CN100510067C - 葡糖淀粉酶变体 - Google Patents
葡糖淀粉酶变体 Download PDFInfo
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- CN100510067C CN100510067C CNB008101329A CN00810132A CN100510067C CN 100510067 C CN100510067 C CN 100510067C CN B008101329 A CNB008101329 A CN B008101329A CN 00810132 A CN00810132 A CN 00810132A CN 100510067 C CN100510067 C CN 100510067C
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- glucoamylase
- variant
- dna
- sequence
- starch
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Abstract
本发明涉及亲本真菌葡糖淀粉酶的变体,其表现出特性改变,具体是热稳定性和/或比活提高。
Description
发明领域
本发明涉及亲本AMG的特性改变的新葡糖淀粉酶变体(突变体),所述特性改变具体是改善了热稳定性和/或提高了比活,所述变体适用于如淀粉转化,具体适于从淀粉生产葡萄糖,和醇类生产,甜味剂生产。更特定地,本发明涉及葡糖淀粉酶变体和此变体酶的应用。
发明背景
葡糖淀粉酶(1,4-α-D-葡聚糖葡萄糖水解酶,EC3.2.1.3)可以催化从淀粉或相关的寡糖和多糖分子的非还原末端释放D-葡萄糖。葡糖淀粉酶可从几种丝状真菌或酵母产生,其中曲霉属在商业上是最重要的。
在商业上,葡糖淀粉酶用于将被α-淀粉酶部分水解的玉米淀粉转化为为葡萄糖。葡萄糖进一步被葡萄糖异构酶转化为含几乎等量的葡萄糖和果糖的混合物。此混合物或果糖含量更高的混合物是通常使用于全世界商业用途的高果糖玉米浆。此糖浆是世界上由酶方法生产的最大产量的产品。在由淀粉转化为果糖中有关的三种酶是生产的最重要的工业用酶。
考虑到在高果糖玉米浆生产中葡糖淀粉酶的商业用途,其中一个主要问题是葡糖淀粉酶的热稳定性相对较低。葡糖淀粉酶不象α-淀粉酶或葡萄糖异构酶一样热稳定,它最活跃和稳定的pH值比α-淀粉酶或葡萄糖异构酶要低。相应地,它必须用于较低温度和pH值的单独的容器中。
黑曲霉的葡糖淀粉酶具有催化活性结构域(氨基酸1-440)和淀粉结合结构域(氨基酸509-616),此二者被一段较长且高度O-糖基化的接头所分隔(Svensson等(1983),Carlsberg Res.Commun.48,529-544,1983和(1986),欧洲生物化学杂志(Eur.J.Biochem),154,497-502)。泡盛曲霉X100的葡糖淀粉酶的催化活性结构域(氨基酸1-471)具有(α/α)6-折叠,其中六个保守的α→α环片段与内桶和外桶相连(Aleshin等(1992),生物学化学杂志(J.Biol.Chem.)167,19291-19298)。葡糖淀粉酶与1-脱氧野尻霉素(Harris等(1993),生物化学,32,1618-1626)和假四糖抑制因子阿卡波糖及D-葡萄糖-二氢阿卡波糖(Aleshin等(1996),生物化学35,8319-8328)的复合物的晶体结构进一步与分别作为全酸和全碱的谷氨酸179和400相符合。已利用蛋白工程对这些残基在催化中的关键作用进行了研究(Sierks等(1990),蛋白质工程(Protein Engng.)3,193-198;Frandsen等(1994),生物化学,33,13808-13816)。在葡糖淀粉酶复合物结构中强调了在四个糖基残基结合亚点,-1,+1,+2,和+3处葡糖淀粉酶-糖的相互作用,利用定点诱变突变体结合动力学分析广泛地调查对结合和催化作用重要的残基(Sierks等(1989),蛋白质工程,2,621-625;Sierks等(1990),蛋白质工程,3,193-198;Berland等(1995),生物化学,34,10153-10161;FrandSen等(1995),生物化学,34,10162-10169。
黑曲霉葡糖淀粉酶中可以增强热稳定性的几种取代描述如下:i)取代α—螺旋甘氨酸:G137A和G139A(Chen等(1996),蛋白质工程,9,499-505);ii)去除脆弱的Asp-X肽键,D257E和D293E/Q(Chen等(1995),蛋白质工程,8,575-582);阻止在N182的脱氨基作用(Chen等(1994),生物化学杂志,301,275-281);iv)通过工程化方法添加二硫键,A246C(Fierobe等(1996),生物化学,35,8698-8704;和v)在A435和S436中引入Pro残基(Li等(1997),蛋白质工程,10,1199-1204。此外,Clark Ford于1997年10月17日发表的论文,酶工程14,北京/中国10月12-17日,1997,文摘号码:文摘第0-61页。此文摘提出在泡盛曲霉葡糖淀粉酶位置G137A,N20C/A27C,和S30P(未公开))进行突变以改进其热稳定性。
有关葡糖淀粉酶的其它信息可在国际互联网主页(http://www.public.iastate.edu/~pedro/glase/glase.html)上由Pedro M.Coutinho负责的“葡糖淀粉酶WWW网页”(最后一次更新97/10/08)披露了有关葡糖淀粉酶的信息,其中包括得自曲霉属菌株的葡糖淀粉酶。列出了在黑曲霉葡糖淀粉酶中的化学修饰和定点修饰。
发明简述
本发明的目的是提供适用于,例如淀粉转化过程中的糖化步骤的葡糖淀粉酶变体。
术语“热稳定的葡糖淀粉酶变体”在本发明中指与相应的亲本葡糖淀粉酶相比有较高T1/2(半衰期)的葡糖淀粉酶变体。在下面的“材料和方法”部分描述了T1/2的测定(方法I和方法II)。
术语“比活提高的葡糖淀粉酶变体”在本发明中指提高了对所述糖分子中α-1,4键的比活。比活测定为kcat或AGU/mg(按下文“材料和方法”部分所述测定)。提高比活意味着kcat或AGU/mg值分别相对于相应地亲本葡糖淀粉酶有了提高。
本发明人提供了亲本葡糖淀粉酶的几种改进了热稳定性和/或提高了比活的变体。热稳定性的改进可通过突变,例如在亲本葡糖淀粉酶所选位置上的取代和/或缺失,插入来实现。下面有对此的详述。
命名法
在本说明书和权利要求书中,使用传统的一字母和三字母代码表示氨基酸残基。
为便于引用,本发明的AMG变体采用下述的命名法:
原氨基酸:位置:用于取代的氨基酸
根据该命名法,用天冬酰胺取代第30位的丙氨酸示为:Ala30Asn或A30N,在相同位置处缺失丙氨酸示为:Ala30*或A30*,而插入另一个氨基酸残基,如赖氨酸示为:Ala30AlaLys或A30AK。缺失一段连续的氨基酸残基,如氨基酸残基30-33示为(30-33)*或Δ(A30-N33)。
当特定的AMG相对于其它AMG而言含有“缺失”,并在该位置插入某个氨基酸,例如在第36位插入天冬氨酸时,示为*36Asp或*36D。
多个突变由加号隔开,即:Ala30Asp+Glu34Ser或A30N+E34S表示分别将第30和34位的丙氨酸和谷氨酸用天冬酰胺和丝氨酸取代。多个突变也可以如下隔开,其意义与加号相同:Ala30Asp/Glu34Ser或A30N/E34S。
当在给定的位置插入一个或多个其它氨基酸残基时,示为A30N,E或A30N/E或A30N或A30E。
另外,当本文鉴定出适于修饰的位置,而没有暗示任何具体的修饰时,应理解为可用任一氨基酸残基取代该位置存在的氨基酸残基。因此,例如,当提到修饰第30位的丙氨酸,但未具体说明时,应理解为丙氨酸可缺失,或用任一其它氨基酸,即下列任一氨基酸所取代:R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V。
附图简述
图1表示了含有黑曲霉G1葡糖淀粉酶基因的质粒pCAMG91。
发明详述
本发明旨在改进特定葡糖淀粉酶的热稳定性和/或提高其比活,所述酶得自真菌生物,特别是黑曲霉属的菌株这些酶本身已根据它们在诸如淀粉转化或乙醇发酵中的适当性质进行了筛选。
本发明人从中惊奇地发现通过修饰亲本葡糖淀粉酶的氨基酸序列上的一或多个氨基酸残基确实有可能改进亲本葡糖淀粉酶热稳定性和/或提高比活。本发明就是基于此发现。
因此,本发明的第一个方面涉及亲本葡糖淀粉酶在下述位置包含一或多个突变的变体。
亲本葡糖淀粉酶
本发明考虑的亲本葡糖淀粉酶包括野生型葡糖淀粉酶,真菌葡糖淀粉酶,特别是得自曲霉属菌株,如黑曲霉或泡盛曲霉的真菌葡糖淀粉酶及其变体或突变体,同源葡糖淀粉酶,和其它与SEQ ID NO:2结构和/或功能相似的葡糖淀粉酶。特别予以考虑的是公开于Boel等(1984),“从两个不同但密切相关的mRNAs合成的黑曲霉葡糖淀粉酶G1和G2”EMBO J.3(5),p.1097-1102中的黑曲霉葡糖淀粉酶G1和G2。G2葡糖淀粉酶公开为SEQ IDNO:2。在另一实施方案中,AMG骨架得自踝节菌属,特别是公开于WO99/28448的T.emersonii(参见WO 99/28448的SEQ ID NO:7)。
市售亲本葡糖淀粉酶
考虑的市售亲本葡糖淀粉酶包括Novo Nordisk的AMG,以及Genencor,Inc.USA和Gist-Brocades,Delft,The Netherlands的葡糖淀粉酶。
本发明的葡糖淀粉酶变体
在第一个方面,本发明涉及亲本葡糖淀粉酶的变体,其包括在下列一或多个位置的改变:59,66,72,119,189,223,227,313,340,342,352,379,386,393,395,402,408,416,425,427,444,486,490,494,
其中(a)所述每一改变是
(i)在占据此位置的氨基酸下游插入氨基酸,
(ii)占据此位置的氨基酸的缺失,或
(iii)占据此位置的氨基酸用另一氨基酸取代,
(b)所述变体具有亲本葡糖淀粉酶活性且(c)每一位置对应于具有SEQID NO:2之氨基酸序列的亲本葡糖淀粉酶氨基酸序列的位置。
此外,本发明涉及这样一种葡糖淀粉酶变体,其亲本葡糖淀粉酶具有与SEQ ID NO:2之氨基酸序列至少约65%,优选至少约70%,更优选至少约80%,更优选至少约90%,最优选至少约95%,还最优选至少约97%同一性的氨基酸序列。
本发明还涉及亲本葡糖淀粉酶的下述变体,其包含一或多个下列改变:V59A,L66V/R,T72I,S119P,I189T,Y223F,F227Y,N313G,S340G,E342A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V,优选E342T,K352R,S356G,T379A,S386K,N,R,P,A393R,S395R,Y402F,E408R,T416A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,E,W,Y,V,优选T416H,A425T,N427S/M,S444G,S486G,T490A,T494P/A,其中(a)所述变体具有葡糖淀粉酶活性且(b)每一位置对应于具有SEQ ID NO:2之氨基酸序列的亲本葡糖淀粉酶的氨基酸序列中位置。
特定的突变组合包括:
L66R+Y402F+N427S+S486G+AlV;N427M+S44G+V470M+T2K+S30P;
T416H+Y402F+312Q+S119P;A425T+E408R+E386K+A495T;
T379A+T2E+S386K+A393R;S386N+E408R;L66V+T2R+S394P+Y402F+RL;
S386R+T2R+A393R;I189T+Y223F+F227Y+S119P+Y402F;
S386P+S340G+D357S+T360V;V59A+S119P;V59A+N313G;V59A+A393R;
V59A+Y402F;V59A+E408R;V59A+S119P+N313G;V59A+N313G+A393R;
V59A+A393R+Y402F;V59A+Y402F+E408R;V59A+S119P+N313G+A393R;
V59A+N313G+A393R+Y402F;V59A+A393R+Y402F+E408R;
V59A+S119P+N313G+A393R+Y402F;V59A+N313G+A393R+Y402F+E408R;
V59A+S119P+L66R;V59A+S119P+S340G;V59A+S119P+S395R;
V59A+S119P+L66R+S340G;V59A+S119P+S340G+S395R;
V59A+S119P+S395R+L66R;V59A+S119P+S395R+L66R+S340G;
V59A+N313G+L66R;V59A+N313G+S340G;V59A+N313G+S395R;
V59A+N313G+L66R+S340G;V59A+N313G+S340G+S395R;
V59A+N313G+S395R+L66R;V59A+N313G+S395R+L66R+S340G;
V59A+A393R+L66R;V59A+A393R+S340G;V59A+A393R+S395R;
V59A+A393R+L66R+S340G;V59A+A393R+S340G+S395R;
V59A+A393R+S395R+L66R+S340G;V59A+Y402F+L66R;
V59A+Y402F+S340G;V59A+Y402F+S395R;V59A+Y402F+L66R+S395R;
V59A+Y402F+L66R+S340G;
V59A+Y402F+L66R+S395R+S340G;V59A+E408R+L66R;
V59A+E408R+S395R;V59A+E408R+S340G;V59A+E408R+S395R+S340G;
V59A+E408R+L66R+S340G;V59A+E408R+L66R+S395R;
V59A+E408R+L66R+S395R+S340G;V59A+S119P+N313G+L66R;
V59A+S119P+N313G+L66R+S340G;V59A+S119P+N313G+L66R+S395R;
V59A+S119P+N313G+L66R+S395R+S340G;V59A+N313G+A393R+L66R;
V59A+N313G+A393R+L66R+S395R;V59A+N313G+A393R+L66R+S340G;
V59A+N313G+A393R+L66R+S340G+S395R;V59A+A393R+Y402F;
V59A+Y402F+E408R;V59A+S119P+N313G+A393R;
V59A+N313G+A393R+Y402F;V59A+A393R+Y402F+E408R;
V59A+S119P+N313G+A393R+Y402F;
V59A+N313G+A393R+Y402F+E408R;
S119P+N313G;N313G+A393R;A393R+Y402F;Y402F+E408R;
S119P+N313G+A393R;N313G+A393R+Y402F;A393R+Y402F+E408R;
V59A+S119P+N313G+A393R+Y402F;N313G+A393R+Y402F+E408R;
S119P+L66R;V59A+S119P+S340G;S119P+S395R;S119P+L66R+S340G;
S119P+S340G+S395R;S119P+S395R+L66R;S119P+S395R+L66R+S340G;
N313G+L66R;N313G+S340G;N313G+S395R;N313G+L66R+S340G;
N313G+S340G+S395R;N313G+S395R+L66R;N313G+S395R+L66R+S340G;
A393R+L66R;A393R+S340G;A393R+S395R;A393R+L66R+S340G;
A393R+S340G+S395R;A393R+S395R+L66R+S340G;Y402F+L66R;
Y402F+S340G;Y402F+S395R;Y402F+L66R+S395R;Y402F+L66R+S340G;
Y402F+L66R+S395R+S340G;E408R+L66R;E408R+S395R;E408R+S340G;
E408R+S395R+S340G;E408R+L66R+S340G;E408R+L66R+S395R;
E408R+L66R+S395R+S340G;S119P+N313G+L66R;
S119P+N313G+L66R+S340G;S119P+N313G+L66R+S395R;
S119P+N313G+L66R+S395R+S340G;N313G+A393R+L66R;
N313G+A393R+L66R+S395R;N313G+A393R+L66R+S340G;N313G+A393R+
L66R+S340G+S395R;A393R+Y402F;Y402F+E408R;
V59A+S119P+N313G+A393R;N313G+A393R+Y402F;A393R+Y402F+E408R;
S119P+N313G+A393R+Y402F;N313G+A393R+Y402F+E408R.
V59A+S119P+S340G;S119P+S395R;S119P+S340G;S119P+S340G+S395R;
S119P+S395R;S119P+S395R+S340G;N313G+S340G;N313P+S395R;
N313G+S340G;N313G+S395R;N313G+S395R+S340G;A393R+S340G;
A393R+S395R+S340G;Y402F+S395R;Y402F+S340G;
Y402F+S395R+S340G;E408R+S340G;E408R+S395R;
E408R+S395R+S340G;S119P+N313G;S119P+N313G+S340G;
S119P+N313G+S395R;S119P+N313G+S395R+S340G;N313G+A393R;
N313G+A393R+S395R;N313G+A393R+S340G;N313G+A393R+
S340G+S395R;.
N313G+A393R;V59A+N313G+A393R+Y402F;V59A+S340G;L66R+S340G;
S340G+S395R;S395R+L66R;S395R+L66R+S340G;N313G+L66R;
N313G+L66R+S340G;N313G+L66R+S395R;N313G+L66R+S395R+S340G;
V59A+N313G+A393R;N313G+A393R+Y402F;
S119P+A393R;A393R+Y402F;V59A+S119P+A393R+Y402F;
A393R+Y402F+E408R;S119P+S395R+L66R+S340G;L66R+S340G;
S340G+S395R;S395R+L66R;S395R+L66R+S340G;S119P+L66R;
S119P+L66R+S340G;S119P+L66R+S395R;S119P+L66R+S395R+S340G;
A393R+L66R;A393R+L66R+S395R;A393R+L66R+S340G;A393R+
L66R+S340G+S395R;V59A+S119P+A393R;A393R+Y402F;
S119P+A393R+Y402F;A393R+Y402F+E408R;
S119P+N313G;N313G+Y402F;Y402F+E408R;V59A+S119P+N313G+Y402F;
N313G+Y402F+E408R;L66R+S340G;S340G+S395R;S395R+L66R+S340G;
N313G+L66R;N313G+L66R+S395R;N313G+L66R+S340G;
N313G+L66R+S340G+S395R;
V59A+S119P+N313G;N313G+Y402F;Y402F+E408R;S119P+N313G+Y402F;
N313G+Y402F+E408R.
S119P+S340G;S119P+L66R;S119P+L66R+S340G;N313G+S340G;
N313G+L66R;N313G+L66R+S340G;A393R+S340G;A393R+L66R+S340G;
Y402F+L66R;Y402F+L66R+S340G;Y402F+L66R+S340G;E408R+S340G;
E408R+L66R;E408R+L66R+S340G;
S119P+N313G+L66R;S119P+N313G+L66R+S340G;N313G+A393R+L66R;
N313G+A393R+L66R+S340G;
N313G+A393R;A393R+E408R;V59A+S119P+N313G+A393R;
N313G+A393R+E408R;
L66R+S395R;L66R+S340G;L66R+S395R+S340G;N313G+A393R;
A393R+E408R;S119P+N313G+A393R;N313G+A393R+E408R.
A393R+Y402F;N313G+A393R+Y402F;S395R+S340G;L66R+S340G;
L66R+S395R;L66R+S395R+S340G;A393R+Y402F;N313G+A393R+Y402F.
S119P+L66R;V59A+S119P;S119P+S395R;S119P+L66R;S119P+S395R;
S119P+S395R+L66R;N313G+L66R;N313G+S395R;N313G+S395R+L66R;
A393R+L66R;A393R+S395R;A393R+S395R+L66R;Y402F+L66R;
Y402F+L66R+S395R;E408R+S395R;E408R+L66R;E408R+L66R+S395R;
S119P+N313G+L66R;S119P+N313G+L66R+S395R;N313G+A393R+L66R;
N313G+A393R+L66R+S395R;
N9A+S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R;
S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R;
V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R;
S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R;
A246T+N313G+E342T+A393R+S394R+Y402F+E408R;
N313G+E342T+A393R+S394R+Y402F+E408R;
E342T+A393R+S394R+Y402F+E408R;
A393R+S394R+Y402F+E408R;S394R+Y402F+E408R;Y402F+E408R;
V59A+L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M;
L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M;
T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M;
S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M;
N313G+S340G+S356G+A393R+Y402F+E408R+N427M;
S340G+S356G+A393R+Y402F+E408R+N427M;
S356G+A393R+Y402F+E408R+N427M;A393R+Y402F+E408R+N427M;
Y402F+E408R+N427M;E408R+N427M;
I189T+Y223F+F227Y+S119P+Y402F;Y223F+F227Y+S119P+Y402F;
F227Y+S119P+Y402F;S119P+Y402F;I189T+Y223F+F227Y+Y402F;
1189T+Y223F+F227Y;I189T+Y223F;I189T+F227Y;I189T+F227Y+S119P;
I189T+F227Y+Y402F;Y223F+F227Y+Y402F;Y223F+F227Y+S119P.
本发明还涉及这样一种葡糖淀粉酶变体,其亲本酶由在中度,优选在高度严谨条件下与SEQ ID NO:1的核酸序列或其互补链杂交的核酸序列编码。
改进的热稳定性
在另一方面,本发明涉及亲本葡糖淀粉酶改进了热稳定性的变体,具体是在40-80℃,优选63-75℃,具体在pH4-5,使用麦芽糖糊精为底物时改进了热稳定性,所述变体在表示为SEQ ID NO:2的氨基酸序列的下列位置含有一或多个突变:59,66,72,119,189,223,227,313,340,342,352,379,386,393,395,402,408,416,425,427,444,486,490,494,或是与SEQ ID NO:2的氨基酸序列有至少60%同源性的葡糖淀粉酶中相应位置。
可改进热稳定性的特定取代包括:V59A,L66V/R,T72I,S119P,I189T,Y223F,F227Y,N313G,S340G,E342A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V,优选E342T,K352R,S356G,T379A,S386K,N,R,P,A393R,S395R,Y402F,E408R,T416A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,E,W,Y,V,优选T416H,A425T,N427S/M,S444G,S486G,T490A,T494P/A。
具体的突变组合包括:
E408R+A425T+S465P+T494A,
A425T+E408R+S386K+A495T,
T379A+T2E+S386K+A393R,
S386N+E408R,
L66V+T2R+S394P+Y402F+RL(N-末端延伸)
S386R+T2R+A393R.
N427S+S486G+A1V+L66R+Y402F,
N427M+S44G+V470M+T2K+S30P,
T490A+V59A++A393R+PLASD(N-末端延伸)
在“本发明葡糖淀粉酶变体”部分列出的所有变体都被认为具有改进的热稳定性。实施例2和4中显示了本发明所选变体的这方面。
提高的比活
在另一方面,本发明涉及亲本葡糖淀粉酶比活已提高的变体,所述变体在表示为SEQ ID NO:2的氨基酸序列的下列位置含有一或多个突变:59,66,72,119,189,223,227,313,340,342,352,379,386,393,395,402,408,416,425,427,444,486,490,494,优选189,223,227或与SEQ ID NO:2的氨基酸序列有至少60%同源性的葡糖淀粉酶中相应位置。
可提高比活的具体突变包括:V59A,L66V/R,T72I,S119P,I189T,Y223F,F227Y,N313G,S340G,K352R,S356G,T379A,S386K,N,R,P,A393R,S395R,Y402F,E408R,T416A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,E,W,Y,V优选T416H,A425T,N427S/M,S444G,S486G,T490A,T494P/A,优选I189T,Y223F,F227Y。
具体的突变组合包括:
I189T+Y223F+F227Y+S119P+Y402F;
Y223F+F227Y+S119P+Y402F;F227Y+S119P+Y402F;S119P+Y402F;
I189T+Y223F+F227Y+Y402F;I189T+Y223F+F227Y;I189T+Y223F;
I189T+F227Y;I189T+F227Y+S119P;I189T+F227Y+Y402F;
Y223F+F227Y+Y402F;Y223F+F227Y+S119P.
在“本发明葡糖淀粉酶变体”部分列出的所有变体都被认为具有已提高的比活。实施例3中显示了本发明所选变体的这方面。
同源性(同一性)
针对上述亲本葡糖淀粉酶的同源性测定为两个蛋白序列之间可指示其中第一个序列衍生自第二个序列的同一性程度。此同源性适于由本领域所熟知的计算机程序测定,如GCG软件包提供的GAP(Wisconsin软件包程序手册,第8版,1994.8,遗传学计算机组,575 Science Drive,Madison,Wisconsin,美国53711)(Needleman,S.B.Wunsch,C.D.,(1970),分子生物学杂志(Journal of Molecular BiologY,48,p.443-453))。采用下列设置以GAP进行多肽序列的比对:GAP产生得分为3.0,GAP延伸得分为0.1,本发明中DNA类似序列编码的多肽的成熟部分显示出与SEQ ID NO:2的氨基酸序列的成熟部分优选至少80%,至少90%,更优选至少95%,更优选至少97%,最优选至少99%的同一性。
在一个实施方案中,亲本葡糖淀粉酶是黑曲霉G1葡糖淀粉酶(Boel等(1984),EMBO J.3(5),p.1097-1102(SEQ ID NO:13)。亲本葡糖淀粉酶可以是截短的葡糖淀粉酶,例如,黑曲霉G2葡糖淀粉酶(SEQ ID NO:2)。
优选地,亲本葡糖淀粉包含SEQ ID NO:2的氨基酸序列;或其等位基因变体;或其具有葡糖淀粉酶活性的片段。
SEQ ID NO:2的片段是在此氨基酸序列的氨基和/或羧基末端具有一或多个氨基酸缺失的多肽。例如,AMG G2(SEQ ID NO:2)是黑曲霉G1葡糖淀粉酶(Boel等(1984)EMBO J.3(5),p.1097-1102)的具有葡糖淀粉酶活性的片段。等位基因变体表示占据同一染色体位点的一个基因的两或多个替代形式中的任一种。等位基因变异通过突变自然产生,可能会产生种群内的多态现象。基因突变可以是沉默的(编码的多肽没有改变)或可以编码氨基酸序列已改变的多肽。多肽的等位基因变体是被基因的等位基因变体编码的多肽。
通过一或多个氨基酸残基的插入或缺失和/或一或多个氨基酸残基被不同氨基酸残基取代,同源的亲本葡糖淀粉酶的氨基酸序列可能与SEQ IDNO:2氨基酸序列不同。优选地,氨基酸改变为较小的性质改变,即并不显著影响蛋白质折叠和/或活性的保守氨基酸的取代;小的缺失,通常为1到约30个氨基酸的缺失;小的氨基-或羧基-末端延伸,如氨基酸末端蛋氨酸残基;最多20-25个残基的小接头肽;或通过改变净电荷或另一基团以利于纯化的小的延伸,如多组氨酸段,抗原表位或结合域。
在另一实施方案中,分离的亲本葡糖淀粉酶活性被在极低度严谨,优选低度严谨,更优选中度严谨,更优选中高度严谨,进而更优选高度严谨,最优选极高度严谨条件下与核苷酸探针杂交的核酸编码,所述探针在同样的条件下与(i)SEQ ID NO:1的核酸序列,(ii)SEQ ID NO:1的cDNA序列,(iii)(i)或(ii)的亚序列,或(iv)(i),(ii)或(iii)的互补链杂交(J.Sambrook,E.F.Fritsch,和T.Maniatus,1989,分子克隆,实验室手册,第二版,冷泉港,纽约)。SEQ ID NO:1的亚序列是至少100个核苷酸或优选至少200个核苷酸。而且,亚序列可以编码具有葡糖淀粉酶活性的多肽片段。亲本多肽也可以是具有葡糖淀粉酶活性的多肽的等位基因变体或片段。
可采用SEQ ID NO:1的核酸序列或其亚序列,以及SEQ ID NO:2的氨基酸序列或其片段设计核酸探针,以便按照本领域熟知的方法从不同属或种的菌株鉴定和克隆编码具有葡糖淀粉酶活性的多肽的DNA。具体地,这样的探针可用于经标准的Southern印迹方法与目的属或种的基因组DNA或cDNA杂交,以便鉴定并分离本文中相应基因。所述探针可以比完整序列短很多,但长度至少为15,优选至少25,和更优选至少35个核苷酸。也可以使用更长的探针。DNA和RNA探针都可使用。通常标记探针以便检测相应基因(例如,用32P、3H、35S、生物素或抗生物素蛋白标记)。这些探针均包括在本发明中。
因此,可在从所述其他生物制备的基因组DNA或cDNA文库中筛选与上述探针杂交并编码具有葡糖淀粉酶活性之多肽的DNA。通过琼脂糖或聚丙烯酰胺凝胶电泳或其它分离技术可分离来自所述其他生物的基因组或其他DNA。可将来自所述文库的DNA或分离的DNA转移至并固定在硝酸纤维素或其他适宜的载体材料上。为了鉴定与SEQ ID NO:1或其亚序列同源的克隆或DNA,在Southern印迹中使用该载体材料。为了达到本发明的目的,杂交指在极低至极高严谨条件下,核酸序列与对应于SEQ ID NO:1或其互补链或其亚序列的核酸序列杂交。用X射线胶片检测在这些条件下与所述核酸探针杂交的分子。
对于至少100个核苷酸的长探针,用2xSSC,0.2%SDS优选至少在45℃(极低严谨度),更优选50℃(低严谨度),更优选55℃(中等严谨度),更优选至少60℃(中等偏高严谨度),更优选至少65℃(高严谨度),最优选至少70℃(极高严谨度)将载体材料最后洗涤3次,每次15分钟。
对于约15个核苷酸至约70个核苷酸的短探针,将严谨度定义为在0.9M NaCl,0.09M Tris-HCl pH7.6,6mM EDTA,0.5% NP-40,1X Denhardt’s溶液,1mM焦磷酸钠,1mM磷酸氢二钠,0.1mM ATP和0.2mg/ml酵母RNA中,在相对于按照Bolton和McCarthy(1962,美国国家科学院进展(Proceedings of The National Academy of Sciences USA)48:1390)计算方法计算的Tm低5℃至10℃的温度下,根据标准Southern印迹方法,进行预杂交、杂交和杂交后洗涤。
对于约15个核苷酸至约70个核苷酸的短探针,在比所计算的Tm低5℃-10℃的温度下,将载体材料用6 X SCC加0.1% SDS洗涤一次,15分钟,用6X SSC洗涤两次,每次15分钟。
本发明还涉及分离的核酸序列,其通过(a)在极低,低,中,中高,高,或极高严谨度下与SEQ ID NO:1或其互补链,或其亚序列杂交;和(b)分离所述核酸序列而产生。亚序列优选至少100个核苷酸的序列,如编码具有葡糖淀粉酶活性的多肽片段的序列。
所涉及的亲本葡糖淀粉酶具有SEQ ID NO:2的成熟葡糖淀粉酶的葡糖淀粉酶活性的至少20%,优选至少40%,更优选60%,更优选80%,更优选90%,最优选100%。
克隆能编码亲本葡糖淀粉酶的DNA序列
用本领域熟知的各种方法,从生产所述葡糖淀粉酶的任何细胞或微生物中可分离编码亲本葡糖淀粉酶的DNA序列。首先应用来自产所述葡糖淀粉酶的生物体的染色体DNA或信使RNA构建基因组DNA和/或cDNA文库。然后,如果所述葡糖淀粉酶的氨基酸序列是已知的,则可合成标记的寡核苷酸探针并用于从制备自目标生物的基因组文库中鉴定编码葡糖淀粉酶的克隆。或者,可用含有与另一种已知葡糖淀粉酶基因同源之序列的标记型寡核苷酸探针作为探针,利用极低到极高严谨度的杂交和洗涤条件鉴定编码葡糖淀粉酶的克隆。如上述。
用于鉴定葡糖淀粉酶编码克隆的另一种方法涉及将基因组DNA的片段插入表达载体,例如质粒中,用所得基因组DNA文库转化葡糖淀粉酶阴性细菌,然后将转化的细菌铺板在含葡糖淀粉酶底物(即麦芽糖)的琼脂上,由此鉴定表达葡糖淀粉酶的克隆。
或者,通过现有标准方法,例如S.L.Beaucage和M.H.Caruthers所述的亚磷酰胺法(1981),Tetrahedron Letters 22,p.1859-1869或者Matthes等(1984),EMBO J.3,p.801-805所述的方法合成编码所述酶的DNA序列。在亚磷酰胺方法中,用例如自动DNA合成仪合成寡核苷酸,使其纯化、退火、连接,然后克隆到适宜的载体中。
最后,所述DNA序列可以是按照标准技术通过连接合成的、基因组的或cDNA来源的序列(根据需要,对应于完整DNA序列各部分的片段)而制备的基因组序列与合成序列的混合序列、合成序列与cDNA序列的混合序列或者基因组序列与cDNA的混合序列。还可通过聚合酶链反应(PCR),用特异性引物,按US4683202或R.K.Saiki等(1988),科学239,1988,pp.487-491所述制备DNA序列。
定点诱变
分离了编码葡糖淀粉酶的DNA序列并鉴定了合乎要求的突变位点后,用合成的寡核苷酸导入突变。这些寡核苷酸含有位于所需突变位点侧的核苷酸序列;在寡核苷酸合成的过程中插入突变核苷酸。在具体的方法中,在携带葡糖淀粉酶基因的载体中产生单链DNA缺口,编码葡糖淀粉酶的序列。然后,将携带所需突变的合成核苷酸与单链DNA的同源部分退火。用DNA聚合酶I(Klenow片段)填平剩余缺口,用T4连接酶连接构建体。Morinaga等(1984),生物技术2,p.646-639描述了该方法的具体实施例。US4760025公开了通过对表达盒进行微小改变来导入编码多个突变的寡核苷酸。但是,通过Morinaga方法可以在任何时间导入更多种的突变,因为可以导入不同长度的众多寡核苷酸。
Nelson和Long(1989),分析生物化学(Analytical Biochemistry)180,p.147-151描述了另一种将突变导入编码葡糖淀粉酶的DNA序列的方法。该方法包括经3步产生PCR片段,该片段含有通过用化学合成的DNA链作为PCR反应中的一个引物而导入的所需突变。通过用限制核酸内切酶裂解从PCR-产生的片段中分离携带突变的DNA片段,然后再插入到表达质粒中。
此外,Sierks等(1989)“在泡盛曲霉葡糖淀粉酶的活性位点Trp120的定点诱变”,蛋白质工程(Protein Eng.),2,621-625;Sierks等(1990)“通过在活性位点Asp176,Glu179,Glu180进行定点诱变来确定泡盛曲霉葡糖淀粉酶的催化机理”蛋白质工程,32,193-198;还记述了曲霉属葡糖淀粉酶中的定点诱变。
定域随机诱变
随机诱变可有利地定域于所述亲代葡糖淀粉酶的一部分。例如,当酶的某些区域被鉴定为对酶的给定特性特别重要时,以及当预期修饰能导致具有改良特性的变体时,定域随机诱变较为有利。当亲代酶的三级结构已被阐明,且所述结构与酶的功能相关时,一般可鉴定出所述区域。
通过使用上述的PCR产生的诱变技术或本领域已知的任何其它适当的技术,可以方便地进行定域的或区域-特异性的随机诱变。或者,通过例如插入适当的载体来分离编码待修饰的DNA序列部分的DNA序列,随后可使用上述任何诱变方法对所述部分进行诱变。
提供本发明变体的备选方法包括基因改组,例如WO 95/22625(来自Affymax Technologies N.V.)或WO96/00343(来自Novo Nordisk A/S)中所述。
葡糖淀粉酶变体的表达
根据本发明,可使用表达载体,以酶的形式表达通过上述方法,或通过本领域已知的任何其它方法产生的编码变体的DNA序列,所述表达载体一般包括编码启动子,操纵子,核糖体结合位点,翻译起始信号的控制序列,并任选包括阻抑基因或多种激活基因。
表达载体
带有编码本发明葡糖淀粉酶变体DNA序列的重组表达载体是任何可以方便地进行重组DNA方法的载体,载体的选择取决于将被引入的宿主细胞。载体可以是,当被引入宿主细胞时可整合进宿主细胞基因组并与其所整合进的染色体一起复制的那些。合适的表达载体例子包括pMT838。
启动子
载体中,DNA序列应该可操作连接适合的启动子序列。启动子可以是在所选宿主细胞中表现出转录活性的任何DNA序列,可以从编码对宿主细胞同源或异源的蛋白的基因获得。
引导编码本发明葡糖淀粉酶变体的DNA序列转录,特别是在细菌宿主中,转录的适合的启动子是例如,大肠杆菌lac操纵子,天蓝色链霉菌琼脂酶基因dagA,地衣芽孢杆菌α-淀粉酶基因(amyL)、嗜热脂肪芽孢杆菌产麦芽淀粉酶基因(amyM)、解淀粉芽孢杆菌α-淀粉酶基因(amyQ)、枯草芽孢杆菌xylA和xylB基因等的启动子。对于在真菌宿主中进行转录,可用的启动子的例子有来自米曲霉TAKA淀粉酶基因,酿酒酵母TPI(丙糖磷酸异构酶)(Alber等(1982),J.Mol.Appl.Genet 1,p.419-434,米赫根毛霉(Rhizomucormiehei)天冬氨酸蛋白酶,黑曲霉中性α-淀粉酶,黑曲霉酸稳定的α-淀粉酶,黑曲霉葡糖淀粉酶,米赫根毛霉脂肪酶,米曲霉碱性蛋白酶,米曲霉丙糖磷酸异构酶或构巢曲霉乙酰胺酶的启动子。
表达载体
本发明表达载体也含有适当的转录终止子,在真核生物中,还含有与编码本发明α-淀粉酶变体的DNA序列可操作相连的聚-腺苷酸化序列。终止和聚腺苷酸化序列可适当地得自与启动子相同的来源。
载体可进一步含有能使载体在所述宿主细胞中复制的DNA序列。所述序列的例子是质粒pUC19,pACYC177,pUB110,pE194,pAMB1和pIJ702的复制起点。
载体也可含有选择标记,例如其产物可以补偿宿主细胞缺陷的基因,如枯草芽孢杆菌或地衣芽孢杆菌的dal基因,或能赋予抗生素抗性的基因,所述抗生素抗性如氨苄青霉素,卡那霉素,氯霉素或四环素抗性。另外,载体可含有曲霉属选择标记,如amdS,argB,niaD和sC,产生潮霉素抗性的标记,或者可通过WO 91/17243中所述的共-转化来完成选择。
分别连接编码葡糖淀粉酶变体的本发明DNA构建体,启动子,终止子和其它元件,并将它们插入含有复制所需信息的适当载体的方法是本领域技术人员众所周知的(参见例如Sambrook等,分子克隆:实验室手册,第2版,冷泉港,1989)。
宿主细胞
含有本发明的上述DNA构建体或表达载体的本发明的细胞,在本发明葡糖淀粉酶变体的重组生产中作为宿主细胞较有利。可以方便地通过将本发明的DNA构建体整合进宿主染色体,从而用该DNA构建体转化该细胞。由于所述DNA序列更可能在细胞中保持稳定,此整合通常被认为是有利的。DNA构建体整合进入宿主染色体可按照常规方法,例如,同源或异源重组来进行。备选地,可用上述与不同类型宿主细胞有关的表达载体转化细胞。
本发明的细胞可以是较高等生物的细胞,如哺乳动物或昆虫的细胞,但优选是微生物细胞,如细菌或真菌(包括酵母)的细胞。
适当细菌的例子如下:革兰氏阳性细菌,如枯草芽孢杆菌,地衣芽孢杆菌,迟缓芽孢杆菌,短芽孢杆菌,嗜热脂肪芽孢杆菌,嗜碱芽孢杆菌,解淀粉芽孢杆菌,凝结芽孢杆菌,环状芽孢杆菌,灿烂芽孢杆菌,巨大芽孢杆菌,苏云金芽孢杆菌,浅青紫链霉菌或鼠灰链霉菌;或革兰氏阴性细菌,如大肠杆菌。细菌的转化可以,例如,通过原生质体转化或使用感受态细胞以其本身已知的方式进行。
酵母优选选自糖酵母属或裂殖糖酵母属,例如酿酒酵母。
宿主细胞可以是丝状真菌,例如曲霉属某个种的菌株,最优选米曲霉或黑曲霉,或镰孢属菌株,如尖孢镰孢(Fusarium oxysporium),禾谷镰孢(完整状态即Gribberella zeae,以前为Sphaeria zeae,又名Gibberella roseum和Gibberella roseum f.sp.cerealis),或硫色镰孢(完整状态即Gribberella puricaris,又名三隔镰孢,杆孢状镰孢,接骨木镰孢,玫瑰色镰孢,玫瑰色镰孢的禾谷镰孢变种),Fusarium cerealis(又名Fusarium crookwellense),或Fusariumvenenatum。
在本发明优选实施方案中宿主细胞是蛋白酶缺陷型菌株或蛋白酶阴性菌株。
例如碱性蛋白酶基因“alp”缺失的米曲霉JaL125就是蛋白酶缺陷型菌株。在WO 97/35956(Novo Nordisk),或欧洲专利429,490中有此菌株的描述。
丝状真菌细胞可通过涉及原生质体形成和原生质体转化及随后使细胞壁以其本身的方式再生的一种方法来转化。在EP238023(Novo Nordisk A/S)中描述了用曲霉作宿主微生物,其内容在此引作参考。
生产葡糖淀粉酶变体的方法
在更进一步的方面,本发明涉及生产本发明葡糖淀粉酶变体的方法,该方法包括在利于生产变体并从细胞及/或培养液中回收该变体的条件下培养宿主细胞。
用于培养细胞的培养基可以是适于目的宿主细胞生长并表达本发明葡糖淀粉酶变体的任何常规培养基。可用的培养基可从销售商购得或按照已公开的配方(例如按美国典型培养物保藏中心的目录中所述)制备。
通过已熟知的方法从培养液中可以方便地回收宿主细胞分泌的葡糖淀粉酶变体,所述方法包括通过离心或过滤从培养液分离细胞,通过盐如硫酸铵沉淀培养基质中的蛋白类成分,然后使用层析的方法,如离子交换层析,亲和层析,等。
淀粉转化
本发明提供一种利用本发明的葡糖淀粉酶变体从淀粉生产葡萄糖及类似物的方法。通常,该方法包括在α-淀粉酶存在下部分水解前体淀粉,然后在葡糖淀粉酶存在下通过切割α-(1→4)和α-(1→6)糖苷键从淀粉或相关寡糖和多糖分子的非还原末端进一步水解释放D-葡萄糖。
利用α-淀粉酶进行的前体淀粉的部分水解使淀粉分子通过水解内部的α-(1→4)键而初步分解。在商业应用中,利用α-淀粉酶的初步水解在约105℃下进行。所处理的淀粉浓度很高,通常30%-40%固含量。在这种提高的温度下的初步水解一般进行5分钟。然后将部分水解的淀粉转移到第二个罐,85-90℃温育约1小时,得到10-15的葡萄糖当量值(D.E.)。
通常在一个单独的罐中,在降低至30-60℃的温度下,在有葡糖淀粉酶时进一步水解从淀粉或相关寡糖和多糖分子的非还原末端释放出的D-葡萄糖。该底物液体的温度优选降至55-60℃之间。该溶液的pH值从6-6.5降至3-5.5。优选pH为4-4.5。将葡糖淀粉酶加入该溶液中,使反应进行24-72小时,优选36-48小时。
通过使用本发明的热稳定的葡糖淀粉酶变体可以在比传统分批糖化过程更高的温度下进行糖化过程。依照本发明,糖化可在60-80℃以上进行,优选63-75℃。其既可用于传统分批方法(如上所述)也可用于连续糖化。
实际上,包括一或多步膜分离步骤,即过滤步骤的连续糖化过程必须在60℃以上进行以保持一个合理的高膜通量。因此,本发明热稳定的葡糖淀粉酶变体为在工业化糖化过程可接受的合理价格和时间内进行大规模连续糖化处理提供了可能。依照本发明甚至可以缩短糖化时间。
本发明葡糖淀粉酶变体(如AMG变体)的活性在60-80℃比在传统采用的温度范围30-60℃时通常要高很多。因此,当所述葡糖淀粉酶实施糖化过程时通过提高温度可以缩短时间。
此外,提高热稳定性改善了T1/2(半衰期,如“材料和方法”部分所定义)。由于本发明葡糖淀粉酶变体改进了热稳定性,所以糖化过程中仅需要添加小量葡糖淀粉酶以替代已失活的酶。依照本发明,更多的葡糖淀粉酶在糖化过程中保持活性。此外,在63℃以上进行糖化可以减少微生物污染的风险。
在JP3-224493;JP1-191693;JP62-272987;EP452,238中记述了使用本发明葡糖淀粉酶变体进行糖化过程的实例。
在本发明方法中,本发明葡糖淀粉酶变体可与一种能水解至少有4个糖基的分子中的α-(1→6)糖苷键的酶联合使用。优选地,本发明葡糖淀粉酶变体可与支链淀粉酶或异淀粉酶联合使用。在G.M.A.van Beynum等,淀粉转化技术,Marcel Dekker,New York,1985,101-142中提出了支链淀粉酶和异淀粉酶的去支链应用,所述酶的分子性质,所述酶和葡糖淀粉酶的潜在用途。
本发明的又一方面涉及本发明葡糖淀粉酶变体在淀粉转化过程中的应用。
此外,本发明葡糖淀粉酶变体可用于包括连续糖化步骤的连续淀粉转化过程。
本发明葡糖淀粉酶变体也可以以固定化形式使用。这适于并经常用于生产麦芽糖糊精或葡萄糖浆,或特定的糖浆,如麦芽糖糖浆,还可用于与果糖浆生产有关的寡糖提余液(raffinate)流。
依照本发明,本发明的AMG变体也可用于生产醇类,例如燃用或食用醇类。US 5231017记述了其中一个方法。
材料和方法
酶:
AMG G1:公开在Boel等,(1984),EMBO J.3(5),1097-1102,(SEQ IDNO:13)中的黑曲霉葡糖淀粉酶G1,可从Novo Nordisk得到。
AMG G2:截短的黑曲霉葡糖淀粉酶G1,表示为SEQ ID NO:2,可从Novo Nordisk得到。
溶液:
缓冲液:0.05M乙酸钠(1L milli-Q-water中6.8g),pH4.5
终止液:0.4M NaOH
GOD-perid,124036,Boehringer Mannheim
底物:
麦芽糖:29mM(100ml50mM乙酸钠中1g麦芽糖,pH4.5)(Sigma)
麦芽庚糖:10mM,115mg/10ml(Sigma)
宿主细胞:
米曲霉JaL125:米曲霉IFO 4177,可从发酵研究所,Osaka;17-25 JusoHammachi 2-Chome Yodogawa-ku,Osaka,日本得到,其采用米曲霉pyrG基因作标记,并通过一步基因取代方法(G.May,“丝状真菌的应用分子遗传学(Applied Molecular Genetics of Filamentous Fungi”(1982),p.1-25.Eds.J.R.Knghorn和G.Turner;Blackie Academic and Professional)使碱性蛋白酶基因“alp”缺失(Murakami K等(1991),Agric.Biol.Chem.55,p.2807-2811)。WO 97/35956(Novo Nordisk)中进一步公开了菌株JaL125。
微生物:
菌株:酿酒酵母(S.cerevisiae)YNG318:MATα1eu2-Δ2 ura3-52 his4-539 pep4-Δ1[cir+]
质粒:
pCAMG91:参见图1,包含黑曲霉G1葡糖淀粉酶(AMG G1)的质粒。Boel等(1984),EMBO J.3(7),p1581-1585中记述了pCAMG91的构建。
pMT838:编码截短的黑曲霉葡糖淀粉酶G2(SEQ ID NO:2)的质粒
pJSO026:(酿酒酵母表达质粒)(J.S.Okkels,(1996)“pYES载体中URA3-启动子的缺失提高了酿酒酵母中真菌脂肪酶的表达水平.DNA重组生物技术III:生物学和工程学的整合,vol.782,Annals o fthe New York Academy ofSciences”)。更特定地,通过用来自酿酒酵母的组成型表达的TPI(丙糖磷酸异构酶)启动子(Albert和karwasaki,(1982),J.Mol.Appl.Genet.,1,419-434),取代pYES2.0的诱导型GAL1-启动子,并缺失URA3启动子的一部分,可从pYES2.0得到表达质粒pJSO37。
方法:
酿酒酵母YNG318的转化
将DNA片段和开放型载体混合并通过标准方法转化进入酿酒酵母YNG318。
测定比活并表示为kcat(sec.-1)
750μL底物(1%麦芽糖,50mM乙酸钠,pH4.3)在所选温度例如37℃或60℃温育5分钟。
加入50μL稀释在乙酸钠中的酶。分别在0、3、6、9、12分钟后取100μL等份试样,转移到100μL0.4M氢氧化钠中终止反应。空白也包括在内。
将20μL移到微滴板上,加入200μL GOD-Perid溶液。室温下温育30分钟后650nm测定吸收值。
用葡萄糖作为标准,比活计算为kcat(sec.-1)。
测定AGU活性并表示为AGU/mg
一个Novo淀粉葡糖苷酶单位(AGU)定义为37℃,pH4.3下每分钟水解1微摩尔麦芽糖的酶量。此分析方法的详述(AEL-SM-0131)可要求NovoNordisk提供。
用Boehringer Mannheim,124036的葡萄糖GOD-Perid试剂盒以修饰的(AEL-SM-0131)方法将活性测定为AGU/ml。标准品:AMG-标准品,批次7-1195,195 AGU/ml。
375μL底物(50mM乙酸钠中加入1%麦芽糖,pH4.3)37℃温育5分钟。添加25μL的酶的乙酸钠稀释液。10分钟后,加入100μL 0.25M NaOH终止反应。取20μL转到96孔微滴板上,加入200μL GOD-Perid溶液。室温30分钟后,测定650nm吸光度,参照AMG标准品以AGU/mg计算活性。
活性(AGU/ml)除以蛋白浓度(mg/ml)可算得以AGU/mg表示的比活。
曲霉的转化(一般方法)
以米曲霉孢子接种100ml YPD(Sherman等,(1981),酵母遗传学方法,冷泉港实验室),振荡培养约24小时。通过微孔布过滤收集菌丝体,用200ml0.6M MgSO4清洗。将菌丝体悬浮在15ml 1.2M MgSO4,10mM NaH2PO4中,pH5.8中。将悬浮液于冰上冷却,加入1ml含120mg NovozymTM 234的缓冲液。5分钟后,加入1ml 12mg/ml BSA(Sigma H25型),37℃下轻微振荡连续培养1.5-2.5小时,直到通过镜检观察到样品中大量原生质体出现。
微孔布过滤悬浮液,滤液移至无菌试管,加5ml 0.6M山梨糖醇,100mMTris-HCl,pH7.0覆盖。1000g下离心5分钟,从MgSO4层上层收集原生质体。2倍体积的STC(1.2M山梨糖醇,10mM Tris-HCl,pH7.5,10mM CaCl2)加入到该原生质体悬浮液中,将该混合物1000g离心5分钟。将原生质体沉淀重悬在3ml STC中,重新沉积。重复该过程。最终,将原生质沉体沉淀重悬在0.2-1ml STC中。
将100μL原生质体悬浮液与5-25μg p3SR2(Hynes等,Mol.And Cel.Biol.,Vol.3,No.8,1430-1439,Aug.1983所述携带构巢曲霉amdS基因的质粒)混合在10μL STC中。混合液室温静置25分钟。加入0.2ml 60% PEG4000(BDH29576),10mM CaCl2,10mMTris-HCl,pH7.5,小心混合(两次),最终加入0.85ml同一溶液,小心混合。使该混合液室温静置25分钟,2500g下旋转15分钟,得到的沉淀重悬于2ml 1.2M山梨糖醇中。再次沉淀后,将原生质体铺板在基本平板(Cove,(1996),Biochem.Biophys.Acta 113,51-56)上,该平板中含有1.0M蔗糖,pH7.0,10mM乙酰胺作氮源,20mM CsC1抑制背景生长。37℃培育4-7天后,拣出孢子,悬在无菌水中,划线接种以获得单菌落。重复该过程,第二次再分离后取单菌落保存为确定的转化体。
补料分批发酵
在含有以麦芽糖糊精为碳源,尿素为氮源,并含有酵母提取物的培养基中进行补料分批发酵。将所需米曲霉宿主细胞的摇瓶培养液接种到含3.5%所述碳源和0.5%所述氮源的培养基中进行补料分批发酵。在34℃pH5.0的条件下培养24小时后,开始连续补加碳源和氮源。保持碳源水平作为限制性因子,要确保氧过量供应。补料分批培养进行4天,然后通过离心,超滤,澄清过滤和除菌过滤来回收酶。
纯化
将培养液过滤,加入硫酸铵(AMS)到1.7M,pH调到5。离心除去沉淀的物质,将含有葡糖淀粉酶活性的溶液上样到Toyo Pearl Butyl柱,该柱已用1.7M AMS,20mM乙酸钠,pH5预平衡。用平衡缓冲液洗出未结合的物质。用10mM乙酸钠,pH4.5,以10倍柱体积中1.7-0M AMS的线性梯度洗脱结合蛋白。收集含葡糖淀粉酶的级分,并对20mM乙酸钠,pH4.5透析。然后将该溶液上样到Q Sepharose柱,该柱已用10mM Piperazin,Sigma,pH5.5预平衡。用平衡缓冲液洗下未结合的物质。以在10mM Piperazin中0-0.3M氯化钠(10倍柱体积)的线性梯度洗脱结合蛋白。收集含葡糖淀粉酶的级分,通过SDS-PAGE确认纯度。
T1/2(半衰期)方法I
变体的热稳定性使用下列方法测定为T1/2:68℃、70℃或75℃下将950μL50mM乙酸钠缓冲液(pH4.3)(NaOAc)温育5分钟。缓冲液中加入50μL酶(4AGU/ml)。在,例如0,5,10,20,30和40分钟时取样2×40μL样品,冰上冷却。用温育前(0分钟)测定的活性(AGU/ml)为参照(100%)。稳定性的降低(百分比)计算为保温时间的函数。在不同时间测定葡糖淀粉酶的百分比残余活性。T1/2表示相对活性降为50%所需的时间。
T1/2(半衰期)方法II
T1/2测定步骤如下:在30%葡萄糖,50mM乙酸钠,pH4.5,所需温度(例如70℃)下温育目标酶(约0.2AGU/ml)。在设定的时间间隔取样,冰上冷却,用pNPG方法(如下所述)测定残余酶活性。
在不同的时间测定葡糖淀粉酶的百分比残余活性。T1/2表示相对活性降为50%所需的时间。
残余酶活的测定(pNPG法)
pNPG试剂:
0.2g pNPG(对-硝基苯葡萄糖吡喃糖苷)溶解在0.1M乙酸盐缓冲液(pH4.3)中,补足100ml。
硼酸盐溶液:
3.8g Na2B4O7·10H2O溶解在Milli-Q water中,补足100ml。
25μL样品中加入50μL底物,50℃下温育2小时。加入150μL硼酸盐溶液终止反应。405nm测定光密度,计算残余活性。
pAMGY的构建
pAMGY载体如下构建:用AMG基因取代pJSO026中的脂肪酶基因,所述AMG基因是用正向引物FG2:5’-CAT CCC CAG GAT CCT TAC TCAGCA ATG-3’(SEQ ID NO:10)和反向引物RG2:5’-CTC AAA CGA CTCACC AGC CTC TAG AGT-3’(SEQ ID NO:11),以AMG基因的质粒pLAC103为模板经PCR扩增获得。用XbaI和SmaI在37℃消化pJSO026质粒2小时,用Klenow片段使PCR扩增子末端钝化,然后用XbaI消化。连接该载体片段和PCR扩增子,通过电转化作用转化进入大肠杆菌。产生的载体为pAMGY。
pLaC103的构建
用黑曲霉AMGII cDNA克隆(Boel等,(1984),同上)作为构建pLaC103的来源以便在酿酒酵母中表达AMG的GII形式。
构建分几步,列于下:
用XbaI切割pT7-212(EP37856/US5162498),用Klenow DNA聚合酶和dNTP进行末端钝化。用EcoRI切割后,得到的载体片段通过琼脂糖凝胶电泳纯化,与pBoel53的2.05kb EcoR1-EcoRV片段连接,从而在所得质粒pG2x的AMG编码片段的EcoRV末端再造XbaI位点。
为除去AMG cds上游的DNA,以及用适当的限制性内切酶识别位点装饰AMG的编码DNA,进行下述的构建:
分离p53的930kb EcoRI-PstI片段并进行AluI切割,将得到的771bpAlu-PstI片段与带有末端钝化型EcoRI位点的pBR322(如上)连接,再用PstI切割。在得到的质粒pBR-AMG’中,在距离AMG cds的起始密码子34bp处再造EcoRI位点。
从pBR-AMG’分离得到775bp的EcoRI-PstI片段,并在包括pT7-212的XbaI-EcoRI载体片段的连接反应中与pG2x中的1151bp PstI-XbaI片段连接。
得到的质粒pT7GII在有碱性磷酸酶的存在下进行BamHI切割,然后在磷酸酶失活后用SphI进行部分切割。得到2489bp的SphI-BamHI片段,其包括连接到AMGII cds的S.c.TPI启动子。上述片段与pT7GII的1052bpBamHI片段一起与经SphI-BamHI消化及碱性磷酸酶处理的pMT743(EP37856/US5162498)载体片段连接。得到的质粒是pLaC103。
筛选热稳定的AMG变体
在下述热稳定性滤膜分析中筛选文库。
用于热稳定性的滤膜分析
酵母文库铺板在含100μg/ml氨苄青霉素的SCFura琼脂板上的醋酸纤维素滤膜(OE67,Schleicher&Schuell,Dassel,德国)-和硝酸纤维素滤膜(Protran-Ba 85,Schleicher&Schuell,Dassel,德国)的夹层上,30℃下至少72小时。将菌落影印到已用甲醇活化1分钟的PVDF滤膜(Immobilon-P,Millipore,Bedford),或Protran滤膜(未活化)上,然后用0.1M NaAc洗膜,再室温下温育2小时。用自来水从PVDF/Protran滤膜上洗下菌落。温育前用针分别标记每一滤膜夹层和PVDF/Protran滤膜,这样在筛选后可以在滤膜上定位阳性变体。将结合有变体的PVDF膜转移到有0.1M NaAc,pH4.5的容器中,47℃或使用Protran膜时可选67-69℃下温育15分钟。将SC ura-琼脂平板上的醋酸纤维素和硝酸纤维素滤膜夹层室温保存以备用。温育后,在含5%麦芽糖,1%琼脂糖,50mMNaAc,pH4.5的平板上测定残余活性。含有PVDF的试验板如滤膜夹层一样标记,并50℃温育2小时。除去PVDF滤膜后,试验板用Glucose GOD perid(Boehringer Mannheim GmbH,德国)染色。具有残余活性的变体在试验板上表现为白色背景上的墨绿点。改进的变体位于保存板上。按照第一次筛选的条件再次筛选改进的变体。
使用DOPE程序进行随机诱变的一般方法
随机诱变可按下列步骤进行:
1.在亲本酶中选择用于修饰的目的区域,
2.在所选目的区域中确定突变和非突变位点,
3.确定可以进行何种突变,例如考虑所要构建的变体的所需稳定性和/或性能,
4.挑选结构合理的突变,
5.参照步骤4调整经步骤3挑选的残基,
6.通过使用适当的DOPE算法分析核苷酸分布,
7.如果需要,调整所要残基以满足遗传密码的实用性,例如考虑由遗传密码引起的约束,如为避免引入终止密码子所引起的约束;本领域技术人员可以知道一些密码子组合并不实用,需要进行调整。
8.制备引物
9.使用引物进行随机诱变
10.通过筛选所需改进的特性来选择所得葡糖淀粉酶变体。
Dope算法
适用于步骤6的Dope算法为本领域所熟知。Tomandl,D.等,1997,计算机辅助分子设计杂志(Journal of Computer-Aided Molecular Design)11:29-38记述一种这样的算法。另一算法是DOPE(Jensen,LJ,Andersen,KV,Sevendsen,A,和Kretzschmar,T(1998)核酸研究26:697-702)。
实施例
实施例1
AMG G2变体的构建
定点诱变
为构建AMG G2酶(SEQ ID NO:2)的变体,参照制造商说明书使用市售试剂盒,Chameleon双链定点诱变试剂盒。
将编码目的AMG G2酶的基因定位在pMT838上,所述质粒是在含AMG G1型的质粒pCAMG91(见图1)上缺失G2 nt.1362和G2 nt.1530间的DNA而得到的。
参照制造商说明书,利用下列引物将pMT838的氨苄青霉素基因的ScaI位点改变为Mlul位点:
7258:5’p gaa tga ctt ggt tga cgc gtc acc agt cac 3’(SEQ ID NO:3)
(这样就改变了氨苄青霉素抗性基因中发现并用于切割至MluI位点的ScaI位点)。然后以含目的AMG基因的pMT838载体作DNA聚合酶的模板并使用oligo 7258(SEQ ID NO:3)及21401(SEQ ID NO:4)。
引物21401(SEQ ID NO:4)用作选择性引物。
21401:5’p gg gga tca tga tag gac tag cca tat taa tga agg gca tat acc acg ccttgg acc tgc gtt ata gcc 3’
(在未改变氨基酸序列的情况下,改变了AMG基因中发现的ScaI位点)。
通过加入含有所要突变的适合的oligo可引入所要的突变(例如,引入半胱氨酸残基)到目的AMG基因中。
用引物107581引入T12P
107581:5’pgc aac gaa gcg ccc gtg gct cgt ac 3’(SEQ ID NO:5)
通过整个基因的测序证实突变。使用上述“材料和方法”部分的方法将质粒转化进入米曲霉。按照上述变体“材料和方法”部分所述进行变体的发酵及纯化。
实施例2
通过定域随机掺入诱变来构建与亲本酶相比稳定性提高了的黑曲霉
AMG变体
为改进黑曲霉AMG的热稳定性,在预选区域进行随机突变。
残基
区域:L19-G35
区域:A353-V374
用DOPE软件(见材料与方法)在上述区域确定每一所需改变所要掺入的密码子以使终止密码子的数量最少(参见表1)。在密码子的三个位置计算确切的核苷酸分布以给出建议的氨基酸变化群。所要掺入的区域特别掺入在指定的位置,这可以有较多的机会获得所要残基,但是也有其它可能。
第一列是要突变的氨基酸,第二列是野生型的百分比,第三列是新的氨基酸。
表1:
在L19-G35中的掺入
L19 90% N
N20 95% T
N21 不变
I22 不变
G23 95% A
A24 90% S,T
D25 93% S,T,R
G26 95% A
A27 90% S,T
W28 <80% R,Y
V29 不变
S30 93% T,N
G31 95% A
A32 95% V
D33 80% R,K,H
S34 90% N
G35 不变
得到的掺入寡核苷酸链在表2中作为有义链表示:表中还有引物序列,野生型核苷酸序列,亲本氨基酸序列和每一掺入位置的核苷酸分布。
表2:
位点: 19 20 21 22 23 24 25 26 27
氨基酸序列: L N N I G A D G A
引物: 12T A3T AAC ATC G4G 5CG 67C G4T 8CT
野生型序列: CTG AAT AAC ATC GGG GCG GAC GGT GCT
位点(续): 28 29 30 31 32 33 34 35
氨基酸序列(续):W V S G A D S G
引物: 91010 GTG 1112C G4C G13G 141516 1718T GGC
野生型序列: TGG GTG TCG GGC GCG GAC TCT GGC
每一掺入位置的核苷酸分布
1:A10,C90
2:A6,T94
3:A95,C5
4:G95,C5
5:G91,A3,T3,C3
6:G95,A3,C2
7:G3,A95,C2
8:G92,A4,T4
9:A3,T97
10:G95,T5
11:G3,A97
12:G95,A2,C3
13:T5,C95
14:G88,A8,C4
15:G7,A93
16:G4,C96
17:G4,A96
18:G95,A2,C3
正向引物(SEQ ID NO:6):
FAMGII′5-C GAA GCG ACC GTG GCT CGT ACT GCC ATC 12T A3T AAC ATC
G4G 5CG 67C G4T 8CT 91010 GTG 1112C G4C G13G 141516 1718T GGC
ATT GTC GTT GCT AGT CCC AGC ACG GAT AAC-3′
反向引物(SEQ ID NO:7):
RAMG1:5′-GAT GGC AGT ACG AGC CAC GGT CGC TTC G-3′
表3
在区域A353-V374中的掺入:
A353 <80% D,E,Q,N,Y
L354 90% Q,E
Y355 90% N,Q
S356 90% T,D,N
G357 80% P,A,S,T
A358 93% S
A359 90% S,T,N
T360 90% R,K
G361 85% A,S,T
T362 90% S
Y363 不变
S364 93% D
S365 93% N,Q,K
S366 93% P,D
S367 不变
S368 93% D,N,T
Y369 93% Q,E
Y370 不变
S371 93% N
S372 93% N,T
I373 不变
V374 93% N,Y,H
得到的掺入寡核苷酸链在表4中作为有义链表示:表中还有引物序列,野生型核苷酸序列,亲本氨基酸序列和每一掺入位置的核苷酸分布。
表4:
位点: 353 354 355 356 357 358 359 360 361 362
氨基酸序列:A L Y S D A A T G T
引物: 123 4SA 6AC 78C 910T 11CT 1213T 1415A 1617C 18CC
野生型序列:GCA CTG TAC AGC GAT GCT GCT ACT GGC ACC
位点(续): 363 364 365 366 367 368 369
370
氨基酸序列(续):Y S S S S S T Y
引物(续): TAC 1920T A2122 2324C AGT 1425C 2627G T28T
野生型序列(续):TAC TCT TCG TCC AGT TCG ACT TAT
位点(续): 371 372 373 374
氨基酸序列(续):S S I V
引物(续): A16T 2930T ATT 313233
野生型序列(续):AGT AGC ATT GTA
每一掺入位置的核苷酸分布
1:G91,A3,T3,C3
2:A13,C87
3:A40,T60
4:G3,A3,C94
5:A6,T94
6:G4,A4,T92
7:G2,A96,C2
8:G93,A3.5,C3.5
9:G87,A8,C5
10:A84,C16
11:G93,T7
12:G92,A5,T3
13:A3,C97
14:G3,A97
15:G2,A2,T4,C92
16:G93,A7
17:G93,C7
18:A90,T10
19:G4,A96
20:G95,A5
21:G96,A4
22:G3,C97
23:G2,A1,T95,C2
24:A3,C97
25:G95,A3,C2
26:G2,A96,C2
27:A5,C95
28:A95,T5
29:G2,A98
30:G94,A4,C2
31:G94,A3,T1,C2
32:A4,T96
33:A20,C80
引物:FAMGIV(SEQ ID NO:8)
5′-GTG TCG CTG GAC TTC TTC AAG 123 45A 6AC 78C 910T 11CT 1213T
1415A 1617C 18CC TAC 1920T A2122 2324C AGT 1425C 2627G T28T
A16T 2930C ATT 313233 GAT GCC GTG AAG ACT TTC GCC GA-3,
引物RAMGVI(SEQ ID NO:9)
5′-ctt gaa gaa gtc cag cga cac-3′
随机诱变
使用表2和表3中可见的掺入的寡核苷酸(通称FAMG)和针对L19-G35区域的反向引物RAMG,以及,覆盖了N-末端(FG2:5’-CAT CCC CAGGAT CCT TAC TCA GCA ATG-3’(SEQ ID NO:10)和C-末端(RG2:5’-CTCAAA CGA CTC ACC AGC CTC TAG AGT(SEQ ID NO:11)的特定SEQ IDNO:2引物,通过重叠延伸方法(Horton等,基因,77(1989),pp.61-68)产生PCR-文库片段,有21个碱基对的重叠。质粒pAMGY用作聚合酶链式反应的模板。在大肠杆菌/酵母穿梭载体pAMGY中通过同源重组克隆PCR片段(参见材料和方法)。
筛选
如上述“材料和方法”部分所述,使用Protran膜在热稳定膜分析中筛选文库,并在67-69℃下温育。
实施例3
在68℃的热稳定性
使用实施例1所述方法构建AMG G2变体。
使用“材料和方法”中的方法I在68℃下测定热稳定性,以T1/2表示,并与相同条件下的野生型黑曲霉AMG G2对比。
酶 | T1/2 |
AMG G2野生型 | 8.5 |
T721+A246T | 11.3 |
A495P | 11.0 |
A425T+S465P+E406R+A495T | 8.6 |
T379A+S386K+A393R+T2E | 16.4 |
L66V+S394P+Y402F+T2R+RL | 11.1 |
S386R+A393R+T2R | 14.1 |
S3b6N+E408R | 12.6 |
A1V+L66R+Y402F+N427S+S486G |
T2K+S30P+N427M+S444G+V470M
A393R+T490A+V59A+PLASD(N-末端延伸)
S119P+Y312Q+Y402F+S416H,
T379A+S386K+A393R+T2E,
S386P+S340G+D357S+T360V.
实施例4
比活
AMG G2变体如实施例1构建。如“材料和方法”部分所述,在37℃pH4.5下测定比活为kcat或AGU/mg,使用麦芽糖为底物。
酶 | AGU/mg | KCat(Sec.-1) |
AMG G2(野生型)I189T+Y223F+F227Y+Y402F+S119P | 5.69.3 |
实施例5
在75℃的热稳定性
采用实施例1所述方法构建AMG G2变体。
使用“材料和方法”中的方法I在75℃和pH4.5下测定热稳定性,以T1/2表示,并与相同条件下的野生型黑曲霉AMG G2对比。
AGRNo. | 突变 | Y1/2(分钟) |
G2(对比) | 4 | |
136 | V59A+A393R+T490A | 6 |
109 | S56A+V59A+N313G+S356G+A393R+S394R+Y402r | 9 |
111 | A11E+V59A+T72I+S119P+F237H+S240G+A246T+N313G+S340G+K352R+A393R+S394R-Y402F+E408R | 10 |
120 | T2H+A11P+V59A+T721+S119P+A246T+N33G+D336S+T360V+A393R+Y402F÷E408R+N427M | 12 |
122 | T2H+V59A+T721+S119P+S240G+N313G÷T360V+S3b8P+A393R+Y402F+E408R+N427M | 10 |
124 | N9A+S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R | 21 |
130 | V59A+L66R+T721+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M | 29 |
132 | T2H+N9A+V59A+S56A+L66R+R721+S119P+N313G+F318+E342T+S356G+T390R+Y402F+E408R+N427M | 9 |
141 | T2H+A1E+V59A+S119P+N313G+E342T+S356P+A393R+S3941+Y402F+L410R+N427S | 13 |
142 | T2H+A11P+V59A+S119P+N313G+S340G÷S356G+E408R+N427M | 9 |
151 | T2H+A11E+V59A+L66R+S119P+N313G+S340G+D357S+A393R+S394R+Y402F+E408R | 20 |
154 | T2H+N9A+S56A+V59A+L66R+721+S119P+S240G+N313G+S340G+K352R+A393R+S394R+Y402F+E408R+N427S | 19 |
实施例6
AMG变体AGR130的糖化性能
如下所述在70℃测定具有改进的热稳定性的变体AGR130(V59A+L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M)的糖化性能。
参比酶是野生型黑曲霉AMG G2。糖化在下列条件下进行:
底物 10DE麦芽糖糊精,约30% DS(w/w)
温度 70℃
初始pH 4.3(在70℃)
酶量 0.24AGU/g DS
糖化
将麦芽糖糊精(从普通淀粉制备)溶解在煮沸的Milli-Q水中,制备糖化底物,将干物质调为约30%(w/w)。pH调到4.3。相当于15g干重的底物样品转移到50ml蓝盖玻璃烧瓶中,水浴搅拌。加入酶,必要时调pH。进行平行实验。定时取样,HPLC分析确定碳水化合物组成。
序列表
<110>诺沃挪第克公司(Novo Nordisk A/S)
<120>葡糖淀粉酶变体
<130>5967.204-W0
<160>13
<170>适用于Windows 3.0的FastSEQ
<210>1
<211>1605
<212>DNA
<213>黑曲霉(Aspergillus niger)
<220>
<221>sig-peptide
<222>(1)...(72)
<221>mat-peptide
<222>(73)...(1602)
<221>CDS
<222>(1)...(1602)
<400>1
<210>2
<211>534
<212>PRT
<213>黑曲霉(Aspergillus niger)
<220>
<221>signal
<222>(1)...(24)
<400>2
<210>3
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物7258
<400>3
<210>4
<211>68
<212>DNA
<213>人工序列
<220>
<223>引物21401
<400>4
<210>5
<211>25
<212>DNA
<213>人工序列
<220>
<223>引物107581
<400>5
<210>6
<211>88
<212>DNA
<213>人工序列
<220>
<223>引物FAMGII
<400>6
<210>7
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物RAMG1
<400>7
<210>8
<211>75
<212>DNA
<213>人工序列
<220>
<223>引物FAMGIV
<400>8
<210>9
<211>21
<212>DNA
<213>引物RAMGVI
<400>9
<210>10
<211>27
<212>DNA
<213>人工序列
<220>
<223>引物FG2
<400>10
<210>11
<211>27
<212>DNA
<213>人工序列
<220>
<223>引物RG2
<400>11
<210>12
<211>2602
<212>DNA
<213>黑曲霉(ASPERGILLUS NIGER)
<400>12
<210>13
<211>640
<212>PRT
<213>黑曲霉(ASpERGILLUS NIGER)
<400>13
Claims (20)
1.来自黑曲霉的序列为SEQ ID NO:2的黑曲霉G2葡糖淀粉酶的变体,其包含在位置342的改变,所述变体选自N9A+S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R,T2H+N9A+V59A+S56A+L66R+T72I+S119P+N313G+F318Y+E342T+S356G+T390R+Y402F+E408R+N427M和T2H+A11E+V59A+S119P+N313G+E342T+S356P+A393R+S394I+Y402F+L410R+N427S。
2.含有编码权利要求1的葡糖淀粉酶变体的DNA序列的DNA构建体。
3.用权利要求2的DNA构建体转化的细胞。
4.权利要求3的细胞,它是包括细菌和真菌的微生物。
5.权利要求3或4的细胞,它是来自包括黑曲霉的曲霉属或者是包括Talaromyces emersonii的踝节菌属的菌株。
6.将淀粉转化或部分水解为含葡萄糖的糖浆的方法,该方法包括在权利要求1所述的葡糖淀粉酶变体存在下糖化淀粉水解产物的步骤。
7.权利要求6的方法,其中葡糖淀粉酶变体的用量范围为0.05-0.5AGU/g干固体。
8.权利要求6或7的方法,包含糖化至少30%重量干固体的淀粉水解产物。
9.权利要求6或7的方法,其中在脱支酶的存在下进行糖化,该脱支酶选自支链淀粉酶和异淀粉酶。
10.权利要求9的方法,其中支链淀粉酶源自Bacillus acidopullulyticus或Bacillus deramificans。
11.权利要求9的方法,其中异淀粉酶源自Pseudomonasamyloderamosa。
12.权利要求6或7的方法,其中糖化在pH3-5.5,温度为60-80℃,pH为4-4.5条件下进行24-72小时。
13.权利要求12的方法,其中温度为63-75℃。
14.权利要求12的方法,其中糖化进行36-48小时。
15.权利要求1的葡糖淀粉酶变体在淀粉转化过程或在连续淀粉转化过程中的应用。
16.权利要求1的葡糖淀粉酶变体在生产寡糖、麦芽糖糊精或葡萄糖糖浆过程中的应用。
17.权利要求1的葡糖淀粉酶变体在生产燃用或食用乙醇或在生产饮料过程中的应用。
18.权利要求1的葡糖淀粉酶变体在生产有机化合物的发酵过程中的应用。
19.权利要求18的应用,其中所述有机化合物为柠檬酸、抗坏血酸、赖氨酸或谷氨酸。
20.通过进行权利要求1中所述的一或多个位点的改变而改进亲本葡糖淀粉酶的热稳定性和/或比活的方法,该亲本葡糖淀粉酶为SEQ ID NO:2的黑曲霉G2葡糖淀粉酶。
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CN101550410A (zh) | 2009-10-07 |
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CN1360630A (zh) | 2002-07-24 |
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