CN109863162A - 工程化亚胺还原酶和用于酮和胺化合物的还原胺化的方法 - Google Patents
工程化亚胺还原酶和用于酮和胺化合物的还原胺化的方法 Download PDFInfo
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Abstract
本申请提供了具有亚胺或肟还原酶活性的工程化多肽、编码该工程化多肽的多核苷酸、能够表达该工程化多肽的宿主细胞、以及使用这些工程化多肽和一系列酮胺底物化合物以制备仲胺和叔胺产物化合物的方法。
Description
本申请要求2016年8月26日提交的美国临时专利申请系列第62/380,165号的优先权，在此通过引用以其整体出于所有目的并入。
技术领域
本发明提供了可用于产生仲胺的具有亚胺和/或肟还原酶活性的工程化多肽，以及利用这些工程化多肽的组合物和方法。
对序列表、表格或计算机程序的引用
序列表的正式副本作为ASCII格式文本文件经由EFS-Web与说明书同时被提交，文件名为“CX2-151USP1_ST25.txt”，创建日期为2016年8月26日，且大小为511千字节。经由EFS-Web提交的序列表为说明书的一部分并且通过引用以其整体并入本文。
背景
手性仲胺是制药行业的重要组成部分。然而，仅存在少量的已知生产这类手性胺化合物的生物催化途径。现有的化学方法使用手性硼试剂、基于过渡金属的还原方法或保护基团策略，它们需要多步骤合成用于全面还原亚胺。
在文献中存在几篇关于稳定环状胺的生物催化合成的报道。亚胺还原酶或“IRED”从链霉菌属种(Streptomyces sp.)GF3587和GF3546纯化和表征，并且示出立体选择性地还原2-甲基-1-吡咯啉(参见，Mitsukura等人,Biosci.Biotech.Biochem.,75:1778-1782[2011)]；Huber等人.Chem.Cat.Chem.,6:2248-2252[2014])。还示出了使用天然亚胺还原酶还原成无环胺和带有烷基基团的氨基酸(Gand等人,J.Mol.Catal.B:Enzym.,110:126-132[2014])。IRED作为用于以工业规模生产胺的起始酶的使用依赖于鉴定具有最高活性的正确变体。
对现有抗生素有抗性的细菌菌株的流行强调了开发新型抗细菌剂的必要性。被配制成靶向多种作用模式的抗生素混合物(antibiotic cocktail)变得越来越受欢迎。通过用β-内酰胺酶水解化合物，细菌在中和β-内酰胺抗生素方面变得非常有效。Relebactam是一种β-内酰胺酶抑制剂，其使酶无效，这允许抗生素作用于其靶上。relebactam的化学合成是一个多步骤的过程，其中之一需要苄氧基胺的立体选择性加成。该化学步骤的低收率使得酶的使用成为一种有吸引力的选择。
概述
本发明提供了新型生物催化剂和用于通过亚胺底物的直接还原来合成手性仲胺的相关方法。本公开内容的生物催化剂是来自Pseudonocardia spinosispora的野生型基因的工程化多肽变体，该野生型基因编码具有SEQ ID NO:2的氨基酸序列的亚胺还原酶“或“IRED”。这些工程化多肽能够催化亚胺转化为仲胺。
本发明提供了一种工程化多肽，所述工程化多肽包含与参考序列SEQ ID NO:2具有至少80％序列同一性的氨基酸序列，包含在选自57/211、87/175、103/212、122、123、124/178、124、132、134、169、171、173、175、176、178/242、178、211、212、213、214、215、217、234和235的一个或更多个位置处的至少一个取代，其中所述位置参考SEQ ID NO:2被编号。在一些另外的实施方案中，工程化多肽包含至少一个选自57T/211L、87S/175G、103H/212C、122E、123G、124A/178I、124G、132H/R、134R、169N/S、171W、173H/I、175G/H、176S、178I/242Y、178M、211L/W、212C/G/V、213P/R/T/V、214M、215L/M、217I/L、234L和235K的取代，其中所述位置参考SEQ ID NO:2被编号。在一些实施方案中，工程化多肽包含至少一个选自A57T/I211L、P87S/M175G、R103H/E212C、L122E、T123G、I124A/V178I、I124G、E132H/R、T134R、A169N/S、Y171W、A173H/I、M175G/H、L176S、V178I/A242Y、V178M、I211L/W、E212C/G/V、A213P/R/T/V、V214M、T215L/M、W217I/L、D234L和A235K的取代，其中所述位置参考SEQ IDNO:2被编号。在一些另外的实施方案中，工程化多肽包含与SEQ ID NO:4至SEQ ID NO:74中列出的任何偶数序列具有至少80％序列同一性的氨基酸序列。
本发明还提供了一种工程化多肽，所述工程化多肽包含与参考序列SEQ ID NO:270具有至少80％序列同一性的氨基酸序列，包含至少两个选自以下取代组的取代：123/124/169/171/173/213/235、123/124/169/212/213/235、123/169/171/173/212/213/235、123/169/171/173/213/235、123/169/173/175/213/235、123/169/173/212/213、123/169/175、123/169/175/212/235、123/169/212/213、123/173/175/212/213/235、123/212/213/235、123/212/235、123/169/171/173/175/212/213、123/169/173、123/169/212/213/235、123/213、124/169、124/169/173/175/212/213、124/169/173/212/213、124/169/173/212/213/235、124/169/175/212/213/235、124/169/212/213/235、124/169/213/235、124/175/212/213/235、124/212/213/235、134/169/176/178/211/214、134/169/176/178/211、134/169/176/178/211/217、134/169/176/211/215/217/234、134/169/176/211、134/169/178/211/214/217、134/169/211/214/217、134/169/211/217、134/176/211/214、134/176/211/215、134/211/214、169/171/173/213/235、169/173/175/213/235、169/175/212/213/235、169/176/178/211/214、169/176/178/211/214/217、169/176/178/211/217、169/176/178、169/176/178/211/214/217、169/176/211/214、169/176/211/214/215/234、169/176/211/214/217、169/176/211/217、169/176/214/215/217、169/178/217、169/178/234、169/211/214/215、169/211/217、169/212/213、169/212/235、169/212/213/235、169/235、171/173/212/213/235、173/175/213、175/213/235、175/235、176/178/211/215/217、176/178/211/217、176/211/214、176/214/217、178/211/214、178/211/217、178/215、211/234、212/213/235和213/235，其中所述位置参考SEQ ID NO:270被编号。在一些实施方案中，工程化多肽包含至少一个选自以下的取代组：123G/124G/169N/171V/173H/213V/235K、123G/124G/169N/212V/213V/235K、123G/169N/171V/173H/213V/235K、123G/169N/171V/173I/212C/213V/235K、123G/169N/173H/175H/213V/235K、123G/169N/173I/212G/213T、123G/169N/175G、123G/169N/175G/212C/235K、123G/169N/212G/213V、123G/173H/175H/212C/213V/235K、123G/212C/213V/235K、123G/212G/235K、123G/169N/171V/173H/175H/212X/213V、123G/169N/173I、123G/169N/212G/213V/235K、123G/213T、124G/169N、124A/169N/173I/175H/212G/213V、124G/169N/173I/212C/213V、124G/169N/173I/212G/213V、124G/169N/173I/212V/213V/235K、124G/169N/175G/212C/213V/235K、124G/169N/212G/213V/235K、124G/169N/212V/213V/235K、124G/175G/212V/213V/235K、124G/169N/213V/235K、124G/212C/213V/235K、124G/212G/213V/235K、134R/169N/176S/178I/211L/214M、134R/169N/176S/178I/211W、134R/169N/176S/178M/211L/217L、134R/169N/176S/211L/215M/217I/234L、134R/169N/176S/211W、134R/169N/178M/211W/214M/217I、134R/169N/211W/214M/217L、134R/169N/211W/217L、134R/176S/211W/214M、134R/176S/211W/215M、134R/211W/214M、169N/171V/173I/213V/235K、169N/173I/175H/213V/235K、169N/175G/212C/213V/235K、169N/M175H/E212G/A213V/A235K、169N/176S/178I/211L/214M、169N/176S/178I/211W/214M/217L、169N/176S/178I/211W/217L、169N/176S/178M、169N/176S/178M/211W/214M/217I、169N/176S/211W/214M、169N/176S/211W/214M/215L/234L、169N/176S/211W/217I、169N/176S/211W/217L、169N/176S/214M/215M/217I、169N/176S/211L/214M/217I、169N/178I/217I、169N/178M/234L、169N/211W/214M/215L、169N/211W/217I、169N/212C/235K、169N/212C/213V/235K、169N/212G/213V、169N/212V/213V/235K、169N/235K、171V/173I/212G/213V/235K、173I/175H/213V、175H/213P/235K、175H/235K、176S/178I/211W/215L/217L、176S/178I/211W/217I、176S/211L/214M、176S/214M/217L、178M/211L/214M、178M/211W/217L、178I/215M、211W/234L、212G/213V/235K、212V/213V/235K、213T/235K和213V/235K，其中所述位置参考SEQ ID NO:270被编号。在一些另外的实施方案中，工程化多肽包含至少一个选自以下的取代组：T123G/I124G/A169N/Y171V/A173H/A213V/A235K、T123G/I124G/A169N/E212V/A213V/A235K、T123G/A169N/Y171V/A173H/A213V/A235K、T123G/A169N/Y171V/A173I/E212C/A213V/A235K、T123G/A169N/A173H/M175H/A213V/A235K、T123G/A169N/A173I/E212G/A213T、T123G/A169N/M175G、T123G/A169N/M175G/E212C/A235K、T123G/A169N/E212G/A213V、T123G/A173H/M175H/E212C/A213V/A235K、T123G/E212C/A213V/A235K、T123G/E212G/A235K、T123G/A169N/Y171V/A173H/M175H/E212X/A213V、T123G/A169N/A173I、T123G/A169N/E212G/A213V/A235K、T123G/A213T、I124G/A169N、I124A/A169N/A173I/M175H/E212G/A213V、I124G/A169N/A173I/E212C/A213V、I124G/A169N/A173I/E212G/A213V、I124G/A169N/A173I/E212V/A213V/A235K、I124G/A169N/M175G/E212C/A213V/A235K、I124G/A169N/E212G/A213V/A235K、I124G/A169N/E212V/A213V/A235K、I124G/M175G/E212V/A213V/A235K、I124G/A169N/A213V/A235K、I124G/E212C/A213V/A235K、I124G/E212G/A213V/A235K、T134R/A169N/L176S/V178I/I211L/V214M、T134R/A169N/L176S/V178I/I211W、T134R/A169N/L176S/V178M/I211L/W217L、T134R/A169N/L176S/I211L/T215M/W217I/D234L、T134R/A169N/L176S/I211W、T134R/A169N/V178M/I211W/V214M/W217I、T134R/A169N/I211W/V214M/W217L、T134R/A169N/I211W/W217L、T134R/L176S/I211W/V214M、134R/L176S/I211W/T215M、T134R/I211W/V214M、A169N/Y171V/A173I/A213V/A235K、A169N/A173I/M175H/A213V/A235K、A169N/M175G/E212C/A213V/A235K、A169N/M175H/E212G/A213V/A235K、A169N/L176S/V178I/I211L/V214M、A169N/L176S/V178I/I211W/V214M/W217L、A169N/L176S/V178I/I211W/W217L、A169N/L176S/V178M、A169N/L176S/V178M/I211W/V214M/W217I、A169N/L176S/I211W/V214M、A169N/L176S/I211W/V214M/T215L/D234L、A169N/L176S/I211W/W217I、A169N/L176S/I211W/W217L、A169N/L176S/V214M/T215M/W217I、A169N/L176S/I211L/V214M/W217I、A169N/V178I/W217I、A169N/V178M/D234L、A169N/I211W/V214M/T215L、A169N/I211W/W217I、A169N/E212C/A235K、A169N/E212C/A213V/A235K、A169N/E212G/A213V、A169N/E212V/A213V/A235K、A169N/A235K、Y171V/A173I/E212G/A213V/A235K、A173I/M175H/A213V、M175H/A213P/A235K、M175H/A235K、L176S/V178I/I211W/T215L/W217L、L176S/V178I/I211W/W217I、L176S/I211L/V214M、L176S/V214M/W217L、V178M/I211L/V214M、V178M/I211W/W217L、V178I/T215M、I211W/D234L、E212G/A213V/A235K、E212V/A213V/A235K、A213T/A235K和A213V/A235K，其中所述位置参考SEQ ID NO:270被编号。在一些另外的实施方案中，工程化多肽包含与SEQ ID NO:76至SEQ ID NO:232中列出的任何偶数序列具有至少80％序列同一性的氨基酸序列。
本发明还提供了一种工程化多肽，所述工程化多肽包含与参考序列SEQ ID NO:229具有至少80％序列同一性的，包含在选自45、127、130、134、174、206、209、212、216、223、227和279的一个或更多个位置处的至少一个取代的氨基酸序列，其中所述位置参考SEQ IDNO:229被编号。在一些另外的实施方案中，工程化多肽包含至少一个选自45I、127R、130R、134L/R、174M、206L、209K/S、212H/K/R、216N/R/S、223N、227V和279V的取代，其中所述位置参考SEQ ID NO:229被编号。在一些另外的实施方案中，工程化多肽包含至少一个选自V45I、G127R、T130R、T134L/R、A174M、M206L、Q209K/S、E212H/K/R、G216N/R/S、D223N、A227V和D279V的取代，其中所述位置参考SEQ ID NO:229被编号。在一些另外的实施方案中，工程化多肽包含与SEQ ID NO:234至SEQ ID NO:270中列出的任何偶数序列具有至少80％序列同一性的氨基酸序列。
本发明还提供了编码至少一种在上文段落中描述的工程化多肽的工程化多核苷酸。在一些实施方案中，工程化多核苷酸包含SEQ ID NO:3至SEQ ID NO:267中列出的奇数序列。
本发明还提供了一种工程化多核苷酸，其编码工程化亚胺或肟还原酶多肽，其中工程化多核苷酸包含在以下位置处的取代：12/15/18/21/27、169/631/633、259/523/524/525、308/634/635/636、364/365/366、367/368/369、370/371、370/371/372/532、394/396、394/395/396、401/402、505/506/507、512/513、517/518/519、523/524/525、526/527/528、532/534、532/724/725、631/633、631/632/633、634/635/636、635/636、637/638、637/639、637/639、638/639、640、643/644/645、644/645、649/650/651、700/701/702和703/704/705，其中所述位置参考SEQ ID NO:1被编号。在一些实施方案中，编码工程化亚胺或肟还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：12A/15T/18G/21C/27A、169A/631C/633T、259T/523G/524G/525T、308A/634T/635G/636T、364G/365A/366A、367G/368G/369T、370G/371G、370G/371C/372A/532A、394C/396T、394A/395G/396G、401G/402G、505A/506A/507T、505A/506G/507T、512G/513G、517A/518T/519T、517C/518A/519T、523C/524A/525T、523G/524G/525T、526A/527G/528T、532A/534G、532A/724T/725A、631C/633T、631T/632G/633G、634T/635G/636T、635G/636T、635T/636T、637A/638G、637A/639A、637C/639A、638T/639T、640A、643T/644T/645G、644T/645G、649A/650T/651T、649C/650T/651T、700T/701T/702G和703A/704A/705A，并且其中所述位置参考SEQ ID NO:1被编号。在一些另外的实施方案中，编码工程化亚胺或肟还原酶的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：C12A/C15T/A18G/T21C/T27A、G169A/A631C/C633T、C259T/A523G/T524G/G525T、G308A/G634T/A635G/A636T、C364G/T365A/G366A、A367G/C368G/C369T、A370G/T371G、A370G/T371C/T372A/G532A、G394C/A396T、G394A/A395G/A396G、C401G/C402G、G505A/C506A/A507T、G505A/C506G/A507T、A512G/T513G、G517A/C518T/A519T、G517C/C518A/A519T、A523C/T524A/G525T、A523G/T524G/G525T、C526A/T527G/G528T、G532A/C534G、G532A/G724T/C725A、A631C/C633T、A631T/T632G/C633G、G634T/A635G/A636T、A635G/A636T、A635T/A636T、G637A/C638G、G637A/G639A、G637C/G639A、C638T/G639T、G640A、A643T/C644T/C645G、C644T/C645G、T649A/G650T/G651T、T649C/G650T/G651T、G700T/A701T/C702G和G703A/C704A/C705A，并且其中所述位置参考SEQ IDNO:1被编号。
本发明还提供了一种工程化多核苷酸，其编码工程化亚胺或肟还原酶多肽，其中工程化多核苷酸包含在以下位置处的取代：367/368/369/505/506/507/513/519/523/524/525/634/635/636/639/703/704/705、369/370/371/372/505/506/507/513/519/638/703/704/705、369/370/371/372/507/513/519/523/524/525/635/638/703/704/705、369/505/506/507/511/512/517/518/519/638/703/704/705、369/505/506/507/513/517/518/519/523/524/525/638/703/704/705、369/505/506/507/513/519/523/524/525/634/635/636/638/703/704/705、369/505/506/507/513/519/635/638/703/704/705、401/402/505/506/507/526/527/532/534/631/632/633/645/702、401/402/505/506/507/526/527/532/534/631/633/640/645/702、401/402/505/506/507/526/527/532/534/631/633/645/649/650/651/702、401/402/505/506/507/526/527/534/631/632/633/645/702、401/402/505/506/507/526/527/534/631/633/644/645/649/650/651/700/701、401/402/505/506/507/526/532/534/631/632/633/640/645/649/650/651/702、401/402/505/506/507/526/534/631/632/633/640/645/649/650/651/702、401/402/505/506/507/526/534/631/632/633/645/649/650/651/702、401/402/507/526/527/534/631/632/633/640/645/702、401/402/507/526/527/534/631/632/633/644/645/702、401/402/526/534/631/632/633/640/645/702、402/505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、402/505/506/507/526/527/532/534/702、402/505/506/507/526/527/532/534/631/633/640/645/702、402/505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、402/505/506/507/526/527/534/631/633/640/645/649/650/651/702、402/505/506/507/526/527/534/631/632/633/640/643/644/645/700/701、402/505/506/507/526/527/534/631/632/633/640/645/702、402/505/506/507/526/527/534/631/632/633/645/649/650/651/702、402/505/506/507/526/527/534/631/632/633/645/649/650/651/702、402/505/506/507/526/527/534/633/640/644/645/649/650/651/702、402/505/506/507/526/532/534/633/645/700/701、402/505/506/507/526/532/534/645/649/650/651、402/505/506/507/526/534/631/632/633/645/649/650/651/702、402/507/526/527/532/534/631/632/633/643/644/645/649/650/651/702、402/507/526/527/532/534/631/632/633/645/649/650/651/702、402/507/526/532/534/631/633/640/645、402/507/526/532/534/631/632/633/645/649/650/651/702、402/526/527/534/631/633/640/645/702、505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、505/506/507/526/527/532/534/631/632/633/645/649/650/651/702、505/506/507/526/534/631/632/633/640/643/644/645、507/526/527/534/633/640/645/649/650/651/702、507/526/532/534/644/645/702和631/632/633/645/700/701，其中所述位置参考SEQ ID NO:269被编号。在一些实施方案中，编码工程化亚胺或肟还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：367G/368G/369T/505A/506A/507T/513C/519C/523G/524G/525C/634T/635G/636C/639T/703A/704A/705G、369G/370G/371G/372C/505A/506A/507T/513C/519C/638T/703A/704A/705G、369G/370G/371G/372C/507C/513C/519C/523G/524G/525C/635T/638T/703A/704A/705G、369G/505A/506A/507T/511G/512T/517A/518T/519T/638T/703A/704A/705G、369G/505A/506A/507T/513C/517A/518T/519T/523C/524A/525C/638T/703A/704A/705G、369G/505A/506A/507T/513C/519C/523G/524G/525C/634T/635G/636C/638T/703A/704A/705G、369G/505A/506A/507T/513C/519C/635T/638T/703A/704A/705G、401G/402G/505A/506A/507C/526T/527C/532A/534G/631C/633A/645T/649C/650T/651A/702T、401G/402G/505A/506A/507C/526T/527C/532A/534T/631C/633A/640A/645T/702T、401G/402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/645T/702T、401G/402G/505A/506A/507C/526T/527C/534G/631C/633A/644T/645G/649A/650T/651A/700C/701T、401G/402G/505A/506A/507C/526T/527C/534T/631T/632G/633G/645T/702T、401G/402G/505A/506A/507C/526T/532A/534G/631T/632G/633G/640A/645T/649A/650T/651A/702T、401G/402G/505A/506A/507C/526T/534G/631T/632G/633G/640A/645T/649C/650T/651A/702T、401G/402G/505A/506A/507C/526T/534T/631T/632G/633G/645T/649C/650T/651A/702T、401G/402G/507C/526T/527C/534G/631T/632G/633G/640A/645T/702T、401G/402G/507C/526T/527C/534G/631T/632G/633G/644T/645G/702T、401G/402G/526T/534G/631T/632G/633G/640A/645T/702T、402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/640A/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/532A/534G/702T、402G/505A/506A/507C/526T/527C/532A/534T/631C/633A/640A/645T/702T、402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/640A/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/534G/631C/633A/640A/645T/649A/650T/651A/702T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/640A/643T/644T/645G/700C/701T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/640A/645T/702T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/534T/631T/632G/633G/645T/649A/650T/651A/702T、402G/505A/506A/507C/526T/527C/534T/633A/640A/644T/645G/649A/650T/651A/702T、402G/505A/506A/507C/526T/532A/534G/633A/645T/700C/701T、402G/505A/506A/507C/526T/532A/534T/645T/649A/650T/651A、402G/505A/506A/507C/526T/534G/631T/632G/633G/645T/649A/650T/651A/702T、402G/507C/526T/527C/532A/534T/631T/632G/633G/643T/644T/645G/649C/650T/651A/702T、402G/507C/526T/527C/532A/534T/631T/632G/633G/645T/649A/650T/651A/702T、402G/507C/526T/532A/534G/631C/633A/640A/645T、402G/507C/526T/532A/534G/631T/632G/633G/645T/649C/650T/651A/702T、402G/526T/527C/534T/631C/633A/640A/645T/702T、505A/506A/507C/526T/527C/532A/534G/631T/632G/633G/640A/645T/649A/650T/651A/702T、505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/645T/649C/650T/651A/702T、505A/506A/507C/526T/534G/631T/632G/633G/640A/643T/644T/645G、507C/526T/527C/534G/633A/640A/645T/649C/650T/651A/702T、507C/526T/527C/534G/633A/640A/645T/649C/650T/651A/702T、507C/526T/532A/534T/644T/645G/702T和631T/632G/633G/645T/700C/701T，其中所述位置参考SEQ ID NO:269被编号。在一些另外的实施方案中，编码工程化亚胺或肟还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：A367G/C368G/C369T/G505A/C506A/A507T/T513C/A519C/A523G/T524G/G525C/G634T/A635G/A636C/G639T/G703A/C704A/C705G、C369G/A370G/T371G/T372C/G505A/C506A/A507T/T513C/A519C/C638T/G703A/C704A/C705G、C369G/A370G/T371G/T372C/A507C/T513C/519C/A523G/T524G/G525C/A635T/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T511G/A512T/G517A/C518T/A519T/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/G517A/C518T/A519T/A523C/T524A/G525C/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/A519C/A523G/T524G/G525C/G634T/A635G/A636C/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/A519C/A635T/C638T/G703A/C704A/C705G、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534G/A631C/C633A/C645T/T649C/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631C/C633A/G640A/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631C/C633A/C644T/C645G/T649A/G650T/G651A/G700C/A701T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/C534T/A631T/T632G/C633G/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/G532A/C534G/A631T/T632G/C633G/G640A/C645T/T649A/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/C534T/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C401G/C402G/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C401G/C402G/A507C/C526T/T527C/C534G/A631T/T632G/C633G/C644T/C645G/C702T、C401G/C402G/C526T/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534G/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631C/C633A/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631C/C633A/G640A/C645T/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/A643T/C644T/C645G/G700C/A701T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534T/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534T/C633A/G640A/C644T/C645G/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/G532A/C534G/C633A/C645T/G700C/A701T、C402G/G505A/C506A/A507C/C526T/G532A/C534T/C645T/T649A/G650T/G651A、C402G/G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/A643T/C644T/C645G/T649C/G650T/G651A/C702T、C402G/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/A507C/C526T/G532A/C534G/A631C/C633A/G640A/C645T、C402G/A507C/C526T/G532A/C534G/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C402G/C526T/T527C/C534T/A631C/C633A/G640A/C645T/C702T、G505A/C506A/A507C/C526T/T527C/G532A/C534G/A631T/T632G/C633G/G640A/C645T/T649A/G650T/G651A/C702T、G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/G640A/A643T/C644T/C645G、A507C/C526T/T527C/C534G/C633A/G640A/C645T/T649C/G650T/G651A/C702T、A507C/C526T/G532A/C534T/C644T/C645G/C702T和A631T/T632G/C633G/C645T/G700C/A701T，其中所述位置参考SEQ ID NO:269被编号。
本发明还提供了一种工程化多核苷酸，其编码工程化亚胺或肟还原酶多肽，其中工程化多核苷酸包含在以下位置处的取代：133、379/381、388/389/390、400/401/402、401/402、520/521/522、616/618、625、625/626/627、634、634/635/636、634/636、646、646/647、646、667、680/681和836/837，其中所述位置参考SEQ ID NO:229被编号。在一些实施方案中，编码工程化亚胺或肟还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：133A、379A/381G、388C/389G/390T、400C/401T/402T、401G/402G、520A/521T/522G、616C/618T、625A、625A/626G/627T、634A、634A/635G/636G、634C/636T、646A、646A/647A、646C、667A、680T/681T和836T/837T，其中位置参考SEQ ID NO:229被编号。在一些另外的实施方案中，编码工程化亚胺或肟还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：G133A、G379A/C381G、A388C/C389G/C390T、A400C/C401T/C402T、C401G/C402G、G520A/C521T/A522G、A616C/G618T、C625A、C625A/A626G/A627T、G634A、G634A/A635G/A636G、G634C/A636T、G646A、G646A/G647A、G646C、G667A、C680T/C681T和A836T/C837T，其中所述位置参考SEQ ID NO:229被编号。
本发明还提供了载体，所述载体包含至少一种上文描述的工程化多核苷酸。在一些实施方案中，载体还包含至少一个控制序列。
本发明还提供了宿主细胞，所述宿主细胞包含本文提供的载体。在一些实施方案中，宿主细胞产生至少一种本文提供的工程化多肽。
本发明还提供了产生工程化亚胺或肟还原酶多肽的方法，所述方法包括在使得工程化多核苷酸被表达和工程化多肽被产生的条件下培养本文提供的宿主细胞的步骤。在一些实施方案中，方法还包括回收工程化多肽的步骤。
发明描述
除非另外定义，本文使用的所有技术和科学术语通常具有与本发明所属领域普通技术人员通常理解的相同的含义。通常，本文使用的命名和下文描述的细胞培养、分子遗传学、微生物学、有机化学、分析化学和核酸化学的实验室程序是本领域熟知且常用的那些。这样的技术是熟知的并且被描述于本领域技术人员熟知的许多教科书和参考著作中。对于化学合成和化学分析使用了标准技术或其修改形式。本文(上文和下文两者)提及的所有专利、专利申请、文章和出版物，在此通过引用明确地并入本文。
尽管本发明的实践中可使用类似或等同于本文描述那些的任何合适的方法和材料，本文描述了一些方法和材料。应理解本发明不限于所描述的特定方法论、方案和试剂，因为取决于本领域技术人员使用其的情况，这些可以改变。因此，下文即将定义的术语通过参考本发明作为整体被更充分地描述。
应理解，上文的一般描述和下文的详细描述两者仅是示例性的和说明性的，而不是限制本发明。本文使用的章节标题仅用于组织目的，并且不被解释为限制所描述的主题。数值范围包括界定该范围的数字。因此，本文公开的每个数值范围意图涵盖落在这样的较宽数值范围内的每一较窄数值范围，如同这样的较窄数值范围在本文被全部清楚地写出。还意图本文公开的每个最大的(或最小的)数值限制包含每个较低(或较高)的数值限制，如同这样的较低(或较高)数值限制在本文中被清楚地写出。
除非上下文另外清楚地指明，否则如该说明书和所附的权利要求书中使用的单数形式“一(a)”、“一(an)”和“该(the)”包括复数指示物。因此，例如对“多肽(apolypeptide)”的提及包括多于一个多肽。类似地，“包括(comprise、comprises、comprising)”、“包括(include、includes和including)”是可互换的，而不意图限制性的。
应理解，在各种实施方案的描述中使用术语“包括(comprising)”的情况下，本领域技术人员将理解，在一些特定的情况下，可以使用“基本上由...组成”或“由...组成”的语言可选择地描述实施方案。还应理解，在各种实施方案的描述中使用术语“任选的(optional)”或“任选地(optionally)”的情况下，随后描述的事件或情况可能发生或可能不发生，并且该描述包括事件或情况发生的情况和其中它不发生的情况。应理解，上文的一般描述和下文的详细描述两者仅是示例性的和说明性的，而不是限制本公开内容。本文使用的章节标题仅用于组织目的，并且不被解释为限制所描述的主题。
缩写：
用于遗传编码的氨基酸的缩写是常规的并且如下所示：
当使用三字母缩写时，除非前面具体地有“L”或“D”，或者从使用缩写的上下文清楚看出，否则氨基酸可以关于α-碳(Cα)是L-构型或D-构型的。例如，“Ala”表示丙氨酸而不指定关于α-碳的构型，而“D-Ala”和“L-Ala”分别表示D-丙氨酸和L-丙氨酸。
当使用单字母缩写时，大写字母表示关于α-碳的L-构型的氨基酸，并且小写字母表示关于α-碳的D-构型的氨基酸。例如，“A”表示L-丙氨酸并且“a”表示D-丙氨酸。当多肽序列以单字母缩写或三字母缩写(或其混合物)串表示时，根据常规惯例将序列呈现为氨基(N)至羧基(C)方向。
用于遗传编码核苷的缩写是常规的并且如下：腺苷(A)；鸟苷(G)；胞苷(C)；胸苷(T)；和尿苷(U)。除非具体描述，否则缩写的核苷酸可以是核糖核苷或2'-脱氧核糖核苷。核苷可以单独地或总体地指定为核糖核苷或2'-脱氧核糖核苷。当核酸序列以单字母缩写串呈现时，序列根据常规惯例呈现为5'至3'方向，并且不示出磷酸。
定义：
参考本发明，本文描述中使用的技术和科学术语将具有本领域普通技术人员通常理解的含义，除非另外具体定义。因此，以下术语意图具有以下含义。
“EC”编号指的是生物化学和分子生物学国际联合命名委员会(NomenclatureCommittee of the International Union of Biochemistry and Molecular Biology)(NC-IUBMB)的酶命名法。该IUBMB生物化学分类是基于酶催化的化学反应的酶数字分类系统。
“ATCC”指的是美国典型培养物保藏中心(American Type Culture Collection)，其生物保藏收集包括基因和菌株。
“NCBI”指的是美国国家生物技术信息中心(National Center for BiologicalInformation)和在其中提供的序列数据库。
“蛋白”、“多肽”和“肽”在本文中可互换使用，来表示通过酰胺键共价连接的至少两个氨基酸的聚合物，而不论长度或翻译后修饰(例如糖基化、磷酸化、脂化、肉豆蔻酸化、泛素化等)如何。该定义中包括D-氨基酸和L-氨基酸，以及D-氨基酸和L-氨基酸的混合物，以及包含D-氨基酸和L-氨基酸以及D-氨基酸和L-氨基酸的混合物的聚合物。
“氨基酸”通过其通常已知的三字母符号或通过IUPAC-IUB生物化学命名委员会推荐的单字母符号在本文被提及。同样地，核苷酸可以通过其通常可接受的单字母代码被提及。
如本文使用的，“多核苷酸”和“核酸”指的是共价连接在一起的两个或更多个核苷。多核苷酸可以完全包含核糖核苷酸(即RNA)、完全包含2'脱氧核糖核苷酸(即DNA)、或包含核糖核苷酸和2'脱氧核糖核苷酸的混合物。虽然核苷典型地经由标准磷酸二酯键连接在一起，但多核苷酸可以包括一个或更多个非标准键。多核苷酸可以是单链或双链的，或者可以包括单链区和双链区两者。此外，虽然多核苷酸典型地由天然存在的编码核苷碱基(即腺嘌呤、鸟嘌呤、尿嘧啶、胸腺嘧啶和胞嘧啶)组成，它可以包含一种或更多种修饰的和/或合成的核苷碱基，诸如例如肌苷、黄嘌呤、次黄嘌呤等。在一些实施方案中，这样的修饰的或合成的核苷碱基是编码氨基酸序列的核苷碱基。
如本文使用的“冠瘿碱脱氢酶活性”指的是其中将2-酮酸(例如丙酮酸)的羰基基团和中性L-氨基酸(例如L-正缬氨酸)的氨基基团转化为仲胺二羧酸化合物(例如N-[1-(R)-(羧基)乙基]-(S)-正缬氨酸)的酶活性。
如本文使用的，“亚胺”指的是包含氮-碳双键的有机化合物或官能团，其中氮与氢或有机基团键合。
如本文使用的“亚胺还原酶活性”指的是如在方案1中说明的，其中在辅因子NAD(P)H的存在下，酮或醛的羰基基团和伯胺或仲胺的氨基基团(其中羰基基团和氨基基团可以在单独的化合物上或在相同化合物上)被转化为仲胺或叔胺产物化合物的酶活性。
如本文使用的“亚胺还原酶”或“IRED”指的是具有亚胺还原酶活性的酶。应理解，亚胺还原酶不限于衍生自来自Pseudonocardia spinosispora的野生型亚胺还原酶的工程化多肽，而且可以包括具有亚胺还原酶活性的其他酶，包括衍生自其他亚胺还原酶的工程化多肽。如本文使用的亚胺还原酶包括天然存在的(野生型)亚胺还原酶以及通过人类操作产生的非天然存在的工程化多肽。
如本文使用的“肟”指的是具有通式R1R2C＝NOR3的亚胺，其中R1是有机侧链，R2和R3可以是氢或另外的有机基团。
如本文使用的“肟还原酶活性”指的是其中肟部分中的氮和碳之间的双键被还原为单键的酶活性。
“编码序列”指的是编码蛋白质的氨基酸序列的核酸部分(例如基因)。
“天然存在的”或“野生型”指的是自然界中发现的形式。例如，天然存在的或野生型多肽或多核苷酸序列是存在于可以从自然资源分离的生物体中并且没有被人类操作有意修饰的序列。
如本文使用的，当关于细胞、核酸或多肽使用时，“重组”、“工程化”和“非天然存在的”指的是材料或相应于该材料的天然或天然形式的材料，其已经以自然中原本不存在的方式修饰。在一些实施方案中，细胞、核酸或多肽与天然存在的细胞、核酸或多肽相同，但产生自或衍生自合成材料和/或通过使用重组技术被操作。非限制性实例包括，除其他以外，表达在天然(非重组)形式的细胞中未发现的基因或表达原本以不同水平表达的天然基因的重组细胞。
“序列同一性百分比”和“同源性百分比”在本文中可互换使用，以指的是多核苷酸或多肽之间的比较，并且通过在比较窗上比较两个最佳对齐(aligned)的序列来确定，其中与用于两个序列的最佳对齐的参考序列相比，比较窗中的多核苷酸或多肽序列的部分可以包括添加或缺失(即，空位)。百分比可以通过如下计算：确定两个序列中出现相同的核酸碱基或氨基酸残基的位置的数目，以产生匹配位置的数目，将匹配位置的数目除以比较窗中位置的总数目，并将结果乘以100以得到序列同一性的百分比。可选择地，百分比可以通过如下计算：确定两个序列中出现相同的核酸碱基或氨基酸残基或者核酸碱基或氨基酸残基与空位对齐的位置的数目，以产生匹配位置的数目，将匹配位置的数目除以比较窗中位置的总数目，并将结果乘以100以得到序列同一性的百分比。本领域技术人员理解，存在许多可用于比对两个序列的已建立的算法。用于比较的序列的最佳比对可以例如通过Smith和Waterman的局部同源性算法(Smith和Waterman,Adv.Appl.Math.,2:482[1981])、通过Needleman和Wunsch的同源性比对算法(Needleman和Wunsch,J.Mol.Biol.,48:443[1970])、通过Pearson和Lipman的搜索相似性方法(Pearson和Lipman,Proc.Natl.Acad.Sci.USA 85:2444[1988])、通过这些算法的计算机化执行(例如GCGWisconsin软件包中的GAP、BESTFIT、FASTA和TFASTA)、或通过如本领域已知的目视检查进行。适合于确定序列同一性和序列相似性百分比的算法的实例包括但不限于BLAST和BLAST2.0算法，其由Altschul等人(分别参见，Altschul等人,J.Mol.Biol.,215:403-410[1990]；和Altschul等人,Nucl.Acids Res.,3389-3402[1977])描述。用于进行BLAST分析的软件是通过美国国家生物技术信息中心网站公共可获得的。该算法包括首先通过鉴定查询序列中长度W的短字来鉴定高得分序列对(HSP)，其在与数据库序列中的相同长度的字比对时匹配或满足一些正值阈值得分T。T被称为相邻字得分阈值(Altschul等人,同上)。这些最初的相邻字击中(word hit)用作用于启动检索的种子来发现更长的包含它们的HSP。然后字击中沿着每个序列的两个方向延伸直到累积比对得分不可以增加。对于核苷酸序列，累积得分使用参数M(对于匹配残基对的奖励得分；总是＞0)和N(对于错配残基的惩罚得分；总是＜0)计算。对于氨基酸序列，评分矩阵用于计算累积得分。当以下情况时，每个方向中的字击中的延伸被终止：累积比对得分从其最大达到值下降了量X；由于一个或更多个负得分残基比对的累积，累积得分达到0或以下；或达到任一序列的末端。BLAST算法参数W、T和X确定比对的灵敏度和速度。BLASTN程序(对于核苷酸序列)使用以下作为缺省值：字长(W)为11、期望值(E)为10、M＝5、N＝-4、以及两个链的比较。对于氨基酸序列，BLASTP程序使用以下作为缺省值：字长(W)为3，期望值(E)为10和BLOSUM62评分矩阵(参见，Henikoff和Henikoff,Proc.Natl.Acad.Sci.USA 89:10915[1989])。序列比对与％序列同一性的示例性确定可以使用GCG Wisconsin软件包(Accelrys,Madison WI)中的BESTFIT或GAP程序，使用提供的缺省参数。
“参考序列”指的是用作序列比较的基础的指定序列。参考序列可以是更大序列的子集，例如，全长基因或多肽序列的区段。通常，参考序列为至少20个核苷酸或氨基酸残基的长度、至少25个残基的长度、至少50个残基的长度，或核酸或多肽的全长。由于两个多核苷酸或多肽可以各自(1)包含两个序列之间相似的序列(即完整序列的一部分)，和(2)还可以包含两个序列之间不同的序列，因此两个(或更多个)多核苷酸或多肽之间的序列比较典型地通过比较两个多核苷酸或多肽在“比较窗”上的序列来鉴定和比较序列相似性的局部区域来进行。在一些实施方案中，“参考序列”可以基于一级氨基酸序列，其中参考序列是可以在一级序列中具有一个或更多个变化的序列。例如，“基于SEQ ID NO:4的在对应于X14的残基处具有缬氨酸的参考序列”或X14V指的是其中在SEQ ID NO:4中X14处的对应残基(酪氨酸)已经改变为缬氨酸的参考序列。
“比较窗”指的是至少约20个连续核苷酸位置或氨基酸残基的概念区段，其中序列可以与至少20个连续核苷酸或氨基酸的参考序列比较，并且其中与用于两个序列的最佳对齐的参考序列(其不包含添加或缺失)相比，比较窗中的序列的部分可以包含20％或更少的添加或缺失(即，空位)。比较窗可以长于20个连续残基，并且任选地包括30个、40个、50个、100个或更长的窗。
如本文使用的，“大体相同”指的是在至少20个残基位置的比较窗中、通常在至少30个-50个残基的窗中，与参考序列相比，具有至少80％序列同一性、至少85％同一性、至少89％至95％之间的序列同一性、或更通常地至少99％序列同一性的多核苷酸或多肽序列，其中序列同一性的百分比通过在比较窗上比较参考序列和包含总计为参考序列的20％或更少的缺失或添加的序列来计算。在应用于多肽的一些具体的实施方案中，术语“大体相同”意指当诸如通过程序GAP或BESTFIT使用默认空位权重进行最佳比对时，两个多肽序列共享至少80％的序列同一性，优选地至少89％的序列同一性、至少95％的序列同一性或更高(例如99％的序列同一性)。在一些实施方案中，在被比较的序列中不相同的残基位置通过保守氨基酸取代而不同。
当在给定氨基酸或多核苷酸序列的编号的上下文中使用时，“对应于”、“参考”和“相对于”指的是给定氨基酸或多核苷酸序列与参考序列相比时，特定参考序列的残基的编号。换言之，给定的聚合物的残基数目或残基位置根据参考序列指定，而不是通过给定的氨基酸或多核苷酸序列内残基的实际数字位置被指定。例如，可以通过引入空位以优化两个序列之间的残基匹配来比对给定氨基酸序列诸如工程化亚胺还原酶的氨基酸序列与参考序列。在这些情况中，尽管存在空位，在给定的氨基酸或多核苷酸序列中残基的编号是根据与其比对的参考序列做出的。
“氨基酸差异”或“残基差异”指的是多肽序列的一个位置的氨基酸残基相对于参考序列中的相应位置的氨基酸残基的变化。氨基酸差异的位置在本文中通常被称为“Xn”，其中n指的是残基差异所基于的参考序列中的对应位置。例如，“与SEQ ID NO:2相比在位置X25的残基差异”指的是在对应于SEQ ID NO:2的位置25的多肽位置处的氨基酸残基的变化。因此，如果SEQ ID NO:2的参考多肽在位置25具有缬氨酸，则“与SEQ ID NO:2相比在位置X25的残基差异”指的是在对应于SEQ ID NO:2的位置25的多肽的位置处除了缬氨酸以外的任何残基的氨基酸取代。在本文的大多数情况下，在一个位置处的特定氨基酸残基差异指示为“XnY”，其中“Xn”指定如上文描述的对应位置，并且“Y”是在工程化多肽中发现的氨基酸(即，与参考多肽不同的残基)的单字母标识符。在一些实施方案中，多于一种氨基酸可以出现在指定的残基位置(即，替代氨基酸可以以XnY/Z的形式列出，其中Y和Z表示替代的氨基酸残基)。在一些情况下(例如，在表2-2、表3-1、表4-1和表5-1中)，本发明还提供由常规符号“AnB”表示的特定氨基酸差异，其中A是参考序列中的残基的单字母标识符，“n”是在参考序列中的残基位置的数字，并且B是工程化多肽的序列中残基取代的单字母标识符。此外，在一些情况下，本发明的多肽可以包括相对于参考序列的一个或更多个氨基酸残基差异，其由相对于参考序列进行改变的指定位置的列表指示。在一些另外的实施方案中，本发明提供了包含保守氨基酸取代和非保守氨基酸取代二者的工程化多肽序列。
如本文使用的，“保守氨基酸取代”指的是用具有相似侧链的不同残基来取代残基，并且因此典型地包括用相同或相似的氨基酸定义类别中的氨基酸取代多肽中的氨基酸。例如但不限于，具有脂肪族侧链的氨基酸被另一个脂肪族氨基酸(例如，丙氨酸、缬氨酸、亮氨酸和异亮氨酸)取代；具有羟基侧链的氨基酸被另一个具有羟基侧链的氨基酸(例如，丝氨酸和苏氨酸)取代；具有芳香族侧链的氨基酸被另一个具有芳香族侧链的氨基酸(例如，苯丙氨酸、酪氨酸、色氨酸和组氨酸)取代；具有碱性侧链的氨基酸被另一个具有碱性侧链的氨基酸(例如，赖氨酸和精氨酸)取代；具有酸性侧链的氨基酸被另一个具有酸性侧链的氨基酸(例如，天冬氨酸或谷氨酸)取代；和/或疏水性氨基酸或亲水性氨基酸分别被另一个疏水性氨基酸或亲水性氨基酸替代。示例性保守取代在下表1中提供。
“非保守取代”指的是用具有显著不同的侧链性质的氨基酸取代多肽中的氨基酸。非保守取代可以使用定义的组之间而非定义的组之内的氨基酸，并且影响(a)取代区域中肽骨架的结构(例如，用脯氨酸取代甘氨酸)，(b)电荷或疏水性，或(c)侧链的体积。例如但不限于，示例性非保守取代可以是用碱性或脂肪族氨基酸取代酸性氨基酸；用小氨基酸取代芳香族氨基酸；和用疏水性氨基酸取代亲水性氨基酸。
“缺失”指的是通过从参考多肽去除一个或更多个氨基酸的多肽修饰。缺失可以包括去除1个或更多个氨基酸、2个或更多个氨基酸、5个或更多个氨基酸、10个或更多个氨基酸、15个或更多个氨基酸或20个或更多个氨基酸、多达构成参考酶的氨基酸总数的10％或多达构成参考酶的氨基酸总数的20％，同时保留酶活性和/或保留工程化亚胺还原酶的改进的性质。缺失可以涉及多肽的内部和/或端部。在多个实施方案中，缺失可以包括连续的区段或可以是不连续的。
“插入”指的是通过从参考多肽添加一个或更多个氨基酸的多肽修饰。在一些实施方案中，改进的工程化亚胺还原酶包括将一个或更多个氨基酸插入到具有亚胺还原酶活性的天然存在的多肽中，以及将一个或更多个氨基酸插入到其他改进的亚胺还原酶多肽中。插入可以在多肽的内部或至羧基末端或氨基末端。如本文使用的插入包括如本领域已知的融合蛋白。插入可以是氨基酸的连续区段或由天然存在的多肽中的一个或更多个氨基酸分开。
如本文使用的“片段”指的是具有氨基末端和/或羧基末端缺失但其余氨基酸序列与序列中相应位置相同的多肽。片段可以是至少14个氨基酸长、至少20个氨基酸长、至少50个氨基酸长或更长、以及多达全长亚胺还原酶多肽例如SEQ ID NO:2的多肽或在SEQ IDNO:4至270的偶数序列中提供的工程化亚胺还原酶的70％、80％、90％、95％、98％和99％。
“分离的多肽”指的是如下多肽：所述多肽与其天然伴随的其他污染物例如蛋白、脂质和多核苷酸基本上分开。该术语包括已经从它们天然存在的环境或表达系统(例如，宿主细胞或体外合成)中取出或纯化的多肽。工程化亚胺还原酶可以存在于细胞内、存在于细胞培养基中，或以各种形式制备，诸如裂解物或分离的制品。因此，在一些实施方案中，工程化亚胺还原酶可以是分离的多肽。
“基本上纯的多肽”指的是其中多肽种类是存在的优势种类的组合物(即，在摩尔或重量基础上，它比在该组合物中的任何其他单独的大分子种类更丰富)，并且当目标种类构成存在的大分子种类的按摩尔或％重量计的至少约50％时，通常为基本上纯的组合物。通常，基本上纯的亚胺还原酶组合物将包含组合物中存在的所有大分子种类的按摩尔或％重量计的约60％或更多、约70％或更多、约80％或更多、约90％或更多、约95％或更多和约98％或更多。在一些实施方案中，目标种类被纯化至基本均质(即，通过常规检测方法不能在组合物中检测出污染物种类)，其中组合物基本上由单个大分子种类组成。溶剂种类、小分子(<500道尔顿)和元素离子种类不被认为是大分子种类。在一些实施方案中，分离的工程化亚胺还原酶多肽是基本上纯的多肽组合物。
“立体选择性”指的是在化学或酶促反应中一种立体异构体相对于另一种立体异构体的优先形成。立体选择性可以是部分的，其中一种立体异构体的形成优于其他种，或者其可以是完全的，其中仅形成一种立体异构体。当立体异构体是对映异构体时，立体选择性被称为对映选择性，即一种对映异构体在两者的总和中的分数(典型地以百分比报告)。可选择地，本领域通常报告其为根据下式从中计算的对映异构体过量(e.e.)(典型地为百分比)：[主要对映异构体-次要对映异构体]/[主要对映异构体+次要对映异构体]。当立体异构体是非对映异构体时，立体选择性被称为非对映选择性，即一种非对映异构体在两种非对映异构体的混合物中的分数(典型地以百分比报告)，通常可选择地报告为非对映异构体过量(d.e.)。对映异构体过量和非对映异构体过量是立体异构体过量的类型。
“高度立体选择性”指的是能够以至少约85％立体异构体过量将一种或更多种底物(例如底物化合物(1e)和(2b))转化为相应的胺产物(例如化合物(3i))的化学反应或酶促反应。
如本文使用的，“改进的酶性质”指的是酶的至少一种改进的性质。在一些实施方案中，本发明提供了工程化亚胺还原酶多肽，其与参考亚胺还原酶多肽和/或野生型亚胺还原酶多肽和/或另一种工程化亚胺还原酶多肽相比呈现出任何酶性质的改进。对于本文描述的工程化亚胺还原酶多肽，比较通常针对亚胺还原酶由其衍生的野生型酶进行，尽管在一些实施方案中，参考酶可以是另一种改进的工程化亚胺还原酶。因此，“改进”的水平可以在各种亚胺还原酶多肽(包括野生型)以及工程化亚胺还原酶之间确定和比较。改进的性质包括但不限于这样的性质如酶活性(其可以用底物转化的百分比表示)、热稳定性、溶剂稳定性、pH活性谱、辅因子需求、对抑制剂的耐性(refractoriness)(例如底物或产物抑制)、立体专一性和立体选择性(包括对映选择性)。
“增加的酶活性”指的是工程化亚胺还原酶多肽的改进性质，其可以通过与参考亚胺还原酶相比，比活性(例如，产生的产物/时间/重量蛋白)的增加或将底物转化为产物的百分比(例如使用指定量的亚胺还原酶，在指定时间段内将起始量的底物转化为产物的转化百分比)的增加来表示。确定酶活性的示例性方法被提供在实施例中。可以影响与酶活性相关的任何性质，包括经典酶性质Km、Vmax或kcat，它们的改变可以导致增加的酶活性。酶活性的改进可以从相应的野生型酶的约1.2倍的酶活性至亚胺还原酶多肽由其衍生的天然存在的或另一种工程化亚胺还原酶活性的多达2倍、5倍、10倍、20倍、25倍、50倍或更多。亚胺还原酶活性可以通过任何一种标准测定来测量，诸如通过监测底物、辅因子或产物的性质的变化。在一些实施方案中，产生的产物的量可以通过液相色谱-质谱(LC-MS)测量。酶活性的比较使用定义的酶制品、在设定条件下使用定义的测定和一种或更多种限定的底物进行，如本文进一步详细描述的。通常，当比较裂解物时，确定细胞数目和测定的蛋白的量，并且使用相同的表达系统和相同的宿主细胞来使由宿主细胞产生并存在于裂解物中的酶的量的变化最小化。
“转化”指的是将底物酶促转化为相应的产物。“转化百分比”指的是在指定的条件下在一定时间段内被转化为产物的底物的百分比。因此，亚胺还原酶多肽的“酶活性”或“活性”可以表示为底物向产物的“转化百分比”。
“热稳定”指的是与暴露于高温(例如，40℃-80℃)的野生型酶相比，在暴露于相同的高温持续一定时间段(例如，0.5小时-24小时)后，保持相似活性(例如超过60％至80％)的亚胺还原酶多肽。
“溶剂稳定”指的是与暴露于不同浓度(例如，5％-99％)的溶剂(乙醇、异丙醇、二甲基亚砜(DMSO)、四氢呋喃、2-甲基四氢呋喃、丙酮、甲苯，乙酸丁酯、甲基叔丁基醚等)的野生型酶相比，在暴露于相同浓度的相同溶剂持续一定时间段(例如，0.5小时-24小时)后，保持相似活性(超过例如60％至80％)的亚胺还原酶多肽。
“热稳定且溶剂稳定”指的是既热稳定也溶剂稳定的亚胺还原酶多肽。
术语“严格杂交条件”在本文中用于指核酸杂合体稳定的条件。如本领域技术人员已知的，杂合体的稳定性反映在杂合体的解链温度(Tm)。通常，杂合体的稳定性是离子强度、温度、G/C含量和离液剂的存在的函数。多核苷酸的Tm值可以使用用于预测解链温度的已知方法来计算(参见例如，Baldino等人,Meth.Enzymol.,168:761-777[1989]；Bolton等人,Proc.Natl.Acad.Sci.USA 48:1390[1962]；Bresslauer等人,Proc.Natl.Acad.Sci.USA83:8893-8897[1986]；Freier等人,Proc.Natl.Acad.Sci.USA 83:9373-9377[1986]；Kierzek等人,Biochem.,25:7840-7846[1986]；Rychlik等人,1990,Nucl.Acids Res.,18:6409-6412[1990](勘误,Nucl.Acids Res.,19:698[1991])；Sambrook等人,同上)；Suggs等人,1981,于Developmental Biology Using Purified Genes,Brown等人.[Eds.],第683-693页,Academic Press,Cambridge,MA[1981]；和Wetmur,Crit.Rev.Biochem.Mol.Biol.,26:227-259[1991])。在一些实施方案中，多核苷酸编码本文公开的多肽，并且在限定的条件下，诸如中度严格或高度严格的条件下，与编码本发明的工程化亚胺还原酶的序列的互补体杂交。
“杂交严格度”指的是在核酸杂交中的杂交条件，诸如洗涤条件。通常，杂交反应在较低严格度的条件下进行，随后是不同的但较高严格度的洗涤。术语“中度严格杂交”指的是允许靶DNA结合以下互补的核酸的条件，所述互补的核酸与靶DNA具有约60％同一性，优选地约75％同一性，约85％同一性，与靶多核苷酸具有大于约90％同一性。示例性中度严格条件是等同于在42℃在50％甲酰胺、5×Denhart溶液、5×SSPE、0.2％SDS中杂交，随后是在42℃在0.2×SSPE、0.2％SDS中洗涤的条件。“高严格度杂交”通常指的是距离如在针对所定义的多核苷酸序列的溶液条件下确定的热解链温度Tm约10℃或更少的条件。在一些实施方案中，高严格度条件指的是仅允许在65℃在0.018M NaCl中形成稳定杂合体的那些核酸序列杂交的条件(即如果杂合体在65℃、在0.018M NaCl中不稳定，它将不会在高严格条件下稳定，如本文预期的)。可以例如通过在等同于在42℃、50％甲酰胺、5×Denhart溶液、5×SSPE、0.2％SDS的条件中杂交，然后在65℃在0.1×SSPE和0.1％SDS中洗涤来提供高严格度条件。另一个高严格度条件是在等同于在65℃、在含有0.1％(w:v)SDS的5X SSC中杂交，并且在65℃在含有0.1％SDS的0.1×SSC中洗涤的条件中洗涤。其他高严格度杂交条件以及中等严格条件在上文引用的参考文献中被描述。
“异源”多核苷酸指的是通过实验室技术被引入宿主细胞的任何多核苷酸，并且包括从宿主细胞中去除、经历实验室操作并且然后重新引入宿主细胞的多核苷酸。
“密码子优化”指的是编码蛋白的多核苷酸的密码子改变为在特定生物体中优先使用的那些密码子，使得编码的蛋白在感兴趣的生物体中有效地表达。虽然因为大多数氨基酸由几个被称为“同义(synonym)”或“同义(synonymous)”密码子的密码子表示，遗传密码是简并的，但熟知的是特定生物体的密码子使用是非随机的，并且偏好于特定的密码子三联体。这种密码子使用偏好在给定基因、共同功能或祖先来源的基因、相比于低拷贝数蛋白的高表达蛋白和生物体基因组的聚集蛋白编码区中可以更高。在一些实施方案中，编码亚胺还原酶的多核苷酸可以被密码子优化用于从选择用于表达的宿主生物体的最佳生产。
如本文使用的，“优选的、最佳的、高密码子使用偏好密码子”可以互换地指的是在蛋白编码区中的使用频率高于编码相同氨基酸的其他密码子的密码子。优选的密码子可以根据单个基因、共同功能或来源的一组基因、高表达基因中的密码子使用、整个生物体的聚集蛋白编码区中的密码子频率、相关生物体的聚集蛋白编码区中的密码子频率或其组合来确定。其频率随着基因表达的水平而增加的密码子典型地是用于表达的最佳密码子。用于确定特定生物体中密码子的频率(例如密码子使用、相对同义密码子使用)和密码子偏好的多种方法是已知的，包括多变量分析，例如使用聚类分析或相关性分析和基因中使用的密码子的有效数目(参见例如，GCG CodonPreference,Genetics Computer Group WisconsinPackage；CodonW,Peden,University of Nottingham；McInerney,Bioinform.,14:372-73[1998]；Stenico等人,Nucl.Acids Res.,222437-46[1994]；Wright,Gene 87:23-29[1990])。对于许多不同的生物体，密码子使用表是可获得的(参见例如，Wada等人,Nucl.Acids Res.,20:2111-2118[1992]；Nakamura等人,Nucl.Acids Res.,28:292[2000]；Duret,等人,同上；Henaut和Danchin,于Escherichia coli and Salmonella,Neidhardt,等人(编著),ASM Press,Washington D.C.,第2047-2066页[1996])。用于获得密码子使用的数据源可以依赖于能够编码蛋白的任何可获得的核苷酸序列。这些数据集包括实际已知编码表达蛋白(例如完整的蛋白编码序列-CDS)、表达序列标签(ESTS)或基因组序列的预测编码区域的核酸序列(参见例如Mount,Bioinformatics:Sequence and Genome Analysis,第8章,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.[2001]；Uberbacher,Meth.Enzymol.,266:259-281[1996]；和Tiwari等人,Comput.Appl.Biosci.,13:263-270[1997])。
“控制序列”在本文中被定义为包括对本发明的多核苷酸和/或多肽的表达是必需或有利的所有组分。每个控制序列可以是对于编码多肽的核酸序列天然的或外来的。这样的控制序列包括但不限于前导序列、多腺苷酸化序列、前体肽序列、启动子、信号肽序列和转录终止子。在最小程度上，控制序列包括启动子和转录及翻译终止信号。控制序列可以设有接头，目的是引入促进控制序列与编码多肽的核酸序列的编码区连接的特定限制性位点。
“可操作地连接”在本文被定义为如下配置：在所述配置中控制序列被适当地放置(即，以功能关系)在相对于感兴趣的多核苷酸的位置处，使得控制序列指导或调节感兴趣的多核苷酸和/或多肽的表达。
“启动子序列”指的是被宿主细胞识别用于感兴趣的多核苷酸诸如编码序列的表达的核酸序列。启动子序列包含介导感兴趣的多核苷酸的表达的转录控制序列。启动子可以是在选择的宿主细胞中示出转录活性的任何核酸序列，包括突变体、截短的和杂合启动子，并且可以从编码与宿主细胞同源或异源的细胞外多肽或细胞内多肽的基因获得。
“合适的反应条件”指的是生物催化反应溶液中的那些条件(例如酶载量的范围、底物载量、辅因子载量、温度、pH、缓冲液、共溶剂等)，在此条件下，本发明的亚胺还原酶多肽能够将底物化合物转化为产物化合物(例如，将化合物(2)转化为化合物(1))。在本发明中提供了并通过实施例说明了示例性的“合适的反应条件”。
“辅因子再生系统”或“辅因子循环系统”指的是参与还原辅因子的氧化形式的反应(例如，NADP+至NADPH)的一组反应物。被亚胺还原酶催化的酮底物的还原胺化反应氧化的辅因子通过辅因子再生系统以还原形式再生。辅因子再生系统包括化学计量的还原剂，其是还原氢等同物的来源并且能够还原辅因子的氧化形式。辅因子再生系统还可以包含催化剂，例如催化由还原剂还原辅因子的氧化形式的酶催化剂。分别从NAD+或NADP+再生NADH或NADPH的辅因子再生系统是本领域已知的，并且可以在本文描述的方法中使用。
“甲酸脱氢酶”和“FDH”在本文中可互换使用，以分别指催化甲酸盐和NAD+或NADP+转化为二氧化碳和NADH或NADPH的NAD+或NADP+依赖性酶。
诸如“化合物载量”或“酶载量”或“辅因子载量”中的“载量”指的是在反应开始时反应混合物中组分的浓度或量。
在生物催化剂介导的过程的上下文中，“底物”指的是生物催化剂所作用的化合物或分子。例如，在本文公开的还原胺化方法中使用的亚胺还原酶生物催化剂中，存在式(I)的酮(或醛)底物诸如环己酮，和式(II)的胺底物诸如丁胺。
在生物催化剂介导的过程的上下文中，“产物”指的是由生物催化剂的作用所产生的化合物或分子。例如，在本文公开的方法中使用的亚胺还原酶生物催化剂的示例性产物是仲胺或叔胺化合物，诸如式(III)的化合物。
“烷基”指的是具有从1个至18个碳原子(包括端点)的，直链的或支链的，更优选地从1个至8个碳原子(包括端点)，并且最优选地1个至6个碳原子(包括端点)的饱和烃基团。具有指定数目的碳原子的烷基在括号中表示(例如(C1-C6)烷基指的是1个至6个碳原子的烷基)。
“烯基”指的是具有从2个至12个碳原子(包括端点)的、直链或支链的、含有至少一个双键但任选地含有多于一个双键的烃基团。
“炔基”指的是具有从2个至12个碳原子(包括端点)的、直链或支链的、含有至少一个三键但任选地含有多于一个三键，并且另外任选地含有一个或更多个双键键合部分的烃基团。
“亚烷基”指的是具有从1个至18个碳原子(包括端点)，更优选地从1个至8个碳原子(包括端点)，并且最优选地1个至6个碳原子(包括端点)的，任选地被一个或更多个合适的取代基取代的直链或支链的二价烃基团。示例性的“亚烷基”包括但不限于亚甲基、亚乙基、亚丙基、亚丁基及类似基团。
“亚烯基”指的是具有2个至12个碳原子(包括端点)和一个或更多个碳-碳双键，更优选地从2个至8个碳原子(包括端点)，并且最优选地2个至6个碳原子(包括端点)的，任选地被一个或更多个合适的取代基取代的直链或支链的二价烃基团。
“杂烷基”、“杂烯基”和“杂炔基”分别指的是其中一个或更多个碳原子各自独立地被相同或不同的杂原子或杂原子基团替代的如本文定义的烷基、烯基和炔基。可以替代碳原子的杂原子和/或杂原子基团包括但不限于-O-、-S-、-S-O-、-NRγ-、-PH-、-S(O)-、-S(O)2-、-S(O)NRγ-、-S(O)2NRγ-及类似物，包括其组合，其中每个Rγ独立地选自氢、烷基、环烷基、杂环烷基、芳基和杂芳基。
“芳基”指的是具有单环(例如苯基)或多个稠环(例如萘基或蒽基)的具有从6个至12个碳原子(包括端点)的不饱和的芳香族碳环基团。示例性的芳基包括苯基、吡啶基、萘基及类似基团。
“芳基烷基”指的是被芳基取代的烷基(即芳基-烷基-基团)，优选地在烷基部分中具有从1个至6个碳原子(包括端点)且在芳基部分中具有从6个至12个碳原子(包括端点)。这样的芳基烷基基团由苄基、苯乙基及类似基团例示。
“芳氧基”指的是–ORλ基团，其中Rλ是可以任选地被取代的芳基基团。
“环烷基”指的是具有从3个至12个碳原子(包括端点)的、具有单个环状环或多个稠环的环状烷基基团，其可以任选地被从1个至3个烷基基团取代。示例性的环烷基基团包括但不限于单环结构和多环结构，所述单环结构诸如环丙基、环丁基、环戊基、环辛基、1-甲基环丙基、2-甲基环戊基、2-甲基环辛基及类似基团，所述多环结构包括桥接的环体系诸如金刚烷基及类似基团。
“环烷基烷基”指的是被环烷基取代的烷基(即环烷基-烷基-基团)，优选地在烷基部分中具有从1个至6个碳原子(包括端点)并且在环烷基部分中具有从3个至12个碳原子(包括端点)。这样的环烷基烷基基团由环丙基甲基、环己基乙基及类似基团例示。
“氨基”指的是基团-NH2。被取代的氨基指的是基团–NHRη、NRηRη和NRηRηRη，其中每个Rη独立地选自被取代的或未被取代的烷基、环烷基、环杂烷基、烷氧基、芳基、杂芳基、杂芳基烷基、酰基、烷氧基羰基、硫烷基、亚磺酰基、磺酰基及类似基团。典型的氨基基团包括但不限于二甲基氨基、二乙基氨基、三甲基铵、三乙基铵、甲基磺酰基氨基、呋喃基-氧基-磺氨基及类似基团。
“氨基烷基”指的是其中一个或更多个氢原子被一个或更多个氨基基团，包括被取代的氨基基团替代的烷基基团。
“氨基羰基”指的是-C(O)NH2。被取代的氨基羰基指的是–C(O)NRηRη，其中氨基基团NRηRη如本文定义的。
“氧基”指的是二价基团-O-，其可以具有各种取代基以形成不同的氧基基团，包括醚和酯。
“烷氧基”或“烷基氧基”在本文中可互换使用以指的是基团–ORζ，其中Rζ是烷基基团，包括任选地被取代的烷基基团。
“羧基”指的是-COOH。
“羰基”指的是-C(O)-，其可以具有多种取代基以形成不同的羰基基团，包括酸、酸性卤化物、醛、酰胺、酯和酮。
“羧基烷基”指的是其中一个或更多个氢原子被一个或更多个羧基基团替代的烷基。
“氨基羰基烷基”指的是被如本文定义的氨基羰基基团取代的烷基。
“卤素(halogen)”或“卤代(halo)”指的是氟、氯、溴和碘。
“卤代烷基”指的是其中一个或更多个氢原子被卤素替代的烷基基团。因此，术语“卤代烷基”意指包括单卤代烷基、二卤代烷基、三卤代烷基等直到全卤代烷基。例如，表述“(C1-C2)卤代烷基”包括1-氟甲基、二氟甲基、三氟甲基、1-氟乙基、1,1-二氟乙基、1,2-二氟乙基、1,1,1-三氟乙基、全氟乙基等。
“羟基”指的是-OH。
“羟基烷基”指的是其中一个或更多个氢原子被一个或更多个羟基基团替代的烷基基团。
“硫基(thio)”或“硫烷基”指的是-SH。被取代的硫基或硫烷基指的是–S-Rη，其中Rη是烷基、芳基或其他合适的取代基。
“烷硫基”指的是–SRζ，其中Rζ是可以任选地被取代的烷基。典型的烷硫基基团包括但不限于甲硫基、乙硫基、正丙硫基及类似基团。
“烷硫基烷基”指的是被烷硫基基团–SRζ取代的烷基，其中Rζ是可以任选地被取代的烷基。
“磺酰基”指的是-SO2-。被取代的磺酰基指的是–SO2-Rη，其中Rη是烷基、芳基或其他合适的取代基。
“烷基磺酰基”指的是–SO2-Rζ，其中Rζ是可以任选地被取代的烷基。典型的烷基磺酰基基团包括但不限于甲基磺酰基、乙基磺酰基、正丙基磺酰基及类似基团。
“烷基磺酰基烷基”指的是被烷基磺酰基基团–SO2-Rζ取代的烷基，其中Rζ是可以任选地被取代的烷基。
“杂芳基”指的是具有从1个至10个碳原子(包括端点)和在环内的选自氧、氮和硫的1个至4个杂原子(包括端点)的芳香族杂环基团。这样的杂芳基基团可以具有单环(例如吡啶基或呋喃基)或多个稠环(例如吲嗪基(indolizinyl)或苯并噻吩基)。
“杂芳基烷基”指的是被杂芳基取代的烷基(即杂芳基-烷基-基团)，优选地在烷基部分中具有从1个至6个碳原子(包括端点)并且在杂芳基部分中具有从5个至12个环原子(包括端点)。这样的杂芳基烷基基团由吡啶基甲基及类似基团例示。
“杂环”、“杂环的”和可互换的“杂环烷基”指的是具有单环或多个稠环的、具有从2个至10个碳环原子(包括端点)和在环内的选自氮、硫或氧的从1个至4个杂环原子(包括端点)的饱和的或不饱和的基团。这样的杂环基团可以具有单环(例如哌啶基或四氢呋喃基)或多个稠环(例如，二氢吲哚基、二氢苯并呋喃或奎宁环基(quinuclidinyl))。杂环的实例包括但不限于呋喃、噻吩、噻唑、噁唑、吡咯、咪唑、吡唑、吡啶、吡嗪、嘧啶、哒嗪、吲嗪、异吲哚、吲哚、吲唑、嘌呤、喹嗪(quinolizine)、异喹啉、喹啉、酞嗪(phthalazine)、萘基吡啶、喹喔啉、喹唑啉、噌啉、蝶啶、咔唑(carbazole)、咔啉(carboline)、菲啶(phenanthridine)、吖啶、菲咯啉(phenanthroline)、异噻唑、吩嗪(phenazine)、异噁唑、酚噁嗪(phenoxazine)、吩噻嗪(phenothiazine)、咪唑烷(imidazolidine)、咪唑啉(imidazoline)、哌啶、哌嗪、吡咯烷、二氢吲哚(indoline)及类似基团。
“杂环烷基烷基”指的是被杂环烷基取代的烷基(即杂环烷基-烷基-基团)，优选地在烷基部分中具有从1个至6个碳原子(包括端点)，并且在杂环烷基部分中具有从3个至12个环原子(包括端点)。
“元环(membered ring)”意指包括任何环状结构。术语“元”之前的数字表示构成环的骨架原子的数目。因此，例如环己基、吡啶、吡喃和噻喃是6元环，并且环戊基、吡咯、呋喃和噻吩是5元环。
如本文使用的“稠合双环(Fused bicyclic ring)”指的是在每个环中具有5个至8个原子的未被取代的和被取代的碳环和/或杂环的环部分，所述环具有2个共用原子。
在本文中关于上述化学基团使用的“任选地被取代的”意指被氢占据的化学基团的位置可以被另一个原子(除非另外说明)例如但不限于碳、氧、氮或硫或例如但不限于以下的化学基团取代：羟基、氧代、硝基、甲氧基、乙氧基、烷氧基、被取代的烷氧基、三氟甲氧基、卤代烷氧基、氟、氯、溴、碘、卤素、甲基、乙基、丙基、丁基、烷基、烯基、炔基、被取代的烷基、三氟甲基、卤代烷基、羟基烷基、烷氧基烷基、硫基、烷硫基、酰基、羧基、烷氧基羰基、甲酰氨基、被取代的甲酰氨基、烷基磺酰基、烷基亚磺酰基、烷基磺酰基氨基、磺酰氨基、被取代的磺酰氨基、氰基、氨基、被取代的氨基、烷基氨基、二烷基氨基、氨基烷基、酰基氨基、脒基、脒肟基(amidoximo)、羟基甲酰基(hydroxamoyl)、苯基、芳基、被取代的芳基、芳氧基、芳基烷基、芳基烯基、芳基炔基、吡啶基、咪唑基、杂芳基、被取代的杂芳基、杂芳氧基、杂芳基烷基、杂芳基烯基、杂芳基炔基、环丙基、环丁基、环戊基、环己基、环烷基、环烯基、环烷基烷基、被取代的环烷基、环烷基氧基、吡咯烷基、哌啶基、吗啉代、杂环基、(杂环)氧基和(杂环)烷基；其中优选的杂原子是氧、氮和硫。另外，在这些取代化学基团上存在开放化合价的情况下，它们可以进一步被烷基、环烷基、芳基、杂芳基和/或杂环基团取代，在这些开放化合价存在于碳上的情况下，它们可以进一步被卤素和被氧-、氮-、或硫-键合的取代基取代，并且在存在多个这样的开放化合价的情况下，这些基团可以通过直接形成键或通过与新的杂原子(优选地氧、氮或硫)形成键而连接以形成环。还预期，可以进行上文的取代，条件是用取代基替代氢不会对本发明的分子带来不可接受的不稳定性，并且另外在化学上是合理的。本领域普通技术人员将理解，对于被描述为任选地被取代的任何化学基团，仅意图包括空间上可实现的和/或合成上可行的化学基团。如本文使用的“任选地被取代的”指的是术语或一系列化学基团中的所有后续修饰对象(modifier)。例如，在术语“任选地被取代的芳基烷基”中，分子的“烷基”部分和“芳基”部分可以被取代或可以不被取代，并且对于一系列“任选地被取代的烷基、环烷基、芳基和杂芳基”，烷基、环烷基、芳基和杂芳基基团彼此独立地可以被取代或可以不被取代。
肟中间体形成和亚胺向仲胺的转化
肟中间体的形成是产生IRED的起始底物的有效方式，然后该底物可以以高立体选择性还原化合物，产生具有大于95％对映异构体过量的产物，如方案1中示出的。该方法的挑战是鉴定与期望的底物反应的酶，并且鉴定增加酶效率的突变。
方案1
本发明提供了新型生物催化剂和使用它们通过亚胺底物的直接还原来合成手性仲胺的相关方法。本公开内容的生物催化剂是来自Pseudonocardia spinosispora的野生型基因的工程化多肽变体，该野生型基因编码具有SEQ ID NO:2的氨基酸序列的亚胺还原酶“或“IRED”。这些工程化多肽能够催化亚胺转化为仲胺。IRED的一般亚胺还原酶活性如下文的方案2所示。
方案2
本发明的具有亚胺还原酶活性的工程化多肽可以接受宽范围的底物。因此，在方案2的生物催化反应中，底物的R1基团选自氢原子或任选地被取代的烷基、烯基、炔基、烷氧基、芳基烷氧基、羟基烷基、羧基、氨基羰基、杂烷基、杂烯基、杂炔基、羧基烷基、氨基烷基、烷基氨基、卤代烷基、烷硫基烷基、环烷基、芳基、芳基烷基、杂环烷基、杂芳基和杂芳基烷基；并且底物的R2和R3基团独立地选自氢原子和任选地被取代的烷基、烯基、炔基、羟基烷基、羧基、氨基羰基、杂烷基、杂烯基、杂炔基、羧基烷基、烷基氨基、卤代烷基、烷硫基烷基、环烷基、芳基、芳基烷基、杂环烷基、杂芳基和杂芳基烷基，条件是R2和R3不能都是氢。任选地，底物的R1基团可以与R2和R3基团的任一基团连接以形成3元至10元环。类似地，底物的R2和R3基团可以连接以形成3元至10元环。
如本文进一步描述的，具有亚胺还原酶活性的工程化多肽呈现出立体选择性，因此，方案2的亚胺还原酶反应可以用于在单一生物催化反应中建立产物的一个、两个或更多个手性中心。
在一些实施方案中，本发明提供了工程化多肽，其包含与SEQ ID NO:2的氨基酸参考序列具有至少80％序列同一性的氨基酸序列，并且还包含与参考氨基序列相比的一个或更多个氨基酸残基差异，其中工程化多肽具有亚胺还原酶活性。在工程化多肽的一些实施方案中，亚胺还原酶活性是方案2的活性。
工程化亚胺还原酶多肽
本发明提供了具有亚胺还原酶活性的多肽、编码该多肽的多核苷酸、制备该多肽的方法以及用于使用该多肽的方法。在描述涉及多肽的情况下，应理解，它可以描述编码多肽的多核苷酸。
工程化多肽的上文描述的改进的性质进行期望的反应的合适的反应条件可以根据多肽、底物、共底物的浓度或量、缓冲液、溶剂、pH，包括温度和反应时间的条件和/或多肽固定于固体支持物上的条件确定，如下文和实施例中进一步描述的。
在一些实施方案中，示例性的具有改进的性质的亚胺还原酶活性(特别地在化合物[1]转化为化合物[2]中)的工程化多肽，包含与SEQ ID NO:2相比在表2-2所示的残基位置处具有一个或更多个残基差异的氨基酸序列。
在一些实施方案中，示例性的具有改进的性质的亚胺还原酶活性(特别地在化合物[1]转化为化合物[2]中)的工程化多肽，包含与SEQ ID NO:270相比在表3-1所示的残基位置处具有一个或更多个残基差异的氨基酸序列。
在一些实施方案中，示例性的具有改进的性质的亚胺还原酶活性(特别地在化合物[1]转化为化合物[2]中)的工程化多肽，包含与SEQ ID NO:230相比在表4-1所示的残基位置处具有一个或更多个残基差异的氨基酸序列。
关于本发明的示例性的非天然存在的(或工程化)多肽的结构和功能信息基于化合物[1]转化向化合物[2]的转换，其结果在下表2-2、表3-1、表4-1和表5-1中示出，并且在实施例中进一步描述。这些表中的奇数序列标识符(即SEQ ID NO)指的是编码由这些表中的偶数SEQ ID NO所提供的氨基酸序列的核苷酸序列。示例性序列被提供在本发明随附的电子序列表文件中，其在此通过引用并入本文。氨基酸残基差异是基于与参考序列SEQ IDNO:2、230和270的比较，如所示的。
Pseudonocardia spinosispora亚胺还原酶的天然存在的氨基酸序列(SEQ IDNO:1)被密码子优化以用于在大肠杆菌(Escherichia coli)中表达并合成(多肽序列被提供在SEQ ID NO:2中)。
每种工程化多肽相对于SEQ ID NO:2、230或270的参考多肽的活性作为本文实施例中描述的底物的转化来确定。在一些实施方案中，摇瓶粉末(SFP)被用作二次筛选，以评估工程化亚胺还原酶的性质，其结果在实施例中提供。在一些实施方案中，SFP形式提供了工程化多肽的更纯的粉末制品，并且可以包含多达总蛋白的约30％的工程化多肽。
在一些实施方案中，特定的酶性质与在本文所示的残基位置处与SEQ ID NO:2、230和270相比的残基差异相关。在一些实施方案中，影响多肽表达的残基差异可以用于增加工程化亚胺还原酶的表达。
鉴于本文提供的指导，还预期包含SEQ ID NO:4-270的偶数序列的示例性工程化多肽中的任一个通过例如随后轮次的演化可用作用于合成其他工程化亚胺还原酶多肽的起始氨基酸序列，所述演化并入来自表2-2、表3-1、表4-1和表5-1中的其他多肽的多种氨基酸差异和本文描述的其他残基位置的新的组合。另外的改进可以通过在较早轮次的演化中维持未改变的残基位置处包含氨基酸差异来生成。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:2具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性的且与SEQ ID NO:2相比在选自以下的残基位置处具有一个或更多个残基差异的氨基酸序列：57/211、87/175、103/212、122、123、124/178、124、132、134、169、171、173、175、176、178/242、178、211、212、213、214、215、217、234和235。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:2具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性且与SEQ ID NO:2相比具有选自以下的一个或更多个残基差异的氨基酸序列：57T/211L、87S/175G、103H/212C、122E、123G、124A/178I、124G、132H/R、134R、169N/S、171W、173H/I、175G/H、176S、178I/242Y、178M、211L/W、212C/G/V、213P/R/T/V、214M、215L/M、217I/L、234L和235K。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:2具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性的且与SEQ ID NO:2相比在选自以下的残基位置处的一个或更多个残基差异的氨基酸序列：A57T/I211L、P87S/M175G、R103H/E212C、L122E、T123G、I124A/V178I、I124G、E132H/R、T134R、A169N/S、Y171W、A173H/I、M175G/H、L176S、V178I/A242Y、V178M、I211L/W、E212C/G/V、A213P/R/T/V、V214M、T215L/M、W217I/L、D234L和A235K。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性且与SEQ ID NO:270相比具有选自以下的一个或更多个残基差异的氨基酸序列：123/124/169/171/173/213/235、123/124/169/212/213/235、123/169/171/173/212/213/235、123/169/171/173/213/235、123/169/173/175/213/235、123/169/173/212/213、123/169/175、123/169/175/212/235、123/169/212/213、123/173/175/212/213/235、123/212/213/235、123/212/235、123/169/171/173/175/212/213、123/169/173、123/169/212/213/235、123/213、124/169、124/169/173/175/212/213、124/169/173/212/213、124/169/173/212/213/235、124/169/175/212/213/235、124/169/212/213/235、124/169/213/235、124/175/212/213/235、124/212/213/235、134/169/176/178/211/214、134/169/176/178/211、134/169/176/178/211/217、134/169/176/211/215/217/234、134/169/176/211、134/169/178/211/214/217、134/169/211/214/217、134/169/211/217、134/176/211/214、134/176/211/215、134/211/214、169/171/173/213/235、69/173/175/213/235、169/175/212/213/235、169/176/178/211/214、169/176/178/211/214/217、169/176/178/211/217、169/176/178、169/176/178/211/214/217、169/176/211/214、169/176/211/214/215/234、169/176/211/214/217、169/176/211/217、169/176/214/215/217、169/178/217、169/178/234、169/211/214/215、169/211/217、169/212/213、169/212/235、169/212/213/235、169/235、171/173/212/213/235、173/175/213、175/213/235、175/235、176/178/211/215/217、176/178/211/217、176/211/214、176/214/217、178/211/214、178/211/217、178/215、211/234、212/213/235和213/235。在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ ID NO:270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性且与SEQ ID NO:270相比具有选自以下的一个或更多个残基差异的氨基酸序列：123G/124G/169N/171V/173H/213V/235K、123G/124G/169N/212V/213V/235K、123G/169N/171V/173H/213V/235K、123G/169N/171V/173I/212C/213V/235K、123G/169N/173H/175H/213V/235K、123G/169N/173I/212G/213T、123G/169N/175G、123G/169N/175G/212C/235K、123G/169N/212G/213V、123G/173H/175H/212C/213V/235K、123G/212C/213V/235K、123G/212G/235K、123G/169N/171V/173H/175H/212X/213V、123G/169N/173I、123G/169N/212G/213V/235K、123G/213T、124G/169N、124A/169N/173I/175H/212G/213V、124G/169N/173I/212C/213V、124G/169N/173I/212G/213V、124G/169N/173I/212V/213V/235K、124G/169N/175G/212C/213V/235K、124G/169N/212G/213V/235K、124G/169N/212V/213V/235K、124G/175G/212V/213V/235K、124G/169N/213V/235K、124G/212C/213V/235K、124G/212G/213V/235K、134R/169N/176S/178I/211L/214M、134R/169N/176S/178I/211W、134R/169N/176S/178M/211L/217L、134R/169N/176S/211L/215M/217I/234L、134R/169N/176S/211W、134R/169N/178M/211W/214M/217I、134R/169N/211W/214M/217L、134R/169N/211W/217L、134R/176S/211W/214M、134R/176S/211W/215M、134R/211W/214M、169N/171V/173I/213V/235K、169N/173I/175H/213V/235K、169N/175G/212C/213V/235K、169N/M175H/E212G/A213V/A235K、169N/176S/178I/211L/214M、169N/176S/178I/211W/214M/217L、169N/176S/178I/211W/217L、169N/176S/178M、169N/176S/178M/211W/214M/217I、169N/176S/211W/214M、169N/176S/211W/214M/215L/234L、169N/176S/211W/217I、169N/176S/211W/217L、169N/176S/214M/215M/217I、169N/176S/211L/214M/217I、169N/178I/217I、169N/178M/234L、169N/211W/214M/215L、169N/211W/217I、169N/212C/235K、169N/212C/213V/235K、169N/212G/213V、169N/212V/213V/235K、169N/235K、171V/173I/212G/213V/235K、173I/175H/213V、175H/213P/235K、175H/235K、176S/178I/211W/215L/217L、176S/178I/211W/217I、176S/211L/214M、176S/214M/217L、178M/211L/214M、178M/211W/217L、178I/215M、211W/234L、212G/213V/235K、212V/213V/235K、213T/235K和213V/235K。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性且与SEQ ID NO:270相比具有选自以下的一个或更多个残基差异的氨基酸序列：T123G/I124G/A169N/Y171V/A173H/A213V/A235K、T123G/I124G/A169N/E212V/A213V/A235K、T123G/A169N/Y171V/A173H/A213V/A235K、T123G/A169N/Y171V/A173I/E212C/A213V/A235K、T123G/A169N/A173H/M175H/A213V/A235K、T123G/A169N/A173I/E212G/A213T、T123G/A169N/M175G、T123G/A169N/M175G/E212C/A235K、T123G/A169N/E212G/A213V、T123G/A173H/M175H/E212C/A213V/A235K、T123G/E212C/A213V/A235K、T123G/E212G/A235K、T123G/A169N/Y171V/A173H/M175H/E212X/A213V、T123G/A169N/A173I、T123G/A169N/E212G/A213V/A235K、T123G/A213T、I124G/A169N、I124A/A169N/A173I/M175H/E212G/A213V、I124G/A169N/A173I/E212C/A213V、I124G/A169N/A173I/E212G/A213V、I124G/A169N/A173I/E212V/A213V/A235K、I124G/A169N/M175G/E212C/A213V/A235K、I124G/A169N/E212G/A213V/A235K、I124G/A169N/E212V/A213V/A235K、I124G/M175G/E212V/A213V/A235K、I124G/A169N/A213V/A235K、I124G/E212C/A213V/A235K、I124G/E212G/A213V/A235K、T134R/A169N/L176S/V178I/I211L/V214M、T134R/A169N/L176S/V178I/I211W、T134R/A169N/L176S/V178M/I211L/W217L、T134R/A169N/L176S/I211L/T215M/W217I/D234L、T134R/A169N/L176S/I211W、T134R/A169N/V178M/I211W/V214M/W217I、T134R/A169N/I211W/V214M/W217L、T134R/A169N/I211W/W217L、T134R/L176S/I211W/V214M、134R/L176S/I211W/T215M、T134R/I211W/V214M、A169N/Y171V/A173I/A213V/A235K、A169N/A173I/M175H/A213V/A235K、A169N/M175G/E212C/A213V/A235K、A169N/M175H/E212G/A213V/A235K、A169N/L176S/V178I/I211L/V214M、A169N/L176S/V178I/I211W/V214M/W217L、A169N/L176S/V178I/I211W/W217L、A169N/L176S/V178M、A169N/L176S/V178M/I211W/V214M/W217I、A169N/L176S/I211W/V214M、A169N/L176S/I211W/V214M/T215L/D234L、A169N/L176S/I211W/W217I、A169N/L176S/I211W/W217L、A169N/L176S/V214M/T215M/W217I、A169N/L176S/I211L/V214M/W217I、A169N/V178I/W217I、A169N/V178M/D234L、A169N/I211W/V214M/T215L、A169N/I211W/W217I、A169N/E212C/A235K、A169N/E212C/A213V/A235K、A169N/E212G/A213V、A169N/E212V/A213V/A235K、A169N/A235K、Y171V/A173I/E212G/A213V/A235K、A173I/M175H/A213V、M175H/A213P/A235K、M175H/A235K、L176S/V178I/I211W/T215L/W217L、L176S/V178I/I211W/W217I、L176S/I211L/V214M、L176S/V214M/W217L、V178M/I211L/V214M、V178M/I211W/W217L、V178I/T215M、I211W/D234L、E212G/A213V/A235K、E212V/A213V/A235K、A213T/A235K和A213V/A235K。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性且与SEQ ID NO:230相比具有选自以下的一个或更多个残基差异的氨基酸序列：45、127、130、134、174、206、209、212、216、223、227和279。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:230具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性且与SEQ ID NO:230相比选自以下的一个或更多个残基差异的氨基酸序列：45I、127R、130R、134L/R、174M、206L、209K/S、212H/K/R、216N/R/S、223N、227V和279V。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:230具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性且与SEQ ID NO:230相比具有选自以下的一个或更多个残基差异的氨基酸序列：V45I、G127R、T130R、T134L/R、A174M、M206L、Q209K/S、E212H/K/R、G216N/R/S、D223N、A227V和D279V。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含与参考序列SEQ IDNO:2具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性的氨基酸序列，并且包含SEQ ID NO:4和74之间的偶数序列。
在一些实施方案中，具有活性的工程化多肽包含与参考序列SEQ ID NO:270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性的氨基酸序列，并且包含SEQ ID NO:76和232之间的偶数序列。
在一些实施方案中，具有活性的工程化多肽包含与参考序列SEQ ID NO:230具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性的氨基酸序列，并且包含SEQ ID NO:234和270之间的偶数序列。
如本领域技术人员将理解的，在一些实施方案中，选择的以上残基差异之一或其组合可以作为核心特征在工程化亚胺还原酶中保持恒定(即维持)，并且在其他残基位置处的另外的残基差异可以被掺入到序列以产生具有改进性质的另外的工程化亚胺还原酶多肽。因此，应理解，对于含有以上残基差异的一个或子集的任何工程化亚胺还原酶，本发明预期了包含所述残基差异的一个或子集并且在本文公开的其他残基位置处另外包含一个或更多个残基差异的其他工程化亚胺还原酶。
如上文提到的，具有亚胺还原酶活性的工程化多肽也能够将底物(例如化合物[1])转化为产物(例如化合物[2])。在一些实施方案中，工程化亚胺还原酶多肽能够以相对于SEQ ID NO:2、230或270的参考多肽的活性至少1.2倍、1.5倍、2倍、3倍、4倍、5倍、10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍、100倍或更高的活性将底物转化为产物化合物。
在一些实施方案中，能够以相对于SEQ ID NO:2、230和/或270的活性至少2倍的活性将底物化合物转化为产物化合物的工程化亚胺还原酶多肽包含选自SEQ ID NO:4至270中的偶数序列的氨基酸序列。
在一些实施方案中，工程化亚胺还原酶具有包含与SEQ ID NO:2、230和/或270相比的一个或更多个残基差异的氨基酸序列，这增加了工程化亚胺还原酶活性在细菌宿主细胞中，特别地在大肠杆菌中的表达。
在一些实施方案中，具有改进的性质的工程化亚胺还原酶多肽具有包含选自SEQID NO:4至270范围内的偶数序列的序列的氨基酸序列。
在一些实施方案中，具有亚胺还原酶活性的工程化多肽包含这样的氨基酸序列：与SEQ ID NO:4-270范围内的偶数序列中的一个具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，且与SEQ ID NO:2、230和/或270相比在SEQ ID NO:4-270范围内的偶数序列中的任一个中存在的氨基酸残基差异，如实施例中提供的。
除了上文指定的残基位置之外，本文公开的任何工程化亚胺还原酶多肽还可以包含相对于SEQ ID NO:2、230和/或270在其他残基位置(即，除了包含在本文中的那些位置之外的残基位置)处的其他残基差异。这些其他残基位置处的残基差异可以提供氨基酸序列的另外的变化，而不会不利地影响多肽进行底物向产物的转化的能力。因此，在一些实施方案中，除了存在于选自SEQ ID NO:4-270范围内的偶数序列的工程化亚胺还原酶多肽的任一个中的氨基酸残基差异之外，该序列还可以包含与SEQ ID NO:2、229和/或270相比在其他氨基酸残基位置处的1个-2个、1个-3个、1个-4个、1个-5个、1个-6个、1个-7个、1个-8个、1个-9个、1个-10个、1个-11个、1个-12个、1个-14个、1个-15个、1个-16个、1个-18个、1个-20个、1个-22个、1个-24个、1个-26个、1个-30个、1个-35个、1个-40个、1个-45个或1个-50个残基差异。在一些实施方案中，与参考序列相比的氨基酸残基差异的数目可以是1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、30个、35个、40个、45个或50个残基位置。在一些实施方案中，与参考序列相比的氨基酸残基差异的数目可以是1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、18个、20个、21个、22个、23个、24个或25个残基位置。在这些其他位置处的残基差异可以是保守变化或非保守变化。在一些实施方案中，与SEQ ID NO:2、229和/或270的天然存在的亚胺还原酶多肽相比，残基差异可以包括保守取代和非保守取代。
在一些实施方案中，本发明还提供了包含保持本文描述的任何工程化亚胺还原酶的功能活性和/或改进的性质的该工程化亚胺还原酶多肽的片段的工程化多肽。因此，在一些实施方案中，本发明提供了在合适的反应条件下能够将底物转化为产物的多肽片段，其中该片段包含本发明的工程化亚胺还原酶多肽的全长氨基酸序列的至少约80％、90％、95％、96％、97％、98％或99％，本发明的工程化亚胺还原酶多肽诸如选自SEQ ID NO:4-270范围内的偶数序列的示例性工程化亚胺还原酶多肽。在一些实施方案中，工程化亚胺还原酶多肽可以具有包含本文描述的工程化亚胺还原酶多肽序列的任一个中的缺失的氨基酸序列，本文描述的工程化亚胺还原酶多肽诸如SEQ ID NO:4-270范围内的偶数序列的示例性工程化多肽。
因此，对于本发明的工程化亚胺还原酶多肽的各个和每一个实施方案，氨基酸序列可以包括1个或更多个氨基酸、2个或更多个氨基酸、3个或更多个氨基酸、4个或更多个氨基酸、5个或更多个氨基酸、6个或更多个氨基酸、8个或更多个氨基酸、10个或更多个氨基酸、15个或更多个氨基酸、或20个或更多个氨基酸、多达亚胺还原酶多肽的氨基酸总数的10％、多达氨基酸总数的20％、或多达氨基酸总数的30％的缺失，其中本文描述的工程化亚胺还原酶的相关的功能活性和/或改进的性质被保持。在一些实施方案中，缺失可以包括1个-2个、1个-3个、1个-4个、1个-5个、1个-6个、1个-7个、1个-8个、1个-9个、1个-10个、1个-15个、1个-20个、1个-21个、1个-22个、1个-23个、1个-24个、1个-25个、1个-30个、1个-35个、1个-40个、1个-45个或1个-50个氨基酸残基。在一些实施方案中，缺失的数目可以是1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、30个、35个、40个、45个或50个氨基酸残基。在一些实施方案中，缺失可以包括1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、18个、20个、21个、22个、23个、24个或25个氨基酸残基的缺失。
在一些实施方案中，本文的工程化亚胺还原酶多肽可以具有与本文描述的工程化亚胺还原酶多肽的任一个相比包含插入的氨基酸序列，本文描述的工程化亚胺还原酶多肽诸如SEQ ID NO:4-270范围内的偶数序列的示例性工程化多肽。因此，对于本发明的亚胺还原酶多肽的各个和每一个实施方案，插入可以包含1个或更多个氨基酸、2个或更多个氨基酸、3个或更多个氨基酸、4个或更多个氨基酸、5个或更多个氨基酸、6个或更多个氨基酸、8个或更多个氨基酸、10个或更多个氨基酸、15个或更多个氨基酸、20个或更多个氨基酸、30个或更多个氨基酸、40个或更多个氨基酸或50个或更多个氨基酸，其中本文描述的工程化亚胺还原酶的相关功能活性和/或改进的性质被保持。插入可以是在亚胺还原酶多肽的氨基末端或羧基末端，或内部部分。
在一些实施方案中，本文的工程化亚胺还原酶多肽可以具有包含选自SEQ ID NO:4-270范围内的偶数序列的序列和任选地一个或数个(例如多达3个、4个、5个或多达10个)氨基酸残基缺失、插入和/或取代的氨基酸序列。在一些实施方案中，氨基酸序列任选地具有1个-2个、1个-3个、1个-4个、1个-5个、1个-6个、1个-7个、1个-8个、1个-9个、1个-10个、1个-15个、1个-20个、1个-21个、1个-22个、1个-23个、1个-24个、1个-25个、1个-30个、1个-35个、1个-40个、1个-45个或1个-50个氨基酸残基缺失、插入和/或取代。在一些实施方案中，氨基酸序列的数目任选地具有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、30个、35个、40个、45个或50个氨基酸残基缺失、插入和/或取代。在一些实施方案中，氨基酸序列任选地具有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、18个、20个、21个、22个、23个、24个或25个氨基酸残基缺失、插入和/或取代。在一些实施方案中，取代可以是保守取代或非保守取代。
在上文的实施方案中，用于工程化多肽的合适的反应条件在表2-2、表3-1、表4-1和/或表5-1中提供，并且如本文实施例中描述的。
在一些实施方案中，本发明的多肽是融合多肽，其中工程化多肽与其他多肽融合，诸如例如但不限于抗体标签(例如myc表位)、纯化序列(例如用于结合金属的His标签)和细胞定位信号(例如分泌信号)。因此，本文描述的工程化多肽可以与其他多肽融合或不融合使用。
应理解，本文描述的多肽不限于遗传编码氨基酸。除了遗传编码氨基酸之外，本文描述的多肽可以完整地或部分地包含天然存在的和/或合成的非编码氨基酸。本文描述的多肽可以包含的某些常见的非编码氨基酸包括但不限于：遗传编码氨基酸的D-立体异构体；2,3-二氨基丙酸(Dpr)；α-氨基异丁酸(Aib)；ε-氨基己酸(Aha)；δ-氨基戊酸(Ava)；N-甲基甘氨酸或肌氨酸(MeGly或Sar)；鸟氨酸(Orn)；瓜氨酸(Cit)；叔丁基丙氨酸(Bua)；叔丁基甘氨酸(Bug)；N-甲基异亮氨酸(MeIle)；苯基甘氨酸(Phg)；环己基丙氨酸(Cha)；正亮氨酸(Nle)；萘基丙氨酸(Nal)；2-氯苯丙氨酸(Ocf)；3-氯苯丙氨酸(Mcf)；4-氯苯丙氨酸(Pcf)；2-氟苯丙氨酸(Off)；3-氟苯丙氨酸(Mff)；4-氟苯丙氨酸(Pff)；2-溴苯丙氨酸(Obf)；3-溴苯丙氨酸(Mbf)；4-溴苯丙氨酸(Pbf)；2-甲基苯丙氨酸(Omf)；3-甲基苯丙氨酸(Mmf)；4-甲基苯丙氨酸(Pmf)；2-硝基苯丙氨酸(Onf)；3-硝基苯丙氨酸(Mnf)；4-硝基苯丙氨酸(Pnf)；2-氰基苯丙氨酸(Ocf)；3-氰基苯丙氨酸(Mcf)；4-氰基苯丙氨酸(Pcf)；2-三氟甲基苯丙氨酸(Otf)；3-三氟甲基苯丙氨酸(Mtf)；4-三氟甲基苯丙氨酸(Ptf)；4-氨基苯丙氨酸(Paf)；4-碘代苯丙氨酸(Pif)；4-氨基甲基苯丙氨酸(Pamf)；2,4-二氯苯丙氨酸(Opef)；3,4-二氯苯丙氨酸(Mpcf)；2,4-二氟苯丙氨酸(Opff)；3,4-二氟苯丙氨酸(Mpff)；吡啶-2-基丙氨酸(2pAla)；吡啶-3-基丙氨酸(3pAla)；吡啶-4-基丙氨酸(4pAla)；萘-1-基丙氨酸(1nAla)；萘-2-基丙氨酸(2nAla)；噻唑基丙氨酸(taAla)；苯并噻吩基丙氨酸(bAla)；噻吩基丙氨酸(tAla)；呋喃基丙氨酸(fAla)；高苯丙氨酸(hPhe)；高酪氨酸(hTyr)；高色氨酸(hTrp)；五氟苯丙氨酸(5ff)；苯乙烯基丙氨酸(styrylkalanine)(sAla)；蒽基丙氨酸(aAla)；3,3-二苯丙氨酸(Dfa)；3-氨基-5-苯基戊酸(Afp)；青霉胺(Pen)；1,2,3,4-四氢异喹啉-3-羧酸(Tic)；β-2-噻吩基丙氨酸(Thi)；甲硫氨酸亚砜(Mso)；N(w)-硝基精氨酸(nArg)；高赖氨酸(hLys)；膦酰基甲基苯丙氨酸(pmPhe)；磷酸丝氨酸(pSer)；磷酸苏氨酸(pThr)；高天冬氨酸(hAsp)；高谷氨酸(hGlu)；1-氨基环戊-(2或3)-烯-4-羧酸；哌啶酸(PA)；氮杂环丁烷-3-羧酸(ACA)；1-氨基环戊烷-3-羧酸；烯丙基甘氨酸(aGly)；炔丙基甘氨酸(pgGly)；高丙氨酸(hAla)；正缬氨酸(nVal)；高亮氨酸(hLeu)、高缬氨酸(hVal)；高异亮氨酸(hIle)；高精氨酸(hArg)；N-乙酰基赖氨酸(AcLys)；2,4-二氨基丁酸(Dbu)；2,3-二氨基丁酸(Dab)；N-甲基缬氨酸(MeVal)；高半胱氨酸(hCys)；高丝氨酸(hSer)；羟基脯氨酸(Hyp)和高脯氨酸(hPro)。本文描述的多肽可以包含的另外的非编码氨基酸对于本领域技术人员将是明显的(参见例如Fasman,CRC Practical Handbook of Biochemistry and Molecular Biology,CRCPress,Boca Raton,FL,第3-70页[1989]以及其中引用的参考文献中提供的各种氨基酸，其全部通过引用并入)。这些氨基酸可以是L-构型或D-构型的。
本领域技术人员将认识到，具有侧链保护基团的氨基酸或残基还可以构成本文描述的多肽。这样的受保护的氨基酸的非限制性实例，其在此情况下属于芳香族类，包括但不限于(保护基团列于括号中)：Arg(tos)、Cys(甲基苄基)、Cys(硝基吡啶亚磺酰基)、Glu(δ-苄基酯)、Gln(呫吨基)、Asn(N-δ-呫吨基)、His(bom)、His(苄基)、His(tos)、Lys(fmoc)、Lys(tos)、Ser(O-苄基)、Thr(O-苄基)和Tyr(O-苄基)。
可以构成本文描述的多肽的构象约束的非编码氨基酸包括但不限于N-甲基氨基酸(L-构型)；1-氨基环戊-(2或3)-烯-4-羧酸；哌啶酸；氮杂环丁烷-3-羧酸；高脯氨酸(hPro)和1-氨基环戊烷-3-羧酸。
在一些实施方案中，工程化多肽可以呈各种形式，例如诸如分离的制品、作为基本上纯的酶、用编码该酶的基因转化的完整细胞和/或作为这样的细胞的细胞提取物和/或裂解物。酶可以被冻干、喷雾干燥、沉淀或呈粗制糊状物的形式，如下文进一步讨论的。
在一些实施方案中，工程化多肽可以被提供于固体支持物诸如膜、树脂、固体载体或其他固相材料上。固体支持物可以由有机聚合物诸如聚苯乙烯、聚乙烯、聚丙烯、聚氟乙烯、聚氧乙烯(polyethyleneoxy)和聚丙烯酰胺以及它们的共聚物和接枝物组成。固体支持物还可以是无机的，诸如玻璃、二氧化硅(silica)、可控孔隙玻璃(CPG)、反相二氧化硅或金属诸如金或铂。固体支持物的构型可以呈珠、球、颗粒(particle)、颗粒(granule)、凝胶、膜或表面的形式。表面可以是平面的、基本上平面的或非平面的。固体支持物可以是多孔的或无孔的，并且可以具有溶胀或非溶胀特性。固体支持物可以被配置为孔、凹陷或其他容器(container)、器皿(vessel)、特征或位置的形式。
在一些实施方案中，本发明的具有亚胺还原酶活性的工程化多肽可以被固定在固体支持物上，使得它们相对于SEQ ID NO:2、230和/或270的参考多肽保留其改进的活性和/或其他改进的性质。在这样的实施方案中，固定的多肽可以促进底物化合物或其他合适的底物向产物的生物催化转化，并且反应完成后容易保留(例如通过保留固定有多肽的珠)并且然后在随后的反应中重新使用或再循环。这样的固定化酶方法允许进一步提高效率和降低成本。因此，还预期，使用本发明的亚胺还原酶多肽的任何方法可以使用结合或固定在固体支持物上的相同的亚胺还原酶多肽进行。
酶固定化的方法是本领域熟知的。工程化多肽可以被非共价地或共价地结合。用于将酶缀合和固定到固体支持物(例如树脂、膜、珠、玻璃等)的各种方法是本领域熟知的(参见例如Yi等人,Proc.Biochem.,42(5):895-898[2007]；Martin等人,Appl.Microbiol.Biotechnol.,76(4):843-851[2007]；Koszelewski等人,J.Mol.Cat.B:Enzymatic,63:39-44[2010]；Truppo等人,Org.Proc.Res.Dev.,published online:dx.doi.org/10.1021/op200157c；Hermanson,Bioconjugate Techniques,第2版,AcademicPress,Cambridge,MA[2008]；Mateo等人,Biotechnol.Prog.,18(3):629-34[2002]；和“Bioconjugation Protocols:Strategies and Methods,”于Methods in Molecular Biology,Niemeyer(编著),Humana Press,New York,NY[2004]；每一个参考文献的公开内容通过引用并入本文)。可用于固定本发明的工程化亚胺还原酶的固体支持物包括但不限于包含具有环氧化物官能团的聚甲基丙烯酸酯、具有氨基环氧化物官能团的聚甲基丙烯酸酯、具有十八烷基官能团的苯乙烯/DVB共聚物或聚甲基丙烯酸酯的珠或树脂。可用于固定本发明的工程化亚胺还原酶多肽的示例性固体支持物包括但不限于壳聚糖珠、Eupergit C和SEPABEAD(Mitsubishi)，包括以下不同类型的SEPABEAD：EC-EP、EC-HFA/S、EXA252、EXE119和EXE120。
在一些实施方案中，本文描述的多肽以试剂盒的形式提供。试剂盒中的酶可以单独存在或作为多种酶存在。试剂盒还可以包括用于进行酶促反应的试剂、用于评估酶的活性的底物以及用于检测产物的试剂。试剂盒还可以包括试剂分配器和用于使用试剂盒的使用说明。
在一些实施方案中，本发明的试剂盒包括在不同可寻址位置上包含多个不同亚胺还原酶多肽的阵列，其中不同多肽是参考序列的各自具有至少一种不同的改进的酶性质的不同变体。在一些实施方案中，固定在固体支持物上的多个多肽被配置在阵列的多个位置上，用于试剂的自动递送或通过检测方法和/或仪器可寻址。阵列可以被用于测试多肽对各种底物化合物的转化。包含多种工程化多肽的这样的阵列及其使用方法是本领域已知的(参见例如WO2009/008908A2)。
编码工程化亚胺还原酶的多核苷酸、表达载体和宿主细胞
在另一个方面中，本发明提供了编码本文描述的工程化亚胺还原酶多肽的多核苷酸。多核苷酸可以可操作地连接至控制基因表达的一个或更多个异源调节序列，以产生能够表达该多肽的重组多核苷酸。包含编码工程化亚胺还原酶的异源多核苷酸的表达构建体被引入到适当的宿主细胞以表达相应的亚胺还原酶多肽。
如对本领域技术人员将是明显的，蛋白序列的可得性和对应于多种氨基酸的密码子的知识提供能够编码主题多肽的所有多核苷酸的说明。遗传密码的简并性——其中相同的氨基酸由可选择的密码子或同义密码子编码——允许制备极大量的核酸，所有这些核酸都编码改进的亚胺还原酶。因此，知道了特定的氨基酸序列后，本领域技术人员可以通过以不改变蛋白的氨基酸序列的方式简单改变一个或更多个密码子的序列来制备任何数目的不同核酸。在这方面，本发明具体地考虑了可以通过基于可能的密码子选择来选择组合来制备编码本文描述的多肽的多核苷酸的每一种可能的改变，并且对于本文描述的任何多肽，所有这样的改变被认为具体地公开，包括表2-2、表3-1、表4-1和表5-1中呈现的和在通过引用并入本文的序列表中作为SEQ ID NO:4-270范围内的偶数序列公开的氨基酸序列。
在多种实施方案中，密码子被优选地选择为适应在其中产生蛋白的宿主细胞。例如，在细菌中使用的优选的密码子被用于在细菌中表达基因；在酵母中使用的优选的密码子被用于在酵母中表达；并且在哺乳动物中使用的优选的密码子被用于在哺乳动物细胞中表达。在一些实施方案中，不需要替代所有密码子以优化亚胺还原酶的密码子使用，因为天然序列将包含优选的密码子并且因为可能不需要对所有氨基酸残基使用优选的密码子。因此，编码亚胺还原酶的密码子优化的多核苷酸可以在全长编码区的约40％、50％、60％、70％、80％或大于90％的密码子位置包含优选的密码子。
在一些实施方案中，多核苷酸包含编码如由SEQ ID NO:2、230和/或270表示的天然存在的亚胺还原酶多肽氨基酸序列的密码子优化的核苷酸序列。在一些实施方案中，多核苷酸具有包含与编码SEQ ID NO:4-270范围内的偶数序列的密码子优化的核酸序列至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同一性的核酸序列。在一些实施方案中，多核苷酸具有包含与SEQ ID NO:3-269范围内的奇数序列的密码子优化的核酸序列至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同一性的核酸序列。在一些实施方案中，SEQ ID NO:3-269范围内的奇数序列的密码子优化的序列增强了编码的野生型亚胺还原酶的表达，提供能够将底物转化为产物的酶的制品。
在一些实施方案中，多核苷酸能够在高严格条件下与选自SEQ ID NO:3-269中的奇数序列的参考序列或其互补体杂交，并且编码具有亚胺还原酶活性的多肽。
在一些实施方案中，如上文描述的，多核苷酸编码与SEQ ID NO:2、230和/或270相比具有改进的性质的具有亚胺还原酶活性的工程化多肽，其中该多肽包含与选自SEQ IDNO:2、230和/或270的参考序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同一性，且与SEQ ID NO:2、230和/或270相比具有一个或更多个残基差异的氨基酸序列，其中该序列选自SEQ ID NO:4-270范围内的偶数序列。在一些实施方案中，参考氨基酸序列选自SEQ ID NO:4-270范围内的偶数序列。在一些实施方案中，参考氨基酸序列是SEQ ID NO:2，而在一些其他实施方案中，参考序列是SEQ ID NO:230，并且在仍一些其他实施方案中，参考序列是SEQ ID NO:270。
在一些实施方案中，多核苷酸编码能够以与SEQ ID NO:2、230和/或270相比改进的性质将底物转化为产物的亚胺还原酶多肽，其中该多肽包含与参考序列SEQ ID NO:2、230和/或270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性的氨基酸序列。
在一些实施方案中，编码工程化亚胺还原酶的多核苷酸包含选自SEQ ID NO:3-269范围内的奇数序列的多核苷酸序列。
在一些实施方案中，多核苷酸能够在高严格条件下与选自SEQ ID NO:3-269范围内的奇数序列的参考多核苷酸序列或其互补体杂交，并且编码具有本文描述的一种或更多种具有改进的性质的具有亚胺还原酶活性的多肽。在一些实施方案中，能够在高严格条件下杂交的多核苷酸编码包含与SEQ ID NO:2具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列的亚胺还原酶多肽，该亚胺还原酶多肽具有包含与SEQ ID NO:2相比在选自以下的残基位置处的一个或更多个残基差异的氨基酸序列：57/211、87/175、103/212、122、123、124/178、124、132、134、169、171、173、175、176、178/242、178、211、212、213、214、215、217、234和235。
在一些实施方案中，多核苷酸能够在高严格条件下与选自SEQ ID NO:3-269范围内的奇数序列的参考多核苷酸序列或其互补体杂交，并且编码具有本文描述的一种或更多种改进性质的具有亚胺还原酶活性的多肽。在一些实施方案中，能够在高严格条件下杂交的多核苷酸编码包含与SEQ ID NO:270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列的亚胺还原酶多肽，该亚胺还原酶多肽具有包含与SEQ ID NO:270相比在选自以下的残基位置处的一个或更多个残基差异的氨基酸序列：123/124/169/171/173/213/235、123/124/169/212/213/235、123/169/171/173/212/213/235、123/169/171/173/213/235、123/169/173/175/213/235、123/169/173/212/213、123/169/175,123/169/175/212/235、123/169/212/213、123/173/175/212/213/235、123/212/213/235、123/212/235、123/169/171/173/175/212/213、123/169/173、123/169/212/213/235、123/213、124/169、124/169/173/175/212/213、124/169/173/212/213、124/169/173/212/213/235、124/169/175/212/213/235、124/169/212/213/235、124/169/213/235、124/175/212/213/235、124/212/213/235、134/169/176/178/211/214、134/169/176/178/211、134/169/176/178/211/217、134/169/176/211/215/217/234、134/169/176/211、134/169/178/211/214/217、134/169/211/214/217、134/169/211/217、134/176/211/214、134/176/211/215、134/211/214、169/171/173/213/235、169/173/175/213/235、169/175/212/213/235、169/176/178/211/214、169/176/178/211/214/217、169/176/178/211/217、169/176/178、169/176/178/211/214/217、169/176/211/214、169/176/211/214/215/234、169/176/211/214/217、169/176/211/217、169/176/214/215/217、169/178/217、169/178/234、169/211/214/215、169/211/217、169/212/213、169/212/235、169/212/213/235、169/235、171/173/212/213/235、173/175/213、175/213/235、175/235、176/178/211/215/217、176/178/211/217、176/211/214、176/214/217、178/211/214、178/211/217、178/215、211/234、212/213/235和213/235。
在一些实施方案中，能够在高严格条件下杂交的多核苷酸编码具有改进性质的具有亚胺还原酶活性的工程化多肽，所述工程化多肽包含与SEQ ID NO:229具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高同一性且与SEQ ID NO:229相比在选自以下的残基位置处具有一个或更多个残基差异的氨基酸序列：45、127、130、134、174、206、209、212、216、223、227和279。
在一些实施方案中，能够在高严格条件下杂交的多核苷酸编码具有改进性质的具有亚胺还原酶活性的工程化多肽，所述工程化多肽包含与SEQ ID NO:2、230和/或270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高同一性的氨基酸序列。在一些实施方案中，多核苷酸编码本文描述的多肽，但在核苷酸水平与编码工程化亚胺还原酶的参考多核苷酸具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性。在一些实施方案中，参考多核苷酸序列选自SEQ ID NO:3-269。
在一些实施方案中，能够在高严格条件下杂交的多核苷酸编码具有改进的性质的具有亚胺还原酶活性的工程化多肽，该工程化多肽包含与SEQ ID NO:2具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列。在一些实施方案中，多核苷酸编码本文描述的多肽，但在核苷酸水平与编码工程化亚胺还原酶的参考多核苷酸具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性。在一些实施方案中，参考多核苷酸序列选自SEQ ID NO:3-73。
在一些实施方案中，能够在高严格条件下杂交的多核苷酸编码具有改进的性质的具有亚胺还原酶活性的工程化多肽，所述工程化多肽包含与SEQ ID NO:270具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列。在一些实施方案中，多核苷酸编码本文描述的多肽，但在核苷酸水平与编码工程化亚胺还原酶的参考多核苷酸具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性。在一些实施方案中，参考多核苷酸序列选自SEQ ID NO:75-231。
在一些实施方案中，能够在高严格条件下杂交的多核苷酸编码具有改进的性质的具有亚胺还原酶活性的工程化多肽，所述工程化多肽包含与SEQ ID NO:229具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同一性的氨基酸序列。在一些实施方案中，多核苷酸编码本文描述的多肽，但在核苷酸水平与编码工程化亚胺还原酶的参考多核苷酸具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性。在一些实施方案中，参考多核苷酸序列选自SEQ ID NO:233-268。
本发明还提供了编码工程化亚胺还原酶多肽的工程化多核苷酸，其中该工程化多核苷酸包含以下位置处的取代：12/15/18/21/27、169/631/633、259/523/524/525、308/634/635/636、364/365/366、367/368/369、370/371、370/371/372/532、394/396、394/395/396、401/402、505/506/507、512/513、517/518/519、523/524/525、526/527/528、532/534、532/724/725、631/633、631/632/633、634/635/636、635/636、637/638、637/639、637/639、638/639、640、643/644/645、644/645、649/650/651、700/701/702和703/704/705，并且其中位置参考SEQ ID NO:1被编号。
在一些实施方案中，编码工程化亚胺还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：12A/15T/18G/21C/27A、169A/631C/633T、259T/523G/524G/525T、308A/634T/635G/636T、364G/365A/366A、367G/368G/369T、370G/371G、370G/371C/372A/532A、394C/396T、394A/395G/396G、401G/402G、505A/506A/507T、505A/506G/507T、512G/513G、517A/518T/519T、517C/518A/519T、523C/524A/525T、523G/524G/525T、526A/527G/528T、532A/534G、532A/724T/725A、631C/633T、631T/632G/633G、634T/635G/636T、635G/636T、635T/636T、637A/638G、637A/639A、637C/639A、638T/639T、640A、643T/644T/645G、644T/645G、649A/650T/651T、649C/650T/651T、700T/701T/702G和703A/704A/705A，并且其中位置参考SEQ ID NO:1被编号。
在一些实施方案中，编码工程化亚胺还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：C12A/C15T/A18G/T21C/T27A、G169A/A631C/C633T、C259T/A523G/T524G/G525T、G308A/G634T/A635G/A636T、C364G/T365A/G366A、A367G/C368G/C369T、A370G/T371G、A370G/T371C/T372A/G532A、G394C/A396T、G394A/A395G/A396G、C401G/C402G、G505A/C506A/A507T、G505A/C506G/A507T、A512G/T513G、G517A/C518T/A519T、G517C/C518A/A519T、A523C/T524A/G525T、A523G/T524G/G525T、C526A/T527G/G528T、G532A/C534G、G532A/G724T/C725A、A631C/C633T、A631T/T632G/C633G、G634T/A635G/A636T、A635G/A636T、A635T/A636T、G637A/C638G、G637A/G639A、G637C/G639A、C638T/G639T、G640A、A643T/C644T/C645G、C644T/C645G、T649A/G650T/G651T、T649C/G650T/G651T、G700T/A701T/C702G和G703A/C704A/C705A，并且其中位置参考SEQ IDNO:1被编号。
本发明还提供了编码工程化亚胺还原酶多肽的工程化多核苷酸，其中该工程化多核苷酸包含以下位置处的取代：367/368/369/505/506/507/513/519/523/524/525/634/635/636/639/703/704/705、369/370/371/372/505/506/507/513/519/638/703/704/705、369/370/371/372/507/513/519/523/524/525/635/638/703/704/705、369/505/506/507/511/512/517/518/519/638/703/704/705、369/505/506/507/513/517/518/519/523/524/525/638/703/704/705、369/505/506/507/513/519/523/524/525/634/635/636/638/703/704/705、369/505/506/507/513/519/635/638/703/704/705、401/402/505/506/507/526/527/532/534/631/632/633/645/702、401/402/505/506/507/526/527/532/534/631/633/640/645/702、401/402/505/506/507/526/527/532/534/631/633/645/649/650/651/702、401/402/505/506/507/526/527/534/631/632/633/645/702、401/402/505/506/507/526/527/534/631/633/644/645/649/650/651/700/701、401/402/505/506/507/526/532/534/631/632/633/640/645/649/650/651/702、401/402/505/506/507/526/534/631/632/633/640/645/649/650/651/702、401/402/505/506/507/526/534/631/632/633/645/649/650/651/702、401/402/507/526/527/534/631/632/633/640/645/702、401/402/507/526/527/534/631/632/633/644/645/702、401/402/526/534/631/632/633/640/645/702、402/505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、402/505/506/507/526/527/532/534/702、402/505/506/507/526/527/532/534/631/633/640/645/702、402/505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、402/505/506/507/526/527/534/631/633/640/645/649/650/651/702、402/505/506/507/526/527/534/631/632/633/640/643/644/645/700/701、402/505/506/507/526/527/534/631/632/633/640/645/702、402/505/506/507/526/527/534/631/632/633/645/649/650/651/702、402/505/506/507/526/527/534/631/632/633/645/649/650/651/702、402/505/506/507/526/527/534/633/640/644/645/649/650/651/702、402/505/506/507/526/532/534/633/645/700/701、402/505/506/507/526/532/534/645/649/650/651、402/505/506/507/526/534/631/632/633/645/649/650/651/702、402/507/526/527/532/534/631/632/633/643/644/645/649/650/651/702、402/507/526/527/532/534/631/632/633/645/649/650/651/702、402/507/526/532/534/631/633/640/645、402/507/526/532/534/631/632/633/645/649/650/651/702、402/526/527/534/631/633/640/645/702、505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、505/506/507/526/527/532/534/631/632/633/645/649/650/651/702、505/506/507/526/534/631/632/633/640/643/644/645、507/526/527/534/633/640/645/649/650/651/702、507/526/532/534/644/645/702和631/632/633/645/700/701，并且其中位置参考SEQ ID NO:269被编号。
在一些实施方案中，编码工程化亚胺还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：367G/368G/369T/505A/506A/507T/513C/519C/523G/524G/525C/634T/635G/636C/639T/703A/704A/705G、369G/370G/371G/372C/505A/506A/507T/513C/519C/638T/703A/704A/705G、369G/370G/371G/372C/507C/513C/519C/523G/524G/525C/635T/638T/703A/704A/705G、369G/505A/506A/507T/511G/512T/517A/518T/519T/638T/703A/704A/705G、369G/505A/506A/507T/513C/517A/518T/519T/523C/524A/525C/638T/703A/704A/705G、369G/505A/506A/507T/513C/519C/523G/524G/525C/634T/635G/636C/638T/703A/704A/705G、369G/505A/506A/507T/513C/519C/635T/638T/703A/704A/705G、401G/402G/505A/506A/507C/526T/527C/532A/534G/631C/633A/645T/649C/650T/651A/702T、401G/402G/505A/506A/507C/526T/527C/532A/534T/631C/633A/640A/645T/702T、401G/402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/645T/702T、401G/402G/505A/506A/507C/526T/527C/534G/631C/633A/644T/645G/649A/650T/651A/700C/701T、401G/402G/505A/506A/507C/526T/527C/534T/631T/632G/633G/645T/702T、401G/402G/505A/506A/507C/526T/532A/534G/631T/632G/633G/640A/645T/649A/650T/651A/702T、401G/402G/505A/506A/507C/526T/534G/631T/632G/633G/640A/645T/649C/650T/651A/702T、401G/402G/505A/506A/507C/526T/534T/631T/632G/633G/645T/649C/650T/651A/702T、401G/402G/507C/526T/527C/534G/631T/632G/633G/640A/645T/702T、401G/402G/507C/526T/527C/534G/631T/632G/633G/644T/645G/702T、401G/402G/526T/534G/631T/632G/633G/640A/645T/702T、402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/640A/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/532A/534G/702T、402G/505A/506A/507C/526T/527C/532A/534T/631C/633A/640A/645T/702T、402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/640A/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/534G/631C/633A/640A/645T/649A/650T/651A/702T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/640A/643T/644T/645G/700C/701T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/640A/645T/702T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/534T/631T/632G/633G/645T/649A/650T/651A/702T、402G/505A/506A/507C/526T/527C/534T/633A/640A/644T/645G/649A/650T/651A/702T、402G/505A/506A/507C/526T/532A/534G/633A/645T/700C/701T、402G/505A/506A/507C/526T/532A/534T/645T/649A/650T/651A、402G/505A/506A/507C/526T/534G/631T/632G/633G/645T/649A/650T/651A/702T、402G/507C/526T/527C/532A/534T/631T/632G/633G/643T/644T/645G/649C/650T/651A/702T、402G/507C/526T/527C/532A/534T/631T/632G/633G/645T/649A/650T/651A/702T、402G/507C/526T/532A/534G/631C/633A/640A/645T、402G/507C/526T/532A/534G/631T/632G/633G/645T/649C/650T/651A/702T、402G/526T/527C/534T/631C/633A/640A/645T/702T、505A/506A/507C/526T/527C/532A/534G/631T/632G/633G/640A/645T/649A/650T/651A/702T、505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/645T/649C/650T/651A/702T、505A/506A/507C/526T/534G/631T/632G/633G/640A/643T/644T/645G、507C/526T/527C/534G/633A/640A/645T/649C/650T/651A/702T、507C/526T/527C/534G/633A/640A/645T/649C/650T/651A/702T、507C/526T/532A/534T/644T/645G/702T和631T/632G/633G/645T/700C/701T，并且其中位置参考SEQ ID NO:269被编号。
在一些实施方案中，编码工程化亚胺还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：A367G/C368G/C369T/G505A/C506A/A507T/T513C/A519C/A523G/T524G/G525C/G634T/A635G/A636C/G639T/G703A/C704A/C705G、C369G/A370G/T371G/T372C/G505A/C506A/A507T/T513C/A519C/C638T/G703A/C704A/C705G、C369G/A370G/T371G/T372C/A507C/T513C/519C/A523G/T524G/G525C/A635T/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T511G/A512T/G517A/C518T/A519T/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/G517A/C518T/A519T/A523C/T524A/G525C/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/A519C/A523G/T524G/G525C/G634T/A635G/A636C/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/A519C/A635T/C638T/G703A/C704A/C705G、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534G/A631C/C633A/C645T/T649C/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631C/C633A/G640A/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631C/C633A/C644T/C645G/T649A/G650T/G651A/G700C/A701T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/C534T/A631T/T632G/C633G/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/G532A/C534G/A631T/T632G/C633G/G640A/C645T/T649A/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/C534T/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C401G/C402G/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C401G/C402G/A507C/C526T/T527C/C534G/A631T/T632G/C633G/C644T/C645G/C702T、C401G/C402G/C526T/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534G/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631C/C633A/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631C/C633A/G640A/C645T/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/A643T/C644T/C645G/G700C/A701T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534T/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534T/C633A/G640A/C644T/C645G/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/G532A/C534G/C633A/C645T/G700C/A701T、C402G/G505A/C506A/A507C/C526T/G532A/C534T/C645T/T649A/G650T/G651A、C402G/G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/A643T/C644T/C645G/T649C/G650T/G651A/C702T、C402G/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/A507C/C526T/G532A/C534G/A631C/C633A/G640A/C645T、C402G/A507C/C526T/G532A/C534G/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C402G/C526T/T527C/C534T/A631C/C633A/G640A/C645T/C702T、G505A/C506A/A507C/C526T/T527C/G532A/C534G/A631T/T632G/C633G/G640A/C645T/T649A/G650T/G651A/C702T、G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/G640A/A643T/C644T/C645G、A507C/C526T/T527C/C534G/C633A/G640A/C645T/T649C/G650T/G651A/C702T、A507C/C526T/G532A/C534T/C644T/C645G/C702T和A631T/T632G/C633G/C645T/G700C/A701T，并且其中位置参考SEQ ID NO:269被编号。
本发明还提供了编码工程化亚胺还原酶多肽的工程化多核苷酸，其中该工程化多核苷酸包含以下位置处的取代：133、379/381、388/389/390、400/401/402、401/402、520/521/522、616/618、625、625/626/627、634、634/635/636、634/636、646、646/647、646、667、680/681和836/837，并且其中位置参考SEQ ID NO:229被编号。
在一些实施方案中，编码工程化亚胺还原酶的工程化多核苷酸：133A、379A/381G、388C/389G/390T、400C/401T/402T、401G/402G、520A/521T/522G、616C/618T、625A、625A/626G/627T、634A、634A/635G/636G、634C/636T、646A、646A/647A、646C、667A、680T/681T和836T/837T，并且其中位置参考SEQ ID NO:229被编号。
在一些实施方案中，编码工程化亚胺还原酶多肽的工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：G133A、G379A/C381G、A388C/C389G/C390T、A400C/C401T/C402T、C401G/C402G、G520A/C521T/A522G、A616C/G618T、C625A、C625A/A626G/A627T、G634A、G634A/A635G/A636G、G634C/A636T、G646A、G646A/G647A、G646C、G667A、C680T/C681T和A836T/C837T，并且其中位置参考SEQ ID NO:229被编号。
在一些实施方案中，编码本文提供的工程化亚胺还原酶多肽中的任一个的分离的多核苷酸以多种方式被操作，以提供多肽的表达。在一些实施方案中，编码多肽的多核苷酸作为表达载体来提供，其中存在一个或更多个控制序列来调节多核苷酸和/或多肽的表达。取决于表达载体，在分离的多核苷酸插入载体之前对分离的多核苷酸的操作可以是期望的或必要的。利用重组DNA方法改变多核苷酸和核酸序列的技术是本领域熟知的。
在一些实施方案中，控制序列包括，除了其他序列以外，启动子、前导序列、多腺苷酸化序列、前肽序列、信号肽序列和转录终止子。如本领域已知的，合适的启动子可以基于使用的宿主细胞来选择。对于细菌宿主细胞，用于指导本申请的核酸构建体的转录的合适的启动子包括，但不限于从以下获得的启动子：大肠杆菌lac操纵子、天蓝色链霉菌(Streptomyces coelicolor)琼脂糖酶基因(dagA)、枯草芽孢杆菌(Bacillus subtilis)果聚糖蔗糖酶基因(sacB)、地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因(amyL)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)麦芽糖淀粉酶基因(amyM)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶基因(amyQ)、地衣芽孢杆菌青霉素酶基因(penP)、枯草芽孢杆菌xylA和xylB基因以及原核β-内酰胺酶基因(参见，例如，Villa-Kamaroff等人,Proc.Natl Acad.Sci.USA75:3727-3731[1978])，以及tac启动子(参见例如，DeBoer等人,Proc.Natl Acad.Sci.USA 80:21-25[1983])。用于丝状真菌宿主细胞的示例性启动子包括从以下的基因获得的启动子：米曲霉(Aspergillus oryzae)TAKA淀粉酶、米黑根毛霉(Rhizomucor miehei)天冬氨酸蛋白酶、黑曲霉(Aspergillus niger)中性α-淀粉酶、黑曲霉酸稳定型α-淀粉酶、黑曲霉或泡盛曲霉(Aspergillus awamori)葡糖淀粉酶(glaA)、米黑根毛霉脂肪酶、米曲霉碱性蛋白酶、米曲霉磷酸丙糖异构酶、构巢曲霉(Aspergillus nidulans)乙酰胺酶和尖孢镰孢菌(Fusarium oxysporum)胰蛋白酶样蛋白酶(参见，例如WO 96/00787)，以及NA2-tpi启动子(来自黑曲霉中性α-淀粉酶基因和米曲霉磷酸丙糖异构酶基因的启动子的杂合体)，和其突变启动子、截短启动子和杂合启动子。示例性酵母细胞启动子可以来自以下的基因：酿酒酵母(Saccharomyces cerevisiae)烯醇酶(ENO-1)、酿酒酵母半乳糖激酶(GAL1)、酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)、和酿酒酵母3-磷酸甘油酸激酶。用于酵母宿主细胞的其他有用的启动子是本领域已知的(参见例如，Romanos等人，Yeast 8:423-488[1992])。
在一些实施方案中，控制序列是合适的转录终止子序列，转录终止子序列是由宿主细胞识别以终止转录的序列。终止子序列被可操作地连接至编码多肽的核酸序列的3'末端。在选择的宿主细胞中有功能的任何终止子可用于本发明中。例如，用于丝状真菌宿主细胞的示例性转录终止子可以从以下的基因获得：米曲霉TAKA淀粉酶、黑曲霉葡糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、黑曲霉α-葡糖苷酶、和尖孢镰刀菌胰蛋白酶样蛋白酶。用于酵母宿主细胞的示例性终止子可以从以下的基因获得：酿酒酵母烯醇酶、酿酒酵母细胞色素C(CYC1)、和酿酒酵母甘油醛-3-磷酸脱氢酶。用于酵母宿主细胞的其他有用的终止子是本领域已知的(参见例如，Romanos等人，同上)。
在一些实施方案中，控制序列是合适的前导序列，前导序列是一种对由宿主细胞的翻译重要的mRNA的非翻译区域。前导序列被可操作地连接至编码多肽的核酸序列的5'末端。可以使用在所选择的宿主细胞中有功能的任何前导序列。用于丝状真菌宿主细胞的示例性前导序列从以下的基因获得：米曲霉TAKA淀粉酶和构巢曲霉磷酸丙糖异构酶。用于酵母宿主细胞的合适的前导序列包括，但不限于，从以下的基因获得的那些：酿酒酵母烯醇酶(ENO-1)、酿酒酵母3-磷酸甘油酸激酶，酿酒酵母α-因子、和酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)。控制序列也可以是多腺苷酸化序列，多腺苷酸化序列是一种可操作地连接至核酸序列的3'末端的序列，并且其在转录时，被宿主细胞识别为将多腺苷残基添加至转录的mRNA的信号。在所选择的宿主细胞中有功能的任何多腺苷酸化序列可以用于本发明中。用于丝状真菌宿主细胞的示例性多腺苷酸化序列包括，但不限于来自以下的基因的那些：米曲霉TAKA淀粉酶、黑曲霉葡糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、尖孢镰刀菌胰蛋白酶样蛋白酶和黑曲霉α-葡糖苷酶。用于酵母宿主细胞的有用的多腺苷酸化序列也是本领域已知的(参见例如，Guo和Sherman,Mol.Cell.Bio.,15:5983-5990[1995])。
在一些实施方案中，控制序列是信号肽编码区域，其编码连接至多肽的氨基末端的氨基酸序列并且将编码的多肽引导到细胞的分泌途径中。核酸序列的编码序列的5'末端可以固有地包含信号肽编码区域，其符合翻译阅读框地(in translation reading frame)与编码分泌多肽的编码区域的区段天然地连接。可选择地，编码序列的5'末端可以包含对编码序列而言外来的信号肽编码区域。将表达的多肽引导到选择的宿主细胞的分泌途径中的任何信号肽编码区域可用于本文提供的工程化亚胺还原酶多肽的表达。用于细菌宿主细胞的有效的信号肽编码区域包括但不限于从以下的基因获得的信号肽编码区域：芽孢杆菌(Bacillus)NClB 11837麦芽糖淀粉酶、嗜热脂肪芽孢杆菌α-淀粉酶、地衣芽孢杆菌枯草蛋白酶、地衣芽孢杆菌β-内酰胺酶、嗜热脂肪芽孢杆菌中性蛋白酶(nprT、nprS、nprM)、和枯草芽孢杆菌prsA。另外的信号肽是本领域已知的(参见例如，Simonen和Palva，Microbiol.Rev.,57:109-137[1993])。用于丝状真菌宿主细胞的有效的信号肽编码区域包括但不限于从以下的基因获得的信号肽编码区域：米曲霉TAKA淀粉酶、黑曲霉中性淀粉酶、黑曲霉葡糖淀粉酶、米黑根毛霉天冬氨酸蛋白酶、特异腐质霉(Humicola insolens)纤维素酶和胎毛腐质霉(Humicola lanuginosa)脂肪酶。用于酵母宿主细胞的有用的信号肽包括但不限于来自以下的基因的那些：酿酒酵母α因子和酿酒酵母转化酶。
在一些实施方案中，控制序列是前肽编码区域，其编码定位在多肽的氨基末端处的氨基酸序列。产生的多肽被称为“前酶(proenzyme)”、“前多肽(propolypeptide)”或在一些情况下被称为“酶原(zymogen)”。前多肽可以通过催化或自动催化前肽从前多肽的裂解被转化为成熟活性多肽。前肽编码区域包括但不限于以下的基因：枯草芽孢杆菌碱性蛋白酶(aprE)、枯草芽孢杆菌中性蛋白酶(nprT)、酿酒酵母α-因子、米黑根毛霉天冬氨酸蛋白酶、和嗜热毁丝霉(Myceliophthora thermophila)乳糖酶(参见，例如WO 95/33836)。当信号肽区域和前肽区域两者存在于多肽的氨基末端时，前肽区域紧邻多肽的氨基末端定位并且信号肽区域紧邻前肽区域的氨基末端定位。
在一些实施方案中，还利用了调节序列。这些序列促进多肽表达相对于宿主细胞的生长的调节。调节系统的实例是促使基因的表达被开启或关闭以响应于化学或物理刺激的那些，所述化学或物理刺激包括调节化合物的存在。在原核宿主细胞中，合适的调节序列包括但不限于lac、tac和trp操纵子系统。在酵母宿主细胞中，合适的调节系统包括但不限于ADH2系统或GAL1系统。在丝状真菌中，合适的调节序列包括但不限于TAKAα-淀粉酶启动子、黑曲霉葡糖淀粉酶启动子和米曲霉葡糖淀粉酶启动子。
本发明还提供了重组表达载体，所述重组表达载体包含编码工程化亚胺还原酶多肽的多核苷酸以及取决于它们将被引入的宿主的类型的一个或更多个表达调节区域，诸如启动子和终止子、复制起点等。在一些实施方案中，上文描述的各种核酸和控制序列可以被组合在一起以产生重组表达载体，该重组表达载体包括一个或更多个方便的限制性位点，以允许在这样的位点插入或取代编码变体亚胺还原酶多肽的核酸序列。可选择地，本发明的多核苷酸序列通过将多核苷酸序列或包含该多核苷酸的核酸构建体插入到适当的表达载体来表达。在创建表达载体时，编码序列位于载体中，使得编码序列与用于表达的适当的控制序列可操作地连接。
重组表达载体可以是任何载体(例如，质粒或病毒)，其可以方便地经历重组DNA程序并且可以导致变体亚胺还原酶多核苷酸序列的表达。载体的选择典型地将取决于载体与待引入载体的宿主细胞的相容性。载体可以是线性质粒或闭合的环状质粒。
在一些实施方案中，表达载体是自主复制载体(即，作为染色体外的实体存在的载体，其复制独立于染色体复制，诸如质粒、染色体外元件、微型染色体或人工染色体)。载体可以包含用于确保自我复制的任何工具(means)。在一些可选择的实施方案中，载体可以是当被引入到宿主细胞中时，被整合到基因组中并且与其被整合进的染色体一起复制的载体。此外，可以使用单一载体或质粒或者一起包含待引入到宿主细胞的基因组中的总DNA的两种或更多种载体或质粒，或者转座子。
在一些实施方案中，表达载体优选地包含一个或更多个可选择的标志物，其允许容易选择转化的细胞。“可选择的标志物”是其产物提供杀生物剂或病毒抗性、对重金属的抗性、对营养缺陷型的原养型等的基因。细菌的可选择的标志物的实例包括但不限于，来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或赋予抗生素抗性诸如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的标志物。用于酵母宿主细胞的合适的标志物包括但不限于ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。用于在丝状真菌宿主细胞中使用的可选择的标志物包括但不限于amdS(乙酰胺酶)、argB(鸟氨酸氨甲酰基转移酶)、bar(膦丝菌素乙酰基转移酶)、hph(潮霉素磷酸转移酶)、niaD(硝酸还原酶)、pyrG(乳清酸核苷-5'-磷酸脱羧酶)、sC(硫酸腺苷酰转移酶(sulfate adenyltransferase))和trpC(邻氨基苯甲酸合酶)，以及其等同物。在另一个方面中，本发明提供了一种宿主细胞，所述宿主细胞包含编码本发明的至少一种工程化亚胺还原酶多肽的多核苷酸，所述多核苷酸被可操作地连接至用于在宿主细胞中表达工程化亚胺还原酶的一个或更多个控制序列。用于在表达由本发明的表达载体编码的多肽中使用的宿主细胞是本领域熟知的，并且包括但不限于细菌细胞，诸如大肠杆菌、河流弧菌(Vibrio fluvialis)、链霉菌属(Streptomyces)和鼠伤寒沙门氏菌(Salmonellatyphimurium)细胞；真菌细胞，诸如酵母细胞(例如，酿酒酵母和巴斯德毕赤酵母(Pichiapastoris)[ATCC保藏号201178])；昆虫细胞，诸如果蝇属(Drosophila)S2和夜蛾属(Spodoptera)Sf9细胞；动物细胞，诸如CHO、COS、BHK、293和Bowes黑素瘤细胞；和植物细胞。示例性宿主细胞为大肠杆菌菌株(诸如，W3110(ΔfhuA)和BL21)。
因此，在另一个方面中，本发明提供了用于产生工程化亚胺还原酶多肽的方法，其中该方法包括将能够表达编码工程化亚胺还原酶多肽的多核苷酸的宿主细胞在适合于该多肽表达的条件下培养。在一些实施方案中，该方法还包括分离和/或纯化如本文描述的亚胺还原酶多肽的步骤。
上文描述的宿主细胞的适当的培养基以及生长条件是本领域熟知的。用于表达亚胺还原酶多肽的多核苷酸可以通过本领域已知的各种方法被引入细胞。技术包括，除了其他以外，电穿孔、生物颗粒轰击、脂质体介导的转染、氯化钙转染和原生质体融合。
具有本文公开的性质的工程化亚胺还原酶可以通过使编码天然存在的或工程化的亚胺还原酶多肽的多核苷酸经历本领域已知的和如本文描述的诱变和/或定向演化方法来获得。示例性的定向演化技术是诱变和/或DNA改组(参见例如，Stemmer,Proc.Natl.Acad.Sci.USA 91:10747-10751[1994]；WO 95/22625；WO 97/0078；WO 97/35966；WO 98/27230；WO 00/42651；WO 01/75767和美国专利6,537,746)。可以使用的其他定向演化程序包括，除了其他以外，交错延伸过程(StEP)、体外重组(参见例如，Zhao等人,Nat.Biotechnol.,16:258–261[1998])、诱变PCR(参见例如，Caldwell等人,PCR MethodsAppl.,3:S136-S140[1994])、和盒式诱变(参见例如，Black等人,Proc.Natl.Acad.Sci.USA93:3525-3529[1996])。
例如，诱变和定向演化方法可以容易地应用至多核苷酸，以生成可以被表达、筛选和测定的变体库。诱变和定向演化方法是本领域熟知的(参见例如美国专利第5,605,793号、第5,830,721号、第6,132,970号、第6,420,175号、第6,277,638号、第6,365,408号、第6,602,986号、第7,288,375号、第6,287,861号、第6,297,053号、第6,576,467号、第6,444,468号、第5,811238号、第6,117,679号、第6,165,793号、第6,180,406号、第6,291,242号、第6,995,017号、第6,395,547号、第6,506,602号、第6,519,065号、第6,506,603号、第6,413,774号、第6,573,098号、第6,323,030号、第6,344,356号、第6,372,497号、第7,868,138号、第5,834,252号、第5,928,905号、第6,489,146号、第6,096,548号、第6,387,702号、第6,391,552号、第6,358,742号、第6,482,647号、第6,335,160号、第6,653,072号、第6,355,484号、第6,303,344号、第6,319,713号、第6,613,514号、第6,455,253号、第6,579,678号、第6,586,182号、第6,406,855号、第6,946,296号、第7,534,564号、第7,776,598号、第5,837,458号、第6,391,640号、第6,309,883号、第7,105,297号、第7,795,030号、第6,326,204号、第6,251,674号、第6,716,631号、第6,528,311号、第6,287,862号、第6,335,198号、第6,352,859号、第6,379,964号、第7,148,054号、第7,629,170号、第7,620,500号、第6,365,377号、第6,358,740号、第6,406,910号、第6,413,745号、第6,436,675号、第6,961,664号、第6,537,746号、第7,430,477号、第7,873,499号、第7,702,464号、第7,783,428号、第7,747,391号、第7,747,393号、第7,751,986号、第6,376,246号、第6,426,224号、第6,423,542号、第6,479,652号、第6,319,714号、第6,521,453号、第6,368,861号、第7,421,347号、第7,058,515号、第7,024,312号、第7,620,502号、第7,853,410号、第7,957,912号、第7,904,249号、第8,383,346号、第8,504,498号、第8,768,871号、第8,762,066号、第8,849,575号，以及所有相关的非美国的对应申请；Ling等人,Anal.Biochem.,254:157-78[1997]；Dale等人,Meth.Mol.Biol.,57:369-74[1996]；Smith,Ann.Rev.Genet.,19:423-462[1985]；Botstein等人,Science,229:1193-1201[1985]；Carter,Biochem.J.,237:1-7[1986]；Kramer等人,Cell,38:879-887[1984]；Wells等人,Gene,34:315-323[1985]；Minshull等人,Curr.Op.Chem.Biol.,3:284-290[1999]；Christians等人,Nat.Biotechnol.,17:259-264[1999]；Crameri等人,Nature,391:288-291[1998]；Crameri,等人,Nat.Biotechnol.,15:436-438[1997]；Zhang等人,Proc.Nat.Acad.Sci.U.S.A.,94:4504-4509[1997]；Crameri等人,Nat.Biotechnol.,14:315-319[1996]；Stemmer,Nature,370:389-391[1994]；Stemmer,Proc.Nat.Acad.Sci.USA,91:10747-10751[1994]；WO 95/22625；WO 97/0078；WO 97/35966；WO 98/27230；WO 00/42651；WO 01/75767；WO 2009/152336、WO 2009/102901、WO 2009/102899、WO 2011/035105、WO 2013/138339、WO 2013/003290、WO 2014/120819、WO 2014/120821、WO 2015/0134315和WO 2015/048573，其全部通过引用并入本文)。
在一些实施方案中，诱变处理后获得的酶克隆通过以下来筛选：使酶经历指定的温度(或其他测定条件，诸如测试酶对宽范围的底物的活性)，并测量热处理或其他测定条件之后剩余的酶活性的量。然后包含编码亚胺还原酶多肽的多核苷酸的克隆被测序以鉴定核苷酸序列的改变(如果有)，并且用于在宿主细胞中表达酶。测量来自表达文库的酶活性可以使用本领域已知的任何合适的方法(例如，标准生物化学技术，诸如HPLC分析)来进行。
在一些实施方案中，可以对在诱变处理后获得的克隆筛选具有一种或更多种期望的改进的酶性质(例如，改进的区域选择性)的工程化亚胺还原酶。测量来自表达文库的酶活性可以使用诸如以下的标准生物化学技术进行：HPLC分析，和/或产物的衍生(分离前或分离后)，例如使用丹磺酰氯或OPA(参见例如，Yaegaki等人,J Chromatogr.356(1):163-70[1986])。
当已知工程化多肽的序列时，编码该酶的多核苷酸可以根据已知的合成方法通过标准固相方法来制备。在一些实施方案中，可以单独地合成多达约100个碱基的片段，然后连接(例如，通过酶促或化学连接方法或聚合酶介导的方法)以形成任何期望的连续序列。例如，编码亚胺还原酶的部分的多核苷酸和寡核苷酸可以通过如本领域已知的化学合成来制备(例如Beaucage等人,Tet.Lett.22:1859-69[1981]的经典的亚磷酰胺法、或由Matthes等人,EMBO J.3:801-05[1984]描述的方法)，因为它典型地以自动合成方法实施。根据亚磷酰胺方法，寡核苷酸被合成(例如，在自动的DNA合成仪中)、纯化、退火、连接并克隆入适当的载体。此外，可以从各种商业来源中的任一个获得基本上任何核酸。在一些实施方案中，通过合成含有缺失、插入和/或取代的寡核苷酸并且以各种排列组合寡核苷酸可以产生另外的变化，以产生具有改进性质的工程化亚胺还原酶。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:4-74的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性，并且与SEQ ID NO:2相比在选自以下的残基位置处具有一个或更多个残基差异的氨基酸序列：57/211、87/175、103/212、122、123、124/178、124、132、134、169、171、173、175、176、178/242、178、211、212、213、214、215、217、234和235；和(b)表达由多核苷酸编码的亚胺还原酶多肽。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:4-74的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性，并且与SEQ ID NO:2相比具有选自以下的一个或更多个残基差异的氨基酸序列：57T/211L、87S/175G、103H/212C、122E、123G、124A/178I、124G、132H/R、134R、169N/S、171W、173H/I、175G/H、176S、178I/242Y、178M、211L/W、212C/G/V、213P/R/T/V、214M、215L/M、217I/L、234L和235K。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:4-74的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性，并且与SEQ ID NO:2相比具有选自以下的一个或更多个残基差异的氨基酸序列：A57T/I211L、P87S/M175G、R103H/E212C、L122E、T123G、I124A/V178I、I124G、E132H/R、T134R、A169N/S、Y171W、A173H/I、M175G/H、L176S、V178I/A242Y、V178M、I211L/W、E212C/G/V、A213P/R/T/V、V214M、T215L/M、W217I/L、D234L和A235K。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:76-232的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性，并且与SEQ ID NO:270相比在选自以下的残基位置处具有一个或更多个残基差异的氨基酸序列：123/124/169/212/213/235、123/169/171/173/212/213/235、123/169/171/173/213/235、123/169/173/175/213/235、123/169/173/212/213、123/169/175、123/169/175/212/235、123/169/212/213、123/173/175/212/213/235、123/212/213/235、123/212/235、123/169/171/173/175/212/213、123/169/173、123/169/212/213/235、123/213、124/169、124/169/173/175/212/213、124/169/173/212/213、124/169/173/212/213/235、124/169/175/212/213/235、124/169/212/213/235、124/169/213/235、124/175/212/213/235、124/212/213/235、134/169/176/178/211/214、134/169/176/178/211、134/169/176/178/211/217、134/169/176/211/215/217/234、134/169/176/211、134/169/178/211/214/217、134/169/211/214/217、134/169/211/217、134/176/211/214、134/176/211/215、134/211/214、169/171/173/213/235、169/173/175/213/235、169/175/212/213/235、169/176/178/211/214、169/176/178/211/214/217、169/176/178/211/217、169/176/178、169/176/178/211/214/217、169/176/211/214、169/176/211/214/215/234、169/176/211/214/217、169/176/211/217、169/176/214/215/217、169/178/217、169/178/234、169/211/214/215、169/211/217、169/212/213、169/212/235、169/212/213/235、169/235、171/173/212/213/235、173/175/213、175/213/235、175/235、176/178/211/215/217、176/178/211/217、176/211/214、176/214/217、178/211/214、178/211/217、178/215、211/234、212/213/235和213/235。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:76-232的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性，并且与SEQ ID NO:270相比具有选自以下的一个或更多个残基差异的氨基酸序列：123G/124G/169N/171V/173H/213V/235K、123G/124G/169N/212V/213V/235K、123G/169N/171V/173H/213V/235K、123G/169N/171V/173I/212C/213V/235K、123G/169N/173H/175H/213V/235K、123G/169N/173I/212G/213T、123G/169N/175G、123G/169N/175G/212C/235K、123G/169N/212G/213V、123G/173H/175H/212C/213V/235K、123G/212C/213V/235K、123G/212G/235K、123G/169N/171V/173H/175H/212X/213V、123G/169N/173I、123G/169N/212G/213V/235K、123G/213T、124G/169N、124A/169N/173I/175H/212G/213V、124G/169N/173I/212C/213V、124G/169N/173I/212G/213V、124G/169N/173I/212V/213V/235K、124G/169N/175G/212C/213V/235K、124G/169N/212G/213V/235K、124G/169N/212V/213V/235K、124G/175G/212V/213V/235K、124G/169N/213V/235K、124G/212C/213V/235K、124G/212G/213V/235K、134R/169N/176S/178I/211L/214M、134R/169N/176S/178I/211W、134R/169N/176S/178M/211L/217L、134R/169N/176S/211L/215M/217I/234L、134R/169N/176S/211W、134R/169N/178M/211W/214M/217I、134R/169N/211W/214M/217L、134R/169N/211W/217L、134R/176S/211W/214M、134R/176S/211W/215M、134R/211W/214M、169N/171V/173I/213V/235K、169N/173I/175H/213V/235K、169N/175G/212C/213V/235K、169N/M175H/E212G/A213V/A235K、169N/176S/178I/211L/214M、169N/176S/178I/211W/214M/217L、169N/176S/178I/211W/217L、169N/176S/178M、169N/176S/178M/211W/214M/217I、169N/176S/211W/214M、169N/176S/211W/214M/215L/234L、169N/176S/211W/217I、169N/176S/211W/217L、169N/176S/214M/215M/217I、169N/176S/211L/214M/217I、169N/178I/217I、169N/178M/234L、169N/211W/214M/215L、169N/211W/217I、169N/212C/235K、169N/212C/213V/235K、169N/212G/213V、169N/212V/213V/235K、169N/235K、171V/173I/212G/213V/235K、173I/175H/213V、175H/213P/235K、175H/235K、176S/178I/211W/215L/217L、176S/178I/211W/217I、176S/211L/214M、176S/214M/217L、178M/211L/214M、178M/211W/217L、178I/215M、211W/234L、212G/213V/235K、212V/213V/235K、213T/235K和213V/235K；和(b)表达由多核苷酸编码的亚胺还原酶多肽。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:76-232的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性的氨基酸序列，并且具有与SEQ ID NO:270相比选自以下的一个或更多个残基差异：T123G/I124G/A169N/Y171V/A173H/A213V/A235K、T123G/I124G/A169N/E212V/A213V/A235K、T123G/A169N/Y171V/A173H/A213V/A235K、T123G/A169N/Y171V/A173I/E212C/A213V/A235K、T123G/A169N/A173H/M175H/A213V/A235K、T123G/A169N/A173I/E212G/A213T、T123G/A169N/M175G、T123G/A169N/M175G/E212C/A235K、T123G/A169N/E212G/A213V、T123G/A173H/M175H/E212C/A213V/A235K、T123G/E212C/A213V/A235K、T123G/E212G/A235K、T123G/A169N/Y171V/A173H/M175H/E212X/A213V、T123G/A169N/A173I、T123G/A169N/E212G/A213V/A235K、T123G/A213T、I124G/A169N、I124A/A169N/A173I/M175H/E212G/A213V、I124G/A169N/A173I/E212C/A213V、I124G/A169N/A173I/E212G/A213V、I124G/A169N/A173I/E212V/A213V/A235K、I124G/A169N/M175G/E212C/A213V/A235K、I124G/A169N/E212G/A213V/A235K、I124G/A169N/E212V/A213V/A235K、I124G/M175G/E212V/A213V/A235K、I124G/A169N/A213V/A235K、I124G/E212C/A213V/A235K、I124G/E212G/A213V/A235K、T134R/A169N/L176S/V178I/I211L/V214M、T134R/A169N/L176S/V178I/I211W、T134R/A169N/L176S/V178M/I211L/W217L、T134R/A169N/L176S/I211L/T215M/W217I/D234L、T134R/A169N/L176S/I211W、T134R/A169N/V178M/I211W/V214M/W217I、T134R/A169N/I211W/V214M/W217L、T134R/A169N/I211W/W217L、T134R/L176S/I211W/V214M、134R/L176S/I211W/T215M、T134R/I211W/V214M、A169N/Y171V/A173I/A213V/A235K、A169N/A173I/M175H/A213V/A235K、A169N/M175G/E212C/A213V/A235K、A169N/M175H/E212G/A213V/A235K、A169N/L176S/V178I/I211L/V214M、A169N/L176S/V178I/I211W/V214M/W217L、A169N/L176S/V178I/I211W/W217L、A169N/L176S/V178M、A169N/L176S/V178M/I211W/V214M/W217I、A169N/L176S/I211W/V214M、A169N/L176S/I211W/V214M/T215L/D234L、A169N/L176S/I211W/W217I、A169N/L176S/I211W/W217L、A169N/L176S/V214M/T215M/W217I、A169N/L176S/I211L/V214M/W217I、A169N/V178I/W217I、A169N/V178M/D234L、A169N/I211W/V214M/T215L、A169N/I211W/W217I、A169N/E212C/A235K、A169N/E212C/A213V/A235K、A169N/E212G/A213V、A169N/E212V/A213V/A235K、A169N/A235K、Y171V/A173I/E212G/A213V/A235K、A173I/M175H/A213V、M175H/A213P/A235K、M175H/A235K、L176S/V178I/I211W/T215L/W217L、L176S/V178I/I211W/W217I、L176S/I211L/V214M、L176S/V214M/W217L、V178M/I211L/V214M、V178M/I211W/W217L、V178I/T215M、I211W/D234L、E212G/A213V/A235K、E212V/A213V/A235K、A213T/A235K和A213V/A235K。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:234-268的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性，并且与SEQ ID NO:230相比在选自以下的残基位置处具有一个或更多个残基差异的氨基酸序列：45、127、130、134、174、206、209、212、216、223、227和279。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:234-268的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性，并且与SEQ ID NO:230相比具有选自以下的一个或更多个残基差异的氨基酸序列：45I、127R、130R、134L/R、174M、206L、209K/S、212H/K/R、216N/R/S、223N、227V和279V；和(b)表达由多核苷酸编码的亚胺还原酶多肽。
因此，在一些实施方案中，用于制备工程化亚胺还原酶多肽的方法包括：(a)合成编码多肽的多核苷酸，该多肽包含与选自SEQ ID NO:234-268的偶数序列的氨基酸序列具有至少约80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性，并且与SEQ ID NO:230相比具有选自以下的一个或更多个残基差异的氨基酸序列：V45I、G127R、T130R、T134L/R、A174M、M206L、Q209K/S、E212H/K/R、G216N/R/S、D223N、A227V和D279V。
在方法的一些实施方案中，多核苷酸编码任选地具有一个或数个(例如多达3个、4个、5个或多达10个)氨基酸残基缺失、插入和/或取代的工程化亚胺还原酶。在一些实施方案中，氨基酸序列任选地具有1个-2个、1个-3个、1个-4个、1个-5个、1个-6个、1个-7个、1个-8个、1个-9个、1个-10个、1个-15个、1个-20个、1个-21个、1个-22个、1个-23个、1个-24个、1个-25个、1个-30个、1个-35个、1个-40个、1个-45个或1个-50个氨基酸残基缺失、插入和/或取代。在一些实施方案中，氨基酸序列任选地具有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、30个、35个、40个、45个或50个氨基酸残基缺失、插入和/或取代。在一些实施方案中，氨基酸序列任选地具有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、18个、20个、21个、22个、23个、24个或25个氨基酸残基缺失、插入和/或取代。在一些实施方案中，取代可以是保守取代或非保守取代。
在一些实施方案中，可以使用用于蛋白纯化的熟知技术中的任何一种或更多种，从细胞和/或培养基中回收在宿主细胞中表达的工程化亚胺还原酶的任一种，用于蛋白纯化的熟知技术除了其他以外包括，溶菌酶处理、超声处理、过滤、盐析、超离心和色谱法。用于裂解和从细菌诸如大肠杆菌高效提取蛋白的合适的溶液是可商购的(例如，CelLyticBTM,Sigma-Aldrich，St.Louis MO)。
用于分离亚胺还原酶多肽的色谱技术包括，除了其他以外，反相色谱法、高效液相色谱法、离子交换色谱法、凝胶电泳和亲和色谱法。用于纯化特定酶的条件将部分地取决于因素诸如净电荷、疏水性、亲水性、分子量、分子形状等，并且对本领域技术人员将是明显的。
在一些实施方案中，亲和技术可以被用于分离改进的亚胺还原酶。对于亲和色谱纯化，可以使用特异性结合亚胺还原酶多肽的任何抗体。为了产生抗体，可以通过注射亚胺还原酶多肽或其片段来免疫各种宿主动物，包括但不限于兔、小鼠、大鼠等。亚胺还原酶多肽或片段可以通过侧链官能团或附接至侧链官能团的接头的方式附接至合适的载体诸如BSA。在一些实施方案中，亲和纯化可以使用结合亚胺还原酶的特异配体或染料亲和柱(参见例如，EP0641862；Stellwagen,“Dye Affinity Chromatography,”于Current Protocols in Protein Science,Unit 9.2-9.2.16[2001])。
使用工程化亚胺还原酶的方法
在一些实施方案中，本文描述的亚胺还原酶可用于将合适的底物转化为其产物的方法中。
在本文提供且在实施例中说明的实施方案中，可以在该方法中使用的合适的反应条件的各种范围包括但不限于底物载量、共底物载量、pH、温度、缓冲液、溶剂体系、多肽载量和反应时间。用于使用本文描述的工程化亚胺还原酶进行底物化合物向产物化合物的生物催化转化的另外合适的反应条件可以根据本文提供的指导通过常规实验容易地优化，其包括但不限于使工程化亚胺还原酶多肽和底物化合物在浓度、pH、温度和溶剂条件的实验反应条件下接触，并检测产物化合物。
考虑到例如产物化合物的期望量、底物浓度对酶活性的影响、酶在反应条件下的稳定性和底物向产物的转化百分比，反应混合物中的底物化合物可以变化。在一些实施方案中，合适的反应条件包括至少约0.5g/L至约25g/L、1g/L至约25g/L、5g/L至约25g/L、约10g/L至约25g/L或20g/L至约25g/L的底物化合物载量。在一些实施方案中，合适的反应条件包括至少约0.5g/L、至少约1g/L、至少约5g/L、至少约10g/L、至少约15g/L、至少约20g/L或至少约30g/L或甚至更大的底物化合物载量。
在进行本文描述的亚胺还原酶介导的方法时，工程化多肽可以以纯化酶、部分纯化的酶、用编码该酶的基因转化的完整的细胞、这样的细胞的提取物和/或裂解物、和/或固定在固体支持物上的酶的形式添加到反应混合物中。用编码工程化亚胺还原酶的基因转化的完整细胞或细胞提取物、其裂解物和分离的酶可以以多种不同的形式使用，包括固体(例如冻干、喷雾干燥等)或半固体(例如粗制糊状物)。细胞提取物或细胞裂解物可以通过沉淀(硫酸铵、聚乙烯亚胺、热处理等)部分纯化，然后在冻干之前进行脱盐程序(例如超滤、透析等)。任何酶制品(包括完整细胞制品)可以通过使用已知的交联剂诸如例如戊二醛进行交联或固定于固相(例如Eupergit C等)来稳定。
编码工程化亚胺还原酶多肽的基因可以被单独地转化到宿主细胞中或一起转化到相同的宿主细胞中。例如，在一些实施方案中，一组宿主细胞可以用编码一种工程化亚胺还原酶多肽的基因转化，并且另一组宿主细胞可以用编码另一工程化亚胺还原酶多肽的基因转化。两组转化的细胞可以以完整细胞的形式或以源自其的裂解物或提取物的形式一起用于反应混合物中。在其他实施方案中，宿主细胞可以用编码多个工程化亚胺还原酶多肽的基因转化。在一些实施方案中，工程化多肽可以以分泌的多肽的形式表达，并且含有分泌的多肽的培养基可以用于亚胺还原酶反应。
在一些实施方案中，本文公开的工程化亚胺还原酶多肽的改进的活性和/或区域选择性和/或立体选择性提供了其中可以用更低浓度的工程化多肽实现更高转化百分比的方法。在方法的一些实施方案中，合适的反应条件包括底物化合物载量的约1％(w/w)、2％(w/w)、5％(w/w)、10％(w/w)、20％(w/w)、30％(w/w)、40％(w/w)、50％(w/w)、75％(w/w)、100％(w/w)或更高的工程化多肽的量。
在一些实施方案中，工程化多肽以约0.01g/L至约50g/L；约0.05g/L至约50g/L；约0.1g/L至约40g/L；约1g/L至约40g/L；约2g/L至约40g/L；约5g/L至约40g/L；约5g/L至约30g/L；约0.1g/L至约10g/L；约0.5g/L至约10g/L；约1g/L至约10g/L；约0.1g/L至约5g/L；约0.5g/L至约5g/L或约0.1g/L至约2g/L存在。在一些实施方案中，亚胺还原酶多肽以约0.01g/L、0.05g/L、0.1g/L、0.2g/L、0.5g/L、1g/L、2g/L、5g/L、10g/L、15g/L、20g/L、25g/L、30g/L、35g/L、40g/L或50g/L存在。
在反应过程期间，反应混合物的pH可以变化。反应混合物的pH可以保持在期望的pH或在期望的pH范围内。这可以通过在反应过程之前和/或期间添加酸或碱来进行。可选择地，可以通过使用缓冲液来控制pH。因此，在一些实施方案中，反应条件包括缓冲液。保持期望的pH范围的合适的缓冲液是本领域已知的，并且包括例如但不限于硼酸盐、磷酸盐、2-(N-吗啉代)乙磺酸(MES)、3-(N-吗啉代)丙磺酸(MOPS)、乙酸盐、三乙醇胺和2-氨基-2-羟甲基-丙烷-1,3-二醇(Tris)及类似物。在一些实施方案中，反应条件包括水作为合适的溶剂，不存在缓冲液。
在方法的实施方案中，反应条件包括合适的pH。期望的pH或期望的pH范围可以通过使用酸或碱、适当的缓冲液或缓冲和酸或碱添加的组合来保持。反应混合物的pH可以在反应过程之前和/或期间控制。在一些实施方案中，合适的反应条件包括pH从约4至约10、pH从约5至约10、pH从约5至约9、pH从约6至约9、pH从约6至约8的溶液。在一些实施方案中，反应条件包括pH约4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5或10的溶液。
在本文的方法的实施方案中，使用合适的温度用于反应条件，例如考虑到反应速率在较高温度的增加和反应时间段期间酶的活性。因此，在一些实施方案中，合适的反应条件包括约10℃至约60℃、约10℃至约55℃、约15℃至约60℃、约20℃至约60℃、约20℃至约55℃、约25℃至约55℃或约30℃至约50℃的温度。在一些实施方案中，合适的反应条件包括约10℃、15℃、20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃或60℃的温度。在一些实施方案中，酶促反应期间的温度可以在整个反应过程中保持在特定温度。在一些实施方案中，酶促反应期间的温度可以在反应过程期间在温度谱上调节。
在一些实施方案中，本发明的方法在溶剂中进行。合适的溶剂包括水、水性缓冲溶液、有机溶剂、聚合物溶剂和/或共溶剂体系，其通常包含水性溶剂、有机溶剂和/或聚合物溶剂。水性溶剂(水或水性共溶剂体系)可以是pH缓冲的或不缓冲的。在一些实施方案中，使用工程化亚胺还原酶多肽的方法可以在包含有机溶剂(例如乙醇、异丙醇(IPA)、二甲基亚砜(DMSO)、二甲基甲酰胺(DMF)、乙酸乙酯、乙酸丁酯、1-辛醇、庚烷、辛烷、甲基叔丁基醚(MTBE)、甲苯及类似物)、离子或极性溶剂(例如1-乙基4-甲基咪唑鎓四氟硼酸盐、1-丁基-3-甲基咪唑鎓四氟硼酸盐、1-丁基3-甲基咪唑鎓六氟磷酸盐、甘油、聚乙二醇及类似物)的水性共溶剂体系中进行。在一些实施方案中，共溶剂可以是极性溶剂诸如多元醇、二甲基亚砜(DMSO)或低级醇。水性共溶剂体系的非水性共溶剂组分可以与水性组分混溶，提供单一液相，或可以与水性组分部分地混溶或不混溶，提供两个液相。示例性的水性共溶剂体系可以包含水和选自有机溶剂、极性溶剂和多元醇溶剂的一种或更多种共溶剂。通常，选择水性共溶剂体系的共溶剂组分，使得其在反应条件下不会不利地使亚胺还原酶失活。通过利用酶活性测定(诸如本文描述的那些)测量特定的工程化亚胺还原酶与限定的感兴趣的底物在候选溶剂体系中的酶活性可以容易地鉴定适当的共溶剂体系。
在方法的一些实施方案中，合适的反应条件包括水性共溶剂，其中共溶剂包含约1％至约50％(v/v)、约1％至约40％(v/v)、约2％至约40％(v/v)、约5％至约30％(v/v)、约10％至约30％(v/v)或约10％至约20％(v/v)的乙醇。在方法的一些实施方案中，合适的反应条件可以包括包含约1％(v/v)、约5％(v/v)、约10％(v/v)、约15％(v/v)、约20％(v/v)、约25％(v/v)、约30％(v/v)、约35％(v/v)、约40％(v/v)、约45％(v/v)或约50％(v/v)的乙醇的水性共溶剂。
在一些实施方案中，反应条件包括用于稳定或增强反应的表面活性剂。表面活性剂可以包括非离子表面活性剂、阳离子表面活性剂、阴离子表面活性剂和/或两亲性表面活性剂。示例性的表面活性剂包括例如但不限于壬基苯氧基聚乙氧基乙醇(NP40)、TRITONTMX-100聚乙二醇叔辛基苯基醚、聚氧乙烯-硬脂酰胺、十六烷基三甲基溴化铵、油烯基酰氨基硫酸钠、聚氧乙烯脱水山梨糖醇单硬脂酸酯、十六烷基二甲基胺等。可以使用任何可以稳定或增强反应的表面活性剂。在反应中使用的表面活性剂的浓度通常可以从0.1mg/ml至50mg/ml，特别地从1mg/ml至20mg/ml。
在一些实施方案中，反应条件包括消泡剂，其有助于减少或防止在反应溶液中形成泡沫，诸如当反应溶液被混合或喷洒时。消泡剂包括非极性油(例如矿物油、硅酮等)、极性油(例如脂肪酸、烷基胺、烷基酰胺、烷基硫酸酯(alkyl sulfate)等)和疏水性(例如处理的二氧化硅、聚丙烯等)，其中一些也充当表面活性剂。示例性的消泡剂包括Y-(DowCorning)、聚二醇共聚物、氧基/乙氧基化醇和聚二甲基硅氧烷。在一些实施方案中，消泡剂可以以约0.001％(v/v)至约5％(v/v)、约0.01％(v/v)至约5％(v/v)、约0.1％(v/v)至约5％(v/v)或约0.1％(v/v)至约2％(v/v)存在。在一些实施方案中，消泡剂可以以约0.001％(v/v)、约0.01％(v/v)、约0.1％(v/v)、约0.5％(v/v)、约1％(v/v)、约2％(v/v)、约3％(v/v)、约4％(v/v)或约5％(v/v)或更高存在，如促进反应所期望的。
在亚胺还原酶反应中使用的反应物的量通常将取决于期望的产物的量以及伴随的所使用的亚胺还原酶底物的量而变化。本领域普通技术人员将容易地理解如何改变这些量以将它们定制到期望的生产率和生产规模的水平。
在一些实施方案中，添加反应物的顺序不是关键的。反应物可以一起同时添加到溶剂(例如单相溶剂、双相水性共溶剂体系及类似物)中，或者可选择地，一些反应物可以分开添加，而一些反应物可以在不同的时间点一起添加。例如，辅因子、共底物和底物可以首先添加到溶剂中。
固体反应物(例如酶、盐等)可以各种不同的形式提供给反应，包括粉末(例如冻干的、喷雾干燥的、及类似形式)、溶液、乳液、悬浮液及类似物。使用本领域普通技术人员已知的方法和设备，可以容易地将反应物冻干或喷雾干燥。例如，蛋白溶液可以在-80℃以小等分试样冷冻，然后添加到预冷的冻干室中，随后施加真空。
在使用水性共溶剂体系时，为了提高混合效率，可以首先将亚胺还原酶和共底物添加并混合到水相中。亚胺还原酶底物可以被添加并混合进去，然后是有机相，或者底物可以被溶解在有机相中并混合进去。可选择地，亚胺还原酶底物可以在添加到水相之前在有机相中预混合。
通常允许本发明的方法进行，直到底物向产物的进一步转化不随反应时间显著变化(例如少于10％的底物被转化或少于5％的底物被转化)。在一些实施方案中，允许反应进行直到底物完全或接近完全转化为产物。底物向产物的转化可以使用已知方法通过检测底物和/或产物(伴随或不伴随衍生化)来监测。合适的分析方法包括气相色谱法、HPLC、MS及类似方法。
在方法的一些实施方案中，合适的反应条件包括至少约5g/L、10g/L、20g/L或更高的底物载量，并且其中该方法在约24h或更少、在约12h或更少、在约6h或更少或在约4h或更少的时间内产生至少约50％、60％、70％、80％、90％、95％或更高的底物化合物向产物化合物的转化。
当在合适的反应条件下在方法中使用时，本发明的工程化亚胺还原酶多肽产生相比于不期望的产物至少90％、95％、96％、97％、98％、99％或更高非对映异构体过量的期望的产物的过量。
在使用工程化亚胺还原酶多肽将底物化合物转化为产物化合物的方法的一些另外的实施方案中，合适的反应条件可以在反应溶液中包括初始底物载量，然后与多肽接触。然后该反应溶液还补充有以连续或分批添加的另外的底物化合物，以至少约1g/L/h、至少约2g/L/h、至少约4g/L/h、至少约6g/L/h或更高的速率随时间添加。因此，根据这些合适的反应条件，多肽被添加到具有至少约1g/L、5g/L或10g/L的初始底物载量的溶液中。添加多肽后，然后以约2g/L/h、4g/L/h或6g/L/h的速率向溶液连续添加另外的底物，直到达到高得多的至少约30g/L或更高的最终底物载量。因此，在方法的一些实施方案中，合适的反应条件包括将多肽添加到具有至少约1g/L、5g/L或10g/L的初始底物载量的溶液中，然后以约2g/L/h、4g/L/h或6g/L/h的速率向溶液添加另外的底物直到达到至少约30g/L或更高的最终底物载量。这些底物补充反应条件允许达到更高的底物载量，同时保持底物向产物的至少约5％、25％、50％、75％、90％或更高的底物转化的高转化率。
在一些实施方案中，进行另外的反应组分或另外的技术以补充反应条件。这些可以包括采取措施以稳定酶或防止酶失活、减少产物抑制、使反应平衡移动到期望的产物形成。
在另外的实施方案中，用于将底物化合物转化为产物化合物的任何上文描述的方法还可以包括一个或更多个选自以下的步骤：提取；分离；纯化；和产物化合物的结晶。用于从通过上文公开的方法产生的生物催化反应混合物提取、分离、纯化和/或结晶产物的方法、技术和方案是普通技术人员已知的和/或通过常规实验获得的。此外，在下文的实施例中提供了说明性的方法。
本发明的各种特征和实施方案在以下代表性实施例中进行了说明，这些实施例意图说明而非限制。
实验
提供以下实施例，包括实验和获得的结果，仅用于说明的目的，而不应被解释为限制本发明。
在下文的实施例中，适用以下缩写：ppm(百万分率)；M(摩尔/升)；mM(毫摩尔/升)，uM和μM(微摩尔/升)；nM(纳摩尔/升)；mol(摩尔)；gm和g(克)；mg(毫克)；ug和μg(微克)；L和l(升)；ml和mL(毫升)；cm(厘米)；mm(毫米)；um和μm(微米)；sec(秒)；min(s)(分钟)；h(s)和hr(s)(小时)；U(单位)；MW(分子量)；rpm(每分钟转数)；psi和PSI(磅/平方英寸)；℃(摄氏度)；RT和rt(室温)；CAM和cam(氯霉素)；DMSO(二甲基亚砜)；PMBS(硫酸多粘菌素B)；IPTG(异丙基β-D-1-硫代半乳糖苷)；LB(Luria-Bertani肉汤)；TB(Terrific肉汤；12g/L细菌用胰蛋白胨、24g/L酵母提取物、4mL/L甘油、65mM磷酸钾，pH 7.0，1mM MgSO4)；HEPES(HEPES两性离子缓冲液；4-(2-羟乙基)-哌嗪乙烷磺酸)；SFP(摇瓶粉末)；CDS(编码序列)；DNA(脱氧核糖核酸)；RNA(核糖核酸)；大肠杆菌W3110(常用的实验室大肠杆菌菌株，从Coli GeneticStock Center[CGSC]，New Haven，CT可获得)；HTP(高通量)；HPLC(高压液相色谱)；FIOPC(相比于阳性对照增加倍数)；Microfluidics(Microfluidics,Corp.,Westwood,MA)；Sigma-Aldrich(Sigma-Aldrich,St.Louis,MO；Difco(Difco Laboratories,BDDiagnostic Systems,Detroit,MI)；Agilent(Agilent Technologies,Inc.,Santa Clara,CA)；Corning(Corning,Inc.,Palo Alto,CA)；Dow Corning(Dow Corning,Corp.,Midland,MI)；和Gene Oracle(Gene Oracle,Inc.,Mountain View,CA)。
实施例1
生产具有亚胺还原酶活性的工程化多肽
基因合成和优化：
将编码来自Pseudonocardia spinosispora的野生型亚胺还原酶的多核苷酸序列(SEQ ID NO:1)密码子优化以用于在大肠杆菌中表达并合成(SEQ ID NO:2)。将SEQ ID NO:2的合成基因克隆到pCK110900载体系统中(参见例如，美国专利申请公布第2006/0195947号，在此通过引用以其整体并入)，并且随后在lac启动子的控制下在大肠杆菌W3110fhuA中表达。
高通量(HTP)生长、表达和裂解物制备：
在96孔格式中，挑取单菌落并且在含有1％葡萄糖和30μg/mL CAM的180μL LB培养基中、在30℃、200rpm、85％湿度生长。在过夜生长后，将20μL的生长的培养物转移到含有具有30μg/mL氯霉素(CAM)的380μL TB的深孔板中。每种变体以一式两份生长，以产生足够的目标酶以用于活性确定。培养物在30℃、250rpm、85％湿度生长。当培养物的光密度(OD600)达到0.6-0.8时，亚胺还原酶基因的表达通过添加IPTG至1mM的最终浓度来诱导。在诱导后，继续生长持续18小时-20小时。在生长后，将重复培养物合并，并且通过在4℃以4000rpm离心持续10分钟来收获细胞，并弃去培养基。将细胞沉淀物储存在-80℃直到准备使用。在进行反应之前，将细胞沉淀物重悬在250μL的含有100mM HEPES，pH 8的具有1g/L溶菌酶和0.5g/L PMBS的裂解缓冲液中。在室温，在微量滴定板振荡器上用中速振荡来搅拌板持续2小时。然后将板在4℃以4000rpm离心持续20分钟，并且将澄清的上清液用于下文描述的HTP测定反应。
摇瓶粉末(SFP)的产生
摇瓶程序可以用于产生可用于二次筛选测定和/或用于本文描述的生物催化方法的工程化亚胺还原酶多肽摇瓶粉末(SFP)。与HTP测定中使用的细胞裂解物相比，酶的摇瓶粉末(SFP)制品提供了更纯的工程化酶制品(例如，多达总蛋白的30％)，并且也允许使用更浓缩的酶溶液。为了开始培养，将含有编码感兴趣的工程化多肽的质粒的大肠杆菌的单菌落接种到具有30μg/ml CAM和1％葡萄糖的50mL LB中。培养物在培养箱中在30℃伴随250rpm振荡生长过夜(至少16小时)。在过夜生长后，测量培养物的OD600。在1L摇瓶中，将生长的培养物稀释到具有30μg/mL CAM的250mL TB中至0.2的最终OD600。250mL培养物在30℃以250rpm生长，直到OD600达到0.6-0.8。亚胺还原酶基因的表达通过添加IPTG至1mM的最终浓度来诱导，并且继续生长持续另外的18小时-20小时。通过将培养物转移到预先称重的离心瓶中，然后在4℃以4000rpm离心持续20分钟来收获细胞。将细胞沉淀物重悬并且用30mL冷的50mM磷酸钾、pH 8缓冲液洗涤，并在4℃以4000rpm再次离心持续20分钟。弃去上清液，并且称重剩余的细胞沉淀物。在裂解之前，将细胞在-80℃保持冷冻持续至少2小时。在一些实施方案中，将细胞储存在-80℃直到准备使用。为了裂解，将细胞沉淀物重悬在每1g细胞沉淀物的6ml冷的50mM磷酸钾pH8.0中。使用-M110L高压匀浆器(Microfluidics)裂解重悬的细胞。通过在4℃以10,000rpm离心持续60分钟除去细胞碎片。收集澄清的裂解物，在-80℃冷冻，并且然后使用本领域已知的标准方法冻干。冷冻的澄清裂解物的冻干提供了包含粗制工程化多肽的干燥摇瓶粉末。
实施例2
针对在制备化合物[2]中改进的稳定性和亚胺还原酶活性对衍生自SEQ ID NO:2的工程化多肽的演化和筛选
使用编码具有SEQ ID NO:2的亚胺还原酶活性的多肽的工程化多核苷酸来产生表2-2的另外的工程化的多肽。工程化基因的文库使用本领域已知的良好建立的技术(例如饱和诱变、先前鉴定的有益突变的重组)产生。在HTP中产生基因编码的多肽，并且产生可溶性裂解物，如实施例1中描述的。测试每种变体对化合物[2]产生的活性。
在96孔格式中以100μL总体积/孔进行酶测定，该100μL总体积包含85％HTP裂解物、16mM肟、100mM HEPES，pH 8，10％DMSO、1g/L GDH-105、3g/L NADP+和100mM葡萄糖。通过向每个孔中添加以下来进行反应：(i)10μL的GDH辅因子再循环预混物(预混物含有在100mMHEPES，pH 8中的1M葡萄糖、30g/L NADP+、10g/L GDH-105)；(ii)85μL的HTP澄清的裂解物(如实施例1中描述的制备)；和(iii)10μL的在DMSO中的160mM肟储备溶液。将反应板热密封并且在30℃以400rpm振荡持续24小时。通过添加具有0.1％甲酸的100μL CH3CN使反应猝灭。然后将样品在室温在微量滴定板振荡器中振荡，并且然后在4℃以4000rpm离心持续10min。在LC/MS分析之前，猝灭的样品还用具有0.1％甲酸的CH3CN稀释5倍。LC/MS4000QTRAP运行参数在下表2-1中描述。
用于分辨化合物[2]的LC-MS分析。
通过使用MRM跃迁：433.3/377的MRM模式的LC-MS分析如实施例2中描述地制备的HTP测定混合物的化合物[2]的形成。另外的相关LC-MS仪器参数和条件如下文示出的。
相对于SEQ ID NO:2的活性被计算为由变体产生的化合物[2]的峰面积相对于由SEQ ID NO:2产生的化合物[2]的峰面积。与起始多肽(即SEQ ID NO:2)相比，表2-2中列出的IRED变体具有改进的亚胺还原酶活性。确定了有益突变，并且选择一个改进最大的变体作为用于下一轮演化的骨架。
实施例3
针对在化合物[2]产生中改进的表达和亚胺还原酶活性对衍生自SEQ ID NO:2的工程化多核苷酸的演化和筛选
使用编码具有SEQ ID NO:2的亚胺还原酶活性的多肽的工程化多核苷酸产生表3-1的另外的工程化的多核苷酸。工程化基因的文库使用本领域已知的良好建立的技术(例如饱和诱变、先前鉴定的有益突变的重组)产生。在HTP中产生基因编码的多肽，并且产生可溶性裂解物，如实施例1中描述的。测试每种变体对化合物[2]产生的活性。
在96孔格式中以100μL总体积/孔进行酶测定，该100μL总体积包含85％HTP裂解物、16mM肟、100mM HEPES，pH 8，10％DMSO、1g/L GDH-105、3g/L NADP+和100mM葡萄糖。通过向每个孔中添加以下来进行反应：(i)10μL的GDH辅因子再循环预混物(预混物含有在100mMHEPES，pH 8中的1M葡萄糖、30g/L NADP+、10g/L GDH-105)；(ii)85μL的HTP澄清的裂解物(如实施例1中描述的制备)；和(iii)10μL的在DMSO中的160mM肟储备溶液。将反应板热密封并且在30℃以400rpm振荡持续24小时。通过添加具有0.1％甲酸的100μL CH3CN使反应猝灭。然后将样品在室温在微量滴定板振荡器中振荡，并且然后在4℃以4000rpm离心持续10min。在LC/MS分析之前，猝灭的样品还用具有0.1％甲酸的CH3CN稀释5倍。LC/MS4000QTRAP运行参数在表2-1中描述。
实施例4
相对于SEQ ID NO:269的产生化合物[2]的改进
在如实施例2和实施例3中描述的筛选变体后，选择多核苷酸序列SEQ ID NO:269作为亲本酶。工程化基因的文库使用本领域已知的良好建立的技术(例如饱和诱变、先前鉴定的有益突变的重组)产生。在HTP中产生每种基因编码的多肽，并且产生可溶性裂解物，如实施例1中描述的。
在96孔格式中以100μL总体积/孔进行酶测定，该100μL总体积包含85％HTP裂解物、16mM肟、100mM HEPES，pH 8，10％DMSO、1g/L GDH-105、3g/L NADP+和100mM葡萄糖。通过向每个孔中添加以下来进行反应：(i)10μL的GDH辅因子再循环预混物(预混物含有在100mMHEPES，pH 8中的1M葡萄糖、30g/L NADP+、10g/L GDH-105)；(ii)85μL的HTP澄清的裂解物(如上文描述的制备)；和(iii)10μL的在DMSO中的160mM肟储备溶液。将反应板热密封并且在30℃以400rpm振荡持续24小时。通过添加具有0.1％甲酸的100μL CH3CN使反应猝灭。然后将样品在室温在微量滴定板振荡器中振荡，并且然后在4℃以4000rpm离心持续10min。在LC/MS分析之前，猝灭的样品还用具有0.1％甲酸的CH3CN稀释5倍。LC/MS 4000QTRAP运行参数在表2-1中描述。
相对于由SEQ ID NO:269编码的酶(即SEQ ID NO:270)的活性被计算为由变体产生的化合物[2]的峰面积相对于由SEQ ID NO:270产生的化合物[2]的峰面积。与起始多肽相比，表4-1中列出的多肽具有改进的亚胺还原酶活性。鉴定了有益突变，并且选择一个改进最大的变体作为用于下一轮演化的亲本。
实施例5
在化合物[2]的生产中相对于由SEQ ID NO:229编码的多肽的改进
在如实施例3中描述的筛选变体后，选择多核苷酸序列SEQ ID NO:229作为亲本序列。工程化基因的文库使用如本领域已知的良好建立的技术(例如饱和诱变、先前鉴定的有益突变的重组)产生。在HTP中使用实施例1中描述的类似方法产生每种基因编码的多肽。因为与先前的亲本酶相比，由SEQ ID NO:229编码的多肽产生显著更高的产物，因此每种变体仅生长单独的400μL培养物。裂解使用实施例1中描述的相同方法进行。
在96孔格式中以100μL总体积/孔进行酶测定，该100μL总体积包含20％HTP裂解物、16mM肟、100mM HEPES，pH 8，10％DMSO、1g/L GDH-105、3g/L NADP+和100mM葡萄糖。通过向每个孔中添加以下来进行反应：(i)70μL的GDH辅因子再循环预混物(预混物含有在100mMHEPES，pH 8中的0.143M葡萄糖、4.28g/L NADP+、1.43g/L GDH-105)；(ii)20μL的HTP澄清的裂解物(如上文描述的制备)；和(iii)10μL的在DMSO中的160mM肟储备溶液。将反应板热密封并且在30℃以400rpm振荡持续24小时。通过添加具有0.1％甲酸的100μL CH3CN使反应猝灭。然后将样品在室温在微量滴定板振荡器中振荡，并且然后在4℃以4000rpm离心持续10min。在LC/MS分析之前，猝灭的样品还用具有0.1％甲酸的CH3CN稀释10倍。LC/MS4000QTRAP运行参数在实施例2中描述。
相对于由SEQ ID NO:229编码的多肽的活性被计算为由变体产生的产物峰面积相对于由SEQ ID NO:229编码的多肽产生的产物峰面积。与起始多肽相比，表5-1中的IRED变体具有改进的亚胺还原酶活性。
出于所有目的，本申请中引用的所有出版物、专利、专利申请和其他文件在此通过引用以其整体并入，其程度如同每个单独的出版物、专利、专利申请或其他文件被单独地指出出于所有目的通过引用并入一样。
虽然已经示出和描述了各种具体的实施方案，但是将理解，可以做出多种改变而不偏离本发明的精神和范围。
序列表
<110> 科德克希思公司
玛吉·塔布加·博拉-加尔斯克
奥斯卡·阿尔维左
美利莎·安·梅奥
斯蒂芬·詹纳
奥里克·安东尼·索厄尔-肯士
卡梅拉·莫利纳罗
<120> 工程化亚胺还原酶和用于酮和胺化合物的还原胺化的方法
<130> CX2-151USP1
<140> 尚未分配
<141> 随附提交
<160> 270
<210> 1
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶
<400> 1
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 2
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶
<400> 2
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 3
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 3
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 4
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 4
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 5
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 5
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccacatgcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 6
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 6
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro His Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 7
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 7
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgaccg caccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag catcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 8
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 8
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ala Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 9
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 9
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgaccg gtccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 10
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 10
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 11
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 11
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatcatg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 12
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 12
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp His Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 13
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 13
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 14
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 14
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 15
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 15
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagact tggccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 16
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 16
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Leu Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 17
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 17
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tgggatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 18
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 18
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Trp Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 19
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 19
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag catcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgttatgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 20
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 20
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Tyr Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 21
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 21
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaaacag tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 22
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 22
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Thr Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 23
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 23
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggtct tattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 24
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 24
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 25
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 25
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaaaggg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 26
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 26
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Arg Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 27
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 27
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccaagggcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 28
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 28
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Arg Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 29
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 29
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgtc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caggtctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 30
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 30
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Ser Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Gly Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 31
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 31
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgagtag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 32
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 32
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 33
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 33
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caggtctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 34
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 34
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Gly Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 35
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 35
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagttg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 36
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 36
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 37
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 37
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 38
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 38
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 39
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 39
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attgaaacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 40
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 40
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Glu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 41
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 41
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaaccag tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 42
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 42
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Pro Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 43
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 43
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggtat tattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 44
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 44
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 45
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 45
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctagtctg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 46
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 46
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ser Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 47
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 47
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag cacatctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 48
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 48
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 49
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 49
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaaaacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 50
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 50
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 51
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 51
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccatg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgtgcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 52
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 52
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala His Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 53
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 53
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgttgggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 54
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 54
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Leu Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 55
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 55
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 56
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 56
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 57
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 57
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgtgcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 58
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 58
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 59
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 59
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 60
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 60
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 61
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 61
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgttgcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 62
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 62
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Val Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 63
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 63
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgatgggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 64
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 64
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Met Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 65
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 65
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 66
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 66
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 67
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 67
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcgac tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt cttgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 68
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 68
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Thr Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 69
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 69
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt cttgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 70
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 70
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 71
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 71
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggtgcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 72
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 72
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 73
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 73
atgagtgatt cccccgtacc tgtgactgtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcga tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 74
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 74
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Met Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 75
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 75
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggtg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg gttgatcatg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 76
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 76
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Val Asp His Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 77
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 77
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgcgacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 78
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 78
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 79
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 79
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caggcctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 80
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 80
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Gly Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 81
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 81
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg gttgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 82
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 82
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Val Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 83
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 83
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 84
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 84
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 85
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 85
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatcatg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 86
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 86
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp His Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 87
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 87
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggaactg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 88
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 88
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Thr Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 89
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 89
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 90
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 90
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 91
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 91
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tacgatcatg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 92
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 92
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp His Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 93
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 93
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 94
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 94
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 95
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 95
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tacgatgccg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 96
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 96
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 97
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 97
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgcgacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 98
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 98
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 99
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 99
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgctg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 100
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 100
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 101
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 101
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagctg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 102
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 102
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 103
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 103
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tacgatattg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgcgacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 104
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 104
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ile Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 105
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 105
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaaactg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 106
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 106
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Thr Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 107
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 107
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tacgatgccg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaacctg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 108
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 108
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Pro Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 109
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 109
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 110
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 110
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 111
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 111
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgtagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 112
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 112
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Val Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 113
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 113
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgcgacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 114
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 114
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 115
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 115
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgtagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 116
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 116
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Val Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 117
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 117
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 118
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 118
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 119
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 119
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg gttgatcatg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 120
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 120
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Val Asp His Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 121
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 121
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 122
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 122
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 123
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 123
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgtagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 124
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 124
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Val Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 125
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 125
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgcgacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 126
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 126
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 127
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 127
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 128
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 128
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 129
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<220>
<221> Misc_Feature
<222> (634)..(634)
<223> n = a、c、g或t
<220>
<221> Misc_Feature
<222> (636)..(636)
<223> n = a、c、g或t
<400> 129
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg gttgatcatg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcngngtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 130
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<220>
<221> Misc_Feature
<222> (212)..(212)
<223> Xaa = Cys、Ter、Ser、Trp、Arg、Gly
<400> 130
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Val Asp His Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Xaa Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 131
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 131
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaaactg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 132
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 132
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Thr Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 133
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 133
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 134
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 134
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 135
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 135
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggtg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgtagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 136
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 136
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Val Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 137
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 137
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 138
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 138
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 139
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 139
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 140
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 140
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 141
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 141
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 142
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 142
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 143
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 143
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg gttgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 144
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 144
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Val Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 145
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 145
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg ccccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatattg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgcgacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 146
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 146
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ala Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ile Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 147
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 147
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcggagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 148
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 148
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Gly Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 149
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 149
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caggcctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagctg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 150
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 150
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Gly Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 151
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 151
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgggta ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caggcctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgctg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 152
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 152
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Gly Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Gly Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 153
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 153
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgtagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 154
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 154
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Val Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 155
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 155
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caggcctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atctgcgtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 156
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 156
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Gly Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Cys Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 157
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 157
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg gttgatattg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 158
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 158
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Val Asp Ile Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 159
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 159
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatgccg caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 160
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 160
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 161
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 161
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacgg gcccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tacgatgccg caggcctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgtagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 162
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 162
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Gly Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Gly Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Val Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 163
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 163
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacga ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaatctg tacgatattg cacacctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagtgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acaagacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 164
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 164
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ile Ala His Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Val Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Lys Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 165
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 165
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 166
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 166
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 167
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 167
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgttgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 168
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 168
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 169
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 169
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgttgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 170
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 170
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 171
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 171
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tatgatgcag caatgttgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 172
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 172
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 173
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 173
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tatgatgcag caatgtcgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgatgggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 174
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 174
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Met Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 175
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 175
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 176
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 176
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 177
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 177
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cgttatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 178
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 178
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 179
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 179
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cgttatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atagaagcga tgatgggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 180
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 180
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Met Met Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 181
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 181
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgttgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 182
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 182
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 183
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 183
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgttgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 184
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 184
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 185
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 185
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tatgatgcag caatgtcgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 186
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 186
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 187
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 187
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt ctagaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 188
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 188
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 189
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 189
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgttgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atagaagcgg tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacc tcgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 190
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 190
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Leu Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 191
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 191
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 192
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 192
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 193
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 193
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 194
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 194
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 195
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 195
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacc tcgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 196
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 196
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Leu Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 197
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 197
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tatgatgcag caatgtcgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atagaagcga tgactggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 198
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 198
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Met Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 199
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 199
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tatgatgcag caatgtcgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgttgggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 200
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 200
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Leu Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 201
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 201
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tatgatgcag caatgtcgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 202
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 202
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 203
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 203
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 204
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 204
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 205
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 205
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgttgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 206
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 206
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 207
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 207
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 208
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 208
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 209
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 209
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgttgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgttgggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 210
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 210
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Leu Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 211
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 211
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tatgatgcag caatgttgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgatgggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 212
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 212
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Met Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 213
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 213
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt ctagaagcgg tgatgggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacc tcgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 214
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 214
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Val Met Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Leu Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 215
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 215
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cgtgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgttgggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacc tcgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 216
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 216
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Leu Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Leu Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 217
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 217
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgccctg tatgatgcag caatgttgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt ctagaagcga tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 218
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 218
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Met Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 219
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 219
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgtcgag cgttatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt ctagaagcga tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 220
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 220
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Met Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 221
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 221
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt ctagaagcgg tgactggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 222
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 222
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Val Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 223
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 223
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgttgag cgttatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggtct aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 224
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 224
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Leu Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 225
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 225
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cgttatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcgg tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 226
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 226
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Val Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 227
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 227
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta cgattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt ctagaagcga tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 228
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 228
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Met Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 229
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 229
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 230
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 230
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 231
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 231
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag cattatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt ctagaagcga tgactggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 232
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 232
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Ile Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Leu Glu Ala Met Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 233
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 233
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcaaatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 234
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 234
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asn Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 235
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 235
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgtttct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 236
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 236
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Val Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 237
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 237
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgt tggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 238
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 238
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Val Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 239
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 239
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcag gattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 240
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 240
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Arg Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 241
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 241
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactagtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 242
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 242
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Ser Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 243
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 243
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcaa tgatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 244
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 244
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Met Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 245
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 245
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tggcatgcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 246
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 246
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp His Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 247
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 247
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tggagggcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 248
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 248
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Arg Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 249
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 249
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tggaaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 250
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 250
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Lys Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 251
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 251
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtagtggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 252
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 252
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Ser Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 253
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 253
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactcgtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 254
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 254
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Arg Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 255
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 255
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgcttct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 256
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 256
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Leu Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 257
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 257
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagctc ttattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 258
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 258
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Leu Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 259
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 259
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggccgt ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 260
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 260
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Arg Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 261
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 261
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactaatat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 262
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 262
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Asn Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 263
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 263
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tgattggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 264
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 264
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Ile Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 265
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 265
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtaaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 266
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 266
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Lys Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 267
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 267
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ggattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctaacctg tatgatgcag caatgtcgag catgatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt tgggaagcga tgactggtat aattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg atgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 268
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 268
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Arg Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Asn Leu Tyr Asp Ala Ala Met Ser
165 170 175
Ser Met Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Trp Glu Ala Met Thr Gly Ile Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
<210> 269
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 269
atgagtgatt cacctgtgcc cgtgacagtt ctgggtcttg gtctgatggg tcaggcctta 60
gcgggagcgt ttctggcagc cggtcaccct acgacggttt ggaatcgcag cgcagctaaa 120
gctgatgaac tggttggtcg cggtgcaacc ctggccgcgt ctcccgcggc tgccgtcgca 180
gcgggtagtc tgatcgttgt ttgcgtcaca gatcatcgtg cggtgcgcga actgctggaa 240
ccacttggcg atgcactgcc aggtcgcgtt ctggtcaacc tgacgagcgg tacgtcgcgt 300
gaagcccgtg agaacgcaga acatactgcg gctcagggag ccacctacct ggatggtgtg 360
attctgacca ttccaagcgg cattggcacc ccagaagcta ccattctgta ttcgggaccg 420
cgcagtacct tcgatgaaca tgaaagcgca ctgcgcgcat taggtccggg ggcgaccttt 480
gtaggggaag atcacggact ggctgcactg tatgatgcag caatgctgag cgtcatgtgg 540
ggcctgctga atggtttcct gcatggaacc gcactgctgg gaaccgcggg cgtttcggca 600
acctctgtgg ttccgatgct gggtcaaggt atcgaagcgg tgaccggttg gattgcgcgc 660
tacgcagatc agattgatgc cggtgtctat ccagcagacg acgccacgat tgatacccag 720
cgtgctgcaa tgcagcacct ggtcgaggag agccgtgcgg cgggcatcaa tggcgaactt 780
ccggcgtttg tgaccaaact ggccgatcgc gcagtggcag aaggtcgtgg tggtgactct 840
tacgctgcac tgattgaaca gtttcgcaag tcagcttaa 879
<210> 270
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来自Pseudonocardia spinosispora的亚胺还原酶变体
<400> 270
Met Ser Asp Ser Pro Val Pro Val Thr Val Leu Gly Leu Gly Leu Met
1 5 10 15
Gly Gln Ala Leu Ala Gly Ala Phe Leu Ala Ala Gly His Pro Thr Thr
20 25 30
Val Trp Asn Arg Ser Ala Ala Lys Ala Asp Glu Leu Val Gly Arg Gly
35 40 45
Ala Thr Leu Ala Ala Ser Pro Ala Ala Ala Val Ala Ala Gly Ser Leu
50 55 60
Ile Val Val Cys Val Thr Asp His Arg Ala Val Arg Glu Leu Leu Glu
65 70 75 80
Pro Leu Gly Asp Ala Leu Pro Gly Arg Val Leu Val Asn Leu Thr Ser
85 90 95
Gly Thr Ser Arg Glu Ala Arg Glu Asn Ala Glu His Thr Ala Ala Gln
100 105 110
Gly Ala Thr Tyr Leu Asp Gly Val Ile Leu Thr Ile Pro Ser Gly Ile
115 120 125
Gly Thr Pro Glu Ala Thr Ile Leu Tyr Ser Gly Pro Arg Ser Thr Phe
130 135 140
Asp Glu His Glu Ser Ala Leu Arg Ala Leu Gly Pro Gly Ala Thr Phe
145 150 155 160
Val Gly Glu Asp His Gly Leu Ala Ala Leu Tyr Asp Ala Ala Met Leu
165 170 175
Ser Val Met Trp Gly Leu Leu Asn Gly Phe Leu His Gly Thr Ala Leu
180 185 190
Leu Gly Thr Ala Gly Val Ser Ala Thr Ser Val Val Pro Met Leu Gly
195 200 205
Gln Gly Ile Glu Ala Val Thr Gly Trp Ile Ala Arg Tyr Ala Asp Gln
210 215 220
Ile Asp Ala Gly Val Tyr Pro Ala Asp Asp Ala Thr Ile Asp Thr Gln
225 230 235 240
Arg Ala Ala Met Gln His Leu Val Glu Glu Ser Arg Ala Ala Gly Ile
245 250 255
Asn Gly Glu Leu Pro Ala Phe Val Thr Lys Leu Ala Asp Arg Ala Val
260 265 270
Ala Glu Gly Arg Gly Gly Asp Ser Tyr Ala Ala Leu Ile Glu Gln Phe
275 280 285
Arg Lys Ser Ala
290
Claims (29)
1.一种工程化多肽，所述工程化多肽包含与参考序列SEQ ID NO:2具有至少80％序列同一性的氨基酸序列，包含在选自57/211、87/175、103/212、122、123、124/178、124、132、134、169、171、173、175、176、178/242、178、211、212、213、214、215、217、234和235的一个或更多个位置处的至少一个取代，其中所述位置参考SEQ ID NO:2被编号。
2.如权利要求1所述的工程化多肽，其中所述多肽包含至少一个选自57T/211L、87S/175G、103H/212C、122E、123G、124A/178I、124G、132H/R、134R、169N/S、171W、173H/I、175G/H、176S、178I/242Y、178M、211L/W、212C/G/V、213P/R/T/V、214M、215L/M、217I/L、234L和235K的取代，其中所述位置参考SEQ ID NO:2被编号。
3.如权利要求1所述的工程化多肽，其中所述多肽包含至少一个选自A57T/I211L、P87S/M175G、R103H/E212C、L122E、T123G、I124A/V178I、I124G、E132H/R、T134R、A169N/S、Y171W、A173H/I、M175G/H、L176S、V178I/A242Y、V178M、I211L/W、E212C/G/V、A213P/R/T/V、V214M、T215L/M、W217I/L、D234L和A235K的取代，其中所述位置参考SEQ ID NO:2被编号。
4.如权利要求1所述的工程化多肽，其中所述多肽包含与SEQ ID NO:4至SEQ ID NO:74中列出的任何偶数序列具有至少80％序列同一性的氨基酸序列。
5.一种工程化多肽，所述工程化多肽包含与参考序列SEQ ID NO:270具有至少80％序列同一性的氨基酸序列，包含至少两个选自以下取代组的取代：123/124/169/171/173/213/235、123/124/169/212/213/235、123/169/171/173/212/213/235、123/169/171/173/213/235、123/169/173/175/213/235、123/169/173/212/213、123/169/175、123/169/175/212/235、123/169/212/213、123/173/175/212/213/235、123/212/213/235、123/212/235、123/169/171/173/175/212/213、123/169/173、123/169/212/213/235、123/213、124/169、124/169/173/175/212/213、124/169/173/212/213、124/169/173/212/213/235、124/169/175/212/213/235、124/169/212/213/235、124/169/213/235、124/175/212/213/235、124/212/213/235、134/169/176/178/211/214、134/169/176/178/211、134/169/176/178/211/217、134/169/176/211/215/217/234、134/169/176/211、134/169/178/211/214/217、134/169/211/214/217、134/169/211/217、134/176/211/214、134/176/211/215、134/211/214、169/171/173/213/235、169/173/175/213/235、169/175/212/213/235、169/176/178/211/214、169/176/178/211/214/217、169/176/178/211/217、169/176/178、169/176/178/211/214/217、169/176/211/214、169/176/211/214/215/234、169/176/211/214/217、169/176/211/217、169/176/214/215/217、169/178/217、169/178/234、169/211/214/215、169/211/217、169/212/213、169/212/235、169/212/213/235、169/235、171/173/212/213/235、173/175/213、175/213/235、175/235、176/178/211/215/217、176/178/211/217、176/211/214、176/214/217、178/211/214、178/211/217、178/215、211/234、212/213/235和213/235，其中所述位置参考SEQ ID NO:270被编号。
6.如权利要求5所述的工程化多肽，其中所述多肽包含至少一个选自以下的取代组：123G/124G/169N/171V/173H/213V/235K、123G/124G/169N/212V/213V/235K、123G/169N/171V/173H/213V/235K、123G/169N/171V/173I/212C/213V/235K、123G/169N/173H/175H/213V/235K、123G/169N/173I/212G/213T、123G/169N/175G、123G/169N/175G/212C/235K、123G/169N/212G/213V、123G/173H/175H/212C/213V/235K、123G/212C/213V/235K、123G/212G/235K、123G/169N/171V/173H/175H/212X/213V、123G/169N/173I、123G/169N/212G/213V/235K、123G/213T、124G/169N、124A/169N/173I/175H/212G/213V、124G/169N/173I/212C/213V、124G/169N/173I/212G/213V、124G/169N/173I/212V/213V/235K、124G/169N/175G/212C/213V/235K、124G/169N/212G/213V/235K、124G/169N/212V/213V/235K、124G/175G/212V/213V/235K、124G/169N/213V/235K、124G/212C/213V/235K、124G/212G/213V/235K、134R/169N/176S/178I/211L/214M、134R/169N/176S/178I/211W、134R/169N/176S/178M/211L/217L、134R/169N/176S/211L/215M/217I/234L、134R/169N/176S/211W、134R/169N/178M/211W/214M/217I、134R/169N/211W/214M/217L、134R/169N/211W/217L、134R/176S/211W/214M、134R/176S/211W/215M、134R/211W/214M、169N/171V/173I/213V/235K、169N/173I/175H/213V/235K、169N/175G/212C/213V/235K、169N/M175H/E212G/A213V/A235K、169N/176S/178I/211L/214M、169N/176S/178I/211W/214M/217L、169N/176S/178I/211W/217L、169N/176S/178M、169N/176S/178M/211W/214M/217I、169N/176S/211W/214M、169N/176S/211W/214M/215L/234L、169N/176S/211W/217I、169N/176S/211W/217L、169N/176S/214M/215M/217I、169N/176S/211L/214M/217I、169N/178I/217I、169N/178M/234L、169N/211W/214M/215L、169N/211W/217I、169N/212C/235K、169N/212C/213V/235K、169N/212G/213V、169N/212V/213V/235K、169N/235K、171V/173I/212G/213V/235K、173I/175H/213V、175H/213P/235K、175H/235K、176S/178I/211W/215L/217L、176S/178I/211W/217I、176S/211L/214M、176S/214M/217L、178M/211L/214M、178M/211W/217L、178I/215M、211W/234L、212G/213V/235K、212V/213V/235K、213T/235K和213V/235K，其中所述位置参考SEQID NO:270被编号。
7.如权利要求5所述的工程化多肽，其中所述多肽包含至少一个选自以下的取代组：T123G/I124G/A169N/Y171V/A173H/A213V/A235K、T123G/I124G/A169N/E212V/A213V/A235K、T123G/A169N/Y171V/A173H/A213V/A235K、T123G/A169N/Y171V/A173I/E212C/A213V/A235K、T123G/A169N/A173H/M175H/A213V/A235K、T123G/A169N/A173I/E212G/A213T、T123G/A169N/M175G、T123G/A169N/M175G/E212C/A235K、T123G/A169N/E212G/A213V、T123G/A173H/M175H/E212C/A213V/A235K、T123G/E212C/A213V/A235K、T123G/E212G/A235K、T123G/A169N/Y171V/A173H/M175H/E212X/A213V、T123G/A169N/A173I、T123G/A169N/E212G/A213V/A235K、T123G/A213T、I124G/A169N、I124A/A169N/A173I/M175H/E212G/A213V、I124G/A169N/A173I/E212C/A213V、I124G/A169N/A173I/E212G/A213V、I124G/A169N/A173I/E212V/A213V/A235K、I124G/A169N/M175G/E212C/A213V/A235K、I124G/A169N/E212G/A213V/A235K、I124G/A169N/E212V/A213V/A235K、I124G/M175G/E212V/A213V/A235K、I124G/A169N/A213V/A235K、I124G/E212C/A213V/A235K、I124G/E212G/A213V/A235K、T134R/A169N/L176S/V178I/I211L/V214M、T134R/A169N/L176S/V178I/I211W、T134R/A169N/L176S/V178M/I211L/W217L、T134R/A169N/L176S/I211L/T215M/W217I/D234L、T134R/A169N/L176S/I211W、T134R/A169N/V178M/I211W/V214M/W217I、T134R/A169N/I211W/V214M/W217L、T134R/A169N/I211W/W217L、T134R/L176S/I211W/V214M、134R/L176S/I211W/T215M、T134R/I211W/V214M、A169N/Y171V/A173I/A213V/A235K、A169N/A173I/M175H/A213V/A235K、A169N/M175G/E212C/A213V/A235K、A169N/M175H/E212G/A213V/A235K、A169N/L176S/V178I/I211L/V214M、A169N/L176S/V178I/I211W/V214M/W217L、A169N/L176S/V178I/I211W/W217L、A169N/L176S/V178M、A169N/L176S/V178M/I211W/V214M/W217I、A169N/L176S/I211W/V214M、A169N/L176S/I211W/V214M/T215L/D234L、A169N/L176S/I211W/W217I、A169N/L176S/I211W/W217L、A169N/L176S/V214M/T215M/W217I、A169N/L176S/I211L/V214M/W217I、A169N/V178I/W217I、A169N/V178M/D234L、A169N/I211W/V214M/T215L、A169N/I211W/W217I、A169N/E212C/A235K、A169N/E212C/A213V/A235K、A169N/E212G/A213V、A169N/E212V/A213V/A235K、A169N/A235K、Y171V/A173I/E212G/A213V/A235K、A173I/M175H/A213V、M175H/A213P/A235K、M175H/A235K、L176S/V178I/I211W/T215L/W217L、L176S/V178I/I211W/W217I、L176S/I211L/V214M、L176S/V214M/W217L、V178M/I211L/V214M、V178M/I211W/W217L、V178I/T215M、I211W/D234L、E212G/A213V/A235K、E212V/A213V/A235K、A213T/A235K和A213V/A235K，其中所述位置参考SEQ ID NO:270被编号。
8.如权利要求5所述的工程化多肽，其中所述多肽包含与SEQ ID NO:76至SEQ ID NO:232中列出的任何偶数序列具有至少80％序列同一性的氨基酸序列。
9.一种工程化多肽，所述工程化多肽包含与参考序列SEQ ID NO:229具有至少80％序列同一性的氨基酸序列，包含在选自45、127、130、134、174、206、209、212、216、223、227和279的一个或更多个位置处的至少一个取代，其中所述位置参考SEQ ID NO:229被编号。
10.如权利要求9所述的工程化多肽，其中所述多肽包含至少一个选自45I、127R、130R、134L/R、174M、206L、209K/S、212H/K/R、216N/R/S、223N、227V和279V的取代，其中所述位置参考SEQ ID NO:229被编号。
11.如权利要求9所述的工程化多肽，其中所述多肽包含至少一个选自V45I、G127R、T130R、T134L/R、A174M、M206L、Q209K/S、E212H/K/R、G216N/R/S、D223N、A227V和D279V的取代，其中所述位置参考SEQ ID NO:229被编号。
12.如权利要求9所述的工程化多肽，其中所述多肽包含与SEQ ID NO:234至SEQ IDNO:270中列出的任何偶数序列具有至少80％序列同一性的氨基酸序列。
13.一种工程化多核苷酸，所述工程化多核苷酸编码权利要求1-12中任一项所述的至少一种工程化多肽。
14.一种工程化多核苷酸，所述工程化多核苷酸包含SEQ ID NO:3至SEQ ID NO:267中列出的奇数序列。
15.一种工程化多核苷酸，所述工程化多核苷酸编码工程化亚胺或肟还原酶多肽，其中所述工程化多核苷酸包含在以下位置处的取代：12/15/18/21/27、169/631/633、259/523/524/525、308/634/635/636、364/365/366、367/368/369、370/371、370/371/372/532、394/396、394/395/396、401/402、505/506/507、512/513、517/518/519、523/524/525、526/527/528、532/534、532/724/725、631/633、631/632/633、634/635/636、635/636、637/638、637/639、637/639、638/639、640、643/644/645、644/645、649/650/651、700/701/702和703/704/705，其中所述位置参考SEQ ID NO:1被编号。
16.如权利要求15所述的编码所述工程化亚胺或肟还原酶多肽的工程化多核苷酸，其中所述工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：12A/15T/18G/21C/27A、169A/631C/633T、259T/523G/524G/525T、308A/634T/635G/636T、364G/365A/366A、367G/368G/369T、370G/371G、370G/371C/372A/532A、394C/396T、394A/395G/396G、401G/402G、505A/506A/507T、505A/506G/507T、512G/513G、517A/518T/519T、517C/518A/519T、523C/524A/525T、523G/524G/525T、526A/527G/528T、532A/534G、532A/724T/725A、631C/633T、631T/632G/633G、634T/635G/636T、635G/636T、635T/636T、637A/638G、637A/639A、637C/639A、638T/639T、640A、643T/644T/645G、644T/645G、649A/650T/651T、649C/650T/651T、700T/701T/702G和703A/704A/705A，并且其中所述位置参考SEQ ID NO:1被编号。
17.如权利要求15所述的编码所述工程化亚胺或肟还原酶多肽的工程化多核苷酸，其中所述工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：C12A/C15T/A18G/T21C/T27A、G169A/A631C/C633T、C259T/A523G/T524G/G525T、G308A/G634T/A635G/A636T、C364G/T365A/G366A、A367G/C368G/C369T、A370G/T371G、A370G/T371C/T372A/G532A、G394C/A396T、G394A/A395G/A396G、C401G/C402G、G505A/C506A/A507T、G505A/C506G/A507T、A512G/T513G、G517A/C518T/A519T、G517C/C518A/A519T、A523C/T524A/G525T、A523G/T524G/G525T、C526A/T527G/G528T、G532A/C534G、G532A/G724T/C725A、A631C/C633T、A631T/T632G/C633G、G634T/A635G/A636T、A635G/A636T、A635T/A636T、G637A/C638G、G637A/G639A、G637C/G639A、C638T/G639T、G640A、A643T/C644T/C645G、C644T/C645G、T649A/G650T/G651T、T649C/G650T/G651T、G700T/A701T/C702G和G703A/C704A/C705A，并且其中所述位置参考SEQ ID NO:1被编号。
18.一种工程化多核苷酸，所述工程化多核苷酸编码工程化亚胺或肟还原酶多肽，其中所述工程化多核苷酸包含在以下位置处的取代：367/368/369/505/506/507/513/519/523/524/525/634/635/636/639/703/704/705、369/370/371/372/505/506/507/513/519/638/703/704/705、369/370/371/372/507/513/519/523/524/525/635/638/703/704/705、369/505/506/507/511/512/517/518/519/638/703/704/705、369/505/506/507/513/517/518/519/523/524/525/638/703/704/705、369/505/506/507/513/519/523/524/525/634/635/636/638/703/704/705、369/505/506/507/513/519/635/638/703/704/705、401/402/505/506/507/526/527/532/534/631/632/633/645/702、401/402/505/506/507/526/527/532/534/631/633/640/645/702、401/402/505/506/507/526/527/532/534/631/633/645/649/650/651/702、401/402/505/506/507/526/527/534/631/632/633/645/702、401/402/505/506/507/526/527/534/631/633/644/645/649/650/651/700/701、401/402/505/506/507/526/532/534/631/632/633/640/645/649/650/651/702、401/402/505/506/507/526/534/631/632/633/640/645/649/650/651/702、401/402/505/506/507/526/534/631/632/633/645/649/650/651/702、401/402/507/526/527/534/631/632/633/640/645/702、401/402/507/526/527/534/631/632/633/644/645/702、401/402/526/534/631/632/633/640/645/702、402/505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、402/505/506/507/526/527/532/534/702、402/505/506/507/526/527/532/534/631/633/640/645/702、402/505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、402/505/506/507/526/527/534/631/633/640/645/649/650/651/702、402/505/506/507/526/527/534/631/632/633/640/643/644/645/700/701、402/505/506/507/526/527/534/631/632/633/640/645/702、402/505/506/507/526/527/534/631/632/633/645/649/650/651/702、402/505/506/507/526/527/534/631/632/633/645/649/650/651/702、402/505/506/507/526/527/534/633/640/644/645/649/650/651/702、402/505/506/507/526/532/534/633/645/700/701、402/505/506/507/526/532/534/645/649/650/651、402/505/506/507/526/534/631/632/633/645/649/650/651/702、402/507/526/527/532/534/631/632/633/643/644/645/649/650/651/702、402/507/526/527/532/534/631/632/633/645/649/650/651/702、402/507/526/532/534/631/633/640/645、402/507/526/532/534/631/632/633/645/649/650/651/702、402/526/527/534/631/633/640/645/702、505/506/507/526/527/532/534/631/632/633/640/645/649/650/651/702、505/506/507/526/527/532/534/631/632/633/645/649/650/651/702、505/506/507/526/534/631/632/633/640/643/644/645、507/526/527/534/633/640/645/649/650/651/702、507/526/532/534/644/645/702和631/632/633/645/700/701，其中所述位置参考SEQ ID NO:269被编号。
19.如权利要求18所述的编码所述工程化亚胺或肟还原酶多肽的工程化多核苷酸，其中所述工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：367G/368G/369T/505A/506A/507T/513C/519C/523G/524G/525C/634T/635G/636C/639T/703A/704A/705G、369G/370G/371G/372C/505A/506A/507T/513C/519C/638T/703A/704A/705G、369G/370G/371G/372C/507C/513C/519C/523G/524G/525C/635T/638T/703A/704A/705G、369G/505A/506A/507T/511G/512T/517A/518T/519T/638T/703A/704A/705G、369G/505A/506A/507T/513C/517A/518T/519T/523C/524A/525C/638T/703A/704A/705G、369G/505A/506A/507T/513C/519C/523G/524G/525C/634T/635G/636C/638T/703A/704A/705G、369G/505A/506A/507T/513C/519C/635T/638T/703A/704A/705G、401G/402G/505A/506A/507C/526T/527C/532A/534G/631C/633A/645T/649C/650T/651A/702T、401G/402G/505A/506A/507C/526T/527C/532A/534T/631C/633A/640A/645T/702T、401G/402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/645T/702T、401G/402G/505A/506A/507C/526T/527C/534G/631C/633A/644T/645G/649A/650T/651A/700C/701T、401G/402G/505A/506A/507C/526T/527C/534T/631T/632G/633G/645T/702T、401G/402G/505A/506A/507C/526T/532A/534G/631T/632G/633G/640A/645T/649A/650T/651A/702T、401G/402G/505A/506A/507C/526T/534G/631T/632G/633G/640A/645T/649C/650T/651A/702T、401G/402G/505A/506A/507C/526T/534T/631T/632G/633G/645T/649C/650T/651A/702T、401G/402G/507C/526T/527C/534G/631T/632G/633G/640A/645T/702T、401G/402G/507C/526T/527C/534G/631T/632G/633G/644T/645G/702T、401G/402G/526T/534G/631T/632G/633G/640A/645T/702T、402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/640A/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/532A/534G/702T、402G/505A/506A/507C/526T/527C/532A/534T/631C/633A/640A/645T/702T、402G/505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/640A/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/534G/631C/633A/640A/645T/649A/650T/651A/702T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/640A/643T/644T/645G/700C/701T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/640A/645T/702T、402G/505A/506A/507C/526T/527C/534G/631T/632G/633G/645T/649C/650T/651A/702T、402G/505A/506A/507C/526T/527C/534T/631T/632G/633G/645T/649A/650T/651A/702T、402G/505A/506A/507C/526T/527C/534T/633A/640A/644T/645G/649A/650T/651A/702T、402G/505A/506A/507C/526T/532A/534G/633A/645T/700C/701T、402G/505A/506A/507C/526T/532A/534T/645T/649A/650T/651A、402G/505A/506A/507C/526T/534G/631T/632G/633G/645T/649A/650T/651A/702T、402G/507C/526T/527C/532A/534T/631T/632G/633G/643T/644T/645G/649C/650T/651A/702T、402G/507C/526T/527C/532A/534T/631T/632G/633G/645T/649A/650T/651A/702T、402G/507C/526T/532A/534G/631C/633A/640A/645T、402G/507C/526T/532A/534G/631T/632G/633G/645T/649C/650T/651A/702T、402G/526T/527C/534T/631C/633A/640A/645T/702T、505A/506A/507C/526T/527C/532A/534G/631T/632G/633G/640A/645T/649A/650T/651A/702T、505A/506A/507C/526T/527C/532A/534T/631T/632G/633G/645T/649C/650T/651A/702T、505A/506A/507C/526T/534G/631T/632G/633G/640A/643T/644T/645G、507C/526T/527C/534G/633A/640A/645T/649C/650T/651A/702T、507C/526T/527C/534G/633A/640A/645T/649C/650T/651A/702T、507C/526T/532A/534T/644T/645G/702T和631T/632G/633G/645T/700C/701T，其中所述位置参考SEQ ID NO:269被编号。
20.如权利要求18所述的编码所述工程化亚胺或肟还原酶多肽的工程化多核苷酸，其中所述工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：A367G/C368G/C369T/G505A/C506A/A507T/T513C/A519C/A523G/T524G/G525C/G634T/A635G/A636C/G639T/G703A/C704A/C705G、C369G/A370G/T371G/T372C/G505A/C506A/A507T/T513C/A519C/C638T/G703A/C704A/C705G、C369G/A370G/T371G/T372C/A507C/T513C/519C/A523G/T524G/G525C/A635T/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T511G/A512T/G517A/C518T/A519T/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/G517A/C518T/A519T/A523C/T524A/G525C/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/A519C/A523G/T524G/G525C/G634T/A635G/A636C/C638T/G703A/C704A/C705G、C369G/G505A/C506A/A507T/T513C/A519C/A635T/C638T/G703A/C704A/C705G、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534G/A631C/C633A/C645T/T649C/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631C/C633A/G640A/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631C/C633A/C644T/C645G/T649A/G650T/G651A/G700C/A701T、C401G/C402G/G505A/C506A/A507C/C526T/T527C/C534T/A631T/T632G/C633G/C645T/C702T、C401G/C402G/G505A/C506A/A507C/C526T/G532A/C534G/A631T/T632G/C633G/G640A/C645T/T649A/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C401G/C402G/G505A/C506A/A507C/C526T/C534T/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C401G/C402G/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C401G/C402G/A507C/C526T/T527C/C534G/A631T/T632G/C633G/C644T/C645G/C702T、C401G/C402G/C526T/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534G/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631C/C633A/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/G640A/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631C/C633A/G640A/C645T/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/A643T/C644T/C645G/G700C/A701T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/G640A/C645T/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534G/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534T/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/T527C/C534T/C633A/G640A/C644T/C645G/T649A/G650T/G651A/C702T、C402G/G505A/C506A/A507C/C526T/G532A/C534G/C633A/C645T/G700C/A701T、C402G/G505A/C506A/A507C/C526T/G532A/C534T/C645T/T649A/G650T/G651A、C402G/G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/A643T/C644T/C645G/T649C/G650T/G651A/C702T、C402G/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/T649A/G650T/G651A/C702T、C402G/A507C/C526T/G532A/C534G/A631C/C633A/G640A/C645T、C402G/A507C/C526T/G532A/C534G/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、C402G/C526T/T527C/C534T/A631C/C633A/G640A/C645T/C702T、G505A/C506A/A507C/C526T/T527C/G532A/C534G/A631T/T632G/C633G/G640A/C645T/T649A/G650T/G651A/C702T、G505A/C506A/A507C/C526T/T527C/G532A/C534T/A631T/T632G/C633G/C645T/T649C/G650T/G651A/C702T、G505A/C506A/A507C/C526T/C534G/A631T/T632G/C633G/G640A/A643T/C644T/C645G、A507C/C526T/T527C/C534G/C633A/G640A/C645T/T649C/G650T/G651A/C702T、A507C/C526T/G532A/C534T/C644T/C645G/C702T和A631T/T632G/C633G/C645T/G700C/A701T，其中所述位置参考SEQ ID NO:269被编号。
21.一种工程化多核苷酸，所述工程化多核苷酸编码工程化亚胺或肟还原酶多肽，其中所述工程化多核苷酸包含在以下位置处的取代：133、379/381、388/389/390、400/401/402、401/402、520/521/522、616/618、625、625/626/627、634、634/635/636、634/636、646、646/647、646、667、680/681和836/837，其中所述位置参考SEQ ID NO:229被编号。
22.如权利要求21所述的编码所述工程化亚胺或肟还原酶多肽的工程化多核苷酸，其中所述工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：133A、379A/381G、388C/389G/390T、400C/401T/402T、401G/402G、520A/521T/522G、616C/618T、625A、625A/626G/627T、634A、634A/635G/636G、634C/636T、646A、646A/647A、646C、667A、680T/681T和836T/837T，其中所述位置参考SEQ ID NO:229被编号。
23.如权利要求21所述的编码所述工程化亚胺或肟还原酶多肽的工程化多核苷酸，其中所述工程化多核苷酸包含选自以下的以下取代/取代组中的至少一个：G133A、G379A/C381G、A388C/C389G/C390T、A400C/C401T/C402T、C401G/C402G、G520A/C521T/A522G、A616C/G618T、C625A、C625A/A626G/A627T、G634A、G634A/A635G/A636G、G634C/A636T、G646A、G646A/G647A、G646C、G667A、C680T/C681T和A836T/C837T，其中所述位置参考SEQ IDNO:229被编号。
24.一种载体，所述载体包含权利要求13至23中任一项所述的工程化多核苷酸。
25.如权利要求24所述的载体，还包含至少一个控制序列。
26.一种宿主细胞，所述宿主细胞包含权利要求24和/或权利要求25所述的载体。
27.如权利要求26所述的宿主细胞，其中所述宿主细胞产生至少一种权利要求1-12中任一项所述的工程化多肽。
28.一种产生工程化亚胺或肟还原酶多肽的方法，所述方法包括在使得所述工程化多核苷酸被表达和所述工程化多肽被产生的条件下培养权利要求26或27所述的宿主细胞的步骤。
29.如权利要求28所述的方法，还包括回收所述工程化多肽的步骤。
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| CN110819601A (zh) * | 2019-11-04 | 2020-02-21 | 江南大学 | 还原胺化酶、编码基因、重组载体、重组细胞及其应用 |
| CN114317472A (zh) * | 2021-12-31 | 2022-04-12 | 长兴制药股份有限公司 | 一种高立体选择性亚胺还原酶及其制备方法和应用 |
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| CN114317472B (zh) * | 2021-12-31 | 2022-07-05 | 长兴制药股份有限公司 | 一种高立体选择性亚胺还原酶及其制备方法和应用 |
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| JP2019526252A (ja) | 2019-09-19 |
| CN109863162B (zh) | 2023-03-17 |
| US20200208123A1 (en) | 2020-07-02 |
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| EP3515926A4 (en) | 2020-04-22 |
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| SG10202111589RA (en) | 2021-11-29 |
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| SG11201900669WA (en) | 2019-03-28 |
| EP3515926B1 (en) | 2023-10-25 |
| CA3032519A1 (en) | 2018-03-01 |
| US11987818B2 (en) | 2024-05-21 |
| US11046936B2 (en) | 2021-06-29 |
| EP4223766A2 (en) | 2023-08-09 |
| EP3515926A1 (en) | 2019-07-31 |
| EP4223766A3 (en) | 2023-10-18 |
| IL264686A (en) | 2019-04-30 |
| JP7316662B2 (ja) | 2023-07-28 |
| US20210284973A1 (en) | 2021-09-16 |
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