CN106715687B - 丙酮酸脱氢酶突变体、包含突变体的微生物和通过使用微生物生产l-氨基酸的方法 - Google Patents

丙酮酸脱氢酶突变体、包含突变体的微生物和通过使用微生物生产l-氨基酸的方法 Download PDF

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CN106715687B
CN106715687B CN201680002649.4A CN201680002649A CN106715687B CN 106715687 B CN106715687 B CN 106715687B CN 201680002649 A CN201680002649 A CN 201680002649A CN 106715687 B CN106715687 B CN 106715687B
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许兰
文准玉
裵贤原
金亨俊
宋成基
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Abstract

本发明涉及：丙酮酸脱氢酶突变体；编码该突变体的多核苷酸；具有产L‑氨基酸的能力并且包含丙酮酸脱氢酶突变体的棒状杆菌属的微生物；和通过使用该微生物生产L‑氨基酸的方法。

Description

丙酮酸脱氢酶突变体、包含突变体的微生物和通过使用微生 物生产L-氨基酸的方法
技术领域
本公开内容涉及丙酮酸脱氢酶变体、编码该丙酮酸脱氢酶变体的多核苷酸、具有生产L-氨基酸能力的包含该变体的棒状杆菌属的微生物、和使用该微生物生产L-氨基酸的方法。
背景技术
丙酮酸脱氢酶多酶复合物(PDHC)是将在糖酵解期间生成的丙酮酸转变为乙酰辅酶A的酶并且是用于测定碳引入三羧酸循环的重要的酶。PDHC由丙酮酸脱氢酶(E1p)、二氢硫辛酰胺乙酰转移酶(E2p)和二氢硫辛酰胺脱氢酶(E3p)组成。在它们之中，E1p酶由aceE基因编码。虽然已经已知由于aceE基因的缺失和弱化而引起的在生产L-赖氨酸的菌株中L-赖氨酸生产的改变(Blombach等,Appl.Microbiol.Biotechnol.,76:615,2007；Buchholz J等,Appl Environ Microbiol.,79(18):5566-75,2013)，但是关于可以提高生产L-氨基酸能力的E1p变体还没有报道。
公开内容
技术问题
本发明人已经进行了大量的努力来研发可用于以高浓度生产L-氨基酸的E1p变体和使用其的微生物，结果，已经研发了E1p变体并发现L-氨基酸可以以高产量从包含该E1p变体的微生物生产，从而完成本公开内容。
技术方案
本公开内容的目标是提供丙酮酸脱氢酶变体。
本公开内容的另一个目标是提供编码该丙酮酸脱氢酶变体的多核苷酸。
本公开内容的进一步目标是提供生产L-氨基酸的棒状杆菌属的微生物，其包含该丙酮酸脱氢酶变体。
本公开内容的仍进一步目标是提供生产L-氨基酸的方法，其包括：(a)在培养基中培养生产L-氨基酸的棒状杆菌属的微生物——其包含丙酮酸脱氢酶变体——以生产L-氨基酸；和(b)从培养的微生物或培养基中回收L-氨基酸。
本发明的有益效果
具有减弱活性的酶可以使用本公开内容的丙酮酸脱氢酶变体获得。因此，L-氨基酸可以使用微生物以高效率生产，该微生物与包含野生型丙酮酸脱氢酶蛋白的产L-氨基酸的微生物相比包含具有减弱活性的丙酮酸脱氢酶变体。另外，与丙酮酸脱氢酶缺失的微生物不同，本公开内容的微生物能够有效生产L-氨基酸同时几乎没有抑制该微生物的生长。例如，作为动物饲料的必需氨基酸的赖氨酸需要从工业方面以大规模生产。因此，如在本公开内容中以高效率生产L-氨基酸可以降低制造动物饲料的成本。
最佳模式
本公开内容的一个方面提供了丙酮酸脱氢酶变体，其包括在SEQ ID NO:1的190至205的位置处的氨基酸区域中或在SEQ ID NO:1的415到440的位置处的氨基酸区域中的至少一个氨基酸置换。
如本文所使用，术语“丙酮酸脱氢酶”指的是组成丙酮酸脱氢酶多酶复合物(PDHC)的酶中的一种，其参与丙酮酸转变为乙酰辅酶A。如本文所使用，丙酮酸脱氢酶不被具体限制，只要其具有相应的活性，并且其可以是衍生自棒状杆菌属——具体地，谷氨酸棒状杆菌——的微生物的丙酮酸脱氢酶，但是不限于此。例如，丙酮酸脱氢酶可以是SEQ ID NO:1的氨基酸序列或者与SEQ ID NO:1的氨基酸序列具有至少75％，具体地至少80％，更具体地85％，并且甚至更具体地90％、91％、92％、93％、94％、95％、96％、97％、98％、或者99％或更高的同源性的氨基酸序列。具有SEQ ID NO:1的氨基酸序列的E1p蛋白可以由具有SEQ IDNO:2的多核苷酸序列的aceE基因编码，但是不限于此。另外，如果氨基酸序列与上述序列具有同源性并且与SEQ ID NO:1的蛋白质具有基本上相同或相应的生物学活性，则具有缺失、修饰、置换或添加的氨基酸序列也应当属于本公开内容的范围是显而易见的。在本公开内容中，编码丙酮酸脱氢酶的任何多核苷酸序列可以属于本公开内容的范围。例如，多核苷酸序列可以是与SEQ ID NO:2的多核苷酸序列具有至少75％，具体地至少80％，更具体地85％，并且甚至更具体地90％、91％、92％、93％、94％、95％、96％、97％、98％、或者99％或更高的同源性的多核苷酸序列。另外，基于密码子简并性或考虑生物体表达蛋白质优选的密码子，编码蛋白质的多核苷酸序列可以在不改变从编码区表达的蛋白质的氨基酸序列的范围内具有编码区上的各种变体。
根据本公开内容的丙酮酸脱氢酶变体可以包括在SEQ ID NO:1的190到205或415到440的位置处的氨基酸区域中的至少一个、至少二个、至少三个、至少四个、至少五个、至少六个、至少七个、至少八个、至少九个或至少十个氨基酸置换。
具体而言，根据本公开内容的丙酮酸脱氢酶变体可以包括在SEQ ID NO:1的190到205的位置处的氨基酸区域中的至少一个、至少二个、至少三个、至少四个氨基酸置换。具体地，在SEQ ID NO:1的190到205的位置处的氨基酸区域中的氨基酸置换可以选自位置190、195、199和201处的氨基酸，但是不限于此。
在SEQ ID NO:1的190到205的位置处的氨基酸区域中的置换可以是用不同种类氨基酸置换在190到205的位置处的氨基酸区域中的至少一个氨基酸，更具体地，用不同种类氨基酸置换在190、195、199和201的位置处的至少一个氨基酸，并且甚至更具体地，为选自如下的至少一种：用缬氨酸置换位置190处的谷氨酸(E190V)、用组氨酸置换位置195处的谷氨酰胺(Q195H)、用丝氨酸置换位置199处的脯氨酸(P199S)和用丙氨酸置换位置201处的酪氨酸(Y201A)，但是不具体地限于此。
另外，根据本公开内容的丙酮酸脱氢酶变体可以包括在SEQ ID NO:1的415到440的位置处的氨基酸区域中的至少一个、至少二个、至少三个、至少四个、至少五个或至少六个氨基酸置换。具体地，在SEQ ID NO:1的415到440的位置处的氨基酸区域中的氨基酸置换可以选自位置418、428、432、435和438处的氨基酸。
SEQ ID NO:1的415到440的位置处的氨基酸区域中的置换可以是用不同种类氨基酸置换在415到440的位置处的氨基酸区域中的至少一个氨基酸，具体地，用不同种类氨基酸置换在位置418、428、432、435和438处的至少一个氨基酸，并且甚至更具体地，为选自如下的至少一种：用组氨酸置换位置418处的酪氨酸(Y418H)、用丙氨酸置换位置428处的天冬酰胺(N428A)、用谷氨酸置换位置432处的谷氨酰胺(Q432E)、用丙氨酸置换位置432处的谷氨酰胺(Q432A)、用丙氨酸置换位置435处的赖氨酸(K435A)和用脯氨酸置换亮氨酸(L438P)，但是不具体地限于此。
更具体地，丙酮酸脱氢酶变体可以包括选自SEQ ID NO:14到33的氨基酸序列。
丙酮酸脱氢酶变体不仅可以包括具有SEQ ID NO:14到33的氨基酸序列的蛋白质而且可以非限制性地包括与SEQ ID NO:14到33的氨基酸序列具有75％或更高，具体地80％或更高，更具体地85％或更高，并且甚至更具体地90％、91％、92％、93％、94％、95％、96％、97％、98％、和99％或更高的同源性的丙酮酸脱氢酶变体，只要它们的丙酮酸脱氢酶活性与野生型的丙酮酸脱氢酶活性相比大幅度减弱。显而易见的是，与具有SEQ ID NO:14到33的氨基酸序列的蛋白质具有基本上相同或相应生物学活性的氨基酸序列也应当属于本公开内容的范围，尽管该氨基酸序列在序列的部分中可以具有缺失、修饰、置换或添加。
如本文所使用，术语“同源性”指的是两个多核苷酸或多肽部分之间的同一性的百分比。一个部分与另一个部分的序列之间的同源性可以通过本领域中已知的技术测定。例如，同源性可以通过使用容易可获得的计算机程序直接排列两个多核苷酸分子或两个多肽分子的序列信息来测定。计算机程序的实例可以包括BLAST(NCBI)、CLC Main Workbench(CLC bio)、MegAlignTM(DNASTAR Inc.)等。另外，多核苷酸之间的同源性可以通过如下步骤来测定：在同源区之间形成稳定双链的条件下杂交多核苷酸，利用单链特异性核酸酶分解，然后对分解的片段进行大小测定。
本公开内容的一个方面提供了编码丙酮酸脱氢酶变体的多核苷酸。
丙酮酸脱氢酶变体与上面解释的相同。具体地，编码丙酮酸脱氢酶变体的多核苷酸可以属于本公开内容的范围，只要该多核苷酸编码具有SEQ ID NO:14到33的氨基酸序列的蛋白质。而且，多核苷酸可以是与上述多核苷酸序列具有75％或更高，具体地80％或更高，更具体地85％或更高，并且甚至更具体地90％、91％、92％、93％、94％、95％、96％、97％、98％、和99％或更高的同源性的任何多核苷酸。
如本文所使用，术语“多核苷酸”指的是通过核苷酸单元的共价键纵向链延伸的核苷酸的聚合物，一般指具有某一长度的DNA或RNA链，并且在本公开内容中，其指的是编码丙酮酸脱氢酶变体的多核苷酸。基于密码子简并性，多核苷酸可以具有编码相同氨基酸的不同核苷酸序列。另外，对于根据宿主细胞类型优化表达，多核苷酸可以具有密码子优化的序列。
本公开内容的仍另一方面提供了生产L-氨基酸的棒状杆菌属的微生物，其包含丙酮酸脱氢酶变体。
具体地，微生物可以通过突变包含丙酮酸脱氢酶变体，或者可以是通过包含编码丙酮酸脱氢酶变体的多核苷酸的载体转化的微生物。
如本文所使用，术语“载体”指的是用于克隆和/或转移碱基至宿主细胞的任何运载体。载体可以是复制子以允许复制与载体结合的其它DNA片段(一种或多种)。“复制子”指的是在体内充当DNA复制单元的任何遗传单元，即，通过自我调节可复制的遗传单元(例如，质粒、噬菌体、粘粒、染色体和病毒)。如本文所使用，术语“载体”不被具体地限制，只要其可以在宿主中复制，并且可以使用本领域中已知的任何载体。在重组载体的构建中使用的载体可以是天然状态的或者处于重组状态的质粒、粘粒、病毒和噬菌体。例如，作为噬菌体载体或粘粒载体，可以使用pWE15、M13、λEMBL3、λEMBL4、λFIXII、λDASHII、λZAPII、λgt10、λgt11、Charon4A、Charon21A等，并且作为质粒载体，可以使用基于pDZ载体、pBR、pUC、pBluescriptII、pGEM、pTZ、pCL、pET等的那些。可以在本公开内容中使用的载体不被具体地限制，但是可以使用任何已知的表达载体。具体地，可以使用pDZ(韩国专利号10-0924065以其全部作为引用并入本公开内容)，但是不限于此。
如本文所使用，术语“转化”指的是将基因引入宿主细胞，从而使得该基因在宿主细胞中能够表达的过程。转化的基因可以非限制性地包括任何基因，不管其被插入宿主细胞的染色体并且位于其中或者位于染色体的外面，只要其在宿主细胞中可以表达。多核苷酸可以以表达盒的形式被引入宿主细胞，该表达盒是包含对于自我表达需要的所有必需元件的多核苷酸构建体。表达盒可以常规地包括可操作地连接至基因的启动子、转录终止信号、核糖体结合结构域和翻译终止信号。表达盒可以是能够自我复制的表达载体的形式。另外，多核苷酸可以被引入宿主细胞，其按原样或者以多核苷酸构建体的形式并可操作地连接至对于其在宿主细胞中表达必需的序列，但是不限于此。
微生物可以是任何原核或真核微生物，只要该微生物包括相应的丙酮酸脱氢酶变体的活性或者该微生物被转化以表达相应蛋白质。微生物的实例可以包括埃希氏杆菌属(genus Escherichia)、欧文氏菌属(genus Erwinia)、沙雷氏菌属(genus Serratia)、普罗维登斯菌属(genus Providencia)、肠道菌属(genus Enterobacteria)、沙门氏菌属(genusSalmonella)、链霉菌属(genus Streptomyces)、假单胞菌属(genus Pseudomonas)、短杆菌属(genus Brevibacterium)、棒状杆菌属等的微生物菌株，具体地，棒状杆菌属的微生物，并且更具体地，谷氨酸棒状杆菌，但是不限于此。
另外，当本公开内容的丙酮酸脱氢酶变体包含在具有生产L-氨基酸能力的微生物中时，与包含野生型丙酮酸脱氢酶的微生物相比，生产L-氨基酸的能力可以显著地提高而不明显地抑制细胞的生长。
如本文所使用，术语“L-氨基酸”指的是可以通过丙酮酸从不同碳源生产的所有L-氨基酸，并且具体地，可以指不经历在生物合成途径中将丙酮酸转变为乙酰辅酶A的过程的L-氨基酸。更具体地，L-氨基酸可以包括L-赖氨酸、L-苏氨酸、L-甲硫氨酸、L-异亮氨酸、L-缬氨酸、L-亮氨酸或L-丙氨酸，并且甚至更具体地，L-赖氨酸或L-缬氨酸。
生产L-氨基酸的微生物可以包括可以在体内生产L-氨基酸的真核和原核微生物二者，并且该微生物的实例与上面描述的相同。生产L-氨基酸的微生物可以非限制性地是任何微生物，只要该微生物具有生产L-氨基酸的能力，其包括野生型菌株和重组菌株。
例如，生产L-赖氨酸的棒状杆菌属的微生物可以被修饰以对L-赖氨酸类似物具有抗性，或者与未修饰的微生物相比具有增强的L-赖氨酸生物合成相关蛋白的活性。具体地，与L-赖氨酸生物合成相关的至少一种基因的表达通过如下改善：基因扩增；序列比如启动子或起始密码子的置换或修饰；引入用于改善表达的修饰，等，但是不限于此。
另外，L-赖氨酸生物合成相关基因的实例可以包括位于L-赖氨酸生物合成途径中的基因，并且具体地，二氢吡啶二羧酸(dihydrodipicolinic acid)合酶基因(dapA)、天冬氨酸激酶基因(asparto kinasegene)(lysC)、二氢吡啶二羧酸还原酶基因(dapB)、二氨基庚二酸脱羧酶基因(lysA)、二氨基庚二酸脱氢酶基因(ddh)、烯醇丙酮酸磷酸羧激酶基因(ppc)、天冬氨酸半醛脱氢酶基因(asd)、天冬氨酸酶基因(aspB)和丙酮酸羧化酶(Pyc)，但是不限于此。另外，L-赖氨酸生物合成相关基因的实例可以包括存在于戊糖磷酸途径中的转酮醇酶(tkt)等，但是不限于此。
具体而言，生产L-赖氨酸的棒状杆菌属的微生物可以通过包含本领域中公开的修饰——其与L-赖氨酸生产有关——展示生产L-赖氨酸的能力，但是不限于此。
生产L-苏氨酸的微生物可以是如此微生物：其具有甲硫氨酸需求，对苏氨酸类似物具有抗性，对赖氨酸类似物具有抗性，对异亮氨酸类似物具有抗性和/或对甲硫氨酸类似物具有抗性，但是不具体地限于此。甲硫氨酸类似物可以是选自D,L-乙硫氨酸、正亮氨酸、α-甲基甲硫氨酸和L-甲硫氨酸-D,L-亚砜亚胺(sulfoximine)的至少一种化合物；苏氨酸类似物可以是选自α-氨基-β-羟基戊酸和D,L-苏氨酸异羟肟酸的至少一种化合物；并且赖氨酸类似物可以是选自S-(2-氨乙基)-L-半胱氨酸和δ-甲基-L-赖氨酸的至少一种化合物。
另外，生产L-苏氨酸的微生物可以包括如此微生物：其中参与将草酰乙酸(OAA)——其是L-苏氨酸生物合成的中间体——转变为磷酸烯醇丙酮酸(PEP)的PckA的活性被减弱或失活；或者如此微生物：其中TyrR——其抑制参与将草酰乙酸转变为天冬氨酸的lysC基因——的活性被减弱或失活；或如此微生物：其中GalR——其抑制参与引入葡萄糖的galP基因的表达——的活性被减弱或失活，但是不限于此。
生产L-异亮氨酸的微生物可以是对L-异亮氨酸或其衍生物具有抗性的微生物，或者被遗传操作以解除L-异亮氨酸或其衍生物的反馈抑制的微生物。L-异亮氨酸的衍生物的实例可以包括4-硫代异亮氨酸(4-thiaisoleucine)(thiaile)和异亮氨酸-异羟肟酸(ileHx)，但是不限于此。
生产L-缬氨酸的微生物可以是对L-缬氨酸或其衍生物具有抗性的微生物，或者其中L-缬氨酸的生物合成途径中的酶被遗传操作以解除L-缬氨酸或其衍生物的反馈抑制的微生物。微生物的实例可以包括包含乙酰羟酸合酶变体的微生物，在其中对L-缬氨酸的反馈抑制被解除，但是不限于此。另外，微生物可以是其中L-缬氨酸操纵子的表达被修饰为增强的微生物，并且例如，微生物可以是其中L-缬氨酸操纵子的表达由于L-缬氨酸操纵子的调控区内编码前导肽的多核苷酸序列的一部分或全部缺失而增强的微生物(韩国专利申请公布号10-2014-0111421，其说明书的全部作为引用可被并入，但是不限于此)。
本公开内容的仍另一个方面提供了用于生产L-氨基酸的方法，其包括：(a)在培养基中培养生产L-氨基酸的棒状杆菌属的微生物——其包含丙酮酸脱氢酶变体——以生产L-氨基酸；和(b)从培养的微生物或培养基回收L-氨基酸。
生产L-氨基酸的棒状杆菌属的微生物等与上面描述的相同。
如本文所使用，术语“培养”指的是在适合和人工控制的环境条件下生长微生物。本公开内容的培养过程可以基于本领域中已知的适合的培养基和培养条件来执行。具体的条件，比如培养温度、培养时间、培养基的pH值等，可以通过本领域普通技术人员的一般知识或者本领域中已知的常规方法来确定，并且相应地适当调节。具体地，这些已知的培养方法在如下文献中详细描述[Chmiel；Bioprozesstechnik 1.Einfuhrung indieBioverfahrenstechnik(Gustav Fischer Verlag,Stuttgart,1991)，和Storhas；Bioreaktoren und periphere Einrichtungen(Vieweg Verlag,Braunschweig/Wiesbaden,1994)]。另外，培养方法可以包括分批培养、连续培养和补料分批培养，并且具体地，培养可以以补料分批或重复补料分批工艺连续地执行，但是不限于此。培养中使用的培养基必须适当地满足具体菌株的需要。在培养基中使用的碳源的实例可以包括糖类和碳水化合物，比如葡萄糖、蔗糖、乳糖、果糖、麦芽糖、淀粉和纤维素；油和脂肪，比如豆油、葵花油、蓖麻油和椰子油；脂肪酸，比如棕榈酸、硬脂酸和亚油酸；醇类比如甘油和乙醇；和有机酸，比如乙酸，但是不限于此。这些碳源可以单独或组合使用，但是不限于此。在培养基中使用的氮源的实例可以包括蛋白胨、酵母提取物、肉汁、麦芽提取物、玉米浆、大豆粉；以及尿素或无机化合物，比如硫酸铵、氯化铵、磷酸铵、碳酸铵和硝酸铵。这些氮源也可以单独或组合使用，但是不限于此。在培养基中使用的磷源的实例可以包括磷酸二氢钾、磷酸氢二钾、相应的含钠盐等，但是不限于此。另外，对于生长需要的金属盐比如硫酸镁或硫酸铁可以包含在培养基中。最后，除了上面描述的物质之外，也可以包含生长的必需物质，比如氨基酸和维生素。另外，可以使用适合于培养基的前体。这些来源可以在培养期间通过分批培养或者连续培养以适合的方式添加至培养物中，但是方法不限于此。
另外，培养物的pH值可以在培养时通过将化合物比如氢氧化铵、氢氧化钾、氨、磷酸和硫酸以适合的方式添加至培养物中来调节。在培养周期中，可以加入消泡剂比如脂肪酸聚乙二醇酯以防止泡沫生成。另外，氧气或含氧气体可以被注入培养物中以便于维持培养物的需氧状态，或者氮气、氢气或二氧化碳气体可以被注入而不注入空气以便于维持培养物的厌氧或微需氧状态。培养温度可以通常在27℃到37℃，并且具体地30℃到35℃的范围内。培养可以继续直到获得期望量的有用物质，并且具体地持续10小时至100小时。L-氨基酸可以被释放至正在培养的培养基中或者可以包含在微生物中。
另外，关于本公开内容的生产L-氨基酸的方法，从培养的微生物或培养物中回收L-氨基酸的方法是本领域中公知的。回收L-氨基酸的方法可以包括过滤、阴离子交换色谱法、结晶、HPLC等，但是不限于此。
实施本发明的模式
在下文中，将参照实施例更详细地描述本公开内容。但是，这些实施例仅出于说明性的目的，并且本公开内容不意欲被这些实施例所限制。
实施例1：通过人工诱变构建E1p变体的文库
在该实施例中，通过下面描述的方法构建染色体内初级交叉插入(crossinsertion)的载体文库，以便于获得E1p变体。对谷氨酸棒状杆菌ATCC13032-衍生的aceE基因(SEQ ID NO:2)——其编码E1p(SEQ ID NO:1)——执行易错PCR，并且获得随机引入有碱基置换的修饰的aceE基因(2852bp)。通过Genemorph II Random Mutagenesis Kit(Stratagene)使用谷氨酸棒状杆菌ATCC13032基因组DNA作为模板连同引物1(SEQ ID NO:3)和引物2(SEQ ID NO:4)一起进行易错PCR。
引物1(SEQ ID NO:3):5’-TGGGA CCGGG AAACC GGG-3’
引物2(SEQ ID NO:4):5’-GATTT ATCTG TCCCT TGA-3’
扩增的基因片段每1kb被引入0到3.5个碱基置换，并且PCR在如下条件下进行总计30个循环：96℃变性30秒，53℃下退火30秒，和72℃下聚合2分钟。
将扩增的片段使用pCR2.1-TOPO TA克隆试剂盒(Invitrogen)连接至pCR2.1-TOPO载体(在下文中，“pCR2.1”)，转化入大肠杆菌DH5α，并且在含有卡那霉素(25mg/L)的LB固体培养基上平板培养。选择20个不同种类的转化的菌落并且然后从其获得质粒。在对质粒的多核苷酸序列进行分析之后，确认了修饰以1.4个突变/kb的频率被引入至相互不同的位置。收集大约20,000个转化的大肠杆菌菌落并且提取它们的质粒并命名为“pCR2.1-aceE(mt)”文库。
另外，构建了具有野生型aceE基因的质粒，其被用作对照菌株。以与上面描述的相同方式使用谷氨酸棒状杆菌ATCC13032基因组DNA作为模板连同引物1(SEQ ID NO:3)和引物2(SEQ ID NO:4)一起执行PCR。对于聚合酶，使用Pfu超高保真(PfuUltra High-Fidelity)DNA聚合酶(Stratagene)并且如此构建的质粒被命名为“pCR2.1-aceE(WT)”。
实施例2：构建aceE-缺失菌株
使用KCCM11016P(上述微生物首次被公布为KFCC10881并且被重新保藏至布达佩斯条约下的国际保藏机构并且被分配登录号KCCM11016P；韩国专利号10-0159812)菌株作为亲本菌株引入pCR2.1-aceE(mt)文库来构建aceE-缺失菌株。
对于aceE-缺失载体的构建，使用野生型谷氨酸棒状杆菌ATCC13032作为模板连同引物3(SEQ ID NO:5)、引物4(SEQ ID NO:6)、引物5(SEQ ID NO:7)和引物6(SEQ ID NO:8)一起执行PCR。
引物3(SEQ ID NO:5):5’-GCAGG TCGAC TCTAG ATGCG ATTCG CGTCA AACGT G-3’
引物4(SEQ ID NO:6):5’-GTCCC TTGAG GTGAT GTGAA TCCAT CCACT-3’
引物5(SEQ ID NO:7):5’-AGTGG ATGGA TTCAC ATCAC CTCAA GGGAC-3’
引物6(SEQ ID NO:8):5’-CCGGG GATCC TCTAG ACGAA GCGCC GTGAG CAATT C-3’
在如下条件下进行PCR：95℃下变性5分钟；95℃下变性30秒，55℃下退火30秒，和72℃下聚合30秒，30个循环；和72℃下聚合7分钟。
结果，获得分别包含5’末端和3’末端的SEQ ID NO:9(521bp)和SEQ ID NO:10(538bp)。
使用SEQ ID NO:9和SEQ ID NO:10的扩增序列作为模板连同引物3(SEQ ID NO:5)和引物6(SEQ ID NO:8)一起进行PCR。
在如下条件下进行PCR：95℃下变性5分钟；95℃下变性30秒，55℃下退火30秒，和72℃下聚合60秒，30个循环；和72℃下聚合7分钟。
结果，扩增了具有1029bp大小的SEQ ID NO:11(在下文中，ΔaceE)——其中aceE基因的5’末端和3’末端被连接。
pDZ载体(韩国专利号10-0924065)——其不能在谷氨酸棒状杆菌中复制——和ΔaceE片段利用限制性内切酶Xba I处理和使用DNA连接酶连接，并克隆以获得质粒。质粒被命名为pDZ-ΔaceE。
将pDZ-ΔaceE通过电脉冲转化入谷氨酸棒状杆菌KCCM11016P，其是产L-赖氨酸的菌株(Appl.Microbiol.Biothcenol.(1999)52:541-545)，并且转化的菌株从包含卡那霉素(25mg/L)的选择培养基获得。获得其中aceE基因通过ΔaceE失活并且经由二次交换将DNA片段插入基因组的菌株并命名为KCCM11016PΔaceE。
实施例3：构建E1p人工变体文库并且筛选具有增强的生产L-赖氨酸的能力的菌株
通过同源重组将构建的pCR2.1-aceE(mt)文库转化入作为亲本菌株的KCCM11016PΔaceE菌株，在包含卡那霉素(25mg/L)的复合平板培养基上平板培养，并且获得大约10,000个菌落，并且每个菌落分别命名为“KCCM11016PΔaceE/pCR2.1-aceE(mt)-1”到“KCCM11016P/pCR2.1-aceE(mt)-10000”。
另外，将如此构建的pCR2.1-aceE(WT)载体转化入KCCM11016PΔaceE以获得对照菌株，并命名为“KCCM11016PΔaceE/pCR2.1-aceE(WT)”。
<复合平板培养基(pH 7.0)>
葡萄糖10g、蛋白胨10g、牛肉膏5g、酵母提取物5g、脑心浸液18.5g、NaCl 2.5g、尿素2g、山梨醇(Sorbitiol)91g和琼脂20g(基于1L蒸馏水)
将如此获得的10,000个菌落分别接种入选择培养基(300μL)并且在维持于32℃、1000rpm速率的96深孔板中培养持续大约24小时。在培养中产生的L-赖氨酸产品的量通过茚三酮方法分析(J.Biol.Chem.1948.176:367-388)。在培养完成之后，使10μL培养上清液和190μL茚三酮反应溶液在65℃下反应30分钟。在波长570nm下的吸光度通过分光光度计测量，并与对照菌株KCCM11016PΔaceE/pCR2.1-aceE(WT)的吸光度进行比较，选择显示吸光度具有至少10％增加的256种修饰的菌株。与对照菌株的吸光度相比，其它菌落显示了相似的或降低的吸光度。
<选择培养基(pH 8.0)>
葡萄糖10g、(NH4)2SO4 5.5g、MgSO4·7H2O 1.2g、KH2PO4 0.8g、K2HPO4 16.4g，生物素100μg、盐酸硫胺素1000μg、钙-泛酸2000μg和烟酰胺2000μg(基于1L蒸馏水)
将选择的256种菌株以与上述相同的方式经历茚三酮反应，并且选择与KCCM11016PΔaceE/pCR2.1-aceE(WT)菌株的生产L-赖氨酸的能力相比具有提高的生产L-赖氨酸的能力的前53种菌株。
实施例4：确认选自E1p人工变体文库的菌株的生产L-赖氨酸的能力
为了比较在实施例3中选择的53种不同菌株的生产L-赖氨酸的能力，通过下面描述的方法培养菌株并且分析其获得的培养物的组分。
分别将每种菌株接种入含25mL种子培养基的250mL角挡板烧瓶中，并且在摇动培养箱(200rpm)中在30℃下培养20小时。将含24mL的产L-赖氨酸的培养物的250mL角挡板烧瓶中的每个接种1mL种子培养液，并在摇动培养箱(200rpm)中在30℃下培养72小时。每个培养物中L-赖氨酸的浓度经由HPLC分析。
<种子培养基(pH 7.0)>
葡萄糖(20g)、蛋白胨(10g)、酵母提取物(5g)、尿素(1.5g)、KH2PO4(4g)、K2HPO4(8g)、MgSO4·7H2O(0.5g)、生物素(100μg)、盐酸硫胺素(1000μg)、钙-泛酸(2000μg)、烟酰胺(2000μg)(基于1L蒸馏水)
<产赖氨酸培养基(pH 7.0)>
葡萄糖(100g)、(NH4)2SO4(40g)、大豆蛋白(2.5g)、玉米饼(Corn Steep Solid)(5g)、尿素(3g)、KH2PO4(1g)、MgSO4·7H2O(0.5g)、生物素(100μg)、盐酸硫胺素(1000μg)、钙-泛酸(2000μg)、烟酰胺(3000μg)和CaCO3(30g)(基于1L蒸馏水)
从53种不同的菌株中选择具有最高L-赖氨酸浓度的前10种菌株，并且重复进行上面描述的培养和分析。分析的L-赖氨酸浓度在下面的表1中示出。
[表1]
Figure GDA0002635683930000091
Figure GDA0002635683930000101
作为L-赖氨酸浓度分析的结果，确认了10种选择的菌株的L-赖氨酸产量与对照菌株KCCM11016PΔaceE/pCR2.1-aceE(WT)菌株相比展示了22％的最大增加。
实施例5：确认选自E1p人工变体文库的菌株的aceE基因修饰
为了确认在实施例4中选择的10种菌株的E1p中引入的置换，分析修饰的aceE的多核苷酸序列。为了确定该多核苷酸序列，使用引物1(SEQ ID NO:3)和引物2(SEQ ID NO:4)进行PCR。
引物1(SEQ ID NO:3):5’-TGGGA CCGGG AAACC GGG-3’
引物2(SEQ ID NO:4):5’-GATTT ATCTG TCCCT TGA-3’
获得的修饰类型的aceE基因片段中的每个的多核苷酸序列通过分析确认，并与SEQ ID NO:2的多核苷酸序列比较，并且因此确认E1p变体的氨基酸序列。关于选择的菌株的E1p氨基酸序列置换的信息在下面的表2中示出。
[表2]
Figure GDA0002635683930000102
Figure GDA0002635683930000111
实施例6：构建用于在染色体上引入E1p变体的载体
为了确认在实施例5中确认的E1p变体的应用效果，构建用于在染色体上引入其的载体。
基于报道的多核苷酸序列，合成引物9(SEQ ID NO:12)和引物10(SEQ ID NO:13)，引物9在5’末端处引入有Xba I限制位点，并且引物10在3’末端处引入有Xba I限制位点。使用选择的10种不同菌株的染色体为模板，通过PCR分别扩增10种不同修饰类型的菌株的aceE(mt)基因片段。
在如下条件下进行PCR：94℃下变性5分钟；94℃下变性30秒，56℃下退火30秒，和72℃下聚合30秒，30个循环；和72℃下聚合7分钟。
引物9(SEQ ID NO:12):5’-AATCT AGATG GGACC GGGAA ACCGG G-3’
引物10(SEQ ID NO:13):5’-AATCT AGAGA TTTAT CTGTC CCTTG A-3’
10种不同菌株的PCR扩增的基因片段用Xba I处理以获得其各自的DNA片段，并且将片段连接至pDZ载体用于引入包含Xba I限制位点的染色体，转化入大肠杆菌DH5α，并且在含有卡那霉素(25mg/L)的固体LB培养基上划线培养。
转化有插入靶基因的载体的转化菌落通过PCR选择并且质粒通过常规已知的质粒提取方法获得。根据在每种质粒的E1p中插入的修饰，分别将质粒命名为pDZ-E1p(Q432E)、pDZ-E1p(E190V)、pDZ-E1p(L438P)、pDZ-E1p(Q195H)、pDZ-E1p(P199S)、pDZ-E1p(K435A)、pDZ-E1p(Q432A)、pDZ-E1p(Y418H)、pDZ-E1p(N428A)和pDZ-E1p(Y201A)。
实施例7：构建引入有E1p变体的KCCM11016P衍生的菌株以及它们的生产L-赖氨酸能力的比较
谷氨酸棒状杆菌KCCM11016P——一种产L-赖氨酸的菌株，通过同源染色体的2步重组使用在实施例6中制备的用于引入E1p变体的10种载体进行转化。然后，通过分析多核苷酸序列选择在染色体上引入有E1p变体的菌株，并且根据插入的E1p变体将质粒分别命名为KCCM11016P::E1p(Q432E)、KCCM11016P::E1p(E190V)、KCCM11016P::E1p(L438P)、KCCM11016P::E1p(Q195H)、KCCM11016P::E1p(P199S)、KCCM11016P::E1p(K435A)、KCCM11016P::E1p(Q432A)、KCCM11016P::E1p(Y418H)、KCCM11016P::E1p(N428A)、和KCCM11016P::E1p(Y201A)。
以与实施例4中相同的方式培养菌株，并且分析培养物的L-赖氨酸浓度。为了测量构建菌株的生长速率，在培养开始之后18小时测量培养物的剩余葡萄糖浓度(表3)。
[表3]
Figure GDA0002635683930000121
10种不同菌株(即，KCCM11016P::E1p(Q432E)、KCCM11016P::E1p(E190V)、KCCM11016P::E1p(L438P)、KCCM11016P::E1p(Q195H)、KCCM11016P::E1p(P199S)、KCCM11016P::E1p(K435A)、KCCM11016P::E1p(Q432A)、KCCM11016P::E1p(Y418H)、KCCM11016P::E1p(Y201A)和KCCM11016P::E1p(N428A))与亲本菌株相比显示了葡萄糖消耗率的轻微减少，同时显示了赖氨酸生产的10％的最大增加。
在这点上，本发明人命名了KCCM11016P::E1p(N428A)——在具有提高的生产L-赖氨酸的能力的菌株中的代表性菌株——为谷氨酸棒状杆菌“CA01-2289”，并且将该菌株在2014年10月23日保藏至为布达佩斯条约下承认的国际保藏机构的韩国微生物保藏中心，登录号为KCCM11590P。
检查之后，确认了10种不同菌株的E1p变体(即，E1p(Q432E)(SEQ ID NO:14)、E1p(E190V)(SEQ ID NO:15)、E1p(L438P)(SEQ ID NO:16)、E1p(Q195H)(SEQ ID NO:17)、E1p(P199S)(SEQ ID NO:18)、E1p(K435A)(SEQ ID NO:19)、E1p(Q432A)(SEQ ID NO:20)、E1p(Y418H)(SEQ ID NO:21)、E1p(Y201A)(SEQ ID NO:22)和E1p(N428A)(SEQ ID NO:23))几乎分布在2个组中(190到201位置处的氨基酸残基，和418到438位置处的氨基酸)。
合成10种不同的菌株，其中组合地包含属于每组的置换(即，(KCCM11016P::E1p(E190V,Q195H)、KCCM11016P::E1p(E190V,P199S)、KCCM11016P::E1p(Q195H,P199S)、KCCM11016P::E1p(E190V,Y201A)、KCCM11016P::E1p(Q195H,Y201A)、KCCM11016P::E1p(P199S,Y201A)、KCCM11016P::E1p(N428A,Q432E)、KCCM11016P::E1p(N428A,K435A)、KCCM11016P::E1p(Y418H,K435A)、KCCM11016P::E1p(Y418H,Q432A))，并且它们生产L-赖氨酸的能力以与上面描述的相同方式测量(表4)。
[表4]
Figure GDA0002635683930000131
Figure GDA0002635683930000141
如上面的表中所示，具有组合置换的所有10种菌株显示了葡萄糖消耗率的轻微降低，同时显示了生产L-赖氨酸能力的15.6％的最大增加。结果确认了引入有组合置换的菌株与引入有单一置换的菌株相比可以展现提高的生产L-赖氨酸的能力。
结果确认具有E1p蛋白的变体的10种菌株与亲本菌株相比是可以显著地增加生产L-赖氨酸的能力同时不显著地降低葡萄糖消耗率的有效菌株，并且还确认190到205或415到440位置处的氨基酸残基是可以展现上述效果的主要区域。
实施例8：关于引入有E1p变体的菌株，测量丙酮酸脱氢酶复合物(PDHC)的活性
关于选择的菌株，通过前述期刊中报道的方法(Schreiner等,J.Bacteriol.187:6005,2005)测量PDHC活性。将对照菌株KCCM11016P和KCCM11016PΔaceE，和10种选择的菌株(KCCM11016P::E1p(Q432E)、KCCM11016P::E1p(E190V)、KCCM11016P::E1p(L438P)、KCCM11016P::E1p(Q195H)、KCCM11016P::E1p(P199S)、KCCM11016P::E1p(K435A)、KCCM11016P::E1p(Q432A)、KCCM11016P::E1p(Y418H)、KCCM11016P::E1p(Y201A)和KCCM11016P::E1p(N428A))接种入实施例4中规定的25mL种子培养基，并培养直到对数生长后期。
细胞通过离心收集，利用100mM Tris-HCl(pH 7.2，3mM L-半胱氨酸、10mM MgCl2)缓冲溶液清洗两次，并最终悬浮在2mL相同缓冲溶液中。细胞的悬浮液通过普通玻璃珠涡流方法物理地压碎10分钟，通过进行离心(13,000rpm，4℃，和30分钟)两次回收上清液，并且将其用作测量PDHC酶的活性的粗提取物。为了测量PDHC酶的活性，向用于测量酶活性的0.95mL的量的反应溶液(在Tris-HCl缓冲液中(pH 7.2)中的10mM MgCl2、3mM半胱氨酸、2mMNAD、0.9mM二磷酸硫胺素、0.25mM氯丙嗪、6mM丙酮酸、0.2mM CoA)加入0.05mL粗提取物，并且在30℃下反应。PDHC的活性单位根据每分钟消耗的NADH的μmole数来限定，并且酶活性的测量结果在下面的表5中显示。
[表5]
Figure GDA0002635683930000142
Figure GDA0002635683930000151
引入有变体的菌株的PDHC活性展示了相对于亲本菌株的35％到56％的活性。
实施例9：与aceE-缺失菌株比较生产L-赖氨酸的能力
为了比较评估KCCM11016PΔaceE、实施例2中构建的aceE-缺失菌株和10种选择的菌株(KCCM11016P::E1p(Q432E)、KCCM11016P::E1p(E190V)、KCCM11016P::E1p(L438P)、KCCM11016P::E1p(Q195H)、KCCM11016P::E1p(P199S)、KCCM11016P::E1p(K435A)、KCCM11016P::E1p(Q432A)、KCCM11016P::E1p(Y418H)、KCCM11016P::E1p(Y201A)和KCCM11016P::E1p(N428A))，使用含有醋酸铵的培养基以与实施例4中相同的方式培养这些菌株。得到的培养物的L-赖氨酸浓度通过HPLC分析，并且为了测量构建的菌株的生长速率，在开始培养后18小时测量剩余葡萄糖的浓度。
<含有醋酸铵的产赖氨酸的培养基(pH 7.0)>
葡萄糖(100g)、CH3COONH3(5g)、(NH4)2SO4(40g)、大豆蛋白(2.5g)、玉米饼(5g)、尿素(3g)、KH2PO4(1g)、MgSO4·7H2O(0.5g)、生物素(100μg)、盐酸硫胺素(1000μg)、钙-泛酸(2000μg)、烟酰胺(3000μg)和CaCO3(30g)(基于1L蒸馏水)
[表6]
Figure GDA0002635683930000152
Figure GDA0002635683930000161
KCCM11016PΔaceE菌株与亲本菌株相比显示了生产L-赖氨酸的能力的9.6％的增加但是显著地降低了生长速率。相反，引入有置换的菌株显示了赖氨酸产量和葡萄糖消耗率的增加，其与不加入醋酸铵时类似。
结果表明引入有aceE变体的菌株可以以高产量生产赖氨酸而对生长速率没有显著影响，与之相比，aceE-缺失菌株可以增加赖氨酸产量同时生长速率显著降低。
实施例10：构建引入有E1p变体的KFCC10750衍生的菌株和它们生产L-赖氨酸能力的比较
为了确认在谷氨酸棒状杆菌属的其它菌株中引入10种变体的效果，以与实施例7中相同的方式构建菌株，其中10种不同E1p变体中的每种分别被引入谷氨酸棒状杆菌KFCC10750(上述微生物首次被公布为KFCC10750并且被重新保藏至布达佩斯条约下的国际保藏机构并且被分配登录号KCCM11347P；韩国专利号10-0073610)——一种产L-赖氨酸的菌株，并且将这些菌株命名为KFCC10750::E1p(Q432E)、KFCC10750::E1p(E190V)、KFCC10750::E1p(L438P)、KFCC10750::E1p(Q195H)、KFCC10750::E1p(P199S)、KFCC10750::E1p(K435A)、KFCC10750::E1p(Q432A)、KFCC10750::E1p(Y418H)、KFCC10750::E1p(Y201A)和KFCC10750::E1p(N428A)。以与实施例4中相同的方式培养包括KFCC10750菌株作为对照菌株的11种菌株，并分析菌株的培养物的L-赖氨酸浓度(表7)。
[表7]
Figure GDA0002635683930000162
Figure GDA0002635683930000171
结果，确认了引入有变体的10种不同的菌株与亲本菌株相比具有生产L-赖氨酸能力的17％的最大增加。
实施例11：构建引入有E1p变体的KCCM10770P衍生的菌株和它们生产L-赖氨酸能力的比较
为了确认在谷氨酸棒状杆菌属的其它菌株中引入10种变体的效果，以与实施例7中相同的方式构建菌株，其中每种E1p变体被引入谷氨酸棒状杆菌KCCM10770P(韩国专利号10-0924065)——一种产L-赖氨酸的菌株，并且将这些菌株命名为KCCM10770P::E1p(Q432E)、KCCM10770P::E1p(E190V)、KCCM10770P::E1p(L438P)、KCCM10770P::E1p(Q195H)、KCCM10770P::E1p(P199S)、KCCM10770P::E1p(K435A)、KCCM10770P::E1p(Q432A)、KCCM10770P::E1p(Y418H)、KCCM10770P::E1p(Y201A)和KCCM10770P::E1p(N428A)。以与实施例4中相同的方式培养包括KCCM10770P菌株作为对照菌株的菌株，并分析菌株的培养物的L-赖氨酸浓度(表8)。
[表8]
Figure GDA0002635683930000172
Figure GDA0002635683930000181
结果，确认了引入有变体的10种菌株与亲本菌株相比具有生产L-赖氨酸能力的17％的最大增加。
实施例12：构建引入有E1p变体的CJ3P衍生的菌株和它们生产L-赖氨酸能力的比较
为了确认在谷氨酸棒状杆菌属的其它菌株中引入E1p变体的效果，以与实施例7中相同的方式构建菌株，其中每种E1p变体被引入谷氨酸棒状杆菌CJ3P(Binder等,GenomeBiology 2012,13:R40)——一种产L-赖氨酸的菌株，并且将这些菌株命名为CJ3P::E1p(Q432E)、CJ3P::E1p(E190V)、CJ3P::E1p(L438P)、CJ3P::E1p(Q195H)、CJ3P::E1p(P199S)、CJ3P::E1p(K435A)、CJ3P::E1p(Q432A)、CJ3P::E1p(Y418H)、CJ3P::E1p(Y201A)和CJ3P::E1p(N428A)。以与实施例4中相同的方式培养菌株，并且分析菌株的培养物的L-赖氨酸浓度(表9)。
[表9]
Figure GDA0002635683930000182
结果，确认了引入有变体的10种不同菌株与亲本菌株相比具有生产L-赖氨酸能力的19.5％的最大增加。
结果表明具有E1p变体(E1p(Q432E)、E1p(E190V)、E1p(L438P)、E1p(Q195H)、E1p(P199S)、E1p(K435A)、E1p(Q432A)、E1p(Y418H)、E1p(Y201A)和E1p(N428A))的新获得的10种不同菌株中的每种分别具有增加生产L-赖氨酸能力的优异效果。
实施例13：构建引入有E1p变体的KCCM11201P衍生的菌株和它们生产L-缬氨酸能力的比较
为了确认在谷氨酸棒状杆菌属的产其它氨基酸的菌株中具有E1p变体的10种选择的菌株的效果，以与实施例7中相同的方式构建菌株，其中E1p变体被引入谷氨酸棒状杆菌KFCC11201P(韩国专利号10-1117022)——一种产L-缬氨酸的菌株，并且将这些菌株命名为KCCM11201P::E1p(Q432E)、KCCM11201P::E1p(E190V)、KCCM11201P::E1p(L438P)、KCCM11201P::E1p(Q195H)、KCCM11201P::E1p(P199S)、KCCM11201P::E1p(K435A)、KCCM11201P::E1p(Q432A)、KCCM11201P::E1p(Y418H)、KCCM11201P::E1p(Y201A)和KCCM11201P::E1p(N428A)。
为了评估菌株，将每种菌株分别接种入含有下面规定的25mL L-缬氨酸的250mL角挡板烧瓶，并且在摇动培养箱(200rpm)中在30℃下培养20小时。每个培养物中L-缬氨酸浓度经由HPLC分析(表10)。
<产缬氨酸培养基(pH 7.2)>
葡萄糖(50g)、(NH4)2SO4(20g)、玉米浆(20g)、KH2PO4(1g)、MgSO4·7H2O(0.5g)、生物素(200μg)和CaCO3(30g)(基于1L蒸馏水)
[表10]
Figure GDA0002635683930000191
结果，确认了引入有变体的10种不同菌株与亲本菌株相比具有生产L-缬氨酸能力的21％的最大增加。
实施例14：构建引入有E1p变体的野生型衍生的菌株和它们生产L-缬氨酸能力的比较
为了重新确认10种选择的E1p变体——其显示了L-缬氨酸产量高度增加的效果——中四种不同变体的生产L-缬氨酸的能力，以与实施例7中相同的方式构建菌株，其中每种E1p变体被引入谷氨酸棒状杆菌ATCC13032，并将这些菌株命名为ATCC13032::E1p(E190V)、ATCC13032::E1p(L438P)、ATCC13032::E1p(Y418H)和ATCC13032::E1p(N428A)。
为了确认上述菌株的生产L-缬氨酸的能力，将这些菌株通过电穿孔转化有pECCG117-DvalS(韩国专利申请公布号10-2014-0111421)——其是用于L-缬氨酸生物合成的过表达载体。转化的菌株获得自含卡那霉素(25mg/L)的选择培养基并被命名为ATCC13032::E1p(E190V)_DvalS、ATCC13032::E1p(L438P)_DvalS、ATCC13032::E1p(Y418H)_DvalS和ATCC13032::E1p(N428A)_DvalS。
以与实施例13中相同的方式培养菌株并且分析每个培养物中L-缬氨酸的浓度(表11)。
[表11]
Figure GDA0002635683930000201
结果，确认了引入有变体的4种不同菌株与对照菌株相比具有生产L-缬氨酸能力的38％的最大增加。
根据上面的描述，本公开内容所属领域的技术人员将能够理解本公开内容可以以其它具体的形式体现而不修改本公开内容的技术概念或必要特征。在这一点上，本文公开的示例性实施方式仅出于说明的目的并且不应当被解释为限制本公开内容的范围。相反，本公开内容意欲不仅覆盖示例性实施方式而且覆盖各种替代方案、修改、等价物和可以包含在本公开内容的精神和范围内的其它实施方式，如由所附权利要求书所限定的。
Figure GDA0002635683930000202
Figure GDA0002635683930000211
<110> CJ第一制糖株式会社
<120> 丙酮酸脱氢酶变体、包含其的微生物和使用该微生物生产L-氨基酸的方法
<130> OPA15316-CN
<150> KR10-2015-0037654
<151> 2015-03-18
<160> 33
<170> KopatentIn 2.0
<210> 1
<211> 922
<212> PRT
<213> 谷氨酸棒状杆菌ATCC13032
<400> 1
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 2
<211> 2769
<212> DNA
<213> 谷氨酸棒状杆菌ATCC13032
<400> 2
atggccgatc aagcaaaact tggtggcaag ccctcggatg actctaactt cgcgatgatc 60
cgcgatggcg tggcatctta tttgaacgac tcagatccgg aggagaccaa cgagtggatg 120
gattcactcg acggattact ccaggagtct tctccagaac gtgctcgtta cctcatgctt 180
cgtttgcttg agcgtgcatc tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac 240
tacgtcaaca ccattccaac ctctatggaa cctgaattcc caggcgatga ggaaatggag 300
aagcgttacc gtcgttggat tcgctggaac gcagccatca tggttcaccg cgctcagcga 360
ccaggcatcg gcgtcggcgg acacatttcc acttacgcag gcgcagcccc tctgtacgaa 420
gttggcttca accacttctt ccgcggcaag gatcacccag gcggcggcga ccagatcttc 480
ttccagggcc acgcatcacc aggtatgtac gcacgtgcat tcatggaggg tcgcctttct 540
gaagacgatc tcgatggctt ccgtcaggaa gtttcccgtg agcagggtgg cattccgtcc 600
taccctcacc cacacggtat gaaggacttc tgggagttcc caactgtgtc catgggtctt 660
ggcccaatgg atgccattta ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc 720
aaggacacct ctgaccagca cgtctgggcc ttccttggcg acggcgaaat ggacgagcca 780
gaatcacgtg gtctcatcca gcaggctgca ctgaacaacc tggacaacct gaccttcgtg 840
gttaactgca acctgcagcg tctcgacgga cctgtccgcg gtaacaccaa gatcatccag 900
gaactcgagt ccttcttccg tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc 960
gagtgggatg aacttctgga gaaggaccag gatggtgcac ttgttgagat catgaacaac 1020
acctccgatg gtgactacca gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac 1080
ttcttcggac gtgacccacg caccgcaaag ctcgttgaga acatgaccga cgaagaaatc 1140
tggaagcttc cacgtggcgg ccacgattac cgcaaggttt acgcagccta caagcgagct 1200
cttgagacca aggatcgccc aaccgtcatc cttgctcaca ccattaaggg ctacggactc 1260
ggccacaact tcgaaggccg taacgcaacc caccagatga agaagctgac gcttgatgat 1320
ctgaagttgt tccgcgacaa gcagggcatc ccaatcaccg atgagcagct ggagaaggat 1380
ccttaccttc ctccttacta ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag 1440
gaacgtcgcg cagcgctcgg tggctacctg ccagagcgtc gtgagaacta cgatccaatt 1500
caggttccac cactggataa gcttcgctct gtccgtaagg gctccggcaa gcagcagatc 1560
gctaccacca tggcgactgt tcgtaccttc aaggaactga tgcgcgataa gggcttggct 1620
gatcgccttg tcccaatcat tcctgatgag gcacgtacct tcggtcttga ctcttggttc 1680
ccaaccttga agatctacaa cccgcacggt cagaactacg tgcctgttga ccacgacctg 1740
atgctctcct accgtgaggc acctgaagga cagatcctgc acgaaggcat caacgaggct 1800
ggttccgtgg catcgttcat cgctgcgggt acctcctacg ccacccacgg caaggccatg 1860
attccgctgt acatcttcta ctcgatgttc ggattccagc gcaccggtga ctccatctgg 1920
gcagcagccg atcagatggc acgtggcttc ctcttgggcg ctaccgcagg tcgcaccacc 1980
ctgaccggtg aaggcctcca gcacatggat ggacactccc ctgtcttggc ttccaccaac 2040
gagggtgtcg agacctacga cccatccttt gcgtacgaga tcgcacacct ggttcaccgt 2100
ggcatcgacc gcatgtacgg cccaggcaag ggtgaagatg ttatctacta catcaccatc 2160
tacaacgagc caaccccaca gccagctgag ccagaaggac tggacgtaga aggcctgcac 2220
aagggcatct acctctactc ccgcggtgaa ggcaccggcc atgaggcaaa catcttggct 2280
tccggtgttg gtatgcagtg ggctctcaag gctgcatcca tccttgaggc tgactacgga 2340
gttcgtgcca acatttactc cgctacttct tgggttaact tggctcgcga tggcgctgct 2400
cgtaacaagg cacagctgcg caacccaggt gcagatgctg gcgaggcatt cgtaaccacc 2460
cagctgaagc agacctccgg cccatacgtt gcagtgtctg acttctccac tgatctgcca 2520
aaccagatcc gtgaatgggt cccaggcgac tacaccgttc tcggtgcaga tggcttcggt 2580
ttctctgata cccgcccagc tgctcgtcgc ttcttcaaca tcgacgctga gtccattgtt 2640
gttgcagtgc tgaactccct ggcacgcgaa ggcaagatcg acgtctccgt tgctgctcag 2700
gctgctgaga agttcaagtt ggatgatcct acgagtgttt ccgtagatcc aaacgctcct 2760
gaggaataa 2769
<210> 3
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物1
<400> 3
tgggaccggg aaaccggg 18
<210> 4
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物2
<400> 4
gatttatctg tcccttga 18
<210> 5
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物3
<400> 5
gcaggtcgac tctagatgcg attcgcgtca aacgtg 36
<210> 6
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物4
<400> 6
gtcccttgag gtgatgtgaa tccatccact 30
<210> 7
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物5
<400> 7
agtggatgga ttcacatcac ctcaagggac 30
<210> 8
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物6
<400> 8
ccggggatcc tctagacgaa gcgccgtgag caattc 36
<210> 9
<211> 521
<212> DNA
<213> 人工序列
<220>
<223> 包含aceE基因的5'端的aceE基因片段
<400> 9
gcaggtcgac tctagatgcg attcgcgtca aacgtgagag aaacatcaca tctcgcggga 60
aactacccga taattctttg caaaactttg caaagcggaa tgaacatgca gctagtttcc 120
gtagaaatgt tctttaaaaa atccacaaca attgccagga agcacaccga ttgatggata 180
cctgaaatcc cagtgagcgc accgctcccc ttacgtcaca gtctgtaaaa caaatcttcg 240
gtgttgcgta tccttgttaa taacttatgc gttgactcat tcgtgcactt cggcgtgtca 300
caattaggta cgaccaagaa tgggaccggg aaaccgggac gtataaacga aataaaacat 360
tccaacagga ggtgtggaaa tggccgatca agcaaaactt ggtggcaagc cctcggatga 420
ctctaacttc gcgatgatcc gcgatggcgt ggcatcttat ttgaacgact cagatccgga 480
ggagaccaac gagtggatgg attcacatca cctcaaggga c 521
<210> 10
<211> 538
<212> DNA
<213> 人工序列
<220>
<223> 包含aceE基因的3'端的aceE基因片段
<400> 10
agtggatgga ttcacatcac ctcaagggac agataaatcc cgccgccaga cgttagtctg 60
gcggcgggat tcgtcgtaaa gcaagctctt tttagccgag gaacgccttg tcagacaatg 120
ttgcgccctt gatgttggcg aactcctgca gcaaatcgcg cacagtcaac ttcgacttgg 180
tagcctgatc tgcctggtag acaatctggc cttcatgcat catgatcagg cgattgccca 240
ggcgaattgc ctgttccatg ttgtgcgtga ccataagcgt agtcagattt ccatctgcca 300
cgatcttttc ggtcaaggtg gtcacaagct ctgcacgctg tggatcaagc gctgcggtgt 360
gctcatccaa cagcatgatt ttaggttgag taaaaccagc catcagcagg gacaatgcct 420
gacgctgacc gccagagagc aaaccaactt tggcagtgag cctgttttcc agacccagct 480
caaggcgctc aagttcctgc ttgaattgct cacggcgctt cgtctagagg atccccgg 538
<210> 11
<211> 1029
<212> DNA
<213> 人工序列
<220>
<223> δ aceE
<400> 11
gcaggtcgac tctagatgcg attcgcgtca aacgtgagag aaacatcaca tctcgcggga 60
aactacccga taattctttg caaaactttg caaagcggaa tgaacatgca gctagtttcc 120
gtagaaatgt tctttaaaaa atccacaaca attgccagga agcacaccga ttgatggata 180
cctgaaatcc cagtgagcgc accgctcccc ttacgtcaca gtctgtaaaa caaatcttcg 240
gtgttgcgta tccttgttaa taacttatgc gttgactcat tcgtgcactt cggcgtgtca 300
caattaggta cgaccaagaa tgggaccggg aaaccgggac gtataaacga aataaaacat 360
tccaacagga ggtgtggaaa tggccgatca agcaaaactt ggtggcaagc cctcggatga 420
ctctaacttc gcgatgatcc gcgatggcgt ggcatcttat ttgaacgact cagatccgga 480
ggagaccaac gagtggatgg attcacatca cctcaaggga cagataaatc ccgccgccag 540
acgttagtct ggcggcggga ttcgtcgtaa agcaagctct ttttagccga ggaacgcctt 600
gtcagacaat gttgcgccct tgatgttggc gaactcctgc agcaaatcgc gcacagtcaa 660
cttcgacttg gtagcctgat ctgcctggta gacaatctgg ccttcatgca tcatgatcag 720
gcgattgccc aggcgaattg cctgttccat gttgtgcgtg accataagcg tagtcagatt 780
tccatctgcc acgatctttt cggtcaaggt ggtcacaagc tctgcacgct gtggatcaag 840
cgctgcggtg tgctcatcca acagcatgat tttaggttga gtaaaaccag ccatcagcag 900
ggacaatgcc tgacgctgac cgccagagag caaaccaact ttggcagtga gcctgttttc 960
cagacccagc tcaaggcgct caagttcctg cttgaattgc tcacggcgct tcgtctagag 1020
gatccccgg 1029
<210> 12
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物9
<400> 12
aatctagatg ggaccgggaa accggg 26
<210> 13
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物10
<400> 13
aatctagaga tttatctgtc ccttga 26
<210> 14
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(Q432E)
<400> 14
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Glu
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 15
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(E190V)
<400> 15
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Val Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 16
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(L438P)
<400> 16
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Pro Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 17
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(Q195H)
<400> 17
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu His Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 18
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(P199S)
<400> 18
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Ser Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 19
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(K435A)
<400> 19
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Ala Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 20
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(Q432A)
<400> 20
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Ala
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 21
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(Y418H)
<400> 21
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly His Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 22
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(Y201A)
<400> 22
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Ala Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 23
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E1p(N428A)
<400> 23
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Ala Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 24
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E190V, Q195H
<400> 24
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Val Val Ser
180 185 190
Arg Glu His Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 25
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E190V, P199S
<400> 25
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Val Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Ser Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 26
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> Q195H, P199S
<400> 26
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu His Gly Gly Ile Ser Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 27
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> E190V, Y201A
<400> 27
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Val Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Ala Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 28
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> Q195H, Y201A
<400> 28
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu His Gly Gly Ile Pro Ser Ala Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 29
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> P199S, Y201A
<400> 29
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Ser Ser Ala Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 30
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> N428A, Q432E
<400> 30
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Ala Ala Thr His Glu
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 31
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> N428A, K435A
<400> 31
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Ala Ala Thr His Gln
420 425 430
Met Lys Ala Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 32
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> Y418H, K435A
<400> 32
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly His Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln
420 425 430
Met Lys Ala Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920
<210> 33
<211> 922
<212> PRT
<213> 人工序列
<220>
<223> Y418H, Q432A
<400> 33
Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn
1 5 10 15
Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp
20 25 30
Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln
35 40 45
Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu
50 55 60
Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp
65 70 75 80
Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp
85 90 95
Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala
100 105 110
Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His
115 120 125
Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn
130 135 140
His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe
145 150 155 160
Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu
165 170 175
Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser
180 185 190
Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys
195 200 205
Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp
210 215 220
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile
225 230 235 240
Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu
245 250 255
Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn
260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu
275 280 285
Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser
290 295 300
Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg
305 310 315 320
Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu
325 330 335
Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn
340 345 350
Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr
355 360 365
Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro
370 375 380
Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala
385 390 395 400
Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys
405 410 415
Gly His Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Ala
420 425 430
Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln
435 440 445
Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro
450 455 460
Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys
465 470 475 480
Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn
485 490 495
Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg
500 505 510
Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg
515 520 525
Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540
Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe
545 550 555 560
Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val
565 570 575
Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile
580 585 590
Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala
595 600 605
Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr
610 615 620
Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp
625 630 635 640
Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala
645 650 655
Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His
660 665 670
Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro
675 680 685
Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg
690 695 700
Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile
705 710 715 720
Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val
725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr
740 745 750
Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala
755 760 765
Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn
770 775 780
Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala
785 790 795 800
Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala
805 810 815
Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val
820 825 830
Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro
835 840 845
Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr
850 855 860
Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val
865 870 875 880
Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser
885 890 895
Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser
900 905 910
Val Ser Val Asp Pro Asn Ala Pro Glu Glu
915 920

Claims (8)

1.一种丙酮酸脱氢酶变体，其中SEQ ID NO:1中的一个或两个氨基酸被置换，并且其中所述置换选自：
E190V；
Q195H；
P199S；
Y201A；
Y418H；
N428A；
Q432E；
Q432A；
K435A；
L438P；
E190V，Q195H；
E190V，P199S；
Q195H，P199S；
E190V，Y201A；
Q195H，Y201A；
P199S，Y201A；
N428A，Q432E；
N428A，K435A；
Y418H，K435A；和
Y418H，Q432A。
2.权利要求1所述的丙酮酸脱氢酶变体，其中所述丙酮酸脱氢酶变体的氨基酸序列选自SEQ ID NO:14到33。
3.一种多核苷酸，其编码权利要求1或2所述的丙酮酸脱氢酶变体。
4.一种生产L-氨基酸的棒状杆菌属的微生物，其中所述微生物包括权利要求1或2所述的丙酮酸脱氢酶变体。
5.权利要求4所述的微生物，其中所述微生物是谷氨酸棒状杆菌。
6.权利要求4或权利要求5所述的微生物，其中所述L-氨基酸包括L-赖氨酸或L-缬氨酸。
7.一种生产L-氨基酸的方法，其包括：
(a)在培养基中培养包括权利要求1或2所述的丙酮酸脱氢酶变体的谷氨酸棒状杆菌的微生物以生产L-氨基酸；和
(b)从所培养的微生物或所述培养基回收所述L-氨基酸。
8.权利要求7所述的方法，其中所述L-氨基酸是L-赖氨酸或L-缬氨酸。
CN201680002649.4A 2015-03-18 2016-03-15 丙酮酸脱氢酶突变体、包含突变体的微生物和通过使用微生物生产l-氨基酸的方法 Active CN106715687B (zh)

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Application Number Priority Date Filing Date Title
KR1020150037654A KR101776375B1 (ko) 2015-03-18 2015-03-18 피루브산 디하이드로게나아제 변이체, 이를 포함하는 미생물 및 이를 이용한 l-아미노산 생산 방법
KR10-2015-0037654 2015-03-18
PCT/KR2016/002594 WO2016148490A1 (ko) 2015-03-18 2016-03-15 피루브산 디하이드로게나아제 변이체, 이를 포함하는 미생물 및 이를 이용한 l-아미노산 생산 방법

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CN106715687A CN106715687A (zh) 2017-05-24
CN106715687B true CN106715687B (zh) 2021-04-27

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