CN112930400A - 改性啤酒花产品的酶法生产 - Google Patents

改性啤酒花产品的酶法生产 Download PDF

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CN112930400A
CN112930400A CN201980071564.5A CN201980071564A CN112930400A CN 112930400 A CN112930400 A CN 112930400A CN 201980071564 A CN201980071564 A CN 201980071564A CN 112930400 A CN112930400 A CN 112930400A
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D·R·伯达尔
K·惠伦
B·P·布芬
M·B·琼斯
K·威廉斯
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Abstract

本发明涉及用于通过啤酒花衍生的异α酸至二氢‑(ρ)‑异α酸的酶催化生物转化来生产啤酒苦味剂的方法。

Description

改性啤酒花产品的酶法生产
序列表、表格或计算机程序的引用
根据37CFR§1.821以计算机可读形式(CRF)通过EFS-Web以文件名KALSEC_75_PCT_SEQ_LISTING.txt同时提交的序列表通过引用并入本文。序列表的电子副本创建于2019年9月25日，文件大小为259千字节。
发明领域
本发明涉及用于通过啤酒花衍生的异α酸至二氢-(ρ)-异α酸的酶催化生物转化来生产啤酒苦味剂的方法。二氢-(ρ)-异α酸具有优良的特性，提高了作为饮料添加剂的效用。消费者可能更喜欢通过该方法生产的二氢-(ρ)-异α酸，所述方法不需要使用苛性的化学试剂，并且使用可以是天然存在的酶。
发明背景
常规啤酒苦味处理方法使用完全新鲜的啤酒花、完全干的啤酒花或在煮沸过程中加入的啤酒花颗粒。通过用超临界二氧化碳提取啤酒花制成的啤酒花提取物，或者是通过在催化剂存在的情况下加热啤酒花制成的异构化啤酒花颗粒，是最近的变苦创新，也已被啤酒酿造者采用。啤酒花颗粒也可以在酿造过程的后期添加，在加干啤酒花法(dryhopping)的情况下，将啤酒花在过滤之前添加到成品啤酒中。这些方法的缺点是啤酒花中存在的苦味化合物的利用率低，这不利地影响了成本。以这种方式生产的啤酒或其它麦芽饮料对光不稳定，必须包装在深棕色的瓶子或罐子中，或放置在避免光诱导形成3-甲基-2-丁烯-1-硫醇(3-MBT)的地方，后者会产生明显的漏过光的(light struck)或臭鼬的气味。将瓶子放在硬纸板盒中，或者用避光或滤光纸、箔片或塑料覆盖物完全包裹它们，是保护这些饮料免受漏过光的风味和气味(light-struck flavor and aroma)影响的另一种昂贵方法。
常规酿造的啤酒中的苦味主要来源于异α酸。这些化合物是在酿造过程中通过葎草酮(humulones)的异构化形成的，葎草酮是啤酒花植物的蛇麻腺中天然存在的化合物。其结果是，鉴于异α酸对啤酒中的光化学反应的天然不稳定性，饮料易于形成漏过光的或臭鼬的风味和气味。
使用所谓的高级或改良啤酒花酸可以制备完全光稳定的啤酒或其它麦芽饮料。使用这些苦味剂制成的啤酒可以装在无色的燧石玻璃瓶中，而不用担心会形成臭鼬气味。二氢-(ρ)-异α酸是异α酸的还原产物，其对光稳定。迄今为止，这些化合物尚未在自然界中发现。常规地，负责光化学的异α酸的部分已经通过使用硼氢化钠还原羰基而被改变。
硼氢化钠是可用于还原酮的无机化合物。在皮肤接触、眼睛接触、吸入或摄入的情况下是极其危险的，口服LD50为160mg/kg(大鼠)。硼氢化钠也是易燃的、腐蚀性的，并且与氧化剂、酸、碱和水份具有极强的反应性(硼氢化钠；MSDS No.S9125；Sigma-Aldrich Co.:Saint Louis,MO，2015年11月1日。
消费者越来越倾向于使用天然材料，而不是合成或半合成材料。因此，不仅需要提供使用天然材料作为啤酒和其他饮料的苦味剂的组合物，还需要提供更天然地生产所述材料的方法。
生物催化生产是新兴技术，其提供高价值化合物的高选择性、安全、清洁和可扩大规模的生产。生物催化生产依靠于天然存在的酶来代替化学催化剂。
酶是能够催化特定化学反应的天然蛋白质。酶存在于自然界中，目前能够在生产改性啤酒花苦味化合物中替代化学催化剂(Robinson,P.K.,Enzymes:principles andbiotechnological applications.Essays Biochem 2015,59,1-41.)。
葎草酮是天然的次生代谢物，其会暴露在与植物啤酒花(Humulus lupulus)共栖的真菌和细菌中。居于土壤和居于植物中的真菌和细菌可能具有能够修饰葎草酮以达到解毒或清除目的的酶。另外，生物体可能已经进化出酶来修饰类葎草酮分子，但由于混杂的活性，这些酶对目标化合物异α酸具有活性(Hult,K.；Berglund,P.,Enzyme promiscuity:mechanism and applications.Trends Biotechnol.2007,25(5),231-238；Nobeli,I.；Favia,A.D.；Thornton,J.M.,Protein promiscuity and its implications for biotechnology.Nat.Biotechnol.2009,27(2),157-167.)。
催化氧化/还原反应(即氢和氧原子或电子从一种物质到另一种物质的转移)的酶被广泛地归类为氧化还原酶。更具体地说，将酮基还原为羟基的酶被称为酮还原酶或羰基还原酶，并且依赖于外源还原等效物(例如辅因子NADH、NADPH)的补充。与本文中表征的酶的现有命名一致，所述酶将被称为“酮还原酶”。
昂贵的辅因子(NADH，NADPH)的成本可以通过包括用于辅因子再循环的另外的酶和底物(例如葡萄糖脱氢酶和葡萄糖)，或者通过利用也能够氧化低成本和天然原料诸如乙醇的酮还原酶来降低。
酶在啤酒酿造中的效用及其对啤酒最终特性的有利影响有着丰富的先例(Pozen,M.,Enzymes in Brewing.Ind.Eng.Chem,1934,26(11),1127-1133.)。已知，在啤酒的自然发酵中酵母酶的存在会产生影响最终饮料的风味和香气的化合物(Praet,T.；Opstaele,F.；Jaskula-Goiris,B.；Aerts,G.；De Cooman,L.,Biotransformations of hop-derivedaroma compounds by Saccharomyces cerevisiae upon fermentation.Cerevisia,2012,36,125-132.)。外源添加的酶为酿造过程提供了多种改进，诸如降低的粘度、增加的可发酵糖、防寒和澄清(Wallerstein,L.(1947)Bentonite and Proteolytic Enzyme Treatmentof Beer,US Patent 2,433,411.；Ghionno,L.；Marconi,O.；Sileoni,V.；De Francesco,G.；Perretti,G.,Brewing with prolyl endopeptidase from Aspergillus niger:theimpact of enzymatic treatment on gluten levels,quality attributes,and sensoryprofile.Int.J.Food Sci.Technol,2017,52(6),1367-1374.)。另外，啤酒花提取物已用用于修饰啤酒花衍生的芳香化合物的酶专门地预处理(Gros,J.；Tran,T.T.H.；Collin,S.,Enzymatic release of odourant polyfunctional thiols from cysteine conjugatesin hop.J.Inst.Brew.2013,119(4),221-227.)。
然而，在本发明之前，在自然界中还没有观察到能够催化异α酸还原为二氢-(ρ)-异α酸的酶，因此文献中也没有描述所述酶。本文公开的方法代表了新型酶促反应。
发明目的
本发明的目的是提供酶法生产二氢-(ρ)-异α酸的方法，所述异α酸是源自啤酒花植物的天然苦味剂的改进形式。本方法旨被设计成替代使用化学试剂硼氢化钠的现有生产方法。
发明内容
本发明涉及可放大到工业水平以用于将异-α酸生物转化为二氢-(ρ)-异α酸的方法，所述二氢-(ρ)-异α酸可以在饮料中用作天然来源且光稳定的苦味剂。
本发明的一个方面是利用酮还原酶或表达编码所述酮还原酶的基因的微生物将异α酸高产率地生物转化为二氢-(ρ)-异α酸的方法。
本发明的另一方面涉及这样的用于生产二氢-(ρ)-异α酸的方法，其中所述方法在温和的温度和pH条件下在含水系统中进行，使其成为环境友好的生产方法。
在本发明的一个实施方案中，异α酸至二氢-(ρ)-异α酸的生物转化包括向异α酸的混合物中加入纯化的酮还原酶和NADPH或NADP，然后孵育直至获得所需的产率。
在本发明的另一个实施方案中，异α酸至二氢-(ρ)-异α酸的生物转化包括在用于辅因子再循环的异丙醇存在下向异α酸的混合物中加入纯化的酮还原酶和NADPH或NADP，随后孵育直至获得所需的产率。
在本发明的另外的实施方案中，异α酸即底物的浓度被最大化，以增加生物转化的体积生产率。
在本发明的另外的实施方案中，混合物中辅因子NADPH或NADP的浓度被最小化，以提高生物转化的经济性。
在本发明的一个实施方案中，异α酸至二氢-(ρ)-异α酸的生物转化包括在用于辅因子再循环的另一种酶(诸如葡萄糖脱氢酶)存在的情况下，将纯化的酮还原酶和NADPH或NADP添加到异α酸的混合物中，随后孵育直至获得所需的产率。
在本发明的另一个实施方案中，异α酸至二氢-(ρ)-异α酸的生物转化包括将全细胞生物催化剂加入到异α酸的混合物中，然后孵育直至获得所需的产率，其中所述全细胞生物催化剂是表达编码酮还原酶的基因的固定化微生物。
在本发明的另一个实施方案中，异α酸到二氢-(ρ)-异α酸的生物转化包括将异α酸供给表达编码酮还原酶的基因的正在生长的微生物。
在本发明的另一个实施方案中，将α酸生物转化为二氢-(ρ)-异α酸包括向α酸提取物中加入热稳定的酮还原酶，其中加热，并将混合物孵育，直至获得所需的二氢-(ρ)-异α酸产量。
在本发明的另一个实施方案中，在根据本发明的方法中使用的酮还原酶显示出对还原异α酸的C(4)处的侧链中的羰基，将光敏偶姻基团(acyloin group)转化为仲醇，并产生光稳定的异α酸衍生物的优先性(图1)。
在本发明的另一个实施方案中，在根据本发明的方法中使用的酮还原酶有利地显示出对催化以下六种主要异α酸中的任一种特定成员的还原的最低优先性或无优先性：顺-异葎草酮、反-异葎草酮、顺-异合葎草酮(cis-isocohumulone)、反-异合葎草酮、顺-异粘果酮(cis-isoadhumulone)和反-异粘果酮。
在本发明的另一个实施方案中，在根据本发明的方法中使用的酮还原酶特异性地还原顺-异葎草酮、顺-异合葎草酮和顺-异粘果酮(cis-isoadhumulone)。
在本发明的另一个实施方案中，在根据本发明的方法中使用的酮还原酶特异性地还原反-异葎草酮、反-异合葎草酮和反-异粘果酮。
在本发明的另一个实施方案中，在根据本发明的方法中使用显示上述底物特异性的2种或更多种酮还原酶的混合物，以将顺式和反式异α酸的混合物还原成它们各自的二氢异α酸。
在本发明的另一个实施方案中，可将显示底物特异性的2种或更多种酮还原酶的混合物加入到反应混合物中，以产生独特的二氢异α酸混合物，所述二氢异α酸混合物不同于通过化学还原剂诸如硼氢化钠产生的混合物。
在另一个实施方案中，本发明涉及如上定义的方法，其中商购可得的酮还原酶选自KRED-P1-B05、KRED-P2-B02、KRED-P2-C02、KRED-P2-C11、KRED-P2-D11、KRED-P2-G03、KRED-P2-G09、KRED-101、KRED-119、KRED-130、KRED-NADH-110、KRED-430、KRED-431、KRED-432、KRED-433、KRED-434、KRED-435和KRED-436。
本发明的另一个实施方案涉及酮还原酶，其包含SEQ ID NO:4、SEQ ID NO:6、SEQID NO:80、SEQ ID NO:104、SEQ ID NO:100、SEQ ID NO:136、SEQ ID NO:116、SEQ ID NO:132、SEQ ID NO:162、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:144、SEQ ID NO:146或SEQ ID NO:158的氨基酸序列。
在另一个实施方案中，本发明涉及如上定义的方法，其中所述酮还原酶或表达编码所述酮还原酶的基因的微生物，与包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:80、SEQID NO:104、SEQ ID NO:100、SEQ ID NO:136、SEQ ID NO:116、SEQ ID NO:132、SEQ ID NO:162、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:144、SEQ ID NO:146或SEQ ID NO:158的氨基酸序列的酮还原酶相比，可以任选地在氨基酸残基处具有一个或多个差异。
在另一个实施方案中，本发明涉及如上定义的方法，其中所述酮还原酶与包含SEQID NO:4、SEQ ID NO:6、SEQ ID NO:80、SEQ ID NO:104、SEQ ID NO:100、SEQ ID NO:136、SEQ ID NO:116、SEQ ID NO:132、SEQ ID NO:162、SEQ ID NO:150、SEQ ID NO:152、SEQ IDNO:144、SEQ ID NO:146或SEQ ID NO:158的氨基酸序列的酮还原酶具有99％、95％、90％、85％、80％、75％或70％同源性。
附图说明
图1显示了异α酸的代表性差向异构体的酶催化还原。
图2显示了在30℃下与异α酸(酸性溶液)一起孵育48小时的Codexis KRED-P1-B05(SEQ ID NO:4)的UPLC色谱图。
图3显示了在30℃下与异α酸(酸性溶液)一起孵育48小时的Codexis KRED-P1-B05(SEQ ID NO:4)的HPLC色谱图和峰值定量。
图4显示了在30℃下与异α酸一起孵育24小时的Codexis KRED-433的UPLC色谱图。
图5显示了在高底物浓度和低NADP浓度下SEQ ID NO:80、104、100、136、116、132、162、150、152、144、146和158的提高的KRED活性。
具体实施方式
在本发明中，酮还原酶替代了硼氢化钠的功能，并允许饮料添加剂二氢-(ρ)-异α酸的更天然的生产方法。所述酶可以是特异性地将酮基还原成异α酸(co-、n-ad-以及顺/反-)的任何或所有异构体的羟基的任何酮还原酶。所述酶可源自但不限于细菌、真菌或植物。所述酶可以是依赖于辅因子的(NADH或NADPH)或不依赖于辅因子的。
在本文中，“异α酸”、“啤酒花异α酸”(hop isoalpha acids)和“啤酒花衍生的异α酸”(hop-derived isoalpha acids)可以互换使用。
使用纯化的酶或含有酶和用于辅因子再循环的任选地另外的酶的混合物对异α酸溶液进行酶促处理。酶的量取决于孵育参数，包括持续时间、温度、底物的量和浓度。
或者，使用含有表达所述酶的微生物的混合物对异α酸溶液进行酶促处理。本发明还提供了根据本发明的还原异α酸的方法，所述方法包括培养产生酮还原酶的微生物，如果合适的话，诱导酮还原酶的表达。可以收获完整的细胞并将其直接加入到反应中，代替分离的酶，用于如上所述的异α酸的还原。此外，可将收获的细胞在加入还原反应之前固定。可通过已知的方法培养微生物并使其发酵。所述微生物可以是细菌或真菌。
顺-和反-异α酸的混合物可以与单一的显示出还原两种异构体的能力的酮还原酶一起孵育。或者，顺-和反-异α酸的混合物可以与2种或更多种显示出不同特异性的酮还原酶一起孵育，其中所得产物是顺-和反-二氢异α酸的混合物。
或者，只含有顺-异α酸的溶液可以与对顺式异构体特异的酮还原酶一起孵育，所得产物是顺-二氢异α酸的溶液。仅有顺-二氢异α酸的溶液可以显示出有利的苦味和/或热稳定性特性。
或者，只含有反-异α酸的溶液可以与对反式异构体特异的酮还原酶一起孵育，所得产物是反-二氢异α酸的溶液。仅有反-二氢异α酸的溶液可以显示出有利的苦味特性。
定制的反-和顺-异α酸的掺混物可以与一种或多种显示可变底物特异性的酮还原酶一起孵育，以产生原本无法获得的二氢异α酸的独特掺混物。
除了用于催化额外所需修饰的酶，诸如但不限于脱氢酶、异构酶、水合酶和裂解酶之外，还可使用酮还原酶对异α酸混合物进行酶促反应。可将不同活性的酶在一锅反应中组合或依次加入。
用于酶孵育的合适溶剂包括水和水与另一种与所述酶相容的溶剂诸如乙醇或异丙醇的混合物。酶促活性得益于水溶液的缓冲。缓冲剂包括但不限于：三(羟基甲基)氨基甲烷(又名Tris)、4-(2-羟基乙基)哌嗪-1-乙磺酸(又名HEPES)、磷酸钠和磷酸钾。
将酶和异α酸在合适的pH范围(例如6-10)和温度范围(例如10-90℃)内孵育，并在此温度下保持足够的时间，以将异α酸转化为所需的二氢-(ρ)-异α酸产量。连续搅拌将确保恒温和底物与酶的接触。反应持续时间(通常为24-48小时)将取决于酶和底物的量及浓度、存在的溶剂和选择的温度。
酶在溶液中可以是游离的，固定在珠粒或类似的可混合的支架上，或者固定在其上方通过异α酸的溶液的薄膜或树脂上。取决于纯化方法，酶的纯度水平可在30％与90+％之间变化。
酶可以通过物理过滤或离心而从终产品中去除。酶也可能因极端温度或pH而失去活性，并保留在最终产品中。
本文所用的酮还原酶包括商购可得的酮还原酶，诸如KRED-P1-B05、KRED-P2-B02、KRED-P2-C02、KRED-P2-C11、KRED-P2-D11、KRED-P2-G03、KRED-P2-G09、KRED-101、KRED-119、KRED-130、KRED-NADH-110、KRED-430、KRED-431、KRED-432、KRED-433、KRED-434、KRED-435和KRED-436(可从Codexis,Inc.,Redwood City,CA获得)。本发明还考虑了在氨基酸残基上体现出一个或多个差异的前述酮还原酶，以及与所述前述酮还原酶具有99％、95％、90％、85％、80％、75％和/或70％的同源性的酮还原酶。
本发明还包括通过已知诱变方法有意产生的酮还原酶，其对单一异α酸或异α酸的混合物显示出不同的活性。与商购可得的酮还原酶相比，一些变体在底物耐受性、温度耐受性、溶剂耐受性和/或周转率方面得到显著改善。
如本文中所用，“序列同源性百分比”、“同源性百分比”和“相一性百分比”是指多核苷酸序列或多肽序列之间的比较，通过在比较窗内比较两个最佳比对的序列来确定，其中与用于两个序列最佳比对的参考序列相比，比较窗内的多核苷酸或多肽序列的部分可包含添加或缺失(即，缺口)。所述百分比通过确定两个序列中出现相同核酸碱基或氨基酸残基的位置数目或者将核酸碱基或氨基酸残基与缺口对齐以产生匹配位置的数目，将匹配位置的数目除以比较窗口中的位置总数，然后将结果乘以100，得到序列同一性百分比来计算。使用BLAST和BLAST 2.0算法进行最佳比对和序列同源性百分比的确定(参见，例如，Altschul等人，J.Mol.Biol.215:403-410[1990]；和Altschul等人，Nucleic AcidsRes.3389-3402[1977])。用于进行BLAST分析的软件可通过国家生物技术信息中心网站公开获得。
实施例
以下实施例说明了本发明而不限制其范围。
实施例1
含有重组KRED基因的大肠杆菌表达宿主
将编码KRED的基因克隆到表达载体pCK110900中(参见美国专利申请公布第2006/0195947号的图3)，与在lac1阻遏物控制下的lac启动子有效连接。所述表达载体还包含P15a复制起始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。如本领域中已知的，通过使细胞进行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
含HTP 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在96孔浅孔微量滴定板的孔中接种到含有1％葡萄糖和30μg/mL氯霉素的190μl Luria-Bertani(LB)液体培养基中。将平板用可渗透O2的密封件密封，将培养物在20℃、200rpm和85％湿度下生长过夜。然后，将20μl的每种细胞培养物转移到含有380μL Terrific肉汤(TB)和30μg/mL氯霉素(CAM)的96孔深孔板的孔中。用可渗透O2的密封件密封深孔板，并在30℃、250rpm和85％湿度下进行孵育，直至达到0.6-0.8的OD600。然后通过添加异丙基β-d-1-硫代吡喃半乳糖苷(IPTG)至终浓度为1mM并在与最初使用的相同的条件下孵育过夜来诱导细胞培养物。然后在4℃、4000rpm下离心10分钟，沉淀细胞。弃去上清液，并在裂解前将沉淀于-80℃冷冻。
实施例3
含HTP KRED的细胞裂解物的制备
首先，通过加入含有100mM pH 8三乙醇胺*H2SO4(含2mM MgSO4)或100mM pH 8磷酸钾(含2mM MgSO4)、1g/L溶菌酶和0.5g/L硫酸多粘菌素B(PMBS)的150μL裂解缓冲液来裂解如实施例2所述生产的细胞沉淀。然后，将细胞沉淀在室温下于台式振荡器上振荡2小时。将平板以4000rpm在4℃下离心15分钟，以除去细胞碎片。然后将上清液用于生物催化反应，以测定其活性水平。
实施例4
由摇瓶(SF)培养物制备冻干裂解物
摇瓶程序可用于产生工程化KRED多肽摇瓶粉末(shake-flask powders，SFP)，其可用于二次筛选测定并且/或者用于本文所述的生物催化过程。与高通量(HTP)测定中使用的细胞裂解物相比，酶的摇瓶粉末(SFP)制备提供了更加纯化的工程化酶制剂(例如，高达总蛋白质的30％)，并且还允许使用更浓的酶溶液。为此，将如上所述生长的选定HTP培养物铺在含有1％葡萄糖和30μg/ml CAM的LB琼脂平板上，并在37℃生长过夜。将每个培养物的单个菌落转移到含有1％葡萄糖和330μg/ml CAM的6ml LB中。将培养物在30℃下以250rpm生长18小时，并以约1:50的比例传代培养至含30μg/ml CAM的250ml TB中，达到0.05的最终OD600。将培养物在30℃下以250rpm生长约3小时，达到0.8-1.0的OD600，并用1mM IPTG诱导。然后培养物在30℃以250rpm生长20小时。将培养物离心(4000rpm 20分钟，4℃)。弃去上清液，将沉淀重新悬浮在35ml含2mM MgSO4的50mM pH 8磷酸钾中。将重新悬浮的细胞离心(4000rpm 20分钟，4℃)。弃去上清液，将沉淀重新悬浮在6ml含2mM MgSO4的50mM pH 8磷酸钾中，用Constant Systems(One Shot)的细胞破裂仪裂解细胞。将裂解物沉淀(以10,000rpm在4℃下进行60分钟)，将上清液冷冻和冻干以产生摇瓶(SF)酶。
实施例5
商购可得的KRED酶小组的筛选
KRED筛选测定
使用商购可得的“KRED筛选试剂盒”(Codexis Inc.,Redwood City,CA)测试了一组商购可得的酮还原酶还原异α酸的能力。对于该筛选中的一部分酶，酶测定在1.5mL体积的试管中以1000μL总体积/管进行，所述1000μL总体积在含有1.7mM MgSO4的128mM pH 7磷酸钠中包含10g/L酶粉、2.9或6.9g/L异α酸底物和0.8g/L的于30体积％异丙醇(IPA)中的NADP。封闭试管，在30℃下以180rpm振荡培养24-48小时。使用10,000MWCO离心过滤装置过滤获得的反应混合物以除去酶。通过UPLC定量异α酸和二氢-(ρ)-异α酸。参见，例如，图4所示的Codexis KRED-433的色谱图。
对于该筛选中的其它部分的酶，酶测定在1.5mL体积的试管中以1000μL总体积/管进行，所述1000μL总体积在含有1.7mM MgSO4的于263mM pH 7磷酸钠中包含10g/L酶粉、1.5g/L异α酸底物、0.8g/L NADP、0.7g/L NAD、14.4g/L D-葡萄糖和4.3U/mL的葡萄糖脱氢酶。封闭试管，在30℃下以180rpm振荡培养24-48小时。使用10,000MWCO离心过滤装置过滤获得的反应混合物以除去酶。通过UPLC定量异α酸和二氢-(ρ)-异α酸。
酮还原酶特征测定
在各种反应条件下进一步表征了产生可检测量的二氢-(ρ)-异α酸的酮还原酶。为此，酶测定在2.0mL体积的试管中以1000μL总体积/试管进行，所述1000μL总体积在含有1.7mM MgSO4的100-263mM pH 7-9磷酸钠(或者可选地，Tris)中包含10-20g/L酶粉、1.5-6.0g/L异α酸底物、0.8g/L NADP(任选地，0.7g/L NAD、14.4g/L D-葡萄糖、4.3U/mL葡萄糖脱氢酶或30体积％异丙醇)。封闭试管，在30-40℃下以180rpm振荡培养24-48小时。过滤所得反应混合物以除去酶。通过UPLC-MS/MS和HPLC检测异α酸和二氢-(ρ)-异α酸。
结果
KRED筛选结果
Codexis“KRED筛选试剂盒”的几种商购可得的酶能够还原异α酸(表1)。最初的试剂盒由24种酮还原酶(称为KRED)组成，所述酶是由制造商选择的(即天然的)或针对广泛的底物范围和增强的活性而加工的。另一种试剂盒由基于KRED-130骨架的7种工程化变体组成。
Figure BDA0003043554180000121
Figure BDA0003043554180000131
Figure BDA0003043554180000141
酮还原酶的表征
如果通过UPLC以比缺乏酶的对照样品大的强度检测到与顺/反-co/ad/n-二氢-(ρ)-异α酸相对应的峰，则确定所述酶能够还原异α酸。
通过定性比较UPLC峰高，KRED-P1-B05(SEQ ID NO:4)在24小时内产生最多的二氢-(ρ)-异α酸(参见图2)。KRED-P1-B05(SEQ ID NO:4)源自由编码来自开菲尔乳杆菌(Lactobacillus kefiri)的天然存在的野生型酮还原酶的氨基酸序列(SEQ ID NO:2)的核苷酸(SEQ ID NO:1)编码的酶。由这种酮还原酶产生的二氢-(ρ)-异α酸以足够高的浓度存在，可通过HPLC定量。在30℃下24小时内，KRED-P1-B05获得了18％的二氢-(ρ)-异α酸的产率。将所述反应以一式两份进行48小时的持续时间，获得42％的二氢-(ρ)-异α酸产率。(参见图3)。当反应温度从30℃升高到37℃持续48小时时，产率为33％。
KRED-P1-B05活性最初使用缓冲液(含1.7mM硫酸镁的128mM磷酸钠pH 7，0.8g/LmM NADP)以及30体积％异丙醇(用于辅因子再循环)进行检测。多种反应条件(温度、持续时间、缓冲液组成、底物浓度等)被确定足以还原异α酸。
底物特异性
用于生物转化目的的理想酮还原酶对异葎草酮同源物没有底物特异性，所述异葎草酮同源物根据侧链组成(得到n-isohumulone、近异葎草酮(ad-isohumulone)和合异葎草酮(co-isohumulone))而变化。另外，酮还原酶对异葎草酮顺式和反式异构体没有特异性，所述异构体在酶促还原位点附近的C4叔醇基团上存在空间差异。底物特异性由氨基酸序列决定，因此由酶的底物结合口袋的几何形状决定。与更受限制的结合袋相比，更大的结合袋容纳更大的基底以及更多种类的底物。
尽管异α酸分子上存在两个额外的酮基，但对于所有表征的酮还原酶，仅观察到C4侧链上的所需还原。
实施例6
对源自SEQ ID NO:4的工程化多肽进行进化和筛选以获得提高的KRED活性
基于对用于还原烯-酸底物的变体进行筛选的结果，将SEQ ID NO:4的酶选为亲本酶。使用成熟的技术(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。如实施例2所述，在HTP中产生由每个基因编码的多肽，并如实施例3所述产生可溶性裂解物。
将表现出优异的KRED活性的编码SEQ ID NO:4的SEQ ID NO:3工程化多核苷酸用于产生表2的进一步工程化的多肽。与起始多肽相比，这些多肽显示出改善的由异α酸至二氢-(ρ)-异α酸的形成。使用如上所述的定向进化方法以及下表2中所述的HTP测定和分析方法由SEQ ID NO:4的“骨架”氨基酸序列产生工程化多肽。
Figure BDA0003043554180000151
Figure BDA0003043554180000161
定向进化始于SEQ ID NO:3所示的多核苷酸。然后选择工程化多肽作为起始“骨架”基因序列。使用各种公知的技术(例如，饱和诱变、先前鉴定的有益氨基酸差异的重组)产生工程化多肽文库，并使用HTP测定和分析方法进行筛选，所述方法测量多肽将异α酸底物转化为所需的二氢-(ρ)-异α酸产物的能力。
酶测定以96孔格式于200μL总体积/孔中进行，所述200μL总体积在含有2mM MgSO4的100mM pH 8三乙醇胺*H2SO4中包含50％v/v的HTP酶裂解物、8g/L异α酸底物和于40％体积的异丙醇(IPA)中的0.1g/L NADP。将平板密封，并在40℃下以600rpm振荡培养20-24小时。
20-24小时后，加入1000μL含0.1％乙酸的乙腈。将板密封，在4℃下以4000rpm离心10分钟。在HPLC分析之前，将淬灭的样品在50:50乙腈:水混合物中进一步稀释4-5倍。HPLC运行参数如下表3所述。
Figure BDA0003043554180000171
Figure BDA0003043554180000181
实施例7
对源自SEQ ID NO:6的工程化多肽进行进化和筛选以获得提高的KRED活性
使用成熟的技术(例如，饱和诱变和先前鉴定的有益突变的重组)产生工程化基因文库。如实施例2所述，在HTP中产生由每个基因编码的多肽，并如实施例3所述产生可溶性裂解物。
将表现出优异的KRED活性的编码SEQ ID NO:6的多肽的SEQ ID NO:5的工程化多核苷酸用于产生表4的进一步工程多肽。与起始多肽相比，这些多肽显示出改善的由异α酸到二氢-(ρ)-异α酸的形成。使用如上所述的定向进化方法以及表3中描述的HTP测定和分析方法由SEQ ID NO:6的“骨架”氨基酸序列产生工程化多肽。
Figure BDA0003043554180000182
Figure BDA0003043554180000191
定向进化始于SEQ ID NO:5所示的多核苷酸。然后选择工程化多肽作为起始“骨架”基因序列。使用各种公知的技术(例如饱和诱变、先前鉴定的有益氨基酸差异的重组)产生工程化多肽的文库，并使用HTP测定和测量多肽将异α酸底物转化为所需的二氢-(ρ)-异α酸产物的能力的分析方法进行筛选。
酶测定以96孔格式于200μL总体积/孔中进行，所述200μL总体积在含有2mM MgSO4的100mM pH 8三乙醇胺*H2SO4中包含50％v/v的HTP酶裂解物、16或40g/L的异α酸底物和于40体积％的异丙醇(IPA)中的0.1g/L NADP。将平板密封，并在40℃下以600rpm振荡培养20-24小时。
20-24小时后，加入1000μL含0.1％乙酸的乙腈。将平板密封，在4℃下以4000rpm离心10分钟。在HPLC分析之前，将淬灭的样品在50:50的乙腈:水混合物中进一步稀释10-20倍。所述HPLC运行参数如表3所述。
实施例8
对源自SEQ ID NO:80的工程化多肽进行进化和筛选以获得提高的KRED活性
使用成熟的技术(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。如实施例2所述，在HTP中产生由每个基因编码的多肽，并如实施例3所述产生可溶性裂解物。
将编码表现出优异KRED活性的SEQ ID NO:80多肽的SEQ ID NO:79的工程化多核苷酸用于产生表5的进一步工程化的多肽。与起始多肽相比，这些多肽显示出改善的由异α酸到二氢-(ρ)-异α酸的形成。使用如上所述的定向进化方法以及下表3中所述的HTP测定和分析方法由SEQ ID NO:80的“骨架”氨基酸序列产生工程化多肽。
Figure BDA0003043554180000201
Figure BDA0003043554180000211
定向进化始于SEQ ID NO:79所示的多核苷酸。然后选择工程化多肽作为起始“骨架”基因序列。使用各种公知的技术(例如饱和诱变、先前鉴定的有益氨基酸差异的重组)产生工程化多肽的文库，并使用HTP测定和测量多肽将异α酸底物转化为所需的二氢-(ρ)-异α酸产物的能力的分析方法进行筛选。
酶测定以96孔格式于200μL总体积/孔中进行，所述200μL总体积在含有2mM MgSO4的100mM pH 8磷酸钾中包含25％v/v HTP酶裂解物、60或80g/L异α酸底物和于40体积％的异丙醇(IPA)中的0.02g/L NADP。将平板密封，并在45℃下以600rpm振荡培养20-24小时。
20-24小时后，加入1000μL含0.1％乙酸的乙腈。将平板密封，在4℃下以4000rpm离心10分钟。在HPLC分析之前，将淬灭的样品在50:50的乙腈:水混合物中进一步稀释20-40倍。所述HPLC运行参数如表3所述。
实施例9
对源自SEQ ID NO:80的工程化多肽进行进化和筛选以获得高底物浓度下的提高的KRED活性
使用成熟的技术(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。如实施例2所述，在HTP中产生由每个基因编码的多肽，并如实施例3所述产生可溶性裂解物。
将编码表现出优异KRED活性的SEQ ID NO:80多肽的SEQ ID NO:79的工程化多核苷酸用于产生表6的进一步工程化的多肽。与起始多肽相比，这些多肽显示出改善的由异α酸到二氢-(ρ)-异α酸的形成。使用如上所述的定向进化方法由SEQ ID NO:80的“骨架”氨基酸序列产生工程化多肽，并且所述工程化多肽描述于下表3中。
Figure BDA0003043554180000221
定向进化始于SEQ ID NO:79所示的多核苷酸。然后选择工程化多肽作为起始“骨架”基因序列。使用各种公知的技术(例如饱和诱变、先前鉴定的有益氨基酸差异的重组)产生工程化多肽的文库，并使用HTP测定和测量多肽将异α酸底物转化为所需的二氢-(ρ)-异α酸产物的能力的分析方法进行筛选。
酶测定以96孔格式于200μL总体积/孔中进行，所述200μL总体积在含有2mM MgSO4的100mM pH 8磷酸钾中包含10-20％v/v HTP酶裂解物、80或160g/L异α酸底物和于40体积％的异丙醇(IPA)中的0.02g/L NADP。将平板密封，并在45℃下以600rpm振荡培养20-24小时。
20-24小时后，加入1000μL含0.1％乙酸的乙腈。将平板密封，在4℃下以4000rpm离心10分钟。在HPLC分析之前，将淬灭的样品在50:50的乙腈:水混合物中进一步稀释20-40倍。所述HPLC运行参数如表3所述。
实施例10
对源自SEQ ID NO:80、104、100、136、116、132、162、150、152、144和146的工程化多肽进行进化和筛选以获得在高底物浓度和低NADP浓度下的提高的KRED活性
通过将100mg酶粉末溶解在500μL的含有2mM MgSO4和0.1g/L NADP的pH8磷酸钾缓冲液中来制备200g/L酶原液溶液。向96孔板中的孔中加入40μL酶/NADP原液溶液、80μL异丙醇和80μL 40重量％的异α酸水溶液。最终的反应组合物为40g/L的酶、160g/L的异α酸和于40％IPA中的0.02g/L NADP。将平板密封并在40℃下孵育24小时，然后淬灭并通过HPLC-UV进行分析。数据如表7和图5所示。
Figure BDA0003043554180000231
实施例11
利用通过异丙醇氧化进行的辅因子再循环对酸化啤酒花衍生的异α酸进行酶处理
以实施例10中所述的方式处理异α酸，其中异α酸酸的来源是高度浓缩的物质(68.9％的异α酸酸)，其pH值小于7。
实施例12
利用通过葡萄糖脱氢酶进行的辅因子再循环对啤酒花衍生的异α酸进行酶处理
除用4.3U/mL葡萄糖脱氢酶、0.7g/L mM NAD和14.4g/L D-葡萄糖代替异丙醇外，以实施例10中所述的方式处理异α酸。
实施例13
在辅因子再循环不存在的情况下对啤酒花衍生的异α酸进行酶处理
除用等摩尔量的NADPH作为底物代替异丙醇外,以实施例10中所述的方式处理异α酸。
实施例14
利用通过乙醇氧化进行的辅因子再循环对啤酒花衍生的异α酸进行酶处理
除用乙醇代替异丙醇外，以实施例10中所述的方式处理异α酸。
实施例15
利用通过SiO2产生的固定化酮还原酶对啤酒花衍生的异α酸进行酶处理
将酮还原酶吸附在SiO2上，并用戊二醛交联，得到固定的酮还原酶材料。以实施例10中描述的方式用固定化酮还原酶处理异α酸。将获得的反应混合物以10,000g离心以除去固定化酶。
实施例16
利用通过DEAE-纤维素产生的固定化酮还原酶对啤酒花衍生的异α酸进行酶处理
将酮还原酶用戊二醛交联并吸附在DEAE纤维素上，得到固定化酮还原酶材料。以实施例10中描述的方式用固定化酮还原酶处理异α酸。将获得的反应混合物以10,000g离心以除去固定化酶。
实施例17
利用通过PEI处理的矾土产生的固定化酮还原酶对啤酒花衍生的异α酸进行酶处 理
将酮还原酶用戊二醛交联并吸附到聚乙基亚胺(PEI)处理的矾土上，得到固定化酮还原酶材料。以实施例10中描述的方式用固定化酮还原酶处理异α酸。将获得的反应混合物以10,000g离心以除去固定化酶。
实施例18
利用NADH辅因子再循环酶对啤酒花衍生的异α酸进行酶处理
其中NADPH辅因子被NADH取代的酶处理。以实施例10中描述的方式处理异α酸，但用NAD代替NADP。
实施例19
对啤酒花衍生的异α酸进行酶处理，随后进行提取
进行酶处理，然后进行提取，以增加二氢-(ρ)-异α酸的终浓度。以实施例10中描述的方式处理异α酸。将获得的反应混合物过滤以除去酶，并用食品级溶剂提取，以获得所需浓度的二氢-(ρ)-异α酸。
实施例20
对啤酒花衍生的异α酸进行酶处理，随后进行热灭活
以实施例10中描述的方式处理异α酸。在30℃下在以180rpm进行轨道摇动的情况下孵育反应物24小时。将所得反应混合物在80-100℃下加热10-30分钟以使酶灭活。
实施例21
对啤酒花衍生的异α酸进行酶处理，随后进行化学灭活
以实施例10中描述的方式处理异α酸。在30℃下在以180rpm进行轨道摇动的情况下孵育反应物24小时。将食品级乙醇添加至终浓度>50％以使酶灭活。
实施例22
利用固定化酮还原酶再循环对啤酒花衍生的异α酸进行酶处理
将酮还原酶用戊二醛交联并吸附在DEAE纤维素上，得到固定化酮还原酶材料。然后以实施例10中所述的方式用固定化酮还原酶处理异α酸。将获得的反应混合物以10,000g离心，以从反应溶液中分离出固定化酮还原酶。回收固定的酮还原酶，用水或含水缓冲液洗涤，并在新的反应混合物中再次使用。
结论
162种酮还原酶已被表征为将异α酸转化为二氢-(ρ)-异α酸。本研究中表征的酮还原酶具有先前未曾描述过的酶促活性。本研究中表征的酮还原酶都将酮基还原成醇，因此是酮还原酶。这些结果表明，在新型生物转化方法中，酮还原酶生物催化剂可用于将异α酸转化为二氢-(ρ)-异α酸。本发明旨在替代目前使用硼氢化钠的方法。
*****
本发明的范围不受本文描述的具体实施方案的限制。实际上，根据上述说明，除了本文中描述的变动外的本发明的各种变动对于本领域技术人员来说是很显然的。此类变动旨在落入所附权利要求书的范围内。
本文引用的所有专利、申请、出版物、测试方法、文献和其它材料据此通过引用并入。
*****
引用的参考文献
1.Sodium Borohydride；MSDS No.S9125；Sigma-Aldrich Co.:Saint Louis,MONovember 01,2015.(2017年8月6日访问的)。
2.Robinson,P.K.,Enzymes:principles and biotechnologicalapplications.Essays Biochem 2015,59,1-41.
3.Hult,K.；Berglund,P.,Enzyme promiscuity:mechanism andapplications.Trends Biotechnol.2007,25(5),231-238.
4.Nobeli,I.；Favia,A.D.；Thornton,J.M.,Protein promiscuity and itsimplications for biotechnology.Nat.Biotechnol.2009,27(2),157-167.
5.Pozen,M.,Enzymes in Brewing.Ind.Eng.Chem,1934,26(11),1127-1133.
6.Praet,T.；Opstaele,F.；Jaskula-Goiris,B.；Aerts,G.；De Cooman,L.,Biotransformations of hop-derived aroma compounds by Saccharomyces cerevisiaeupon fermentation.Cerevisia,2012,36,125-132.
7.Wallerstein,L.(1947)Bentonite and Proteolytic Enzyme Treatment ofBeer,US Patent 2,433,411.
8.Ghionno,L.；Marconi,O.；Sileoni,V.；De Francesco,G.；Perretti,G.,Brewing with prolyl endopeptidase from Aspergillus niger:the impact ofenzymatic treatment on gluten levels,quality attributes,and sensoryprofile.Int.J.Food Sci.Technol,2017,52(6),1367-1374.
Gros,J.；Tran,T.T.H.；Collin,S.,Enzymatic release of odourantpolyfunctional thiols from cysteine conjugates in hop.J.Inst.Brew.2013,119(4),221-227.
序列表
<110> KALAMAZOO HOLDINGS, INC.
<120> 改性啤酒花产品的酶法生产
<130> KALSEC 75 PCT
<140>
<141> 2019-09-26
<150> US 62/736,555
<151> 2018-09-26
<160> 162
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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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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
<220>
<223> 来自开菲尔乳杆菌的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

Claims (21)

1.一种用于制备二氢-(ρ)-异α酸的方法，所述方法包括用酮还原酶或表达编码所述酮还原酶的基因的微生物处理异α酸。
2.根据权利要求1所述的方法，其中所述方法在含水系统中进行。
3.根据权利要求2所述的方法，其中所述方法在温和的温度和pH条件下进行。
4.根据权利要求1所述的方法，所述方法包括将所述酮还原酶和NADPH或NADP加入到异α酸的混合物中，然后进行孵育。
5.根据权利要求1所述的方法，所述方法包括在异丙醇存在下，将所述酮还原酶和NADPH或NADP加入到异α酸的混合物中以用于辅因子循环，然后进行孵育。
6.根据权利要求1所述的方法，其中将异α酸即底物的浓度最大化以增加生物转化的体积生产率。
7.根据权利要求1所述的方法，其中将所述混合物中所述辅因子NADPH或NADP的浓度最小化以提高所述生物转化的经济性。
8.根据权利要求1所述的方法，所述方法包括在另一种酶存在的情况下，将所述酮还原酶和NADPH或NADP加入到异α酸的混合物中以用于辅因子循环，然后进行孵育。
9.根据权利要求1所述的方法，所述方法包括将全细胞生物催化剂加入到异α酸的混合物中，然后进行孵育，其中所述全细胞生物催化剂是表达编码酮还原酶的基因的固定化微生物。
10.根据权利要求1所述的方法，所述方法包括用表达编码所述酮还原酶的基因的正在生长的微生物处理异α酸。
11.根据权利要求1所述的方法，所述方法包括将所述酮还原酶加入到异α酸的提取物中，其中所述酮还原酶是热稳定的，其中进行加热，并孵育所述混合物。
12.根据权利要求1所述的方法，其中所述酮还原酶特异性还原顺-异葎草酮、顺-异合葎草酮(cis-isocohumulone)和顺-异粘果酮(cis-isoadhumulone)。
13.根据权利要求1所述的方法，其中所述酮还原酶特异性还原反-异葎草酮、反-异合葎草酮(trans-isocohumulone)和反-异粘果酮(trans-isoadhumulone)。
14.根据权利要求1所述的方法，所述方法包括加入2种或更多种酮还原酶的混合物，以将顺-和反-异α酸的混合物还原成它们各自的二氢异α酸。
15.根据权利要求14所述的方法，其中2种或更多种酮还原酶的所述混合物产生独特的二氢异α酸混合物，所述混合物不同于通过化学还原剂诸如硼氢化钠产生的混合物。
16.根据任一前述权利要求所述的方法，其中所述酮还原酶选自KRED-P1-B05、KRED-P2-B02、KRED-P2-C02、KRED-P2-C11、KRED-P2-D11、KRED-P2-G03、KRED-P2-G09、KRED-101、KRED-119、KRED-130、KRED-NADH-110、KRED-430、KRED-431、KRED-432、KRED-433、KRED-434、KRED-435和KRED-436。
17.根据权利要求16所述的方法，其中所述酮还原酶或表达编码所述酮还原酶的基因的微生物与选自由KRED-P1-B05、KRED-P2-B02、KRED-P2-C02、KRED-P2-C11、KRED-P2-D11、KRED-P2-G03、KRED-P2-G09、KRED-101、KRED-119、KRED-130、KRED-NADH-110、KRED-430、KRED-431、KRED-432、KRED-433、KRED-434、KRED-435和KRED-436组成的组的酮还原酶相比，可以任选地在氨基酸残基处具有一个或多个差异。
18.根据权利要求17所述的方法，其中所述酮还原酶与选自由KRED-P1-B05、KRED-P2-B02、KRED-P2-C02、KRED-P2-C11、KRED-P2-D11、KRED-P2-G03、KRED-P2-G09、KRED-101、KRED-119、KRED-130、KRED-NADH-110、KRED-430、KRED-431、KRED-432、KRED-433、KRED-434、KRED-435和KRED-436组成的组的酮还原酶具有99％、95％、90％、85％、80％、75％或70％的同源性。
19.根据任一前述权利要求所述的方法，其中所述酮还原酶包含选自由SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:80、SEQ ID NO:104、SEQ ID NO:100、SEQ ID NO:136、SEQ ID NO:116、SEQ ID NO:132、SEQ ID NO:162、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:144、SEQID NO:146和SEQ ID NO:158组成的组的氨基酸序列。
20.根据权利要求19所述的方法，其中所述酮还原酶或表达编码所述酮还原酶的基因的微生物与选自由SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:80、SEQ ID NO:104、SEQ ID NO:100、SEQ ID NO:136、SEQ ID NO:116、SEQ ID NO:132、SEQ ID NO:162、SEQ ID NO:150、SEQID NO:152、SEQ ID NO:144、SEQ ID NO:146和SEQ ID NO:158组成的组的酮还原酶相比，可以任选地在氨基酸残基上具有一个或多个差异。
21.根据权利要求20所述的方法，其中所述酮还原酶与选自由SEQ ID NO:4、SEQ IDNO:6、SEQ ID NO:80、SEQ ID NO:104、SEQ ID NO:100、SEQ ID NO:136、SEQ ID NO:116、SEQID NO:132、SEQ ID NO:162、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:144、SEQ ID NO:146和SEQ ID NO:158组成的组的酮还原酶具有99％、95％、90％、85％、80％、75％或70％的同源性。
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