CN114292828A - 具有提高的温度稳定性的四聚体α/β水解酶变体及其使用和生产方法 - Google Patents
具有提高的温度稳定性的四聚体α/β水解酶变体及其使用和生产方法 Download PDFInfo
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Abstract
本发明涉及相比于亲代α/β水解酶而言性能得到改进的α/β水解酶变体,例如具有提高的温度稳定性,本发明还涉及使用和生产该变体的方法,包括指导由同源二聚体向同源四聚体的四级结构形成的方法。
Description
本申请包括计算机可读形式的序列表,其通过引用包括在本文中。
技术领域
本发明涉及与SEQ ID NO:1的亲代α/β水解酶相比性能得到改进的α/β水解酶变体,例如具有提高的温度稳定性,本发明还涉及编码该变体的多核苷酸、使用和生产该变体的方法,包括指导由同源二聚体到同源四聚体的四级结构形成的方法。尤其,本发明涉及使用该变体降解玉米赤霉烯酮(ZEN)的方法。
背景技术
真菌毒素是丝状真菌产生的次级代谢产物。真菌毒素的一种重要的代表物是玉米赤霉烯酮(ZEN),其以前被称为F-2毒素,由多种镰刀菌产生,在世界各地都能找到。这些真菌感染栽培植物,尤其是诸如各种谷物,其中,真菌感染通常发生于收获之前,那时真菌生长和/或真菌毒素的产生可在存储之前发生,或者,真菌感染甚至可以发生于收获之后,在存储之前或在不恰当的存储条件下。联合国粮农组织(FAO)已估计,全世界25%的农产品受真菌污染,造成严重经济损失。在一项历时8年的国际研究中,从2004年1月到2011年12月对总计19757份样本进行了分析;它们中的72%对至少一种真菌检测为阳性,39%被发现受到污染,37%对ZEN检测为阳性(Schatzmayr和Streit(2013)‘Global occurrence ofmycotoxins in the food and feed chain:Facts and figures.’World MycotoxinJournal 6(3):213-222)。在世界所有地区和检测的全部类型的谷物和饲料作物诸如玉米、大豆粉、小麦、麦麸、DDGS(干酒糟及可溶物)中以及在成品动物饲料混合物中都已经发现了ZEN,发生率达到100%。
ZEN结合至雌激素受体并且会造成激素紊乱,ZEN在口服之后会立即被吸收并被动物转化为两种立体异构代谢产物α-玉米赤霉烯醇(α-ZEL)和/或β-玉米赤霉烯醇(β-ZEL)。例如,α-ZEL,并且α-玉米赤霉醇(α-ZAL)和/或玉米赤霉酮(ZAN),比ZEN具有更强的雌激素效应。虽然共轭ZEN衍生物的雌激素活性比ZEN本身低,但是ZEN在消化道中会从这些共轭ZEN衍生物再次释放,从而恢复其完整的雌激素活性。
ZEN具有高达20000mg/kg体重的经口LD50,亚急性和/或亚慢性毒性作用诸如致畸作用、致癌作用、雌激素效应和免疫抑制作用会在长时间接触的动物或人中发生。被ZEN污染的饲料会在哺乳动物中引起发育障碍。猪,特别是小猪,对ZEN极其敏感。饲料中超过0.5ppm的ZEN浓度会引起发育障碍,超过1.5ppm的浓度会在猪中引起高雌激素。在牛中,浓度是12ppm的ZEN会造成自然流产。
由于ZEN通过粘膜(尤其是通过胃粘膜以及口腔粘膜)能被快速吸收,因此立即定量失活是必须的。口服施用之后30分钟,能够在血液中检测到ZEN。由于ZEN的有害作用,欧盟给出了食品中ZEN的限制上限以及动物饲料产品中ZEN上限的推荐值(EC No.1881/2006)。
降低食物和动物饲料产品中ZEN的主要策略是例如通过保持“良好的农作实践”来限制真菌生长。这包括保证种子不受昆虫和真菌的侵害或及时从田地移除农业废弃物在内的很多措施。此外,田地中的真菌生长能够通过使用杀真菌剂来降低。在收获之后,应当在残余湿度低于15%和低温条件下储存收获的物料以便防治真菌生长。同样地,应当在深加工之前去除真菌感染的物料。尽管有这么多的预防措施,但是,在2004至2011年间,甚至在农业标准最高的地区诸如北美和中欧,仍然发现有高达37%的检测玉米样本被ZEN污染(Schatzmayr和Streit(2013))。
另一种有关的霉菌毒素是赭曲霉毒素A(OTA;也可称作N-{[(3R)-5-氯代-8-羟基-3-甲基-1-氧代-3,4-二氢-1H-2-苯并吡喃-7-基]羰基}-L-苯丙氨酸,(-)-N-((5-氯代-8-羟基-3-甲基-1-氧代-7-异苯并二氢吡喃基)羰基)-3-苯丙氨酸,(2S)-2-{[(3R)-5-氯代-8-羟基-3-甲基-1-氧代-3,4-二氢-1H-2-苯并吡喃-7-羰基]氨基}-3-苯丙酸,(R)-N-((5-氯代-3,4-二氢-8-羟基-3-甲基-1-氧代-1H-2-苯并吡喃-7-基)羰基)苯丙氨酸,N-(((3R)-5-氯代-8-羟基-3-甲基-1-氧代-7-异苯并二氢吡喃基)羰基)-3-苯基-L-丙氨酸,N-[(3R)-5-氯代-8-羟基-3-甲基-1-氧代-3,4-二氢-1H-2-苯并吡喃-7-羰基]-L-苯丙氨酸,N-{[(3R)-5-氯代-8-羟基-3-甲基-1-氧代-3,4-二氢-1H-异苯并吡喃-7-基]羰基}-L-苯丙氨酸),CAS号是303-47-9,它由真菌大量产生并且是包括赭曲霉毒素B、赭曲霉毒素C等在内的赭曲霉毒素组中毒性最强的成员。尤其,由于其对人类和动物产生的严重不良影响,包括肾毒性、免疫毒性和致癌性(Carballo et al.,2019;Malier et al.,2016),OTA受到食品和饲料安全方面的密切关注。在这方面,国际癌症研究机构(IARC)已经将OTA归类于2B组的可能的人类致癌物(IARC,2012)。
为了解决上述问题和缺陷,需要研发在较高温度能够除去ZEN毒性并且适合用作食物或饲料添加剂或者食物或饲料产品的α/β-水解酶变体。
鉴于上述现有技术,作出本发明。因此,本发明的目的是尤其可以表述为提供除去ZEN毒性的改进的方式和方法(例如,除其他外,提供还能够除去其它有关的霉菌毒素的组合物)。
本申请在下文描述的基础上通过提供变体、组合物和方法来满足该需求,以权利要求书为特征并通过所附实施例和附图来描述。
发明概述
本发明涉及亲代α/β水解酶的变体,该变体包括在对应于如下位置的一个或多个位置处的置换:SEQ ID NO:1(优选使用SEQ ID NO:1的编号)的167,168,174,218,155,175,179,182,183,186,187,268,306,10,17,28,39,47,50,57,58,59,63,65,69,126,139,225,230,245,251,281,287,289,293和304,其中所述变体具有α/β水解酶活性,并且,其中该变体是与SEQ ID NO:1的多肽具有至少71%(优选:至少72%,至少73%,至少74%,至少75%,至少76%,至少77%,至少78%,至少79%,至少80%,至少81%,至少82%,至少83%,至少84%,至少85%,至少86%,至少87%,至少88%,至少89%,至少90%,至少91%,至少92%,至少93%,至少94%,至少95%,至少95.5%,至少96%,至少96.5%,至少97%,至少97.5%,至少98%,至少98.5%,至少99%,或至少99.5%)但小于100%序列同一性的多肽,该变体具有与亲代α/β水解酶相比得以提高的温度稳定性。
本发明还涉及降解玉米赤霉烯酮(zearalenone)和/或其衍生物的方法,包括:(a)提供如下物质的一种或多种:本发明的变体和/或亲代α/β水解酶,优选与SEQ ID NO:1的多肽具有至少95%序列同一性的所述亲代α/β水解酶;(b)使(a)的一种或多种多肽与玉米赤霉烯酮和/或其衍生物进行接触(例如,形成酶-底物混合物)。
本申请通过提供下文描述的变体和组合物满足了该需求,以权利要求书为特征,并通过所附实施例和附图加以说明。
序列表概述
SEQ ID NO:1是亲代α/β水解酶氨基酸序列。
SEQ ID NO:2-35是变体α/β水解酶氨基酸序列。
SEQ ID NO:36是共同氨基酸基序1。
SEQ ID NO:37是共同氨基酸基序2。
SEQ ID NO:38是共同氨基酸基序3。
SEQ ID NO:39是共同氨基酸基序4。
发明详述
定义
如本文所述的“EC编号”(酶学委员会编号)可用于指根据2020年2月26日发布的酶命名数据库的酶活性(例如,可从https://enzyme.expasy.org/获得)。EC编号指的是NC-IUBMB的酶命名法1992(学术出版社,圣地亚哥,加利福尼亚州),包括了分别发表于如下刊物的增刊1-5:Eur.J.Biochem.1994,223,1-5;Eur.J.Biochem.1995,232,1-6;Eur.J.Biochem.1996,237,1-5;Eur.J.Biochem.1997,250,1-6;and Eur.J.Biochem.1999,264,610-650。
如本文所述,可参考UniProtKB登记号(http://www.uniprot.org/,可获得自2020年12月2日公布的UniProtKB版2020_06)
术语“多肽”在本文中与术语“蛋白质”等同使用。蛋白质(包括其片段,优选生物活性片段,和肽,通常小于30个氨基酸)包括通过共价肽键彼此连接的一个或多个氨基酸(结果是得到一连串的氨基酸)。如本文使用的术语“多肽”描述了一组分子,这组分子例如由超过30个氨基酸组成。多肽可以进一步形成多聚体,例如二聚体、三聚体和更高级的低聚物,即,由超过一个多肽分子组成。形成这样的二聚体、三聚体等的多肽分子可以是相同的或不同的。因此,这样的多聚体的相应高阶结构被称为同源二聚体或异源二聚体、同源三聚体或异源三聚体等。异源多聚体的一个例子是抗体分子,在其天然存在的形式中,它是由两个相同的轻肽链和两个相同的重肽链组成。术语“多肽”和“蛋白质”还指天然修饰的多肽/蛋白质,其中修饰是由例如翻译后修饰(例如糖基化、乙酰化、磷酸化等)造成的。这样的修饰在本领域中是已知的。
序列同一性:两个氨基酸序列之间或两个核苷酸序列之间的关联性由参数“序列同一性”来描述。就本发明而言,使用Needleman-Wunsch算法(Needleman和Wunsch,1970,J.Mol.Biol.48:443-453)来确定两个氨基酸序列之间的序列同一性,在EMBOSS软件包(EMBOSS:欧洲分子生物学开放软件包,Rice et al.,2000,Trends Genet.16:276-277)(优选5.0.0或更高的版本)的Needle程序中实施Needleman-Wunsch算法。使用的参数可以是缺口开放罚分10、缺口延伸罚分0.5和EBLOSUM62(BLOSUM62的EMBOSS版本)取代矩阵。标记为“最长同一性”(“longest identity”)的Needle输出(使用非-简单(no-brief)选项得到)用作同一性百分比并按照如下公式计算:
(相同残基×100)/(比对的长度-比对中缺口的总数)。
可选地,使用的参数可以是缺口开放罚分10、缺口延伸罚分0.5和EDNAFULL(NCBINUC4.4的EMBOSS版本)取代矩阵。标记为“最长同一性”的Needle输出(使用非-简单选项得到)用作同一性百分比并按照如下公式计算:
(相同脱氧核糖核苷酸×100)/(比对的长度-比对中缺口的总数)。
表达:术语“表达”包括产生变体(多肽)涉及的任何步骤,包括但不限于,转录、转录后修饰、翻译、翻译后修饰和分泌。
表达载体:术语“表达载体”指的是线性或环状DNA分子,其包括编码变体(多肽)的多核苷酸并可操作地连接于提供其表达的调控序列。
片段:术语“片段”指的是在成熟多肽的氨基和/或羧基端缺少一个或多个(例如,几个)氨基酸的多肽;其中,片段仍具有本文描述的活性(例如α/β水解酶活性,例如具有将ZEN水解为HZEN的活性)。
如本文使用的术语“EC:3.1.1.-”意思是“玉米赤霉烯酮水解酶”(也被称为“玉米赤霉烯酮内酯酶”),例如,能够将ZEN水解为HZEN的酶。
宿主细胞:术语“宿主细胞”指的是具有包括本发明多核苷酸的核酸构建体或表达载体,对转化、转染、转导等敏感的任何细胞类型。术语“宿主细胞”包含亲代细胞的任何后代,该后代因为复制过程中发生的突变而与亲代细胞不相同。
核酸构建体:术语“核酸构建体”指的是单链或双链的核酸分子,其与天然存在的基因分离,或者以自然界中不存在的方式或合成的方式将其修饰为含有核酸片段,其包括一个或多个调控序列。
可操作地连接:术语“可操作地连接”指的是如下构型:在该构型中,调控序列位于相对于多核苷酸编码序列的合适位置,如此,调控序列指导编码序列的表达。
调控序列:如本文使用的术语“调控序列”(“control sequences”)指的是编码本发明的变体(多核苷酸)的多核苷酸进行表达所必需的核酸序列。每个调控序列对于编码变体的多核苷酸来说可以是天然的(即来自相同基因)或外源的(即来自不同基因),或者,彼此是天然的或外源的。这样的调控序列,包括但不限于,前导序列、聚腺苷酸化序列、前肽序列、启动子、信号肽序列和转录终止子。至少,调控序列包括启动子以及转录和翻译终止信号。为了引入促进调控序列与本发明多核苷酸编码区域进行连接的特定限制性位点,向调控序列提供接头(linker)。
如本文使用,术语“对应于”指的是序列特定氨基酸的测定方法,其中参考了特定氨基酸序列(例如US2020071638)。例如,就本发明而言,当参考特定氨基酸位置时,本领域技术人员能够将另一氨基酸序列与所述参考的氨基酸序列进行比对,以便确定哪个特定氨基酸在所述另一氨基酸序列中是有意义的。另一α/β水解酶中对应氨基酸残基的识别可以使用几种计算机程序通过多个多肽序列的比对来确定,前述计算机程序包括但不限于MUSCLE(基于对数期望的多序列比较;3.5或更高版本;Edgar,2004,Nucleic AcidsResearch 32:1792-1797)、MAFFT(6.857或更高版本;Katoh and Kuma,2002,NucleicAcids Research 30:3059-3066;Katoh et al.,2005,Nucleic Acids Research 33:51 1-518;Katoh and Toh,2007,Bioinformatics 23:372-374;Katoh et al.,2009,Methods inMolecular Biology 537:39-64;Katoh and Toh,2010,Bioinformatics 26:1899-1900)和使用ClustalW的EMBOSS EMMA(1.83或更高版本;Thompson et al.,1994,Nucleic AcidsResearch 22:4673-4680),分别使用它们各自的罚分参数。
就本发明而言,SEQ ID NO:1公开的成熟多肽用于确定另一α/β水解酶中对应氨基酸残基。另一α/β水解酶的氨基酸序列与SEQ ID NO:1公开的成熟多肽进行比对,基于比对,使用Needleman-Wunsch算法(Needleman and Wunsch,1970,J.Mol.Biol.48:443-453)来确定与SEQ ID NO:1公开的成熟多肽中任何氨基酸残基相对应的氨基酸位置编号,前述Needleman-Wunsch算法使用EMBOSS软件包(优选5.0.0或更高版本)的Needle程序来实施(EMBOSS:欧洲分子生物学开放软件包,Rice et al.,2000,Trends Genet.16:276-277)。使用的参数是缺口开放罚分10、缺口延伸罚分0.5和EBLOSUM62(BLOSUM62的EMBOSS版本)取代矩阵。
根据本发明使用的术语“位置”指的是本文描述的氨基酸序列中的氨基酸的位置。在本文中,术语“对应”可包括一个位置不仅取决于前面的核苷酸/氨基酸的编号。
如本文使用,“沉默”突变的意思是核酸序列中碱基置换,其不改变核酸序列编码的氨基酸序列。“保守或等效”置换(或突变)是指如下面表1中“典型置换”所列举的置换。如本文使用的“高度保守”置换在下面表1中标题“优选的置换”下显示。
表1.氨基酸置换
变体:术语“变体”指的是具有本文描述的比活力(specific activity)的多肽,其在一个或多个(例如,几个)位置包括改变,即置换、插入和/或缺失。置换的意思是占据一个位置的氨基酸被不同的氨基酸所取代;缺失的意思是占据一个位置的氨基酸被去掉;插入的意思是紧接着占据一个位置的氨基酸再增加一个氨基酸。
在描述本发明的变体中,为了便于参考而调整下述命名法。采用了公认的lUPAC单字母或三字母氨基酸缩写。
置换。对于氨基酸置换,使用下述命名法:原始氨基酸,位置,置换的氨基酸。因此,将位置167的Asn(N)被Thr(T)置换表示为“N167T”或“Asn167Thr”。多个突变可以使用附加标记(“+”)或(“,”)来分开,例如,“N167T+F168Y+S174C+F218Y”或“N167T,F168Y,S174C,F218Y”,表示给定位置的多个置换。在本申请的实施例中,多个突变可以使用逗号来分开,例如,N167T,F168Y,S174C,F218Y。此外,本文使用的“X”或“Xaa”可以意指任何氨基酸(例如,如上面表1所描述)。因此,本文使用的“X167T”意思是位置167的任何氨基酸被T(Thr)置换。假使原始氨基酸残基可以是任何氨基酸残基,也可使用简写来只表明位置和置换的氨基酸。因此,在标明置换时可以省略“X”或“Xaa”,例如,“167T”名称可用来表示位置167的任何氨基酸被T(Thr)置换。此外,本文使用的“X167G,A,S,C,U,I,L,V,T”意思是位置167的任何氨基酸被G,A,S,C,U,I,L,V或T的任一个置换。假使置换氨基酸残基可以是任何氨基酸残基,也可使用简写来只表明原始氨基酸及其位置,例如“N167”。
如本文使用,术语“转基因的”指的是基因组通过引入外源遗传物质或额外拷贝的自体遗传物质(例如,通过转化或重组(例如,US7410800B2))而被改变的生物体。转基因生物可以是植物、哺乳动物、真菌、细菌或病毒。如本文使用,“转基因植物、种子或花粉粒”指的是植物、种子或花粉粒,或者由其衍生的任何下一代的后代植物、种子或花粉粒,其中,植物、种子或花粉粒或者其后代包含引入的外源性DNA,该外源性DNA是相同品种的非转基因植物、种子或花粉粒中最初并不存在的。此外,转基因植物、种子或花粉粒可包含被改造的植物中原有的序列,但是其中,外源性DNA已经被改变,以便改变编码序列的表达水平或模式。
术语“回收变体”指的是例如通过亲和纯化从细菌裂解物中纯化变体。
术语“食品”指的是具有食用价值的物质。
术语“草料”(fodder)指的是喂给家畜的物质。
术语“饲料”(feed)指的是用作牲畜的食品的物质。
术语“添加剂”指的是例如以少量加入到其它产品或物质中以便影响期望的性质和/或特性的化合物或物质。
术语“益生元”指的是能够诱导有益微生物生长和/或活性的化合物或物质。
术语“解毒剂”指的是能够降低和/或抑制毒性的化合物或物质。
术语“营养补充剂”指的是能够补足膳食营养含量的化合物或物质,例如,维生素和矿物质。
术语“中间物”指的是在获得本发明的最终产品的过程中(例如,在过程的中间阶段)产生的化合物或物质,例如,本发明的食品、草料、饲料、添加剂(例如,食品添加剂、草料添加剂或饲料添加剂)、解毒剂、营养补充剂或益生元。
术语“藻类物质”(phycophytic substance)指的是来源于海藻物种的物质。
氨基酸基序:如本文使用的术语“氨基酸基序”或“基序”指的是多肽的特别限定的氨基酸片段(stretch)。因此,本发明的氨基酸基序指的是给定多肽中氨基酸的短序列。
如本文使用的术语“OTA衍生物”指的是具有如下化学结构的化合物或物质:
在OTA衍生物的分子结构中,R1、R2和R3可以是任何原子或原子基团。尤其,R1选自H和OH构成的组,R2选自H和CH2-CH3构成的组,R3选自H和Cl构成的组。OTA衍生物可以是:赭曲霉毒素B,其中R1是H、R2是H且R3是H;赭曲霉毒素C,R1是H、R2是CH2-CH3且R3是Cl;或者,赭曲霉毒素TA,R1是OH、R2是H且R3是Cl。尤其,术语“OTA衍生物”指的是从赭曲霉毒素B和赭曲霉毒素C构成的组选出的化合物或物质。
必须注意的是,如本文使用,除非在文中有明确的指出,否则单数形式“一个”(a)、“一种”(an)和“该”(the)包括复数引用。因此,例如,对“一种试剂”的引用包括一种或多种此类的不同试剂,对“该方法”的引用包括对本领域技术人员已知的等同步骤和方法的引用,本领域技术人员能够对本文描述的方法进行修改或替代。
除非指出,否则,在一系列要素之前的术语“至少”应理解为指的是该系列中的每个元素。本领域技术人员将意识到或者能够仅使用常规实验来确定与本文描述的发明的实施方案等同的众多方案。这些等同方案也旨在包括在本发明中。
不管在本文中哪里使用,术语“和/或”包括“和”、“或”和“与所述术语有关的要素的全部或者任何其它组合”。
如本文使用的术语“约”或“大约”意思是给定数值或范围的20%以内、优选10%以内且更优选5%以内。
在整个说明书中以及所附的权利要求书中,除非上下文中有要求,否则,词语“包括”(comprise)以及其变型诸如“包含”(comprises)和“含有”(comprising),将被理解为意指包括规定的整数或步骤或者整数或步骤的组,但是不排除其它任何整数或步骤或者整数或步骤的组。当在本文中使用时,术语“含有”(comprising)可被术语“包括”(containing)或“包含”(including)代替,或者,有时候在本文中使用时,可被术语“具有”(having)代替。
当在本文中使用时,“由...组成”排除权利要求的要素中没有指明的任何要素、步骤或成分。当在本文中使用时,“主要由...组成”并不排除对权利要求的基本的和新颖的特征不产生实质影响的材料或步骤。
在本文的每个例子中,术语“包括”、“主要由...组成”和“由...组成”中的任何一个可以被其它两个术语中的任一个所代替。
SEQ ID NO:1的野生型亲代α/β水解酶能够将ZEN降解为无毒的HZEN,其显示低熔点(Tm=50℃)并且具有二聚同源二聚体四级结构。由于在工业酶产品的生产和应用中高的酶稳定性是关键点,因此需要进一步研发改良的α/β水解酶变体,其能够在较高温度解毒ZEN并且特别适合用作食物或饲料添加剂或者食物或饲料产品。
在本发明的过程中,在具有SEQ ID NO:1、指导由同源二聚体向同源四聚体的四级结构形成且因此尤其增强了温度稳定性的亲代α/β水解酶中识别出特定的氨基酸置换。因此,本发明涉及本文描述的用于降解玉米赤霉烯酮(ZEN)的改良水解酶变体的用途。
ZEN(CAS注册号#17924-92-4,(4S,12E)-16,18-二羟基-4-甲基-3-氧杂双环[12.4.0]十八碳-1(14),12,15,17-四烯-2,8-二酮)是具有如下结构式的非甾体雌激素大环内酯,通过聚酮代谢途径合成:
然而,多种ZEN衍生物也会发生在自然界中,可通过ZEN的酶修饰或化学修饰来形成。例子包括糖苷ZEN缀合物(conjugate)或者包含硫酸盐的那些,由真菌、植物或哺乳动物代谢以及人类或动物体等中形成的ZEN代谢物来形成。下面将ZEN衍生物理解为自然发生的或者通过化学合成或生物化学合成来合成的ZEN缀合物或ZEN代谢物,尤其是,α-玉米赤霉烯醇(α-ZEL;CAS注册号#36455-72-8;(2E,7R,11S)-7,15,17-三羟基-11-甲基-12-氧杂双环[12.4.0]-十八碳-1(18),2,14,16-四烯-13-酮),β-玉米赤霉烯醇(β-ZEL;CAS注册号#71030-11-0;(2E,7S,11S)-7,15,17-三羟基-11-甲基-12-氧杂双环[12.4.0]十八碳-1(18),2,14,16-四烯-13-酮),α-玉米赤霉醇(α-ZAL;CAS注册号#26538-44-3;(7R,11S)-7,15,17-三羟基-11-甲基-12-氧杂双环[12.4.0]十八碳-1(18),14,16-三烯-13-酮),β-玉米赤霉醇(β-ZAL;CAS注册号#42422-68-4;(7S,11S)-7,15,17-三羟基-11-甲基-12-氧杂双环[12.4.0]-十八碳-1(14),15,17-三烯-13-酮),玉米赤霉烯酮14-硫酸盐(Z14S;[(2E,11S)-15-羟基-11-甲基-7,13-二氧-12-氧杂双环[12.4.0]十八碳-1(18),2,14,16-四烯-17-yl]硫酸氢盐),玉米赤霉烯酮-14-糖苷(Z14G;(2E,11S)-15-羟基-11-甲基-17-[(3R,4S,5S,6R)-3,4,5-三羟基-6-(羟甲基)-四氢吡喃-2-基]氧-12-氧杂双环[12.4.0]十八碳1(18)2,14,16-四烯-7,13-二酮),以及玉米赤霉酮(ZAN;CAS注册号#5975-78-0;(11S)-15,17-二羟基-11-甲基-12-氧杂双环-[12.4.0]十八碳-1(18),14,16-三烯-7,13-二酮)。
ZEN以及ZEN衍生物尤其是α-ZEL、β-ZEL、Z14S、α-ZAL、β-ZAL、Z14G和ZAN也可在加工的食物或动物饲料产品中检测到,诸如面包或啤酒,原因在于它们高度的化学和物理稳定性。
序列号是1至35的任何多肽都能成功水解ZEN或ZEN衍生物。认为,ZEN或其衍生物的水解按照以下反应机理在酯基处发生:
ZEN水解形成无毒的水解玉米赤霉烯酮(HZEN)和/或水解ZEN衍生物可以利用本发明的α/β-水解酶来发生。HZEN进一步脱羧得到脱羧水解ZEN(DHZEN)和/或脱羧水解ZEN衍生物被认为是自发的。
本文描述的α/β-水解酶和变体能够并适合降解ZEN。例如,α/β-水解酶能够适合于ZEN和/或其衍生物的酯基的无辅因子水解切割(例如,水解依赖于H2O)。
可以按照如下方法来测量ZEN降解:制备玉米赤霉烯酮降解分析缓冲液(118.5mMNaCl,8.55mM醋酸,14.9mM醋酸盐,0.1mg/ml BSA,pH5.0,(Jantratid,E.,Janssen,N.,Reppas,C.&Dressman,J.B.(2008):Dissolution media simulating conditions in theproximal human gastrointestinal tract:An update.Pharmaceutical Research 25(7):1663-1576),含有0.1mg/ml牛血清白蛋白和0.52ppm玉米赤霉烯酮作为底物),在37.0℃预热,将960μl试样的分析缓冲液转移至96深井板的反应管中。用活动盖密封该板,37.0℃保存在DWP-热振动筛中直至玉米赤霉烯酮降解分析开始。使用纯化的玉米赤霉烯酮-切割多肽通过在样本缓冲液(Teorell Stenhagen缓冲液pH 7.5,含有0.1mg/ml牛血清白蛋白)中稀释至比最终玉米赤霉烯酮降解实验中分析的浓度高25倍来制备酶工作液,玉米赤霉烯酮-切割多肽的浓度可在给定条件下有效降解玉米赤霉烯酮。为了开始玉米赤霉烯酮降解分析,将40.0μl酶工作液加入到含有960.0μl分析缓冲液的试管中,从而在分析反应中实现0.5ppm的最终ZEN浓度。加入pH 7.5的酶工作液不会改变pH 5.0的玉米赤霉烯酮降解反应。37.0℃,持续振动的DWP-热振动筛中孵育玉米赤霉烯酮降解反应。玉米赤霉烯酮降解反应开始之后立即用吸管头再悬浮混合,将120.0μl的0.0h样本转移至新的PCR板的管中,用管帽密封。在几个时间点(例如5.0,10.0,20.0,30.0,45.0,60.0,90.0分钟)之后从玉米赤霉烯酮降解反应取出额外样本。一旦从降解反应中取出样本,就通过在99.0℃在热块(thermo-block)(例如,Thermal Shake lite,460-029,VWR)孵育10.0分钟来热灭活该样本中的玉米赤霉烯酮切割多肽。随后,对管离心(3.0分钟,室温,2500x g),将90.0μl上清液转移至有边缘的PCR板(例如,PCR板,0030128648,Eppendorf AG),其用热封膜(例如,热封膜,0030127838,Eppendof AG)封闭。这些样本板储存在4.0℃,直至HPLC-FLD测量。按照Vekiru et al.(Vekiru et al.(2016)‘Isolation and characterization ofenzymatic zearalenone hydrolysis reaction products’World Mycotoxin Journal 9:353-363)描述的改进的HPLC-FLD方法测量玉米赤霉烯酮浓度。278.0nm消光和465.0nm发射,在HPLC-FLD上进行分析。当在40.0℃在Kinetex 5μm EVO C18 100A 50.0 x 2.1mm柱(00B-4633-AN,Phenomenex Inc.)上完成分离时,玉米赤霉烯酮的保留时间是1.8分钟,前述分离采用了如下方法:使用溶剂A(95.0%乙腈+4.9%水+0.1%甲酸)和溶剂B(5.0%乙腈+94.9%水+0.1%甲酸),梯度:0.0-0.5分钟10%相B,0.5-2.0分钟线性增加至50.0%相B,然后在0.1分钟内减至10%相B,持续进行,总运行时间2.3分钟。流速设置为1.5ml/min,进样体积是5.0μl。对各种分析采用了类似的采集设置。基于玉米赤霉烯酮外标物的校准,对玉米赤霉烯酮进行定量。所取反应样品的玉米赤霉烯酮浓度(μM)对取样时间点作图。线性玉米赤霉烯酮降解随时间的斜率以酶促反应体积中每分钟减少的μmol玉米赤霉烯酮计算。考虑到纯化的玉米赤霉烯酮-切割多肽的可能的稀释并将这些合适的稀释因素包括在计算中,计算以每分钟每mg纯化的酶降解的μmol玉米赤霉烯酮(μmol ZEN/min/mg)表示的比酶活(specific enzymatic activity)。
在本文中,应注意,术语“单位”(unit)或“U”指的是测量酶的催化活性,定义为在设定条件下每分钟发生反应或切割的底物(在本发明中即玉米赤霉烯酮)的微摩尔(μmol)数。利用酶或多肽溶液的“活性”定义了酶或多肽溶液的酶浓度,表示为溶液的单位每毫升(U/ml)或单位每升(U/l)。
在一些实施方案中,本发明提供了亲代α/β水解酶的变体,该变体包括在对应于如下位置的一个或多个位置的置换:SEQ ID NO:1(优选使用SEQ ID NO:1的编号)的167,168,174,218,155,175,179,182,183,186,187,268,306,10,17,28,39,47,50,57,58,59,63,65,69,126,139,225,230,245,251,281,287,289,293和304,其中,所述变体具有α/β水解酶活性,并且其中,该变体是与SEQ ID NO:1的多肽具有至少71%(优选:至少72%,至少73%,至少74%,至少75%,至少76%,至少77%,至少78%,至少79%,至少80%,至少81%,至少82%,至少83%,至少84%,至少85%,至少86%,至少87%,至少88%,至少89%,至少90%,至少91%,至少92%,至少93%,至少94%,至少95%,至少95.5%,至少96%,至少96.5%,至少97%,至少97.5%,至少98%,至少98.5%,至少99%,或至少99.5%)但小于100%序列同一性的多肽,优选地,所述变体具有水解酶EC:3.1.1.-活性。
在其它实施方案中,本发明的变体具有如下一种或多种特性:(i)相对于亲代α/β水解酶得到改进的性质,其中所述改进的性质包括提高的温度稳定性,优选地,相比于SEQID NO:1,提高的温度稳定性;(ii)能够四聚体化,优选地,具有四聚体四级结构,进一步优选地,所述四聚体化是同源四聚体化;(iii)具有大于50℃的熔点(Tm),优选地,所述Tm范围是大约51℃至大约70℃;(iv)能够降解玉米赤霉烯酮和/或其衍生物;(v)具有至少一个(优选至少两个,更优选至少三个,最优选至少四个)氨基酸基序,所述氨基酸基序与从如下选出的氨基酸序列具有至少80%(优选至少83%,更优选至少90%)序列同一性:(a)SEQ IDNO:36(QVDLGEX1X2MN),其中,X1是V或I,优选V,并且其中,X2是V或T,优选V;(b)SEQ ID NO:37(EYDPEWX3RX4FX5EGTV),其中,X3是G或A,优选A,并且其中,X4是A或V,优选A,并且其中,X5是F或Y;(c)SEQ ID NO:38(MLX6QVKTPX7LX8THH),其中,X6是S或G,优选S,并且其中,X7是I或V,优选I,并且其中,X8是I或L,优选I;和(d)SEQ ID NO:39(PAX9LLX10PEQTGSWWSYEX11X12X13GLLX14EX15FHVX16AVDX17RGQGRSX18WTPX19RYSLDNFGNDLVRFIX20LVX21KRPVX22VX23GNSSGGX24LAAWLSAYX25MPGQX26RX27X28LCEDX29X30FFASELVPAX31GHSVX32QX33AGPX34FELX35R),其中,X9是V或L,优选L,并且其中,X10是L或I,优选L,并且其中,X11是P或E,优选P,并且其中,X12是V或A,优选V,并且其中,X13是I或M,优选I,并且其中,X14是A或S,优选A,并且其中,X15是S,N或H,优选S,并且其中,X16是F或Y,优选F,并且其中,X17是I或L,优选I,并且其中,X18是T或S,优选T,并且其中,X19是K或R,优选K,并且其中,X20是S,A或N,优选S,并且其中,X21是V或I,优选V,并且其中,X22是I或V,优选I,并且其中,X23是S或A,优选S,并且其中,X24是V或L,优选V,并且其中,X25是A或S,优选A,并且其中,X26是I或L,优选I,并且其中,X27是A或G,优选A,并且其中,X28是A或V,优选A,并且其中,X29是T,A或P,优选T,并且其中,X30是P或A,优选P,并且其中,X31是Y或H,优选H,并且其中,X32是L或R,优选R,并且其中,X33是A或G,优选A,并且其中,X34是A或V,优选A,并且其中,X35是Y或F,优选Y。
在其它实施方案中,本发明的变体包括如下置换的一种或多种,或其等效(例如保守性)氨基酸置换(例如,如本文表1所述):
在其它实施方案中,本发明的亲代α/β水解酶(优选具有EC:3.1.1.-水解酶活性)选自如下多肽构成的组:(i)与SEQ ID NO:1的多肽具有至少60%(优选地,至少81%,至少82%,至少83%,至少84%,至少85%,至少86%,至少87%,至少88%,至少89%,至少90%,至少91%,至少92%,至少93%,至少94%,至少95%,至少95.5%,至少96%,至少96.5%,至少97%,至少97.5%,至少98%,至少98.5%,至少99%,或至少99.5%,或100%)序列同一性的多肽;(ii)SEQ ID NO:1的多肽的片段,其中所述片段具有α/β水解酶活性,优选EC:3.1.1.-水解酶活性。
在其它实施方案中,本发明的变体(i)包括从如下置换构成的组中选出的置换或者由如下置换构成的组中选出的置换所构成:
和/或(ii)选自SEQ ID NO:2-35构成的组。
在其它实施方案中,本发明提供了获得亲代α/β水解酶的变体(优选具有EC:3.1.1.-水解酶活性)和/或提高所述亲代α/β水解酶的稳定性的方法,所述方法包括:在与SEQ ID NO:1的位置167,168,174,218,155,175,179,182,183,186,187,268,306,10,17,28,39,47,50,57,58,59,63,65,69,126,139,225,230,245,251,281,287,289,293和304(优选使用SEQ ID NO:1的编号)相对应的一个或多个位置处,向亲代α/β水解酶引入置换,其中,变体具有α/β水解酶活性;和,回收变体;优选地,所述亲代α/β水解酶是依据前述任一权利要求,进一步优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%序列同一性。
在其它实施方案中,本发明涉及编码本发明的变体的多核苷酸。在其它实施方案中,本发明涉及包括本发明的多核苷酸的核酸构建体。在其它实施方案中,本发明涉及包括本发明的多核苷酸的核酸构建体。在其它实施方案中,本发明涉及重组宿主细胞,其包括如下至少一者:(i)本发明的变体和/或亲代α/β水解酶;(ii)本发明的多核苷酸;(iii)本发明的核酸构建体;和/或,(iv)本发明的表达载体。
因此,本发明还涉及编码本文描述的α/β水解酶变体的核酸分子。核酸可被引入或插入表达载体。术语“表达载体”指的是能够在体内或体外表达基因的核酸分子构建体。尤其,它可以包括DNA构建体,该DNA构建体适合将多肽-编码核苷酸序列转入宿主细胞(例如重组宿主细胞)以便整合至基因组或游离于染色体外空间,并且在细胞内表达多肽-编码核苷酸序列,并且可选地,将多肽运输至细胞外。如本文描述的表达载体可在宿主细胞中表达。术语“宿主细胞”指的是含有待表达核苷酸序列或表达载体的所有细胞,其能够产生根据本发明的酶或多肽。尤其,这指的是原核细胞和/或真核细胞,优选地,毕赤酵母、大肠杆菌、枯草芽孢杆菌、链霉菌、汉逊酵母、木霉菌、乳酸菌、曲霉菌、植物细胞和/或芽孢杆菌的孢子、木霉或曲霉。本文使用的名称毕赤酵母(P.pastoris)与名称毕赤酵母(Komagataellapastoris)意思相同,毕赤酵母(P.pastoris)是较旧名称,毕赤酵母(K.pastoris)是较新的系统名称(Yamada et al.(1995)‘The Phylogenetic Relationships of Methanol-assimilating Yeasts Based on the Partial Sequences of 18S and 26S RibosomalRNAs:The Proposal of Komagataella Gen.Nov.(Saccharomycetaceae)’Bioscience,Biotechnology and Biochemistry,Vol.59,issue 3,pp.439-444)。尤其,毕赤酵母(Komagataella pastoris)的种最近已经重新界定为是法夫驹形氏酵母(Komagataellaphaffii)(Kurtzman(2009)“Biotechnological strains of Komagataella(Pichia)pastoris are Komagataella phaffii as determined from multigene sequenceanalysis.”J Ind Microbiol Biotechnol.36(11):1435-8)。本文使用的法夫驹形氏酵母(Komagataella phaffii)例如涉及菌株Komagataella phaffii CBS 7435、Komagataellaphaffii GS115或Komagataella phaffii JC308。
在其它实施方案中,本发明涉及转基因植物、转基因种子或转基因花粉粒,其包括如下的一种或多种(优选多个):(i)本发明的变体和/或亲代α/β水解酶;(ii)本发明的多核苷酸;(iii)本发明的核酸构建体;(iv)本发明的表达载体;和/或(v)本发明的重组宿主细胞。
在其它实施方案中,本发明涉及食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物,和/或其混合物,它们包括如下的一种或多种:(i)本发明的变体和/或亲代α/β水解酶;优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性;(ii)本发明的多核苷酸;(iii)本发明的核酸构建体;(iv)本发明的表达载体;(v)本发明的重组宿主细胞;(vi)本发明的转基因植物、转基因种子和/或转基因花粉粒。
在其它实施方案中,本发明涉及组合物或试剂盒,其包括如下的一种或多种:(i)本发明的变体和/或亲代α/β水解酶;优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性;(ii)本发明的多核苷酸;(iii)本发明的核酸构建体;(iv)本发明的表达载体;(v)本发明的重组宿主细胞;(vi)本发明的转基因植物、转基因种子和/或转基因花粉粒;和/或(vii)本发明的食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物,和/或其混合物。
因此,本发明还涉及包括本文描述的α/β水解酶变体的组合物。优选地,组合物可以是食物或饲料添加剂或者食物或饲料产品。制备这些食物组合物和/或饲料组合物的方法对本领域技术人员来说是已知的,尤其是在WO 99/35240中被描述。
在其它实施方案中,本发明的组合物或试剂盒还包括营养学上可接受的载体(例如水)和/或本发明的亲代α/β水解酶,优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性。
在一些其它实施方案中,本发明的组合物或试剂盒还包括如下的一种或多种:(i)能够去除OTA和/或至少一种OTA衍生物(例如赭曲霉毒素B和/或赭曲霉毒素C)的毒性的一种或多种其它多肽,进一步优选地,所述一种或多种其它多肽属于M20肽酶氨基酰化酶1-样蛋白2-样氨基水解酶亚家族(例如,M20肽酶ACY1L2氨基水解酶亚家族,例如,根据保守结构域数据库(例如https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid= cd05672)标识符是cd05672,和/或,具有氨肽酶活性(例如EC 3.4.11.10),和/或,包括羧肽酶活性(例如羧肽酶A和/或B活性,例如分别具有EC 3.4.17.1和/或EC 3.4.17.2),和/或,嗜热菌蛋白酶活性(例如具有EC 3.4.24.27));(ii)能够去除一种或多种霉菌毒素(例如,ZEN和/或单端孢真菌毒素诸如脱氧雪腐镰刀菌烯醇(deoxynivalenol)、瓜萎镰菌醇(nivalenol)、新茄病镰刀菌烯醇(neosolaniol)、毛霉素(trichotecin)、扁虫毒素(crotocin)、杆孢菌素A(roridin A)、葡萄穗霉毒素H(satratoxin H)、双乙酸基草镰刀菌醇(diacetoxyscirpenol)、HT-2毒素或T-2毒素;黄曲霉毒素,诸如黄曲霉毒素B1、B2、G1或G2;伏马毒素,诸如伏马毒素B1、B2、B3或B4;多肽霉菌毒素,诸如白僵菌素或恩镰孢菌素;玉米赤霉烯酮;桔霉素;展青霉素;麦角生物碱,诸如麦角胺)和/或一种或多种植物源-和/或细菌源-毒素(例如内毒素等)的毒性的一种或多种其它多肽,尤其,所述一种或多种其它多肽能够去除一种或多种其它霉菌毒素和/或一种或多种植物源-和/或细菌源-毒素,例如,伏马毒素酯酶(例如,如WO 2016/134387 A1所公开的)和/或玉米赤霉烯酮内酯酶(例如,如WO 2020/025580 A1所公开的)和/或麦角肽水解酶(例如,如WO 2014/056006 A1所公开的);(iii)一种或多种有机吸附剂(例如,活的、灭活的、冻干的、休眠的和/或死的完整酵母,或者酵母衍生产品诸如酵母细胞壁,或者酵母寡聚体诸如甘露聚糖)和/或一种或多种无机吸附剂(例如,硅藻土和/或粘土矿物诸如高岭土或高岭石,蒙脱石诸如蒙脱土、伊利石或绿泥石;尤其,膨润土);(iv)能够去除一种或多种其它霉菌毒素(例如,单端孢真菌毒素诸如脱氧雪腐镰刀菌烯醇、瓜萎镰菌醇、新茄病镰刀菌烯醇、毛霉素、扁虫毒素、杆孢菌素A、葡萄穗霉毒素H、双乙酸基草镰刀菌醇、HT-2毒素或T-2毒素;黄曲霉毒素,诸如黄曲霉毒素B1、B2、G1或G2;伏马毒素,诸如伏马毒素B1、B2、B3或B4;多肽霉菌毒素,诸如白僵菌素或恩镰孢菌素;玉米赤霉烯酮;桔霉素;展青霉素;麦角生物碱,诸如麦角胺)和/或一种或多种植物源-和/或细菌源-毒素(例如内毒素等)的毒性的一种或多种活的、灭活的、冻干的和/或休眠的微生物,尤其,所述微生物选自如下构成的组:毛孢子菌属和Apiotrichum属(例如,如WO 03/053161 A1公开的)和红蝽菌科(例如,如EP 3 501 526 A1公开的);(v)一种或多种植物产品(例如,海藻,优选海藻粉(seaweed meal);和/或,藻类,优选海藻粉(algaemeal);和/或,蓟,优选蓟籽;和/或,甘草制剂,优选甘草粉和/或甘草提取物,例如,如WO2018/121881 A1所公开);(vi)一种或多种调味化合物(例如,植物提取物,例如来自牛至、百里香、冬青、香菜、墨角兰、薄荷、胡椒薄荷、茴香、柑橘、柠檬、小茴香、八角茴香、丁香、肉桂和/或大蒜;和/或,精油,诸如D-柠檬烯、γ-萜烯、对伞花烃(p-cymene)、2-蒈烯、氧化芳樟醇、异薄荷酮(isomenthone)、樟脑、芳樟醇、松油烯-4-醇、2-异丙基-1-甲氧基-4-甲苯、L-薄荷醇、乙胺、α-松油醇、β-石竹烯、D-香芹酮、水杨酸甲酯、α-石竹烯、薰衣草乙酸酯、氧化石竹烯、丁香酚百里香酚和/或香芹酚);(vii)一种或多种维生素(例如,维生素A、D、E、K、C、B1、B2、B3、B4、B5、B6、B7、B8、B9和/或B12;尤其是维生素E)。
在特别的实施方案中,本发明的组合物或试剂盒还包括如下的一种或多种:膨润土、伏马毒素酯酶和/或玉米赤霉烯酮内酯酶,能够去除一种或多种霉菌毒素的毒性的红蝽菌科微生物(例如,选自红蝽菌科的微生物,例如https://lpsn.dsmz.de/family/coriobacteriaceae)、硅藻土、酵母(尤其是灭活酵母)、海藻粉、蓟籽、和一种或多种调味化合物。
在一些其它实施方案中,本发明的组合物(例如,如下表2列出的典型组合物1-24)或试剂盒(对应于下表2列出的典型组合物1-24)包括一种或多种(例如,除根据本发明的至少一种多肽之外)的其它组分。这样的其它典型组合物或试剂盒在下表2中明确公开,其描述了本发明的实施方案。
表2.本发明的包括一种或多种其它组分的典型组合物或试剂盒。
在其它实施方案中,本发明的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、重组宿主细胞、转基因植物、转基因种子、转基因花粉粒、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物、组合物和/或试剂盒,适合用作药物和/或用于治疗中,优选地,在疾病的预防或治疗中使用,进一步优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性。
在其它实施方案中,本发明的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、重组宿主细胞、转基因植物、转基因种子、转基因花粉粒、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物、组合物和/或试剂盒,适合用于预防和/或治疗霉菌中毒,优选地,适合用于预防和/或治疗玉米赤霉烯酮或其衍生物引起的激素紊乱。
在其它实施方案中,本发明涉及本发明的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、重组宿主细胞、转基因植物、转基因种子、转基因花粉粒、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物用于或用作如下一种或多种:(i)玉米赤霉烯酮和/或其衍生物的降解;(ii)食物、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物;(iii)如下一种或多种的制造/生产:食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物;(iv)青储饲料、沼气、生物乙醇或糖的制造/生产,优选由甘蔗或甜菜制造/生产;(v)霉菌中毒的预防和/或治疗。
在其它实施方案中,本发明提供了降解玉米赤霉烯酮和/或其衍生物的方法,包括:(a)提供如下的一种或多种:(i)本发明的变体和/或亲代α/β水解酶,优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性;(ii)本发明的多核苷酸;(iii)本发明的核酸构建体;(iv)本发明的表达载体;(v)本发明的重组宿主细胞;(vi)本发明的转基因植物、转基因种子和/或转基因花粉粒;(vii)本发明的食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物;和/或(vii)本发明的组合物或试剂盒;(b)使来自(a)的一种或多种物质接触玉米赤霉烯酮和/或其衍生物(例如,形成酶-底物混合物)。
应当理解的是,本发明不限于本文描述的特定方法、过程和试剂等,其可以改变。本文使用的专业术语目的仅在于描述特定的实施方案,并不旨在限制本发明的保护范围,本发明的保护范围仅由权利要求界定。
本说明书全文所引用的出版物和专利(包括所有专利、专利申请、科技出版物、制造商说明书、使用说明等),不管是在上文中还是在后面,都通过引用将其整体合并入本文中。本文中的没有内容被解释为承认本发明由于在先发明而无权提前披露。在一定程度上,通过引用包括的材料与本说明书存在矛盾或不一致,本说明书将取代任何此类材料。
本发明的特征还在于如下项:
1.一种亲代α/β水解酶的变体,所述变体包括对应于如下位置的一个或多个位置处的置换:SEQ ID NO:1(优选使用SEQ ID NO:1的编号)167,168,174,218,155,175,179,182,183,186,187,268,306,10,17,28,39,47,50,57,58,59,63,65,69,126,139,225,230,245,251,281,287,289,293和304,其中所述变体具有α/β水解酶活性(例如玉米赤霉烯酮水解酶),并且,其中所述变体是与SEQ ID NO:1的多肽具有至少71%(优选:至少72%,至少73%,至少74%,至少75%,至少76%,至少77%,至少78%,至少79%,至少80%,至少81%,至少82%,至少83%,至少84%,至少85%,至少86%,至少87%,至少88%,至少89%,至少90%,至少91%,至少92%,至少93%,至少94%,至少95%,至少95.5%,至少96%,至少96.5%,至少97%,至少97.5%,至少98%,至少98.5%,至少99%,或至少99.5%)但小于100%序列同一性的多肽,优选地,所述变体具有水解酶EC:3.1.1.-活性。
2.根据前述任一项所述的变体,其中所述变体具有如下一种或多种特性:
i)相对于亲代α/β水解酶的改进的性质,其中所述改进的性质包括提高的温度稳定性,优选是相比于SEQ ID NO:1的提高的温度稳定性;
ii)所述变体能够四聚体化,优选具有四聚体四级结构,进一步优选地,所述四聚体化是同源四聚体化;
iii)熔点(Tm)大于50℃,优选地,所述Tm的范围是大约51℃至大约70℃;
iv)能够降解玉米赤霉烯酮和/或其衍生物;
v)包括与选自如下的氨基酸序列具有至少80%(优选至少83%、更优选至少90%)序列同一性的至少一个(优选至少两个、更优选至少三个、最优选至少四个)氨基酸基序:
a)QVDLGEX1X2MN(SEQ ID NO:36),其中X1是V或I,优选V,并且,其中X2是V或T,优选V;
b)EYDPEWX3RX4FX5EGTV(SEQ ID NO:37),其中X3是G或A,优选A,并且,其中X4是A或V,优选A,并且,其中X5是F或Y;
c)MLX6QVKTPX7LX8THH(SEQ ID NO:38),其中X6是S或G,优选S,并且,其中X7是I或V,优选I,并且,其中X8是I或L,优选I;
d)
PAX9LLX10PEQTGSWWSYEX11X12X13GLLX14EX15FHVX16AVDX17RGQGRSX18WTPX19RYSLDNFGNDLVRFIX20LVX21KRPVX22VX23GNSSGGX24LAAWLSAYX25MPGQX26RX27X28LCEDX29X30FFASELVPAX31GHSVX32QX33AGPX34FELX35R(SEQ ID NO:39)其中X9是V或L,优选L,并且,其中X10是L或I,优选L,并且,其中X11是P或E,优选P,并且,其中X12是V或A,优选V,并且,其中X13是I或M,优选I,并且,其中X14是A或S,优选A,并且,其中X15是S、N或H,优选S,并且,其中X16是F或Y,优选F,并且,其中X17是I或L,优选I,并且,其中X18是T或S,优选T,并且,其中X19是K或R,优选K,并且,其中X20是S、A或N,优选S,并且,其中X21是V或I,优选V,并且,其中X22是I或V,优选I,并且,其中X23是S或A,优选S,并且,其中X24是V或L,优选V,并且,其中X25是A或S,优选A,并且,其中X26是I或L,优选I,并且,其中X27是A或G,优选A,并且,其中X28是A或V,优选A,并且,其中X29是T、A或P,优选T,并且,其中X30是P或A,优选P,并且,其中X31是Y或H,优选H,并且,其中X32是L或R,优选R,并且,其中X33是A或G,优选A,并且,其中X34是A或V,优选A,并且,其中X35是Y或F,优选Y。
3.根据前述任一项所述的变体,其中置换的数目是1-30,优选1-20和1-10,诸如1,2,3,4,5,6,7,8,9或10个置换。
4.根据前述任一项所述的变体,其中所述变体包括对应于SEQ ID NO:1如下位置的一个或多个位置处的置换(例如,下表的各行代表根据本发明的特征的单个典型组合,例如,特定实施方案的变体):
5.根据前述任一项所述的变体,其中所述变体包括如下置换的一个或多个(例如,下表的各行代表根据本发明的特征的单个典型组合,例如,特定实施方案的变体)或者其等效氨基酸置换:
6.根据前述任一项所述的变体,其中所述变体包括如下置换的一种或多种:
X167G,A,S,C,U,I,L,V,T;
X174G,A,S,C,U,I,L,V,T;
X155R,H,K;
X175G,A,S,C,U,V,T;
X218Y,F,H;和/或
X168Y,F,H
7.根据前述任一项所述的变体,其中所述亲代α/β水解酶,优选具有EC:3.1.1.-水解酶活性,选自如下肽构成的组:
i)与SEQ ID NO:1的多肽具有至少60%(优选至少81%,至少82%,至少83%,至少84%,至少85%,至少86%,至少87%,至少88%,至少89%,至少90%,至少91%,至少92%,至少93%,至少94%,至少95%,至少95.5%,至少96%,至少96.5%,至少97%,至少97.5%,至少98%,至少98.5%,至少99%,或至少99.5%,或100%)序列同一性的多肽;
ii)SEQ ID NO:1的多肽的片段,其中所述片段具有α/β水解酶活性,优选EC:3.1.1.-水解酶活性。
8.根据前述任一项所述的变体,其中所述亲代α/β水解酶包括SEQ ID NO:1的多肽或由SEQ ID NO:1的多肽组成。
9.根据前述任一项所述的变体,其中所述变体:
i)包括从如下置换构成的组中选出的置换或者由从如下置换构成的组中选出的置换所组成(例如,下表的各行代表根据本发明的特征的单个典型组合,例如,特定实施方案的变体):
和/或
ii)选自SEQ ID NOs:2-35构成的组。
10.一种获得亲代α/β水解酶的变体(所述变体优选具有EC:3.1.1.-水解酶活性)和/或提高所述亲代α/β水解酶的稳定性的方法,所述方法包括:在与SEQ ID NO:1(优选使用SEQ ID NO:1的编号)的位置167,168,174,218,155,175,179,182,183,186,187,268,306,10,17,28,39,47,50,57,58,59,63,65,69,126,139,225,230,245,251,281,287,289,293和304对应的一个或多个位置处向亲代α/β水解酶引入置换,其中所述变体具有α/β水解酶活性;和,回收所述变体(例如从裂解物回收,例如采用亲和纯化的方法);优选地,所述亲代α/β水解酶如前述任一项所述,进一步优选地,所述亲代α/β水解酶与SEQ ID NO:1具有至少95%序列同一性。
11.根据前述任一项所述的方法,其中所述变体是与SEQ ID NO:1的多肽具有至少71%(优选:至少72%,至少73%,至少74%,至少75%,至少76%,至少77%,至少78%,至少79%,至少80%,至少81%,至少82%,至少83%,至少84%,至少85%,至少86%,至少87%,至少88%,至少89%,至少90%,至少91%,至少92%,至少93%,至少94%,至少95%,至少95.5%,至少96%,至少96.5%,至少97%,至少97.5%,至少98%,至少98.5%,至少99%,或至少99.5%)但小于100%序列同一性的多肽。
12.一种编码前述任一项所述变体的多核苷酸。
13.一种包括前述任一项所述多核苷酸的核酸构建体。
14.一种包括前述任一项所述多核苷酸和/或核酸构建体的表达载体。
15.一种宿主细胞,包括如下至少一种:i)根据前述任一项所述的变体和/或亲代α/β水解酶;ii)根据前述任一项所述的多核苷酸;iii)根据前述任一项所述的核酸构建体;和/或iv)根据前述任一项所述的表达载体。
16.根据前述任一项所述的宿主细胞,其中所述宿主细胞是重组宿主细胞,优选是分离的重组宿主细胞。
17.根据前述任一项所述的宿主细胞,其中所述宿主细胞选自细菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞和植物细胞,优选地,选自细菌细胞和酵母细胞。
18.一种转基因植物、转基因种子或转基因花粉粒,包括如下的一种或多种(优选多种):
i)前述任一项所述的变体和/或亲代α/β水解酶;
ii)前述任一项所述的多核苷酸;
iii)前述任一项所述的核酸构建体和/或表达载体;和/或
iv)前述任一项所述的重组宿主细胞。
19.根据前述任一项所述的转基因植物、转基因种子或转基因花粉粒,其能够生产根据前述任一项所述的一种或多种变体,优选地,对所述转基因植物、转基因种子或转基因花粉粒进行基因修饰以便生产前述任一项所述的一种或多种变体。
20.根据前述任一项所述的转基因植物、转基因种子或转基因花粉粒,其中所述转基因植物、转基因种子或转基因花粉粒是所述(ii)和/或(iii)的纯合子。
21.一种生产前述任一项所述变体的方法,包括:(i)在适合所述变体表达的条件下培养根据前述任一项所述的宿主细胞;和(ii)回收所述变体。
22.采用前述任一项所述方法获得的变体。
23.前述任一项所述变体和/或亲代α/β水解酶在如下一种或多种中的用途:
i)降解玉米赤霉烯酮或其衍生物;
ii)食物、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物;
iii)如下一种或多种的制造/生产:食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物,和/或其混合物;
iv)青储饲料、沼气、生物乙醇或糖的制造/生产,优选由甘蔗或甜菜制造/生产;
v)霉菌中毒的预防或治疗。
24.一种组合物或试剂盒,包括如下的一种或多种:
(i)前述任一项所述的变体和/或亲代α/β水解酶,优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性;
(ii)前述权利要求任一项所述的多核苷酸;
(iii)前述权利要求任一项所述的核酸构建体;
(iv)前述权利要求任一项所述的表达载体;
(v)前述权利要求任一项所述的宿主细胞。
25.根据前述任一项所述的组合物或试剂盒,其中所述组合物或试剂盒是兽药组合物、药物组合物或试剂盒。
26.根据前述任一项所述的组合物或试剂盒,进一步包括兽药组合物、药学上可接受的载体。
27.根据前述任一项所述的组合物或试剂盒,其中所述组合物或试剂盒是食品或饲料添加剂,或者食品或饲料产品。
28.根据前述任一项所述的组合物或试剂盒,进一步包括营养学上可接受的载体和/或前述任一项所述的亲代α/β水解酶,优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%序列同一性。
29.根据前述任一项所述的组合物或试剂盒,进一步包括如下一种或多种(例如,如本文表2所示):能够去除赭曲霉毒素A(OTA)和/或至少一种OTA衍生物(例如赭曲霉毒素B和/或赭曲霉毒素C)的毒性(例如修饰和/或水解)和/或结合赭曲霉毒素A(OTA)和/或至少一种OTA衍生物(例如赭曲霉毒素B和/或赭曲霉毒素C)的一种或多种其它多肽,所述一种或多种其它多肽属于M20肽酶氨基酰化酶1-样蛋白2-样氨基水解酶亚家族(例如,M20肽酶ACY1L2氨基水解酶亚家族,例如,根据保守结构域数据库(例如https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=cd05672)标识符是cd05672,和/或,具有氨肽酶活性(例如EC 3.4.11.10),和/或,包括羧肽酶活性(例如羧肽酶A和/或B活性,例如分别具有EC 3.4.17.1和/或EC 3.4.17.2),和/或,嗜热菌蛋白酶活性(例如具有EC 3.4.24.27));(ii)能够去除一种或多种霉菌毒素(例如,ZEN)和/或一种或多种植物源-和/或细菌源-毒素(例如,单端孢真菌毒素诸如脱氧雪腐镰刀菌烯醇、瓜萎镰菌醇、新茄病镰刀菌烯醇、毛霉素、扁虫毒素、杆孢菌素A、葡萄穗霉毒素H、双乙酸基草镰刀菌醇、HT-2毒素或T-2毒素;黄曲霉毒素,诸如黄曲霉毒素B1、B2、G1或G2;伏马毒素,诸如伏马毒素B1、B2、B3或B4;多肽霉菌毒素,诸如白僵菌素或恩镰孢菌素;玉米赤霉烯酮;桔霉素;展青霉素;麦角生物碱,诸如麦角胺)和/或一种或多种植物源-和/或细菌源-毒素(例如内毒素等)的毒性的一种或多种其它多肽,尤其,所述一种或多种其它多肽能够去除一种或多种其它霉菌毒素和/或一种或多种植物源-和/或细菌源-毒素,例如,伏马毒素酯酶(例如,如WO 2016/134387 A1所公开的)和/或玉米赤霉烯酮内酯酶(例如,如WO 2020/025580 A1所公开的)和/或麦角肽水解酶(例如,如WO 2014/056006 A1所公开的);(iii)一种或多种有机吸附剂(例如,活的/灭活的/冻干的/休眠的/死的完整酵母,或者酵母衍生产品诸如酵母细胞壁,或者酵母寡聚体诸如甘露聚糖)和/或无机吸附剂(例如,硅藻土和/或粘土矿物诸如高岭土或高岭石,蒙脱石诸如蒙脱土、伊利石或绿泥石;尤其,膨润土);(iv)能够去除一种或多种其它霉菌毒素(例如,单端孢真菌毒素诸如脱氧雪腐镰刀菌烯醇、瓜萎镰菌醇、新茄病镰刀菌烯醇、毛霉素、扁虫毒素、杆孢菌素A、葡萄穗霉毒素H、双乙酸基草镰刀菌醇、HT-2毒素或T-2毒素;黄曲霉毒素,诸如黄曲霉毒素B1、B2、G1或G2;伏马毒素,诸如伏马毒素B1、B2、B3或B4;多肽霉菌毒素,诸如白僵菌素或恩镰孢菌素;玉米赤霉烯酮;桔霉素;展青霉素;麦角生物碱,诸如麦角胺)和/或一种或多种植物源-和/或细菌源-毒素(例如内毒素等)的毒性的一种或多种活的、灭活的、冻干的和/或休眠的微生物,尤其,所述微生物是如下的一员:毛孢子菌属和Apiotrichum属(例如,如WO 03/053161 A1公开的)或者红蝽菌科(例如,如EP 3501 526 A1公开的);(v)一种或多种植物产品(例如,海藻,优选海藻粉(seaweed meal);和/或,藻类,优选海藻粉(algae meal);和/或,蓟,优选蓟籽;和/或,甘草制剂,优选甘草粉和/或甘草提取物,例如,如WO 2018/121881 A1所公开);(vi)一种或多种调味化合物(例如,植物提取物,例如来自牛至、百里香、冬青、香菜、墨角兰、薄荷、胡椒薄荷、茴香、柑橘、柠檬、小茴香、八角茴香、丁香、肉桂和/或大蒜;和/或,精油,诸如D-柠檬烯、γ-萜烯、对伞花烃、2-蒈烯、氧化芳樟醇、异薄荷酮(isomenthone)、樟脑、芳樟醇、松油烯-4-醇、2-异丙基-1-甲氧基-4-甲苯、L-薄荷醇、乙胺、α-松油醇、β-石竹烯、D-香芹酮、水杨酸甲酯、α-石竹烯、薰衣草乙酸酯、氧化石竹烯、丁香酚百里香酚和/或香芹酚);(vii)一种或多种维生素(例如,维生素A、D、E、K、C、B1、B2、B3、B4、B5、B6、B7、B8、B9和/或B12;尤其是维生素E)。
30.前述任一项所述的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、宿主细胞、转基因植物、转基因种子、转基因花粉粒、组合物或试剂盒用作药物(例如,兽药用途和/或药物用途,例如用于动物和/或人)和/或用于治疗的用途,优选地,用于预防或治疗疾病,进一步优选地,所述亲代α/β水解酶与SEQ ID NO:1具有至少95%序列一致性。
31.变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、宿主细胞、转基因植物、转基因种子、转基因花粉粒、组合物或试剂盒,用于预防或治疗霉菌中毒,优选由玉米赤霉烯酮或其衍生物引发的激素紊乱。
32.一种预防或治疗霉菌中毒的方法,该方法包括:向存在风险或有需要的受试者施用如下的一种或多种:前述任一项所述的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、宿主细胞、转基因植物、转基因种子、转基因花粉粒、组合物或试剂盒。
33.根据前述任一项所述的方法或用途,其中以治疗有效量施用所述变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、宿主细胞、转基因植物、转基因种子、转基因花粉粒、组合物或试剂盒。
34.一种食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂),中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂,中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物,和/或其混合物,它们包括如下的一种或多种:前述任一项所述的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、宿主细胞、转基因植物、转基因种子、转基因花粉粒、组合物或试剂盒,优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%序列同一性。
35.一种降解玉米赤霉烯酮或其衍生物的方法,包括:
i)提供前述任一项所述的一种或多种变体,和/或,优选地前述任一项所述的一种或多种亲代α/β水解酶,优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%序列同一性;
ii)使一种或多种变体和/或亲代接触玉米赤霉烯酮或其衍生物(例如形成酶-底物混合物)。
36.根据前述任一项所述的降解玉米赤霉烯酮或其衍生物的方法,其中,当底物浓度是0.5ppm且pH是5.0时,变体和/或亲代α/β水解酶的比活力至少是0.6μmol/min/mg(例如,至少0.7,至少0.8,至少0.9,至少1.0,至少1.1,至少1.2,至少1.3或至少1.4),优选地,变体的比活力至少是1.35μmol/min/mg(诸如1.39或1.57)。
本发明通过以下实施例来进一步说明,但不限于实施例或实施例的任何特定实施方式。
本发明的实施例
实施例1:编码玉米赤霉烯酮-切割多肽的多核苷酸的修饰、克隆、表达和玉米赤霉烯酮-切割多肽的纯化
按照核苷酸序列合成完整的玉米赤霉烯酮-切割多肽基因(包括多肽C-末端融合的6x组氨酸标签),随后由上市公司(例如Twist Bioscience)将其整合入表达载体。
用包含完整的玉米赤霉烯酮-切割多肽基因的表达载体转化感受态E.coli BL21(DE3)。通过表达载体的抗生素耐药性来筛选转化子。任何其它合适的宿主细胞也都可以用于本工作中。
为了制备感受态E.coli BL21(DE3),将细胞接种于LB培养基(例如,Luria/Miller,颗粒状,6673,Carl Roth GmbH+CoKG,25.0g/L)并在37.0℃持续震荡过夜生长。在LB培养基中接种过夜培养物作为主要培养物,在37.0℃持续震荡生长直至OD600是0.65。在收获之前15.0分钟,将主要培养物冷却至4.0℃,只要有可能或另有说明,在随后的所有步骤中,在4.0℃保存细胞。在4.0℃,2000.0×g进行10分钟,收获细胞。按照每100.0ml主要培养物使用5.0ml TSS缓冲液(LB培养基,10.0%聚乙二醇-6000,5.0%二甲亚砜,20.0mM氯化镁)重悬细胞颗粒。
对于感受态E.coli BL21(DE3)的转化,使用5×KCM缓冲液(500.0mM氯化钾,150.0mM氯化钙,250.0mM氯化镁)1:1.25稀释感受态细胞溶液。向50.0μl制备的细胞溶液提供5.0μl包含完整的玉米赤霉烯酮-切割多肽基因的表达载体,4.0℃孵育10.0分钟。42.0℃热击1.5分钟,随后在4.0℃孵育1.0分钟。37.0℃,细胞在1.0ml LB培养基中再生1.5小时。将100.0μl细胞接种于具有抗生素(例如,卡那霉素50.0μg/ml)的900.0μl LB培养基中,37.0℃持续震荡过夜培养。
使用市售自诱导培养基由阳性转化子细胞内表达完整的玉米赤霉烯酮-切割多肽基因。
将500.0μl过夜培养物接种于盛装在50.0ml反应管中的9.5ml含有抗生素(例如,卡那霉素50.0μg/ml)的自诱导培养基(例如Novagen Overnight ExpressTM Instant TBMedium,71491,Sigma-Aldrich Handels GmbH)中,用密封膜(例如Breathe.Easier密封膜,Z763624,Sigma-Aldrich Handels GmbH)覆盖,以实现高气体交换率。37.0℃持续震荡孵育表达培养物24.0小时。室温,2000.0×g进行10分钟,收获细胞。
通过超声处理来裂解表达步骤收获的细胞。利用6x组氨酸标签亲和纯化从清除的裂解物获得玉米赤霉烯酮-切割多肽。通过SDS-PAGE分析来确认分子量和纯度。通过NanoDropTM分析来测定玉米赤霉烯酮-切割多肽的浓度。
在4.0℃,将收获的细胞重悬于2.0ml结合缓冲液(20.0mM磷酸钠,500.0mM氯化钠,60.0mM咪唑,pH 7.4)。在超声波仪上处理悬浮液直至细胞在4.0℃完全裂解(例如,Q700-220+Q4579,振幅50.0–处理时间8.0分钟–脉冲20.0秒–暂停40.0秒,QSonica L.L.C)。
4.0℃,2000.0×g离心15分钟来使裂解物澄清。按照制造商规程,将1.4ml上清液在HIS-Spin trap柱(例如,HIS-Spin Trap,28-4013-53,GE Healthcare GmbH)上进行纯化。玉米赤霉烯酮-切割多肽在400.0μl洗脱缓冲液(20.0mM磷酸钠,500.0mM氯化钠,500.0mM咪唑,pH 7.4)中进行洗脱,该制剂用于研究酶学特性。
为了确认纯化的玉米赤霉烯酮-切割多肽的分子量和纯度,洗脱物的稀释液中加入加样染料(例如,2×加样染料:0.125M三羟甲基氨基甲烷/盐酸pH 6.8,20.0%甘油,4.0%十二烷基硫酸钠,0.02%溴酚蓝,2%β-巯基乙醇)、煮沸并在12.0%聚丙烯酰胺凝胶(例如,Tetra Cell System,1658004EDU+12%TGXTM Precast Protein Gels,4561043EDU,Bio-Rad Laboratories GmbH)(10×运行缓冲液:0.125M三羟甲基氨基甲烷,1.25M甘氨酸,0.5%十二烷基硫酸钠,200V,40分钟)上分离。肽的条带通过考马斯亮蓝染色而可视化并与标准肽标记物进行比较。
为了测定纯化的玉米赤霉烯酮-切割多肽的浓度,检测280.0nm处的吸光度(例如,NanoDropTM One,ND-ONE-W,Thermo Fisher Scientific Inc.),按照Lambert-Beer法则计算浓度。
酶低聚度(degree of enzyme oligomerisation)的分析(Blue-Native-PAGE–BN-PAGE)
通过Blue-Native-PAGE对单个纯化的玉米赤霉烯酮-切割多肽之间的天然分子量(寡聚构象)进行比较。洗脱物采用非变性加样染料(例如,2×加样染料:0.126M三羟甲基氨基甲烷/盐酸pH 6.8,20.0%甘油,4.0%考马斯蓝G-250)稀释,并在自然条件下在4-15%聚丙烯酰胺凝胶上分离(例如,Tetra Cell System,1658004EDU+4-15%TGXTM Precast Protein Gels,4561083EDU,Bio-Rad LaboratoriesGmbH)(5×阳极缓冲液:0.125M三羟甲基氨基甲烷,0.960M甘氨酸,1×阴极缓冲液:阳极缓冲液+0.02%考马斯蓝G250;200V,40分钟)。通过考马斯染色可以看见肽的条带。采用尺寸排除色谱法、SEC-MALS或X-射线晶体衍射来测定选择的玉米赤霉烯酮-切割多肽的寡聚状态。同源二聚体(例如SEQ ID NO.1)在BN-PAGE凝胶基质上迁移得较快,而同源四聚体(例如SEQ ID NO.2-35)以较高程度保留在凝胶基质。依据同源二聚体肽和同源四聚体肽来确定玉米赤霉烯酮-切割多肽的寡聚构象。
玉米赤霉烯酮-切割多肽熔点(Tm)的测定:使用热位移分析,基于蛋白质变性过程中染料SYPRO Orange的荧光性,测定纯化的玉米赤霉烯酮-切割多肽的熔点。虽然水会淬灭它的荧光,但是,与变性蛋白质上未折叠的疏水表面相结合会引起荧光增加。
在pH7.5的Teorell Stenhagen缓冲液(and Virtama,Acta PhysiologicaScandinavica,1946,11:4,289-293)中将纯化的纯化的玉米赤霉烯酮-切割多肽稀释至0.2mg/ml。Sypro Orange 5000×储备液(Sypro Orange,S5692,Sigma-Aldrich HandelsGmbH)1:100稀释于pH7.5的Teorell Stenhagen缓冲液。在使用适合荧光检测的透明热封膜密封的96-孔PCR-板中,15.0μl稀释的玉米赤霉烯酮-切割多肽中加入3.0μl稀释的SyproOrange溶液和12.0μl pH7.5的Teorell Stenhagen缓冲液。采用在20分钟内20.0℃至95.0℃的线性增加温度在qPCR-循环仪上测量熔解曲线,每条测量曲线147个测量点(460.0nm消光,550.0nm发射)(例如,Mastercycler qPCR ep realplex 2s,6300000604,EppendorfAG)。荧光信号拐点处(换言之,荧光信号增加率最高的温度处)测定熔点。147个测量点的荧光强度相对温度梯度绘图。按照两个随后测量点的d荧光信号/d温度,计算每147个测量点的微分。计算的dF/dT最高值的温度是一阶导数的极大值,表示拐点且因此是玉米赤霉烯酮-切割多肽的熔点。
表3.玉米赤霉烯酮-切割多肽的熔点(Tm)
比活力的测定
制备玉米赤霉烯酮降解分析缓冲液(118.5mM NaCl,8.55mM醋酸,14.9mM醋酸盐,0.1mg/ml BSA,pH 5.0,(Jantratid,E.,Janssen,N.,Reppas,C.&Dressman,J.B.(2008):Dissolution media simulating conditions in the proximal humangastrointestinal tract:An update.Pharmaceutical Research 25(7):1663-1576),含有0.1mg/ml牛血清白蛋白和0.52ppm玉米赤霉烯酮作为底物),在37.0℃预热,将960μl试样的分析缓冲液转移至96深井板的反应管中。用活动盖密封该板,37.0℃保存在DWP-热振动筛中直至玉米赤霉烯酮降解分析开始。
使用纯化的玉米赤霉烯酮-切割多肽通过在样本缓冲液(Teorell Stenhagen缓冲液pH 7.5,含有0.1mg/ml牛血清白蛋白)中稀释至比最终玉米赤霉烯酮降解实验中分析的浓度高25倍来制备酶工作液,玉米赤霉烯酮-切割多肽的浓度可在给定条件下有效降解玉米赤霉烯酮。
为了开始玉米赤霉烯酮降解实验,将40.0μl酶工作液加入到含有960.0μl分析缓冲液的试管中,从而在分析反应中实现0.5ppm的最终ZEN浓度。加入pH 7.5的酶工作液不会改变pH 5.0的玉米赤霉烯酮降解反应。
在DWP-热振动筛中在37.0℃持续震荡条件下孵育玉米赤霉烯酮降解反应。玉米赤霉烯酮降解反应开始之后,立即用吸管头再悬浮混合,将120.0μl的0.0h样本转移至新的PCR板的管中,用管帽密封。在几个时间点(例如5.0,10.0,20.0,30.0,45.0,60.0,90.0分钟)之后从玉米赤霉烯酮降解反应取出额外样本。一旦从降解反应中取出样本,就通过在99.0℃在热块(thermo-block)(例如,Thermal Shake lite,460-029,VWR)孵育10.0分钟来热灭活该样本中的玉米赤霉烯酮切割多肽。随后,对管离心(3.0分钟,室温,2500x g),将90.0μl上清液转移至有边缘的PCR板(例如,PCR板,0030128648,Eppendorf AG),其用热封膜(例如,热封膜,0030127838,Eppendof AG)封闭。这些样本板储存在4.0℃,直至HPLC-FLD测量。按照Vekiru et al.(Vekiru et al.(2016)‘Isolation andcharacterization of enzymatic zearalenone hydrolysis reaction products’WorldMycotoxin Journal 9:353-363)描述的改进的HPLC-FLD方法测量玉米赤霉烯酮浓度。
278.0nm消光和465.0nm发射的HPLC-FLD上进行分析。当在40.0℃在Kinetex 5μmEVO C18 100A 50.0x2.1mm柱(00B-4633-AN,Phenomenex Inc.)上完成分离时,玉米赤霉烯酮的保留时间是1.8分钟,前述分离采用了如下方法:使用溶剂A(95.0%乙腈+4.9%水+0.1%甲酸)和溶剂B(5.0%乙腈+94.9%水+0.1%甲酸),梯度:0.0-0.5分钟10%相B,0.5-2.0分钟线性增加至50.0%相B,然后在0.1分钟内减至10%相B,持续进行,总运行时间2.3分钟。流速设置为1.5ml/min,进样体积是5.0μl。对各种分析采用了类似的采集设置。
基于玉米赤霉烯酮外标物的校准,对玉米赤霉烯酮进行定量。所取反应样品的玉米赤霉烯酮浓度(μM)对取样时间点作图。线性玉米赤霉烯酮降解随时间的斜率以酶促反应体积中每分钟减少的μmol玉米赤霉烯酮计算。考虑到纯化的玉米赤霉烯酮-切割多肽的可能的稀释并将这些合适的稀释因素包括在计算中,计算以每分钟每mg纯化的酶降解的μmol玉米赤霉烯酮(μmol ZEN/min/mg)表示的比酶活(specific enzymatic activity)。
表4:pH5.0时的比酶活
酶鉴定 | 比活力(μmol ZEN/min/mg) |
现有技术比较序列1 | 0.00 |
现有技术比较序列2 | 0.00 |
本发明的SEQ ID NO.1 | 1.57 |
本发明的SEQ ID NO.28 | 1.39 |
本领域技术人员将容易理解,本发明适于实现所述目的并获得所述目的和优点以及其中固有的那些目的和优点。此外,对本领域技术人员来说显而易见的是,在不脱离本发明的范围和精神的情况下,可以对本文公开的发明进行不同的替换和修改。本文所述的组合物、方法、过程、处理、分子和特定化合物目前是代表某些实施方案,是示例性的,不旨在限制本发明的范围。对于本领域技术人员来说,在本发明的精神范围内所包含的变化和其他用途将由权利要求书的范围限定。本说明书中对先前发布的文件的列出或讨论不一定被视为承认该文件是现有技术的一部分或是公知常识。
本文示例性描述的发明可以在没有任何元素或要素、限制或限定的情况下被合适地实践,而本发明未在本文中具体披露。因此,例如,术语“包含”、“包括”、“含有”等应扩展性的解读,而非限制性的。此外,本文中使用的术语和表述已被用作描述术语而非限制术语,并且,使用此类术语和表述时,无意排除所示和描述的特征或其部分的任何等价物,但是,应当认识到,在所要求保护的发明的范围内可以进行各种修改。因此,应当理解,尽管已经通过示例性实施方案和可选特征具体地公开了本发明,但是本领域技术人员可以求助于对本文所体现的发明的修改和变化,并且,这种修改和变化被认为在本发明的范围内。
本文已经对本发明进行了广泛地和一般地描述。落入一般性公开内的每种较窄的种类和亚组也构成本发明的部分。这包括对本发明的一般性描述,限制条款或负面限制是从属中去除任何主题,而不管离体物质在本文中是否进行了明确列举。所有文献,包括专利申请和科学出版物,都通过引用整体包括在本文中。
其他实施方案在以下权利要求中。此外,当以马库什群组来描述本发明的特征或方面时,本领域技术人员将意识到,本发明因此还以马库什群组的任何单个成员或亚组成员来描述。
序列表
<110> 爱尔伯股份公司
<120> 具有提高的温度稳定性的四聚体α/β水解酶变体及其使用和生产方法
<130> ERB17181PCT
<150> EP20200918.9
<151> 2020-10-08
<160> 39
<170> PatentIn version 3.5
<210> 1
<211> 316
<212> PRT
<213> 气微菌属(Aeromicrobium sp.)
<400> 1
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Asn Phe Leu Gly Asp Gln Trp Ser Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 2
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 2
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 3
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 3
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Phe Leu Gly Asp Gln Trp Ser Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 4
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 4
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Ser Phe Leu Gly Asp Gln Trp Ser Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 5
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 5
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Ala Phe Leu Gly Asp Gln Trp Ser Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 6
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 6
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Cys Phe Leu Gly Asp Gln Trp Ser Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 7
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 7
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Leu Phe Leu Gly Asp Gln Trp Ser Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 8
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 8
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Phe Leu Gly Asp Gln Trp Thr Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 9
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 9
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Phe Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 10
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 10
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Lys Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Phe Leu Gly Asp Gln Trp Cys Ala Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 11
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 11
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Lys Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Phe Leu Gly Asp Gln Trp Cys Thr Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 12
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 12
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Ser Phe Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 13
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 13
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Phe Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 14
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 14
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Asp Gly Phe Ala Ala Ala Ala Lys Ala Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Phe Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 15
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 15
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 16
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 16
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Leu Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Ile Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 17
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 17
Met Thr Asp Gln Ser Pro Glu Thr Arg Asn Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 18
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 18
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Tyr Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Met Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 19
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 19
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Thr Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 20
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 20
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp Arg Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 21
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 21
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Ser Gln
35 40 45
Thr Glu Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 22
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 22
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 23
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 23
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ser Glu
50 55 60
His Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 24
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 24
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Tyr Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 25
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 25
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 26
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 26
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 27
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 27
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
Ser Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 28
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 28
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Arg Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 29
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 29
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Val Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 30
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 30
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro Glu Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 31
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 31
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Glu Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 32
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 32
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Thr His
275 280 285
Ser Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 33
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 33
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Ser Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Ser
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 34
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 34
Met Thr Asp Gln Ser Pro Glu Thr Arg Asp Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Val Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp His Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Pro Val Ile Gly Leu Leu Ala Glu
50 55 60
Ser Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Thr Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
His Cys Pro His Asp Leu Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Ala Ser Phe Pro Asp Ala Leu His
275 280 285
Met Met His Gln Phe Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Arg
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 35
<211> 316
<212> PRT
<213> 人工序列
<220>
<223> 变体α/β水解酶
<400> 35
Met Thr Asp Gln Ser Pro Glu Thr Arg Asn Ile Phe Val Pro His Ala
1 5 10 15
Phe Glu Glu Gln Gln Val Asp Leu Gly Glu Val Thr Met Asn Tyr Val
20 25 30
Glu Ala Gly Ser Ala Asp Arg Pro Ala Leu Leu Leu Leu Pro Glu Gln
35 40 45
Thr Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ser Glu
50 55 60
His Phe His Val Tyr Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr
65 70 75 80
Trp Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val
85 90 95
Arg Phe Ile Ser Leu Val Val Lys Arg Pro Val Ile Val Ser Gly Asn
100 105 110
Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro
115 120 125
Gly Gln Ile Arg Ala Ala Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser
130 135 140
Glu Leu Val Pro Ala His Gly His Ser Val Arg Gln Ala Ala Gly Pro
145 150 155 160
Ala Phe Glu Leu Tyr Arg Thr Tyr Leu Gly Asp Gln Trp Cys Val Ala
165 170 175
Asp Trp Glu Gly Phe Cys Arg Ala Ala Arg Arg Ser Pro Ser Lys Ile
180 185 190
Met Ser Leu Phe Pro Ser Ser Asp Glu Pro Pro Gln Asn Leu Lys Glu
195 200 205
Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Ala Leu
210 215 220
Ser Cys Pro His Asp Arg Met Leu Ser Gln Val Lys Thr Pro Ile Leu
225 230 235 240
Ile Thr His His Ala Arg Ala Val Asp Pro His Ser Gly Asp Leu Leu
245 250 255
Gly Ala Leu Ser Asp Phe Gln Ala Glu Arg Ala Gln Ala Ile Ile Glu
260 265 270
Ser Ala Gly Val Pro Val Glu Tyr Glu Ser Phe Pro Asp Ala Thr His
275 280 285
Ser Met His Gln Ser Glu Pro Glu Arg Phe Ala Glu Val Leu Thr Arg
290 295 300
Trp Val Ala Arg Leu Pro Ala Asp Thr Pro Gly Ala
305 310 315
<210> 36
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 共同氨基酸基序1
<220>
<221> 变体
<222> (7)..(7)
<223> Xaa 是 Val 或 Ile
<220>
<221> 变体
<222> (8)..(8)
<223> Xaa 是 Val 或 Thr
<400> 36
Gln Val Asp Leu Gly Glu Xaa Xaa Met Asn
1 5 10
<210> 37
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 共同氨基酸基序2
<220>
<221> 变体
<222> (7)..(7)
<223> Xaa 是 Gly 或 Ala
<220>
<221> 变体
<222> (9)..(9)
<223> Xaa 是 Ala 或 Val
<220>
<221> 变体
<222> (11)..(11)
<223> Xaa 是 Phe 或 Tyr
<400> 37
Glu Tyr Asp Pro Glu Trp Xaa Arg Xaa Phe Xaa Glu Gly Thr Val
1 5 10 15
<210> 38
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 共同氨基酸基序3
<220>
<221> 变体
<222> (3)..(3)
<223> Xaa 是 Ser 或 Gly
<220>
<221> 变体
<222> (9)..(9)
<223> Xaa 是 Ile 或 Val
<220>
<221> 变体
<222> (11)..(11)
<223> Xaa 是 Ile 或 Leu
<400> 38
Met Leu Xaa Gln Val Lys Thr Pro Xaa Leu Xaa Thr His His
1 5 10
<210> 39
<211> 127
<212> PRT
<213> 人工序列
<220>
<223> 共同氨基酸基序4
<220>
<221> 变体
<222> (3)..(3)
<223> Xaa 是 Val 或 Leu
<220>
<221> 变体
<222> (6)..(6)
<223> Xaa 是 Leu 或 Ile
<220>
<221> 变体
<222> (18)..(18)
<223> Xaa 是 Pro 或 Glu
<220>
<221> 变体
<222> (19)..(19)
<223> Xaa 是 Val 或 Ala
<220>
<221> 变体
<222> (20)..(20)
<223> Xaa 是 Ile 或 Met
<220>
<221> 变体
<222> (24)..(24)
<223> Xaa 是 Ala 或 Ser
<220>
<221> 变体
<222> (26)..(26)
<223> Xaa 是 Ser 或 Asn 或 His
<220>
<221> 变体
<222> (30)..(30)
<223> Xaa 是 Phe 或 Tyr
<220>
<221> 变体
<222> (34)..(34)
<223> Xaa 是 Ile 或 Leu
<220>
<221> 变体
<222> (41)..(41)
<223> Xaa 是 Thr 或 Ser
<220>
<221> 变体
<222> (45)..(45)
<223> Xaa 是 Lys 或 Arg
<220>
<221> 变体
<222> (61)..(61)
<223> Xaa 是 Ser 或 Ala 或 Asn
<220>
<221> 变体
<222> (64)..(64)
<223> Xaa 是 Val 或 Ile
<220>
<221> 变体
<222> (69)..(69)
<223> Xaa 是 Ile 或 Val
<220>
<221> 变体
<222> (71)..(71)
<223> Xaa 是 Ser 或 Ala
<220>
<221> 变体
<222> (78)..(78)
<223> Xaa 是 Val 或 Leu
<220>
<221> 变体
<222> (87)..(87)
<223> Xaa 是 Ala 或 Ser
<220>
<221> 变体
<222> (92)..(92)
<223> Xaa 是 Ile 或 Leu
<220>
<221> 变体
<222> (94)..(94)
<223> Xaa 是 Ala 或 Gly
<220>
<221> 变体
<222> (95)..(95)
<223> Xaa 是 Ala 或 Val
<220>
<221> 变体
<222> (100)..(100)
<223> Xaa 是 Thr 或 Ala 或 Pro
<220>
<221> 变体
<222> (101)..(101)
<223> Xaa 是 Pro 或 Ala
<220>
<221> 变体
<222> (111)..(111)
<223> Xaa 是 Tyr 或 His
<220>
<221> 变体
<222> (116)..(116)
<223> Xaa 是 Leu 或 Arg
<220>
<221> 变体
<222> (118)..(118)
<223> Xaa 是 Ala 或 Gly
<220>
<221> 变体
<222> (122)..(122)
<223> Xaa 是 Ala 或 Val
<220>
<221> 变体
<222> (126)..(126)
<223> Xaa 是 Tyr 或 Phe
<400> 39
Pro Ala Xaa Leu Leu Xaa Pro Glu Gln Thr Gly Ser Trp Trp Ser Tyr
1 5 10 15
Glu Xaa Xaa Xaa Gly Leu Leu Xaa Glu Xaa Phe His Val Xaa Ala Val
20 25 30
Asp Xaa Arg Gly Gln Gly Arg Ser Xaa Trp Thr Pro Xaa Arg Tyr Ser
35 40 45
Leu Asp Asn Phe Gly Asn Asp Leu Val Arg Phe Ile Xaa Leu Val Xaa
50 55 60
Lys Arg Pro Val Xaa Val Xaa Gly Asn Ser Ser Gly Gly Xaa Leu Ala
65 70 75 80
Ala Trp Leu Ser Ala Tyr Xaa Met Pro Gly Gln Xaa Arg Xaa Xaa Leu
85 90 95
Cys Glu Asp Xaa Xaa Phe Phe Ala Ser Glu Leu Val Pro Ala Xaa Gly
100 105 110
His Ser Val Xaa Gln Xaa Ala Gly Pro Xaa Phe Glu Leu Xaa Arg
115 120 125
Claims (18)
1.一种亲代α/β水解酶的变体,所述变体包括对应于如下位置的一个或多个位置处的置换:SEQ ID NO:1(优选使用SEQ ID NO:1的编号)167、168、174、218、155、175、179、182、183、186、187、268、306、10、17、28、39、47、50、57、58、59、63、65、69、126、139、225、230、245、251、281、287、289、293和304,其中所述变体具有α/β水解酶活性,并且,其中所述变体是与SEQ ID NO:1的多肽具有至少71%(优选:至少72%、至少73%、至少74%、至少75%、至少76%、至少77%、至少78%、至少79%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少95.5%、至少96%、至少96.5%、至少97%、至少97.5%、至少98%、至少98.5%、至少99%或至少99.5%)但小于100%序列同一性的多肽,优选地,所述变体具有水解酶EC:3.1.1.-活性。
2.根据前述权利要求任一项所述的变体,其中所述变体具有如下一种或多种特性:
i)相对于亲代α/β水解酶的改进的性质,其中所述改进的性质包括提高的温度稳定性,优选是相比于SEQ ID NO:1的提高的温度稳定性;所述变体能够四聚体化,优选具有四聚体四级结构,进一步优选地,所述四聚体化是同源四聚体化;
ii)熔点(Tm)大于50℃,优选地,所述Tm的范围是大约51℃至大约70℃;
iii)能够降解玉米赤霉烯酮和/或其衍生物;
iv)包括与选自如下的氨基酸序列具有至少80%(优选至少83%、更优选至少90%)序列同一性的至少一个(优选至少两个、更优选至少三个、最优选至少四个)氨基酸基序:
a)SEQ ID NO:36(QVDLGEX1X2MN),其中X1是V或I,优选V,并且,其中X2是V或T,优选V;
b)SEQ ID NO:37(EYDPEWX3RX4FX5EGTV),其中X3是G或A,优选A,并且,其中X4是A或V,优选A,并且,其中X5是F或Y;
c)SEQ ID NO:38(MLX6QVKTPX7LX8THH),其中X6是S或G,优选S,并且,其中X7是I或V,优选I,并且,其中X8是I或L,优选I;
d)SEQ ID NO:39
(PAX9LLX10PEQTGSWWSYEX11X12X13GLLX14EX15FHVX16AVDX17RGQGRSX18WTPX19RYSLDNFGNDLVRFIX20LVX21KRPVX22VX23GNSSGGX24LAAWLSAYX25MPGQX26RX27X28LCEDX29X30FFASELVPAX31GHSVX32QX33AGPX34FELX35R)其中X9是V或L,优选L,并且,其中X10是L或I,优选L,并且,其中X11是P或E,优选P,并且,其中X12是V或A,优选V,并且,其中X13是I或M,优选I,并且,其中X14是A或S,优选A,并且,其中X15是S、N或H,优选S,并且,其中X16是F或Y,优选F,并且,其中X17是I或L,优选I,并且,其中X18是T或S,优选T,并且,其中X19是K或R,优选K,并且,其中X20是S、A或N,优选S,并且,其中X21是V或I,优选V,并且,其中X22是I或V,优选I,并且,其中X23是S或A,优选S,并且,其中X24是V或L,优选V,并且,其中X25是A或S,优选A,并且,其中X26是I或L,优选I,并且,其中X27是A或G,优选A,并且,其中X28是A或V,优选A,并且,其中X29是T、A或P,优选T,并且,其中X30是P或A,优选P,并且,其中X31是Y或H,优选H,并且,其中X32是L或R,优选R,并且,其中X33是A或G,优选A,并且,其中X34是A或V,优选A,并且,其中X35是Y或F,优选Y。
4.根据前述权利要求任一项所述的变体,其中所述亲代α/β水解酶,优选具有EC:3.1.1.-水解酶活性,选自如下肽构成的组:
i)与SEQ ID NO:1的多肽具有至少60%(优选至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少95.5%、至少96%、至少96.5%、至少97%、至少97.5%、至少98%、至少98.5%、至少99%或至少99.5%或100%)序列同一性的多肽;
ii)SEQ ID NO:1的多肽的片段,其中所述片段具有α/β水解酶活性,优选EC:3.1.1.-水解酶活性。
6.一种获得亲代α/β水解酶的变体(所述变体优选具有EC:3.1.1.-水解酶活性)和/或提高所述亲代α/β水解酶的稳定性的方法,所述方法包括:在与SEQ ID NO:1(优选使用SEQID NO:1的编号)的位置167、168、174、218、155、175、179、182、183、186、187、268、306、10、17、28、39、47、50、57、58、59、63、65、69、126、139、225、230、245、251、281、287、289、293和304对应的一个或多个位置处向所述亲代α/β水解酶引入置换,其中所述变体具有α/β水解酶活性;和,回收所述变体;优选地,所述亲代α/β水解酶如前述权利要求任一项所述,进一步优选地,所述亲代α/β水解酶与SEQ ID NO:1具有至少95%序列同一性。
7.一种编码前述权利要求任一项所述变体的多核苷酸。
8.一种包括前述权利要求任一项所述多核苷酸的核酸构建体。
9.一种包括前述权利要求任一项所述多核苷酸和/或核酸构建体的表达载体。
10.一种重组宿主细胞,包括如下至少一种:
i)前述权利要求任一项所述的变体和/或亲代α/β水解酶;
ii)前述权利要求任一项所述的多核苷酸;
iii)前述权利要求任一项所述的核酸构建体;和/或
iv)前述权利要求任一项所述的表达载体。
11.一种转基因植物、转基因种子或转基因花粉粒,包括如下的一种或多种(优选多种):
i)前述权利要求任一项所述的变体和/或亲代α/β水解酶;
ii)前述权利要求任一项所述的多核苷酸;
iii)前述权利要求任一项所述的核酸构建体;
iv)前述权利要求任一项所述的表达载体;和/或
v)前述权利要求任一项所述的重组宿主细胞。
12.一种食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂),中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂,中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物,和/或其混合物,它们包括如下的一种或多种:
i)前述权利要求任一项所述的变体和/或亲代α/β水解酶;优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性;
ii)前述权利要求任一项所述的多核苷酸;
iii)前述权利要求任一项所述的核酸构建体;
iv)前述权利要求任一项所述的表达载体;
v)前述权利要求任一项所述的重组宿主细胞;
vi)前述权利要求任一项所述的转基因植物、转基因种子和/或转基因花粉粒。
13.一种组合物或试剂盒,包括如下的一种或多种:
i)前述权利要求任一项所述的变体和/或亲代α/β水解酶,优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性;
ii)前述权利要求任一项所述的多核苷酸;
iii)前述权利要求任一项所述的核酸构建体;
iv)前述权利要求任一项所述的表达载体;
v)前述权利要求任一项所述的重组宿主细胞;
vi)前述权利要求任一项所述的转基因植物、转基因种子或转基因花粉粒;和/或
vii)前述权利要求任一项所述的食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物,和/或其混合物。
14.根据前述权利要求任一项所述的组合物或试剂盒,进一步包括营养学上可接受的载体和/或前述权利要求任一项所述的亲代α/β水解酶,优选地,所述亲代α/β水解酶与SEQID NO:1的多肽具有至少95%的序列同一性。
15.前述权利要求任一项所述的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、重组宿主细胞、转基因植物、转基因种子、转基因花粉粒、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物、组合物和/或试剂盒,用作药物和/或用于治疗,优选地,用于疾病的预防或治疗,进一步优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性。
16.前述权利要求任一项所述的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、重组宿主细胞、转基因植物、转基因种子、转基因花粉粒、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物、组合物和/或试剂盒,用于预防或治疗霉菌中毒,优选地,适合用于预防或治疗玉米赤霉烯酮或其衍生物引起的激素紊乱。
17.前述权利要求任一项所述的变体、亲代α/β水解酶、多核苷酸、核酸构建体、表达载体、重组宿主细胞、转基因植物、转基因种子、转基因花粉粒、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物在如下一种或多种中的用途:
i)玉米赤霉烯酮和/或其衍生物的降解;
ii)食物、食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物;
iii)如下一种或多种的制造/生产:食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物;
iv)青储饲料、沼气、生物乙醇或糖的制造/生产,优选由甘蔗或甜菜制造/生产;
v)霉菌中毒的预防或治疗。
18.一种降解玉米赤霉烯酮和/或其衍生物的方法,包括:
a)提供如下的一种或多种:
i)前述权利要求任一项所述的变体和/或亲代α/β水解酶,优选地,所述亲代α/β水解酶与SEQ ID NO:1的多肽具有至少95%的序列同一性;
ii)前述权利要求任一项所述的重组宿主细胞;
iii)前述权利要求任一项所述的转基因植物;
iv)前述权利要求任一项所述的食品、中间食品;草料、中间草料;饲料、中间饲料;添加剂(优选地,食品添加剂、草料添加剂或饲料添加剂)、中间添加剂(优选地,食品中间添加剂、草料中间添加剂或饲料中间添加剂);解毒剂、中间解毒剂;营养补充剂、中间营养补充剂;益生元、中间益生元、青储饲料接种菌;解毒药;兽用组合物;药物组合物和/或其混合物;和/或
v)前述权利要求任一项所述的组合物或试剂盒;
b)使来自(a)的一种或多种物质接触玉米赤霉烯酮和/或其衍生物。
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CN117757698B (zh) * | 2024-01-25 | 2024-06-11 | 浙江大学 | 一株枯草芽孢杆菌dyf4-3及其在降解多种霉菌毒素中的应用 |
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AT413540B (de) | 2001-12-20 | 2006-03-15 | Erber Ag | Mikroorganismus, welcher ochratoxine sowie ochratoxine und zearalenone entgiftet, sowie verfahren und verwendung hiefür |
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