CN114134133A - 比活和热稳定性提高的腈水合酶变体及其应用 - Google Patents

比活和热稳定性提高的腈水合酶变体及其应用 Download PDF

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CN114134133A
CN114134133A CN202111030266.7A CN202111030266A CN114134133A CN 114134133 A CN114134133 A CN 114134133A CN 202111030266 A CN202111030266 A CN 202111030266A CN 114134133 A CN114134133 A CN 114134133A
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王小龙
林洁
何玉兰
麦倩婷
杨志雄
谢文平
李文佳
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Abstract

本发明提供一种比活和热稳定性提高的腈水合酶变体，所述的腈水合酶变体是由Klebsiella oxytoca来源的腈水合酶通过点突变获得的。本发明还涉及该酶在生物酶催化技术制备右旋酰胺酮洛芬的应用；使用本发明的腈水合酶变体催化生产右旋酰胺酮洛芬，底物转化率高，产物e.e.值>99％，酶投入量少，有效降低生物酶合成法生产右旋酮洛芬的成本，同时该酶的热稳定性大幅提高，有利于贮藏和使用。

Description

比活和热稳定性提高的腈水合酶变体及其应用
技术领域
本发明涉及分子生物学领域，更具体地，本发明涉及一种比活和热稳定性提高的腈水合酶变体及其在催化合成酰胺酮洛芬中的应用。
背景技术
酮洛芬是2-芳基丙酸类非甾体抗炎药(NSAIDs)，此类药物均具有一个手性中心，只有右旋体才具有抗炎抗风湿和镇痛作用，左旋体几乎没有药理活性且具有毒副作用{何凤慈等.酮洛芬及其手性对映体的研究进展[J].中国药房,2004,15(4):244-246；梅之南等.右旋酮洛芬的药动学和药效学性质[J].中国新药杂志,1998,7(005):339-341}。
目前，市售(S)-(+)-酮洛芬主要合成方法有传统的化学合成法、手性拆分以及生物酶合成法。化学合成法主要是不对称合成可通过6步反应，由烯丙醇通过sharpless环氧化制备得到(2S，3S)环氧化合物，再加入手性位移试剂，经过对映体选择性氢解后生成二醇，在RuO4/NaIO4的催化下，最终得到右旋酮洛芬，e.e.值达到98％这种方法的产率和对映体选择性较为理想，但是均需要用到传统的化学催化剂以促使反应快速进行，而大部分催化剂毒性较高、易燃且会造成环境污染，还有可能引入有害的副产物刘继东.酮基布洛芬的合成新方法及拆分新方法研究[D].2000；胡琦蔚等.生物催化法制备手性酮基布洛芬的研究进展[J].发酵科技通讯,2017,46(3):153-157}。
另有手性拆分方法，专利CN101928214A采用国内廉价易得的(-)-葡辛胺作为拆分剂拆分(RS)-酮洛芬，按一定比例混合后升温回流后降温结晶，对酮洛芬利用率达70％，不过该方法所需时间长，工艺繁杂。目前也有采用高分子印迹聚合物、色谱法对酮洛芬对映体进行拆分{姜吉刚等.分子印迹聚合物拆分消旋体酮洛芬[J].分析试验室,2004,23(003):56-58，专利CN102516068B}。此类方法无成本优势，难以直面市场。也有研究利用微生物酶法进行酮洛芬拆分。马燕等人利用紫外诱变得到的微生物菌株在一定条件下进行发酵，利用菌体拆分(RS)-酮洛芬，同时，还利用诺维信脂肪酶435催化酮洛芬酯拆分{马燕等.Novozym435催化酮洛芬酯化拆分研究[J].南京工业大学学报(自然科学版),2015,37(6):54-60,68.}。专利CN103194467A在大肠杆菌中表达来自于古细菌的嗜热酯酶，利用菌体催化酮洛芬乙酯反应生成右旋酮洛芬。该方法最大的不足就是底物浓度低、反应时间长且产物e.e.值仅为90％。利用酯酶的缺点在于拆分率和产物e.e.值都不理想，底物浓度偏低问题也难以解决。
生物酶合成法可以一定程度上解决底物浓度和产物e.e.值偏低的问题。目前也有利用腈水合酶和酰胺酶双酶体系制备光学纯酮洛芬的报道。Norman Layh等以酮洛芬腈为唯一氮原筛选得到的菌株Rhodococcus sp.C3II能将酮洛芬腈转化为(S)-(+)-酰胺酮洛芬，e.e.值为99％，但转化率只有12％{Layh N,Knackmuss H J,StolzA.Enantioselective hydrolysis of ketoprofen amide by Rhodococcus sp.C3II andRhodococcus erythropolis MP 50[J].Biotechnology Letters,1995,17(2):187-192.}。
R.Bauer等利用从根癌农杆菌中纯化得到的腈水合酶催化酮洛芬腈(氰基酮洛芬)，(S)-(+)-酰胺酮洛芬的产率为30％，光学纯度大于90％；转化率为50％时，光学纯度下降至80％，转化率达到100％时生成的是外消旋酰胺酮洛芬{Bauer R,Knackmuss H J,Stolz A.Enantioselective hydration of2-arylpropionitriles by a nitrilehydratase from Agrobacterium tumefaciens strain d3[J].Applied Microbiologyand Biotechnology,1998,49(1):89-95.}。
上述所报道的方法转化率或光学纯度仍达不到生产要求，且光学纯度与转化率难以兼得。
腈水合酶是一种多亚基的金属酶类，由α亚基、β亚基组成多聚体结构。根据腈水合酶中心所含金属离子的不同，可将其分为铁型和钴型两类。另外，腈水合酶的正确表达还需要组装蛋白(accessory protein)的参与。来源于Klebsiella oxytoca的腈水合酶属于钴型腈水合酶，能够催化多种腈类的水解反应。Vojtěch Vejvoda等报道Klebsiella oxytoca的腈水合酶能够水解二苯腈、3-甲苯腈、3-氯苯腈、4-氯苯腈、3-羟基苯腈、4-氨基苯腈、丙腈、丁腈、戊腈、2-甲基-3-丁腈[Vojtěch Vejvoda,Ludmila Martínková,Alicja B.Veselá,Kaplan,O.,Lutz-Wahl,S.,&Fischer,L.,et al.(2011).Biotransformation ofnitriles to hydroxamic acids via a nitrile hydratase–amidase cascadereaction.journal of molecular catalysis b enzymatic,71(1-2),51-55.]。EP1842907A1公开了Klebsiella oxytoca的腈水合酶能够水解2-苯基丙腈、扁桃腈、苄腈、苯基乙腈、2-苯基丁腈、2-氨基苯乙腈、丁腈、甲基丙烯腈，并且具有良好的S手性选择性。Fa-Mou Guo等报道Klebsiella oxytoca的腈水合酶能够水解丙腈、丁腈、异丁腈、丙烯腈、甲基丙烯腈、2-氰基吡啶、3-氰基吡啶、4-氰吡啶、苄腈、苯乙腈、α-甲基苯腈、扁桃腈、2，2-二甲基环丙腈、2-(4-氯苯基)3-甲基丁腈，同时对α-甲基苯腈、扁桃腈、2，2-二甲基环丙腈、2-(4-氯苯基)3-甲基丁腈表现出良好的S手性选择性[Guo,F.M.,Wu,J.P.,Yang,L.R.,&Xu,G..(2015).Overexpression of a nitrile hydratase from klebsiella oxytoca kctc1686in escherichia coli and its biochemical characterization.Biotechnologyand bioprocess engine.e.ring,20(6),995-1004.]。
本研究首次发现来源于Klebsiella oxytoca的腈水合酶NH3能够催化氰基酮洛芬的水解反应，并且e.e.值高达98％以上。在(S)-(+)-酰胺酮洛芬的生产中具有较大的应用潜力。但是，腈水合酶NH3在催化氰基酮洛芬到(S)-(+)-酰胺酮洛芬的过程中，酶活力和稳定性较低，使得催化反应中酶加入量较大，生产成本高。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本申请的发明人基于Klebsiella oxytoca来源的腈水合酶上，通过理性设计和定向进化的方法，筛选到多个比活和热稳定性提高的腈水合酶变体，这些变体比活为腈水合酶亲本比活的110.1％～526.2％，室温保存稳定性也大幅提高。
在本发明的第一方面，本发明提出了一种比活和热稳定性提高的腈水合酶变体。
根据本发明的实施例，所述腈水合酶变体包含一个α亚基和一个β亚基，α亚基氨基酸序列如SEQ ID NO:1所示或包含选自以下对应于SEQ ID NO:1的一个或多个位置的突变：第8位、第36位、第127位、第149位、第172位、第199位，β亚基氨基酸序列如SEQ ID NO:2所示或包含选自以下对应于SEQ ID NO:2的一个或多个位置的突变：第35位、第37位、第41位、第78位、第101位、第122位、第127位、第170位、第181位、第204位、第208位，且α亚基和β亚基的序列中至少有一个序列为突变后获得；
其中，所述腈水合酶变体的α亚基和β亚基的氨基酸位置分别通过于SEQ ID NO:1和SEQ ID NO:2的氨基酸序列相对来编号的。
根据本发明的实施例，所述腈水合酶变体的α亚基氨基酸序列包含选自以下对应于SEQ ID NO:1的一个或多个位置处的突变：第8位氨基酸突变成精氨酸，第36位氨基酸突变成半胱氨酸，第149位氨基酸突变成脯氨酸，第172位氨基酸突变成脯氨酸，第199位氨基酸突变成脯氨酸；所述腈水合酶变体的β亚基氨基酸序列包含选自以下对应于SEQ ID NO:2的一个或多个位置处的突变：第35位氨基酸突变成半胱氨酸，第37位氨基酸突变成丙氨酸、半胱氨酸、甘氨酸、亮氨酸、异亮氨酸、蛋氨酸、丝氨酸或色氨酸，第41位氨基酸突变成丙氨酸，第78位氨基酸突变成精氨酸，第101位氨基酸突变成精氨酸，第122位氨基酸突变成半胱氨酸，第127位氨基酸突变成丙氨酸，第170位氨基酸突变成半胱氨酸，第181位氨基酸突变成半胱氨酸，第204位氨基酸突变成精氨酸，第208位氨基酸突变成亮氨酸。
根据本发明的实施例，所述腈水合酶变体包括以下突变点至少两个的组合：
α亚基第8位、第36位、第149位、第199位，β亚基第35位、第78位、第101位、第122位、第127位、第170位、第181位、第208位。
根据本发明的实施例，所述腈水合酶变体的突变选自下组之一：
αL36C,βA35C；
βA122C,βV170C；
βV170C,βE181C；
αL36C,βA35C,βA122C,βV170C；
αL36C,βA35C,βV170C,βE181C；
αT8R/αA149P/αA172P/αN199P/βT78R/βA101R/βD127A/βT204R/βR208L/βF41A中任一个；
βF37A/βF37C/βF37G/βF37I/βF37L/βF37M/βF37S/βF37W中任一个；
αT8R,αN199P；
αT8R,βD127A；
αA172P,βT78R；
αA149P,βA101R；
βT78R,βA101R；
βR208L,βA101R。
根据本发明的实施例，所述腈水合酶变体的α亚基氨基酸序列选自SEQ ID NO:1、5、11、12、13、14、29所示的序列，β亚基氨基酸序列选自SEQ ID NO:2、6、7、8、9、10、15、16、17、18、19、20、21、22、23、24、25、26、27、28、30、31所示的序列。
根据本发明的的实施例，SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列来源于Klebsiella oxytoca。
在本发明的第二方面，本发明提供一种编码上述腈水合酶变体的核苷酸。
本发明还提供包含编码腈水合酶变体核苷酸和辅助蛋白(accessory protein)的核苷酸的表达载体及转化的宿主细胞。
根据本发明的实施例，所述辅助蛋白选自任意来源的腈水合酶的辅助蛋白，优选氨基酸序列如SEQ ID NO:32所示序列的辅助蛋白。
根据本发明的实施例，所述表达载体为pET28a载体；
任选地，所述的宿主细胞包括枯草芽孢杆菌、地衣芽孢杆菌、解淀粉芽孢杆菌、大肠杆菌、毕赤酵母、酿酒酵母、里氏木霉、黑曲霉和米曲霉。
在本发明的第三方面，本发明提供了腈水合酶变体制备右旋酰胺酮洛芬的方法。根据本发明的实施例，所述方法包括使氰基酮洛芬在本发明所述的腈水合酶变体的催化下进行酰胺化反应，以便获得右旋酰胺酮洛芬。
根据本发明实施例，上述制备右旋酰胺酮洛芬的方法可表示如下：
Figure BDA0003244884080000041
发明人发现，α亚基或β亚基上具有某些氨基酸位点突变的腈水合酶NH3变体对比腈水合酶NH3亲本比活和热稳定性有不同程度的提升，使用腈水合酶变体制备右旋酰胺酮洛芬，可大幅减少催化反应中酶加入量，有效降低生产成本，同时该酶贮藏稳定性也有大幅提升。
根据本发明的实施例，上述制备右旋酰胺酮洛芬的方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述腈水合酶变体α亚基氨基酸序列选自SEQ ID NO:1、5、11、12、13、14、29所示的序列，β亚基氨基酸序列选自SEQ ID NO:2、6、7、8、9、10、15、16、17、18、19、20、21、22、23、24、25、26、27、28、30、31所示的序列。
根据本发明的实施例，所述腈水合酶变体是以腈水合酶发酵菌体的湿菌体、喷干酶粉或冻干酶粉的形式提供的。
根据本发明的具体实施例，所述腈水合酶变体发酵菌体是通过如下方式获得的：将转化有质粒的大肠杆菌菌体进行活化处理，所述质粒中携带表达腈水合酶变体及辅助蛋白的核酸序列；将活化处理后菌液进行基础发酵培养，以便获得OD600为30～35的基础发酵培养菌液；将所述基础发酵培养菌液进行诱导培养，所述诱导培养是在IPTG和CoCl2存在的条件下进行的，所述诱导培养的时间为4～24h。
根据本发明的实施例，所述质粒是以包含腈水合酶NH3核苷酸序列为模板，通过定点突变将一个或者多个氨基酸突变或取代引入腈水合酶NH3序列中，得到的包含腈水合酶变体核酸序列的表达质粒。
根据本发明的实施例，所述辅助蛋白为腈水合酶NH3的辅助蛋白。
根据本发明的实施例，所述腈水合酶变体的湿菌体、喷干酶粉或冻干酶粉的投料量与氰基酮洛芬质量比为(0.15～4):1，如0.2:1,0.5:1,1:1。
根据本发明的实施例，湿菌体是通过将腈水合酶变体发酵液经过离心、洗涤得到的。
根据本发明的实施例，所述喷干酶粉是通过将腈水合酶变体发酵液经过喷雾干燥得到的。
根据本发明的实施例，所述冻干酶粉是通过将腈水合酶变体发酵液经过离心、洗涤、超低温预冷、冷冻干燥得到的。
根据本发明的实施例，所述腈水合酶变体是分散在有机溶剂中进行催化反应的；
任选地，所述有机溶剂选自甲苯、乙酸乙酯和乙酸异丙酯，优选乙酸乙酯；
任选地，所述有机溶剂浓度为5％～15％，优选10％。
根据本发明的实施例，所述氰基酮洛芬是溶在缓冲溶液中进行催化反应的；
任选地，所述缓冲溶液选自磷酸钠、磷酸钾和Tris-HCl，优选磷酸钠缓冲溶液；
任选地，所述缓冲溶液浓度为0.03～0.5M，pH为7.0-8.0。
根据本发明的实施例，所述催化反应温度为25℃～35℃，反应时间为6h～24h。
定义
在本发明中，将Klebsiella oxytoca来源的腈水合酶命名为NH3，如本文中所用的术语“腈水合酶亲本”和“腈水合酶NH3亲本”意指Klebsiella oxytoca来源的腈水合酶。
如本文所用的术语“腈水合酶”意指一类可以催化腈类物质转化成相应酰胺类物质的酶。
如本文所用的术语“腈水合酶变体”意指氨基酸序列经修饰的腈水合酶，其中该腈水合酶的一个或多个氨基酸残基已经被其它氨基酸残基置换，并且/或者其中一个或多个氨基酸残基已从该腈水合酶中删除，并且/或者其中一个或多个氨基酸残基已被添加和/或插入到该腈水合酶中。
如本文所用的术语“IPTG”意指异丙基-β-D-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside)，为常用分子生物学试剂，是β-半乳糖苷酶和β-半乳糖透酶的诱导剂。
在本文中，所用的术语“e.e.”意指对映体过量(enantiomeric excess)，表示一个对映体对另一个对映体的过量，e.e.值用来描述物质光学纯度。
在本文突变点的命名中，第一位字母：α代表α亚基，β代表β亚基；第二位字母为腈水合酶NH3亲本氨基酸；第三位开始直到倒数第二位的数字代表对应于亲本序列编号的位置；最后一位字母代表突变后变体的氨基酸；例如文中突变点“αL36C”，即表示该突变点是由腈水合酶NH3的α亚基氨基酸序列编号的第36位的亮氨酸突变成半胱氨酸所获得的。
附图说明
图1显示了pET28a-NH3质粒图谱；
图2显示了腈水合酶NH3蛋白的SDS-PAGE电泳图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
本发明提供了含腈水合酶变体的发酵菌体的制备方法，并利用发酵菌体进行酶催化反应，其中利用腈水合酶变体催化右旋酰胺酮洛芬合成的途径为：
Figure BDA0003244884080000061
下面将更加详细地描述本发明的实施例。
实施例1腈水合酶NH3亲本大肠杆菌表达菌株的构建
根据腈水合酶NH3亲本的α亚基氨基酸序列(SEQ ID NO:1)、β亚基氨基酸序列(SEQID NO:2)、辅助蛋白的氨基酸序列(SEQ ID NO:32)，分别按照大肠杆菌密码子进行优化得到核苷酸序列。α亚基核苷酸序列如SEQ ID NO:3所示，β亚基核苷酸序列如SEQ ID NO:4所示，辅助蛋白核苷酸序列如SEQ ID NO:33所示。在上述三条核苷酸序列前面均添加上RBS位点，根据本发明的实施例，选择的RBS序列为AAAGAGGAGAAA，然后将他们拼接起来得到一个串联基因(本实施例按照RBS-α亚基-RBS-β亚基-RBS-辅助蛋白的顺序拼接)，提交通用生物系统(安徽)有限公司进行合成。
然后通过基因重组技术，将合成串联基因克隆到pET28a载体的XbaI和HindIII位点之间，得到pET28a-NH3质粒，质粒图谱如图1所示。采用热激法将pET28a-NH3质粒转化BL21(DE3)感受态细胞，转化液涂布LB+Kan平板(10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，50μg/mL卡那霉素，15g/L琼脂粉),37℃过夜培养。得到的转化子经PCR和测序验证正确后，命名为BL21-NH3菌株。BL21-NH3菌株经LB培养基(10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，50μg/mL卡那霉素，15g/L琼脂粉)中37℃培养扩增后，加入终浓度15～20％的无菌甘油后-80℃冻存。
实施例2腈水合酶NH3亲本的诱导表达
将-80℃冻存的BL21-NH3菌株在LB+Kan平板划线分离单菌落。挑取转化平板上的单菌落至5mL液体LB+Kan(10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，50μg/mL卡那霉素)培养基中，37℃250g/min条件下过夜培养，约12h。
取过夜活化好的菌液，2mL转接至新鲜的200mL液体LB+Kan中，37℃250g/min条件下培养直至OD达到0.5-0.8。
取培养好的菌液，加入终浓度为1mM的IPTG以及终浓度为1mM的CoCl2，30℃250g/min条件下培养5h。
诱导结束后，收集全部菌液在4000g/min 4℃条件下离心收集菌体。取适当菌体进行超声波破壁处理，10000g/min、4℃离心10min，取上清液通过SDS-PAGE检测蛋白表达情况，如图2所示。
实施例3腈水合酶NH3变体大肠杆菌表达菌株的构建及其诱导表达
使用本领域已知的分子生物学技术，以pET28a-NH3质粒为模板，采用TAKARA的定点突变试剂盒TaKaRa MutanBEST Kit(Code NO.R401)，将一个或者多个氨基酸突变(取代)引入亲本NH3中，得到各种NH3变体表达质粒。采用热激法分别将NH3变体表达质粒转化BL21(DE3)感受态细胞，转化液涂布LB+Kan平板(10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，50μg/mL卡那霉素，15g/L琼脂粉)，37℃过夜培养。得到的转化子经PCR和测序验证正确后，再经LB培养基(10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，50μg/mL卡那霉素，15g/L琼脂粉)中37℃培养扩增，加入终浓度15～20％的无菌甘油后-80℃冻存。构建的腈水合酶NH3变体见表1所示。
按照实施例2同样的方式分别对腈水合酶NH3变体进行诱导表达。
实施例4腈水合酶NH3亲本和NH3变体的酶活、稳定性检测以及比活计算。
酶活检测和比活计算：按照实施例2和实施例3的方式分别制备腈水合酶NH3亲本和NH3变体菌液，分别将菌液4000g 30min 4℃离心收集湿菌体，采用高压匀浆机在压力800-1000bar，4℃下对菌体进行破壁。破壁液在4000g 30min 4℃离心收集上清液。用柠檬酸调节上清液pH 4.0-4.5，过滤，测电导<10mS/CM。过滤液经离子交换层析(UniGel 80-SP填料)和疏水层析柱(Monomix MC30-Hic Butyl填料)，分别得到纯度98％以上的腈水合酶NH3亲本和NH3变体蛋白。
分别加入适量纯化后的腈水合酶NH3亲本和NH3变体蛋白于100mL反应瓶中，分别加入40mL0.1mol/L pH＝7.5磷酸缓冲液搅拌均匀，于30℃下保温10min。分别称取2.4g氰基酮洛芬于10mL EP管中，加入4mL乙酸乙酯旋涡10min至充分溶解，然后滴加至反应瓶中，控制滴加时间为1min左右。30℃保温反应30min，分别加入20mL二氯甲烷萃取，取萃取相50uL，30～40℃干燥，HPLC检测。HPLC条件为：将干燥样品采用适量乙腈溶解，采用Welch Topsil5u C18 100A，4.6×150mm色谱柱，控制25℃柱温，以2％磷酸盐缓冲液-43％ACN-55％H2O流动相等度洗脱25min，255nm波长下进行检测。酶活的定义为30℃下1分钟内转化1微摩尔底物所需的酶量为一个活力单位(U)。按照说明书采用TAKARA Bradford Protein Assay Kit(Code NO.T9310A)分别对纯化的NH3亲本和NH3变体进行蛋白浓度测定。然后通过公式(比活＝酶活/酶蛋白浓度)计算比活。
稳定性检测：将纯化得到的NH3亲本和NH3变体蛋白溶液，通过透析将溶剂置换为0.1M pH＝7.5磷酸钠缓冲液，测量初始比活。然后分别将其在25℃放置3天后测量放置后比活。通过公式(剩余比活百分比＝25℃放置3天后比活/初始比活*100％)计算剩余比活百分比。剩余比活百分比越高表明稳定性越好，或反之。
腈水合酶NH3亲本和NH3变体的相对比活、稳定性数据见表1所示。NH3变体相比NH3亲本，比活和/或稳定性均有不同程度的提高。
表1 NH3亲本和NH3变体编号、突变位点、比活、稳定性和氨基酸序列SEQ ID NO
Figure BDA0003244884080000081
Figure BDA0003244884080000091
*突变位点说明：第一位字母为NH3亲本氨基酸；第二位：α代表α亚基，β代表β亚基；第三位开始直到倒数第二位的数字代表对应于亲本序列编号的位置；最后一位字母代表突变后变体的氨基酸。多个突变位点以逗号分隔。
实施例5腈水合酶NH3亲本和NH3变体的50L罐发酵
分别取实施例1的腈水合酶NH3亲本和实施例3的腈水合酶NH3变体在-80℃冻存的菌株，进行过夜活化。然后分别取1mL转接至新鲜的1000mL液体LB+Kan培养基中，37℃250g/min条件下培养约10h后，分别接种50L发酵罐(发酵培养基：一水葡萄糖121g，酵母浸粉500g，酵母蛋白胨250g，NaCl 100g，Na2HPO4 25g，MgSO4.7H2O 20g，KH2PO4 100g，(NH4)2SO450g，一水柠檬酸55g，NaOH 75g，GPE消泡剂25mL。118℃灭菌30min，灭菌后体积约25L)。然后分别按照下述工艺进行发酵。
基础培养阶段：控制培养温度37.0℃，通气量1:1vvm，通过搅拌和通气调节发酵液溶氧水平高于50％，罐压40～70Kpa。
补葡萄糖阶段：发酵液pH值反弹至7.15时开始补加葡萄糖溶液，初始补糖速率6.6g/L.h，每0.5h提升0.7g/L.h，葡萄糖补加速率提升至10.0g/L.h，维持此速率补加至诱导阶段。补葡萄糖阶段培养温度34.0℃，通气量1:1.3vvm，通过搅拌和通气调节发酵液溶氧水平高于30％。
诱导阶段：发酵液OD600值在30～35时，开始进行诱导，诱导时一次性打入终浓度1mM的IPTG溶液和终浓度1mM的CoCl2溶液。诱导前降低培养温度至30℃，诱导后葡萄糖补加速率3.3～3.6g/L.h，发酵液溶氧水平20％～50％。维持该速率直至发酵结束，诱导约15h。
发酵结束后得到的发酵液立即制成湿菌体和酶粉，4℃保存。
实施例6腈水合酶NH3亲本和NH3变体的湿菌体和酶粉的制备
湿菌体的制备：分别利用实施例5中获得的腈水合酶NH3亲本和腈水合酶NH3变体发酵液500mL，于4000g 30min 4℃离心收集湿菌体。将菌体用生理盐水洗涤菌体后再次按照上述方法离心移去多余生理盐水，得到获得腈水合酶湿菌体。湿菌体置于4℃保存。
喷干酶粉的制备：分别利用实施例5中获得的腈水合酶NH3亲本和腈水合酶NH3变体发酵液500mL，于喷雾干燥箱(设置参数为进口温度170℃、出口温度80℃、进料流量700mL/h)中进行喷干，获得腈水合酶喷干酶粉。喷干酶粉置于4℃保存。
冻干酶粉的制备：分别利用实施例5中获得的腈水合酶NH3亲本和腈水合酶NH3变体发酵液500mL，于4000g 30min 4℃离心保留菌体，将菌体用生理盐水洗涤菌体后再次按照上述方法离心移去多余生理盐水，加入5％蔗糖溶液做保护剂。重悬菌体并置于冷冻舱里的隔板上，在超低温冰箱(-70℃)中预冷12h。随后在冷冻干燥剂中冷冻干燥48h即得冻干酶粉。冻干酶粉置于4℃保存。
实施例7腈水合酶NH3亲本和NH3变体的湿菌体催化氰基酮洛芬合成右旋酰胺酮洛芬
分别取实施例6中的腈水合酶NH3亲本和腈水合酶NH3变体湿菌体2.0～20g均匀分散到100mL 0.1MpH＝7.5磷酸钠缓冲液中，30℃下温浴10min，再将6g氰基酮洛芬底物溶于10mL乙酸乙酯滴加入反应瓶，保温反应。反应24h取样2mL，加4mL二氯甲烷萃取，取萃取相50uL，干燥，HPLC检测各组分含量。HPLC条件如下：
(1)样品处理：干燥的萃取相采用乙腈溶解，配制成1mg/ml。
(2)检测条件：采用HPLC正相，色谱柱(Welch Topsil 5u C18 100A，4.6×150mm)，检测波长：250nm，柱温：25℃，进样量5ul，流动相：磷酸盐缓冲液(取磷酸二氢钾68.0g，加水溶解并稀释至1000ml，用磷酸调节pH值至3.5±0.1)-乙睛-水(2：43：55)，洗脱条件：等度洗脱，运行时间：25min。
(3)e.e.值计算：e.e.＝(右旋KPA摩尔含量-左旋KPA摩尔含量)/(右旋KPA摩尔含量+左旋摩尔KPA含量)*100％。
(4)转化率计算：转化率＝KPA摩尔含量/初始KPN摩尔含量*100％。
(5)其中，KPN为底物氰基酮洛芬，KPA为产物酰胺酮洛芬。
最终，摸索腈水合酶NH3亲本和每个腈水合酶NH3变体转化率达到45％需要加入的湿菌体量，并检测此时的e.e.值。结果见表2所示。
结果表明NH3变体相比NH3亲本，反应相同时间达到45％的转化率时，NH3变体使用的湿菌体用量均降低，并且e.e.值仍保持在98％以上，能够有效降低生产成本。
实施例8腈水合酶NH3亲本和NH3变体的喷干粉催化氰基酮洛芬合成右旋酰胺酮洛芬
分别取实施例6中的腈水合酶NH3亲本和腈水合酶NH3变体喷干粉0.2～10g均匀分散到100mL 0.1MpH＝7.5磷酸钠缓冲液中，30℃下温浴10min，再将6g氰基酮洛芬底物溶于10mL乙酸乙酯滴加入反应瓶，保温反应。反应24h取样2mL，加4mL二氯甲烷萃取，取萃取相50uL，干燥。按照实施例7的HPLC方法进行检测。最终摸索腈水合酶NH3亲本和每个腈水合酶NH3变体转化率达到45％需要加入的湿菌体量，并按照实施例7的方法计算此时的e.e.值。结果见表2所示。
结果表明NH3变体相比NH3亲本，反应相同时间达到45％的转化率时，NH3变体使用的喷干粉用量大大降低，并且e.e.值仍保持在98％以上，能够有效降低生产成本。
实施例9腈水合酶NH3亲本和NH3变体的冻干粉催化氰基酮洛芬合成右旋酰胺酮洛芬
分别取实施例6中的腈水合酶NH3亲本和腈水合酶NH3变体冻干粉0.2～2g均匀分散到100mL 0.1MpH＝7.5磷酸钠缓冲液中，30℃下温浴10min，再将6g氰基酮洛芬底物溶于10mL乙酸乙酯滴加入反应瓶，保温反应。反应24h取样2mL，加4mL二氯甲烷萃取，取萃取相50uL，干燥。按照实施例7的HPLC方法进行检测。最终摸索腈水合酶NH3亲本和每个腈水合酶NH3变体转化率达到45％需要加入的湿菌体量，并按照实施例7的方法计算此时的e.e.值。结果见表2所示。
结果表明NH3变体相比NH3亲本，反应相同时间达到45％的转化率时，NH3变体使用的冻干粉用量均降低，并且e.e.值仍保持在98％以上，能够有效降低生产成本。
表2NH3亲本和NH3变体催化氰基酮洛芬湿菌体、喷干粉、冻干粉用量和对应e.e.值
Figure BDA0003244884080000111
Figure BDA0003244884080000121
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
序列表
>SEQ ID NO 1
MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH
>SEQ ID NO 2
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 3
ATGAGCCATGATCATGATCATACCGAACCGCCGACCGAAATTGCCCTGCGTGTTAAAGCCCTGGAAAGCCTGCTGACCGAAAAAGGCCTGGTTGATCCGGCCGCCCTGGATGAACTGGTTGATACCTATGAAAATCGCATTGGTCCGCGTAATGGTGCCCTGGTTGTTGCAAAAGCCTGGACCGATCCGGCATATAAACAGCGTCTGCTGACCAATGCAACCGAAGCAATTGCAGAACTGGGTTTTAGCGGTGTTCAGGGCGAAGATATGCTGGTGGTTGAAAATAGTCCGACCGTGCATAATATGACCGTGTGTACCCTGTGCAGTTGTTATCCGTGGCCGACCCTGGGCCTGCCGCCTGCATGGTATAAAAGCGCCCCGTATCGCAGTCGCGTTGTTATTGATCCGCGTGGCGTTCTGGCCGAATTTGGTGTGAGTGTTCCGGCAGATAAAGAAGTGCGCGTTTGGGATAGTAGCGCCGAACTGCGCTATCTGGTGCTGCCGGAACGTCCGGCAGGCACCGAAGGCTGGAGCGAAGAACAGCTGGTGGAACTGGTTACCCGTGATAGTATGATTGGCACCGGCTTTCCGAAAAATCCGGCAGATCTGCATTAA
>SEQ ID NO 4
ATGAACGGTGTGCATGATCTGGGTGGTATGCATGGTCTGGGTCCGATTGCCCCGCCGGCAGATGAACCGGTGTTTGCACATCAGTGGGAACGCCGCATTTTTGCCCTGTTTGTGCCGCTGTTTGGTGGTGGTCATTTTAATGTGGATCAGTTTCGCCATGCAATTGAACGTATGGATCCGGCCCATTATCTGCAGGGCACCTATTATGAACATTGGCTGCATGCCTTTGAAACCCTGCTGATTGAAGGTGGCGCAATTAGTCGTGCCGAACTGGATGCACGTATTAAGCAGATTGGTGGTGCACAGATTATGGCAGTGGTTACCCGCGATATGATTGAACCGATTGTTCGTACCGGTGCCAGTGCACGTGTGGCCGCAGATGTTGCCGCCCGTTTTAAAGTTGGCGATACCGTTCGTGCAAAAAATATTAATCCGACCACCCATACCCGCCTGCCGCGCTATGTTCGCGGCCGTGTTGGTACCATTGAAATTGATCATGGCGTTTTTGTGACCCCGGATACCGTGGCCCACGGTAAAGGTGAACATCCGCAGCATGTGTATTGCGTGCGCTTTGCCGCAGTGGAACTGTGGGGCAGCGATGTGAGTGGTACCGATAATGTTCGCATTGATCTGTGGGATGATTATCTGGAAAAAGCCTAA
>SEQ ID NO 5
MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAACDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH
>SEQ ID NO 6
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFCLFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 7
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASCRVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFCTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 8
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFCTPDTVAHGKGCHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 9
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFCLFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASCRVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFCTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 10
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFCLFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFCTPDTVAHGKGCHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 11
MSHDHDHREPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH
>SEQ ID NO 12
MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPPDKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH
>SEQ ID NO 13
MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPPGTEGWSEEQLVELVTRDSMIGTGFPKNPADLH
>SEQ ID NO 14
MSHDHDHTEPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKPPADLH
>SEQ ID NO 15
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFERLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 16
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGRQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 17
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAAAVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 18
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGRDNVRIDLWDDYLEKA
>SEQ ID NO 19
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVLIDLWDDYLEKA
>SEQ ID NO 20
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALAVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 21
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALCVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 22
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALGVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 23
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALIVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 24
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALLVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 25
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALMVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 26
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALSVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 27
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALWVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 28
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLAGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGAQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 29
MSHDHDHREPPTEIALRVKALESLLTEKGLVDPAALDELVDTYENRIGPRNGALVVAKAWTDPAYKQRLLTNATEAIAELGFSGVQGEDMLVVENSPTVHNMTVCTLCSCYPWPTLGLPPAWYKSAPYRSRVVIDPRGVLAEFGVSVPADKEVRVWDSSAELRYLVLPERPAGTEGWSEEQLVELVTRDSMIGTGFPKPPADLH
>SEQ ID NO 30
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFERLLIEGGAISRAELDARIKQIGGRQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVRIDLWDDYLEKA
>SEQ ID NO 31
MNGVHDLGGMHGLGPIAPPADEPVFAHQWERRIFALFVPLFGGGHFNVDQFRHAIERMDPAHYLQGTYYEHWLHAFETLLIEGGAISRAELDARIKQIGGRQIMAVVTRDMIEPIVRTGASARVAADVAARFKVGDTVRAKNINPTTHTRLPRYVRGRVGTIEIDHGVFVTPDTVAHGKGEHPQHVYCVRFAAVELWGSDVSGTDNVLIDLWDDYLEKA
>SEQ ID NO 32
MAETIDYELVNLPRDDEGPVFEEPWQAQVFSLTVHLHKAGHFTWPQWVQTFSREIGRSPALPGESVNAAYYRQWAAALEHMVAKIGLAGLVDVAGRTEEWRQAYINTPHGHPVLLANAACPPAHDHHHHVPERSPVAISPACPV
>SEQ ID NO 33
ATGGCCGAAACCATTGATTATGAACTGGTGAATCTGCCGCGCGATGATGAAGGTCCGGTTTTTGAAGAACCGTGGCAGGCACAGGTTTTTAGCCTGACCGTGCATCTGCATAAAGCCGGCCATTTTACCTGGCCGCAGTGGGTTCAGACCTTTAGTCGTGAAATTGGCCGTAGTCCGGCACTGCCGGGTGAAAGCGTTAATGCCGCATATTATCGCCAGTGGGCAGCCGCCCTGGAACATATGGTTGCCAAAATTGGTCTGGCAGGCCTGGTTGATGTTGCAGGCCGCACCGAAGAATGGCGTCAGGCCTATATTAATACCCCGCATGGTCATCCGGTGCTGCTGGCAAATGCAGCATGTCCGCCGGCACATGATCATCATCATCATGTTCCGGAACGTAGTCCGGTGGCCATTAGTCCGGCCTGTCCGGTGTAA
SEQUENCE LISTING
<110> 宜昌东阳光生化制药有限公司
<120> 比活和热稳定性提高的腈水合酶变体及其应用
<130> PP1175CN
<160> 33
<170> PatentIn version 3.5
<210> 1
<211> 204
<212> PRT
<213> Klebsiella oxytoca
<400> 1
Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu
1 5 10 15
Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp
20 25 30
Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly
35 40 45
Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala
50 55 60
Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu
65 70 75 80
Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser
85 90 95
Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro
100 105 110
Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr
115 120 125
Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly
130 135 140
Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala
145 150 155 160
Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly
165 170 175
Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile
180 185 190
Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His
195 200
<210> 2
<211> 219
<212> PRT
<213> Klebsiella oxytoca
<400> 2
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 3
<211> 615
<212> DNA
<213> 人工序列
<400> 3
atgagccatg atcatgatca taccgaaccg ccgaccgaaa ttgccctgcg tgttaaagcc 60
ctggaaagcc tgctgaccga aaaaggcctg gttgatccgg ccgccctgga tgaactggtt 120
gatacctatg aaaatcgcat tggtccgcgt aatggtgccc tggttgttgc aaaagcctgg 180
accgatccgg catataaaca gcgtctgctg accaatgcaa ccgaagcaat tgcagaactg 240
ggttttagcg gtgttcaggg cgaagatatg ctggtggttg aaaatagtcc gaccgtgcat 300
aatatgaccg tgtgtaccct gtgcagttgt tatccgtggc cgaccctggg cctgccgcct 360
gcatggtata aaagcgcccc gtatcgcagt cgcgttgtta ttgatccgcg tggcgttctg 420
gccgaatttg gtgtgagtgt tccggcagat aaagaagtgc gcgtttggga tagtagcgcc 480
gaactgcgct atctggtgct gccggaacgt ccggcaggca ccgaaggctg gagcgaagaa 540
cagctggtgg aactggttac ccgtgatagt atgattggca ccggctttcc gaaaaatccg 600
gcagatctgc attaa 615
<210> 4
<211> 660
<212> DNA
<213> 人工序列
<400> 4
atgaacggtg tgcatgatct gggtggtatg catggtctgg gtccgattgc cccgccggca 60
gatgaaccgg tgtttgcaca tcagtgggaa cgccgcattt ttgccctgtt tgtgccgctg 120
tttggtggtg gtcattttaa tgtggatcag tttcgccatg caattgaacg tatggatccg 180
gcccattatc tgcagggcac ctattatgaa cattggctgc atgcctttga aaccctgctg 240
attgaaggtg gcgcaattag tcgtgccgaa ctggatgcac gtattaagca gattggtggt 300
gcacagatta tggcagtggt tacccgcgat atgattgaac cgattgttcg taccggtgcc 360
agtgcacgtg tggccgcaga tgttgccgcc cgttttaaag ttggcgatac cgttcgtgca 420
aaaaatatta atccgaccac ccatacccgc ctgccgcgct atgttcgcgg ccgtgttggt 480
accattgaaa ttgatcatgg cgtttttgtg accccggata ccgtggccca cggtaaaggt 540
gaacatccgc agcatgtgta ttgcgtgcgc tttgccgcag tggaactgtg gggcagcgat 600
gtgagtggta ccgataatgt tcgcattgat ctgtgggatg attatctgga aaaagcctaa 660
<210> 5
<211> 204
<212> PRT
<213> 人工序列
<400> 5
Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu
1 5 10 15
Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp
20 25 30
Pro Ala Ala Cys Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly
35 40 45
Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala
50 55 60
Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu
65 70 75 80
Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser
85 90 95
Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro
100 105 110
Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr
115 120 125
Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly
130 135 140
Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala
145 150 155 160
Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly
165 170 175
Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile
180 185 190
Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His
195 200
<210> 6
<211> 219
<212> PRT
<213> 人工序列
<400> 6
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Cys Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 7
<211> 219
<212> PRT
<213> 人工序列
<400> 7
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Cys Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Cys Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 8
<211> 219
<212> PRT
<213> 人工序列
<400> 8
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Cys Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Cys His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 9
<211> 219
<212> PRT
<213> 人工序列
<400> 9
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Cys Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Cys Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Cys Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 10
<211> 219
<212> PRT
<213> 人工序列
<400> 10
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Cys Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Cys Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Cys His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 11
<211> 204
<212> PRT
<213> 人工序列
<400> 11
Met Ser His Asp His Asp His Arg Glu Pro Pro Thr Glu Ile Ala Leu
1 5 10 15
Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp
20 25 30
Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly
35 40 45
Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala
50 55 60
Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu
65 70 75 80
Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser
85 90 95
Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro
100 105 110
Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr
115 120 125
Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly
130 135 140
Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala
145 150 155 160
Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly
165 170 175
Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile
180 185 190
Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His
195 200
<210> 12
<211> 204
<212> PRT
<213> 人工序列
<400> 12
Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu
1 5 10 15
Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp
20 25 30
Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly
35 40 45
Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala
50 55 60
Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu
65 70 75 80
Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser
85 90 95
Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro
100 105 110
Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr
115 120 125
Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly
130 135 140
Val Ser Val Pro Pro Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala
145 150 155 160
Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly
165 170 175
Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile
180 185 190
Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His
195 200
<210> 13
<211> 204
<212> PRT
<213> 人工序列
<400> 13
Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu
1 5 10 15
Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp
20 25 30
Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly
35 40 45
Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala
50 55 60
Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu
65 70 75 80
Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser
85 90 95
Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro
100 105 110
Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr
115 120 125
Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly
130 135 140
Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala
145 150 155 160
Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Pro Gly Thr Glu Gly
165 170 175
Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile
180 185 190
Gly Thr Gly Phe Pro Lys Asn Pro Ala Asp Leu His
195 200
<210> 14
<211> 204
<212> PRT
<213> 人工序列
<400> 14
Met Ser His Asp His Asp His Thr Glu Pro Pro Thr Glu Ile Ala Leu
1 5 10 15
Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp
20 25 30
Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly
35 40 45
Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala
50 55 60
Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu
65 70 75 80
Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser
85 90 95
Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro
100 105 110
Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr
115 120 125
Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly
130 135 140
Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala
145 150 155 160
Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly
165 170 175
Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile
180 185 190
Gly Thr Gly Phe Pro Lys Pro Pro Ala Asp Leu His
195 200
<210> 15
<211> 219
<212> PRT
<213> 人工序列
<400> 15
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Arg Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 16
<211> 219
<212> PRT
<213> 人工序列
<400> 16
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Arg Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 17
<211> 219
<212> PRT
<213> 人工序列
<400> 17
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Ala Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 18
<211> 219
<212> PRT
<213> 人工序列
<400> 18
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Arg Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 19
<211> 219
<212> PRT
<213> 人工序列
<400> 19
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Leu
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 20
<211> 219
<212> PRT
<213> 人工序列
<400> 20
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Ala Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 21
<211> 219
<212> PRT
<213> 人工序列
<400> 21
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Cys Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 22
<211> 219
<212> PRT
<213> 人工序列
<400> 22
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Gly Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 23
<211> 219
<212> PRT
<213> 人工序列
<400> 23
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Ile Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 24
<211> 219
<212> PRT
<213> 人工序列
<400> 24
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Leu Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 25
<211> 219
<212> PRT
<213> 人工序列
<400> 25
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Met Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 26
<211> 219
<212> PRT
<213> 人工序列
<400> 26
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Ser Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 27
<211> 219
<212> PRT
<213> 人工序列
<400> 27
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Trp Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 28
<211> 219
<212> PRT
<213> 人工序列
<400> 28
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Ala Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Ala Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 29
<211> 204
<212> PRT
<213> 人工序列
<400> 29
Met Ser His Asp His Asp His Arg Glu Pro Pro Thr Glu Ile Ala Leu
1 5 10 15
Arg Val Lys Ala Leu Glu Ser Leu Leu Thr Glu Lys Gly Leu Val Asp
20 25 30
Pro Ala Ala Leu Asp Glu Leu Val Asp Thr Tyr Glu Asn Arg Ile Gly
35 40 45
Pro Arg Asn Gly Ala Leu Val Val Ala Lys Ala Trp Thr Asp Pro Ala
50 55 60
Tyr Lys Gln Arg Leu Leu Thr Asn Ala Thr Glu Ala Ile Ala Glu Leu
65 70 75 80
Gly Phe Ser Gly Val Gln Gly Glu Asp Met Leu Val Val Glu Asn Ser
85 90 95
Pro Thr Val His Asn Met Thr Val Cys Thr Leu Cys Ser Cys Tyr Pro
100 105 110
Trp Pro Thr Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Ala Pro Tyr
115 120 125
Arg Ser Arg Val Val Ile Asp Pro Arg Gly Val Leu Ala Glu Phe Gly
130 135 140
Val Ser Val Pro Ala Asp Lys Glu Val Arg Val Trp Asp Ser Ser Ala
145 150 155 160
Glu Leu Arg Tyr Leu Val Leu Pro Glu Arg Pro Ala Gly Thr Glu Gly
165 170 175
Trp Ser Glu Glu Gln Leu Val Glu Leu Val Thr Arg Asp Ser Met Ile
180 185 190
Gly Thr Gly Phe Pro Lys Pro Pro Ala Asp Leu His
195 200
<210> 30
<211> 219
<212> PRT
<213> 人工序列
<400> 30
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Arg Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Arg Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Arg
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 31
<211> 219
<212> PRT
<213> 人工序列
<400> 31
Met Asn Gly Val His Asp Leu Gly Gly Met His Gly Leu Gly Pro Ile
1 5 10 15
Ala Pro Pro Ala Asp Glu Pro Val Phe Ala His Gln Trp Glu Arg Arg
20 25 30
Ile Phe Ala Leu Phe Val Pro Leu Phe Gly Gly Gly His Phe Asn Val
35 40 45
Asp Gln Phe Arg His Ala Ile Glu Arg Met Asp Pro Ala His Tyr Leu
50 55 60
Gln Gly Thr Tyr Tyr Glu His Trp Leu His Ala Phe Glu Thr Leu Leu
65 70 75 80
Ile Glu Gly Gly Ala Ile Ser Arg Ala Glu Leu Asp Ala Arg Ile Lys
85 90 95
Gln Ile Gly Gly Arg Gln Ile Met Ala Val Val Thr Arg Asp Met Ile
100 105 110
Glu Pro Ile Val Arg Thr Gly Ala Ser Ala Arg Val Ala Ala Asp Val
115 120 125
Ala Ala Arg Phe Lys Val Gly Asp Thr Val Arg Ala Lys Asn Ile Asn
130 135 140
Pro Thr Thr His Thr Arg Leu Pro Arg Tyr Val Arg Gly Arg Val Gly
145 150 155 160
Thr Ile Glu Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Val Ala
165 170 175
His Gly Lys Gly Glu His Pro Gln His Val Tyr Cys Val Arg Phe Ala
180 185 190
Ala Val Glu Leu Trp Gly Ser Asp Val Ser Gly Thr Asp Asn Val Leu
195 200 205
Ile Asp Leu Trp Asp Asp Tyr Leu Glu Lys Ala
210 215
<210> 32
<211> 144
<212> PRT
<213> 人工序列
<400> 32
Met Ala Glu Thr Ile Asp Tyr Glu Leu Val Asn Leu Pro Arg Asp Asp
1 5 10 15
Glu Gly Pro Val Phe Glu Glu Pro Trp Gln Ala Gln Val Phe Ser Leu
20 25 30
Thr Val His Leu His Lys Ala Gly His Phe Thr Trp Pro Gln Trp Val
35 40 45
Gln Thr Phe Ser Arg Glu Ile Gly Arg Ser Pro Ala Leu Pro Gly Glu
50 55 60
Ser Val Asn Ala Ala Tyr Tyr Arg Gln Trp Ala Ala Ala Leu Glu His
65 70 75 80
Met Val Ala Lys Ile Gly Leu Ala Gly Leu Val Asp Val Ala Gly Arg
85 90 95
Thr Glu Glu Trp Arg Gln Ala Tyr Ile Asn Thr Pro His Gly His Pro
100 105 110
Val Leu Leu Ala Asn Ala Ala Cys Pro Pro Ala His Asp His His His
115 120 125
His Val Pro Glu Arg Ser Pro Val Ala Ile Ser Pro Ala Cys Pro Val
130 135 140
<210> 33
<211> 435
<212> DNA
<213> 人工序列
<400> 33
atggccgaaa ccattgatta tgaactggtg aatctgccgc gcgatgatga aggtccggtt 60
tttgaagaac cgtggcaggc acaggttttt agcctgaccg tgcatctgca taaagccggc 120
cattttacct ggccgcagtg ggttcagacc tttagtcgtg aaattggccg tagtccggca 180
ctgccgggtg aaagcgttaa tgccgcatat tatcgccagt gggcagccgc cctggaacat 240
atggttgcca aaattggtct ggcaggcctg gttgatgttg caggccgcac cgaagaatgg 300
cgtcaggcct atattaatac cccgcatggt catccggtgc tgctggcaaa tgcagcatgt 360
ccgccggcac atgatcatca tcatcatgtt ccggaacgta gtccggtggc cattagtccg 420
gcctgtccgg tgtaa 435

Claims (10)

1.一种比活和热稳定性提高的腈水合酶变体，所述腈水合酶变体包含一个α亚基和一个β亚基，其特征在于，α亚基氨基酸序列如SEQ ID NO:1所示或包含选自以下对应于SEQ IDNO:1的一个或多个位置的突变：第8位、第36位、第127位、第149位、第172位、第199位；β亚基氨基酸序列如SEQ ID NO:2所示或包含选自以下对应于SEQ ID NO:2的一个或多个位置的突变：第35位、第37位、第41位、第78位、第101位、第122位、第127位、第170位、第181位、第204位、第208位，且α亚基和β亚基的序列中至少有一个序列为突变后获得。
2.如权利要求1所述腈水合酶变体，其特征在于，所述α亚基氨基酸序列包含选自以下对应于SEQ ID NO:1的一个或多个位置处的突变：第8位氨基酸突变成精氨酸，第36位氨基酸突变成半胱氨酸，第149位氨基酸突变成脯氨酸，第172位氨基酸突变成脯氨酸，第199位氨基酸突变成脯氨酸；所述β亚基氨基酸序列包含选自以下对应于SEQ ID NO:2的一个或多个位置处的突变：第35位氨基酸突变成半胱氨酸，第37位氨基酸突变成丙氨酸、半胱氨酸、甘氨酸、亮氨酸、异亮氨酸、蛋氨酸、丝氨酸或色氨酸，第41位氨基酸突变成丙氨酸，第78位氨基酸突变成精氨酸，第101位氨基酸突变成精氨酸，第122位氨基酸突变成半胱氨酸，第127位氨基酸突变成丙氨酸，第170位氨基酸突变成半胱氨酸，第181位氨基酸突变成半胱氨酸，第204位氨基酸突变成精氨酸，第208位氨基酸突变成亮氨酸。
3.如权利要求1或2所述腈水合酶变体，其特征在于，包括以下突变位点至少两个的组合：
α亚基第8位、第36位、第149位、第199位，β亚基第35位、第78位、第101位、第122位、第127位、第170位、第181位、第208位。
4.如权利要求1-3任一项所述腈水合酶变体，其特征在于，所述突变选自下组之一：
αL36C,βA35C；
βA122C,βV170C；
βV170C,βE181C；
αL36C,βA35C,βA122C,βV170C；
αL36C,βA35C,βV170C,βE181C；
αT8R/αA149P/αA172P/αN199P/βT78R/βA101R/βD127A/βT204R/βR208L/βF41A中任一个；
βF37A/βF37C/βF37G/βF37I/βF37L/βF37M/βF37S/βF37W中任一个；
αT8R,αN199P；
αT8R,βD127A；
αA172P,βT78R；
αA149P,βA101R；
βT78R,βA101R；
βR208L,βA101R。
5.如权利要求1-4任一项所述腈水合酶变体，其特征在于，所述腈水合酶变体的α亚基氨基酸序列选自SEQ ID NO:1、5、11、12、13、14、29所示的序列，β亚基氨基酸序列选自SEQID NO:2、6、7、8、9、10、15、16、17、18、19、20、21、22、23、24、25、26、27、28、30、31所示的序列。
6.编码权利要求1-5任一项腈水合酶变体的核苷酸。
7.包含权利要求6所述核苷酸的表达载体，该载体进一步包含辅助蛋白(accessoryprotein)的核苷酸，所述辅助蛋白选自任意来源的腈水合酶的辅助蛋白，优选地，所述辅助蛋白氨基酸序列如SEQ ID NO:32所示。
8.包含权利要求7所述表达载体的宿主细胞。
9.如权利要求8所述的宿主细胞，包括枯草芽孢杆菌、地衣芽孢杆菌、解淀粉芽孢杆菌、大肠杆菌、毕赤酵母、酿酒酵母、里氏木霉、黑曲霉和米曲霉。
10.采用权利要求1-5任一项所述的腈水合酶变体制备右旋酰胺酮洛芬的方法，其特征在于，使氰基酮洛芬在腈水合酶变体的催化下进行酰胺化反应，以便获得右旋酰胺酮洛芬。
CN202111030266.7A 2020-09-04 2021-09-03 比活和热稳定性提高的腈水合酶变体及其应用 Pending CN114134133A (zh)

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