，其中所述工程化酮还原酶包含编码表5-1、表6-1、表7-1、表8-1、表10-1、表11-1、表16-1、表17-2、表18-1、表19-1、表19-2、表20-1、表20-2、表21-1、表22-1和/或表24-1中提供的变体的多肽序列。
21.根据权利要求1-20中任一项所述的工程化酮还原酶变体，其中所述工程化酮还原酶包含选自SEQ ID NO:6至SEQ ID NO:412中列出的偶数编号的序列的多肽序列。
22.根据权利要求1所述的工程化酮还原酶变体，其中所述工程化酮还原酶多肽能够将一种或更多种异-α-酸底物转化为一种或更多种相应的二氢-(ρ)-异-α-酸产物。
23.根据权利要求1所述的工程化酮还原酶变体，所述工程化酮还原酶变体能够以参考多肽SEQ ID NO:4活性的至少10倍将一种或更多种异-α-酸底物转化为一种或更多种相应的二氢-(ρ)-异-α-酸产物。
24.根据权利要求1-23中任一项所述的工程化酮还原酶变体，其中所述工程化酮还原酶包含与SEQ ID NO:4的酮还原酶相比改进的特性。
25.根据权利要求24所述的工程化酮还原酶变体，其中所述改进的特性包括与SEQ IDNO:4的酮还原酶相比改进的将异-α-酸转化为相应的二氢-(ρ)-异-α-酸的活性。
26.根据权利要求24所述的工程化酮还原酶变体，其中所述改进的特性包括与SEQ IDNO:4的酮还原酶相比改进的在高底物浓度的活性。
27.根据权利要求24所述的工程化酮还原酶变体，其中所述改进的特性包括与SEQ IDNO:4的酮还原酶相比改进的在低辅因子浓度的活性。
28.一种工程化多核苷酸序列，所述工程化多核苷酸序列编码权利要求1-27中任一项所述的工程化酮还原酶变体。
29.根据权利要求28所述的工程化多核苷酸序列，其中所述序列包含与选自SEQ IDNO:5至411中所列的奇数编号的序列的序列至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多地相同的多核苷酸序列。
30.一种载体，所述载体包含权利要求28和/或29所述的工程化多核苷酸序列。
31.根据权利要求30所述的载体，所述载体还包含至少一种控制序列。
32.根据权利要求30和/或31所述的载体，其中所述载体包括SEQ ID NO:413或414。
33.一种宿主细胞，所述宿主细胞包含权利要求30、31和/或32中任一项所述的载体。
34.一种用于产生权利要求1-27中任一项所述的工程化酮还原酶变体的方法，所述方法包括在权利要求33所述的宿主细胞产生所述工程化酮还原酶变体的条件下培养所述宿主细胞。
35.根据权利要求34所述的方法，所述方法还包括回收由所述宿主细胞产生的所述工程化酮还原酶变体的步骤。
36.根据权利要求34和/或35所述的方法，其中所述工程化酮还原酶变体由包含SEQ IDNO:413和/或414的载体的宿主细胞产生。
37.一种组合物，所述组合物包含至少一种权利要求1-27中任一项中提供的工程化酮还原酶变体。
CN202080067803.2A 2019-09-26 2020-09-24 酮还原酶多肽及多核苷酸 Pending CN114555795A (zh)

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