CN101240269A - α-淀粉酶变体 - Google Patents
α-淀粉酶变体 Download PDFInfo
- Publication number
- CN101240269A CN101240269A CNA2008100033029A CN200810003302A CN101240269A CN 101240269 A CN101240269 A CN 101240269A CN A2008100033029 A CNA2008100033029 A CN A2008100033029A CN 200810003302 A CN200810003302 A CN 200810003302A CN 101240269 A CN101240269 A CN 101240269A
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- Prior art keywords
- gly
- asn
- asp
- val
- ala
- Prior art date
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- C—CHEMISTRY; METALLURGY
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38618—Protease or amylase in liquid compositions only
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Detergent Compositions (AREA)
Abstract
本发明涉及亲代Termamyl-样α-淀粉酶的变体,该变体在2,3,4,5,或6个区域/位置中含有突变。所述变体在高温下(相对于其亲代)具有增加的稳定性。本发明还涉及含有编码本发明α-淀粉酶变体之DNA序列的DNA构建体,携带本发明DNA构建体的重组表达载体,用本发明的DNA构建体转化的细胞,本发明的α-淀粉酶变体在洗涤和/或洗碟、织物退浆、淀粉液化方面的用途,含有本发明的α-淀粉酶变体的去污剂添加剂,含有本发明的α-淀粉酶变体的手洗或机洗洗碟剂组合物,产生亲代Termamyl-样α-淀粉酶的变体的方法,所述变体显示增加。
Description
本发明申请是基于申请日为1999年11月16日,申请号为99815544.6(国际申请号为PCT/DK99/00628),发明名称为“α-淀粉酶变体”的专利申请的分案申请。
发明领域
本发明涉及亲代Termamyl-样α-淀粉酶的新变体,相对于亲代α-淀粉酶而言,所述变体具有经改变的特性。所述特性包括例如在酸性pH,低钙浓度和/或高温下增加的稳定性。所述变体适于多种用途,特别是工业淀粉处理(如淀粉液化或糖化)。
发明背景
α-淀粉酶(α-1,4-葡聚糖-4-葡聚糖水解酶,EC 3.2.1.1)构成一组能催化淀粉和其它线性和分支1,4-葡糖苷寡-和多糖水解的酶。
大量专利和科学文献中涉及这一类在工业上非常重要的酶。可从例如WO 90/11352,WO 95/10603,WO 95/26397,WO 96/23873和WO 96/23874中了解多种α-淀粉酶(如Termamyl-样α-淀粉酶)的变体。
WO 96/23874提供了Termamyl-样α-淀粉酶的三维,X-射线晶体结构数据,该α-淀粉酶由解淀粉芽孢杆菌α-淀粉酶的300个N-末端氨基酸残基,和含有氨基酸序列的地衣芽孢杆菌α-淀粉酶C-末端的氨基酸301-483组成(后者有商品TermamylTM(商标名)),因此,与工业上重要的芽孢杆菌α-淀粉酶(在本发明的上下文中,包含在术语“Termamyl-样α-淀粉酶”的定义中,它特别地包括地衣芽孢杆菌,解淀粉芽孢杆菌和嗜热脂肪芽孢杆菌的α-淀粉酶)密切相关。WO 96/23874进一步描述了在分析亲代Termamyl-样α-淀粉酶结构的基础上设计亲代Termamyl-样α-淀粉酶变体的方法学,相对于亲代而言,所述变体表现出有所改变的特性。
发明简述
本发明涉及Termamyl-样α-淀粉酶的新的α-淀粉分解变体(突变体),尤其是在高温和酸性pH下(相对于亲代而言)表现出增加的稳定性的变体,如美国专利3,912,590和欧洲专利公开号252,730和63,909所述,所述变体有利于对淀粉进行工业处理(淀粉液化,糖化等)。
淀粉转化
“传统”的淀粉转化方法将淀粉降解为较低分子量的碳水化合物成分,如糖或脂肪替代物,该方法包括脱支步骤。
“淀粉至糖”的转化
当将淀粉转化为糖时,淀粉被解聚。所述解聚过程由预处理步骤和两个或三个连续处理步骤(即液化处理,糖化处理,根据所需终产物,还任选包括异构化处理)组成。
预-处理天然淀粉
天然淀粉由室温下不溶于水的微粒组成。加热水淀粉浆液时,微粒膨胀,最后胀破,将淀粉分子分散至溶液中。在“凝胶化”处理的过程中,粘度显著增加。由于一般工业过程中的固体水平为30-40%,因此不得不稀释或“液化”淀粉以使其便于处理。目前,主要通过酶促降解使粘度降低。
液化
在液化步骤中,通过α-淀粉酶(如本文的TermamylTM SEQ ID NO:4)将长链淀粉分解为分支的和线性的较短单位(麦芽糖糊精)。液化过程在105-110℃进行5至10分钟,接着在95℃进行1-2小时。pH为5.5至6.2之间。为了确保这些条件下最佳的酶稳定性,需加入1mM钙(40ppm游离的钙离子)。如此处理之后,液化淀粉的“葡萄糖当量”(DE)为10-15。
糖化
液化过程之后,通过加入葡糖淀粉酶(如AMGTM)和脱支酶,如异淀粉酶(美国专利4,335,208)或支链淀粉酶(如PromozymeTM)(美国专利4,560,651),将麦芽糖糊精转变为葡萄糖。在此步骤之前,将pH值降低为4.5以下,维持高温(95℃以上)以灭活液化α-淀粉酶,减少不能被脱支酶正确水解的短寡糖(被称为“潘糖前体”)的形成。
将温度降低为60℃,加入葡糖淀粉酶和脱支酶。将糖化过程进行24-72小时。
通常,当在液化步骤之后变性α-淀粉酶时,约0.2-0.5%的糖化产物是分支的三糖62-α-葡糖基麦芽糖(潘糖),该糖不能被支链淀粉酶降解。如果糖化过程中存在得自液化步骤的活性淀粉酶(即未变性),该水平可以高至1-2%,这非常不合乎需要,因为它显著降低了糖化产量。
异构化
当所需的最终糖产物是例如高果糖浆时,葡萄糖浆可被转变为果糖。糖化过程之后,将pH值增加为6-8,优选为pH7.5,通过离子交换除去钙。然后使用例如固定化的葡萄糖异构酶(如SweetzymeTM)将葡萄糖浆转变为高果糖浆。
在本发明的上下文中,术语“酸性pH”指的是pH低于7.0,尤其是低于上述传统工业淀粉液化过程中所用的pH范围,即为pH5.5-6.2。
在本发明的上下文中,术语“低钙浓度”指的是浓度低于传统的工业淀粉液化过程中所用的正常水平,如浓度为0-40ppm,优选为10-30ppm,如15-25ppm钙。正常浓度根据谷物中游离Ca2+的浓度而变化。通常加入相当于1mM(40ppm)的剂量,该剂量与谷物中的钙水平一起给出40-60ppm游离的Ca2+。
在本发明的上下文中,术语“高温”指的是温度为95至160℃,尤其是正常进行工业淀粉液化处理所用的温度范围,所述范围是95至105℃。
本发明进一步涉及编码本发明变体的DNA构建体,制备本发明变体的方法,和本发明变体单独或与其它α-淀粉分解酶联合用于多种工业过程,尤其是淀粉液化的用途。
命名法
在本说明书和权利要求书中,使用了常规的氨基酸残基一字母和三字母密码。
为了易于参照,利用下述命名法描述本发明的α-淀粉酶变体:原来的氨基酸:位置:取代的氨基酸。
根据该命名法,用天冬酰胺取代第30位的丙氨酸被表示为:Ala30Asn或A30N,在相同位置处缺失丙氨酸被表示为:Ala30*或A30*,而插入另一个氨基酸残基,如赖氨酸被表示为:Ala30AlaLys或A30AK。缺失连续的一段氨基酸残基,如氨基酸残基30-33被表示为(30-33)*或Δ(A30-N33)。
当特定的α-淀粉酶相对于其它α-淀粉酶而言含有“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,被表示为*36Asp或*36D。
多个突变由加号隔开,即:Ala30Asp+Glu34Ser或A30N+E34S分别表示在第30和34位,丙氨酸和谷氨酸分别用天冬酰胺和丝氨酸取代。多个突变也可以由下列与加号意义相同的符号隔开: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,被表示为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,应理解为可用任何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,应理解为丙氨酸可被缺失,或用任何其它氨基酸取代,即下列任何一个氨基酸所取代:R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V。
具体地,本发明涉及如下方面:
1 )亲代Termamyl-样α-淀粉酶的变体,与亲代α-淀粉酶相比,所述α-淀粉酶变体中,表面有一个或多个暴露于溶剂的氨基酸残基发生变化以使该α-淀粉酶的总体亲水性增加和/或表面上所述暴露于溶剂的氨基酸残基的侧链中甲基总数增加。
2)1)的变体,其中向内弯曲的凹面上的一个或多个暴露于溶剂的氨基酸残基变为更具疏水性的氨基酸残基。
3)1)的变体,其中凸面上的一个或多个暴露于溶剂的氨基酸残基经改变后增加了侧链中的甲基数目。
4)亲代Termamyl-样α-淀粉酶的变体,该变体在选自下列的一个或多个位置处具有变化:E376,S417,A420,S356,Y358;
其中:(a)所述变化各为:
(i)在占用上述位置的氨基酸的下游插入一个氨基酸,
(ii)缺失占用上述位置的氨基酸,或
(iii)用不同的氨基酸取代占用上述位置的氨基酸,
(b)所述变体具有α-淀粉酶活性,和(c)每个位置对应于具有SEQ IDNO:4氨基酸序列的亲代Termamyl-样α-淀粉酶的氨基酸序列位置。
5)4)的变体,所述变体的一个或多个暴露于溶剂的氨基酸残基具有如1)-3)中任一项所定义的变化。
6)1)-5)中任一项的变体,其中亲代Termamyl-样α-淀粉酶得自地衣芽孢杆菌,解淀粉芽孢杆菌,嗜热脂肪芽孢杆菌的菌株,芽孢杆菌菌种NCIB12289,NCIB 12512,NCIB 12513或DSM 9375。
7)6)的变体,其中亲代α-淀粉酶得自地衣芽孢杆菌菌株ATCC 27811。
8)1)-6)的变体,其中亲代Termamyl-样α-淀粉酶是选自SEQ ID NO:1,2,3,4,5,6,7和8所示的任何α-淀粉酶。
9)1)-8)中任一项的变体,其中亲代Termamyl-样α-淀粉酶具有的氨基酸序列与SEQ ID NO:4的同一性程度至少为65%,优选为70%,更优选至少为80%,甚至更优选至少约为90%,甚至更优选至少为95%,甚至更优选至少为97%,甚至更优选至少为99%。
10)1)-10)中任一项的变体,其中亲代Termamyl-样α-淀粉酶由能在中度严紧,优选为高度严紧的条件下与SEQ ID NO:12的核酸序列杂交的核酸序列编码。
11)1)-10)的变体,其中亲代Termamyl-样α-淀粉酶是SEQ ID NO:4所示地衣芽孢杆菌α-淀粉酶和SEQ ID NO:5所示解淀粉芽孢杆菌α-淀粉酶的杂合体。
12)11)的变体,其中亲代杂合Termamyl-样α-淀粉酶是LE174。
13)1)-12)中任一项的变体,其中亲代α-淀粉酶还在下列的一个或多个位置处具有突变:K176,I201和H205(使用SEQ ID NO:4的编号)。
14)13)的变体,其中亲代α-淀粉酶具有一个或多个下列取代:K176R,1201F和/或H205N(使用SEQ ID NO:4的编号)。
15)14)的变体,其中亲代α-淀粉酶具有下列取代:K176R+I201F+H205N(使用SEQ ID NO:4的编号)。
16)1)至15)的变体,其中相对于亲代α-淀粉酶而言,所述变体在低于7.0的pH(酸性pH)和/或低钙浓度和/或95-160℃的温度范围(高温)下具有增加的稳定性。
17)1)至16)中任一项的变体,所述变体具有一个或多个下列取代:E376K,S417T,A420Q,R,S356A,Y358F。
18)DNA构建体,其含有编码1)至17)中任一项的α-淀粉酶变体的DNA序列。
19)重组表达载体,其携有18)的DNA构建体。
20)被18)的DNA构建体或19)的载体转化的细胞。
21)20)的细胞,其为微生物。
22)21)的细胞,其为细菌或真菌。
23)22)的细胞,其为革兰氏阳性细菌,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,灿烂芽孢杆菌或苏云金芽孢杆菌。
24)去污剂添加剂,其含有1)至17)中任一项的α-淀粉酶变体,所述变体任选为不成粉末的颗粒,稳定化的液体或被保护的酶的形式。
25)24)的去污剂添加剂,其含有0.02-200mg酶蛋白质/g添加剂。
26)24)或25)的去污剂添加剂,其中还含有另一种酶,例如蛋白酶,脂肪酶,过氧化物酶,另一种淀粉分解酶和/或纤维素酶。
27)去污剂组合物,其含有1)至17)中任一项的α-淀粉酶变体。
28)27)的去污剂组合物,其中还含有另一种酶,例如蛋白酶,脂肪酶,过氧化物酶,另一种淀粉分解酶和/或纤维素酶。
29)手用或机用洗碟去污剂组合物,其含有1)至17)中任一项的α-淀粉酶变体。
30)29)的洗碟去污剂组合物,其中还含有另一种酶,例如蛋白酶,脂肪酶,过氧化物酶,另一种淀粉分解酶和/或纤维素酶。
31)手用或机用洗衣组合物,其含有1)至17)中任一项的α-淀粉酶变体。
32)31)的洗衣组合物,其中还含有另一种酶,例如蛋白酶,脂肪酶,过氧化物酶,淀粉分解酶和/或纤维素酶。
33)组合物,其含有:
(i)具有SEQ ID NO:4所示序列的地衣芽孢杆菌α-淀粉酶与得自具有SEQ ID NO:3所示序列的嗜热脂肪芽孢杆菌α-淀粉酶(作为亲代Termamyl-样α-淀粉酶)的1)至17)中任一项的一种或多种变体的混合物;或
(ii)具有SEQ ID NO:3所示序列的嗜热脂肪芽孢杆菌α-淀粉酶与得自一种或多种其它亲代Termamyl-样α-淀粉酶的1)至17)中任一项的一种或多种变体的混合物;或
(iii)得自具有SEQ ID NO:3所示序列的嗜热脂肪芽孢杆菌α-淀粉酶(作为亲代Termamyl-样α-淀粉酶)的1)至17)中任一项的一种或多种变体与得自一种或多种其它亲代Termamyl-样α-淀粉酶的本发明的一种或多种变体的混合物。
34)含有1)至17)中任一项的变体的组合物,其中亲代α-淀粉酶是含有SEQ ID NO:5所示解淀粉芽孢杆菌α-淀粉酶的N-末端部分和SEQ ID NO:4所示地衣芽孢杆菌α-淀粉酶的C-末端部分的杂合α-淀粉酶。
35)34)的组合物,其中亲代杂合Termamyl-样α-淀粉酶是LE174。
36) 35)的组合物,其中亲代Termamyl-样α-淀粉酶是在一个或多个下列位置具有改变的LE174:K176,I201和H205。
37)36)的组合物,其中亲代Termamyl-样α-淀粉酶是在一个或多个下列取代的LE174:K176R,I201F和H205N。
38)1)至17)中任一项的α-淀粉酶变体或33)至37)的组合物用于洗涤和/或洗碟的用途。
39)1)至17)中任一项的α-淀粉酶变体或33)至37)的组合物用于织物脱浆的用途。
40)1)至17)中任一项的α-淀粉酶变体或33)至37)的组合物用于淀粉液化的用途。
41)产生亲代Termamyl-样α-淀粉酶的变体的方法,其中变体相对于亲代而言,在高温下表现出增加的稳定性,所述方法包括:
(a)对编码亲代Termamyl-样α-淀粉酶的DNA序列进行随机诱变,
(b)在宿主细胞中表达步骤(a)中得到的突变DNA序列,和
(c)筛选表达突变α-淀粉酶的宿主细胞,相对于亲代Termamyl-样α-淀粉酶而言,所述变体在高温下具有增加的稳定性。
附图简述
图1是本发明上下文中6个亲代Termamyl-样α-淀粉酶的氨基酸序列对比。最左边的数字表示以下各个氨基酸序列:
1:SEQ ID NO:2,
2:淀粉酶
3:SEQ ID NO:1,
4:SEQ ID NO:5,
5:SEQ ID NO:4,
6:SEQ ID NO:3。
图2表示实施例1中所用的PCR策略。
发明详述
Termamyl-样α-淀粉酶
众所周知,通过芽孢杆菌种产生的多种α-淀粉酶在氨基酸水平上高度同源。例如,已发现含有SEQ ID NO:4所示氨基酸序列的地衣芽孢杆菌α-淀粉酶(商品为TermamylTM)与含有SEQ ID NO:5所示氨基酸序列的解淀粉芽孢杆菌α-淀粉酶约89%同源,与含有SEQ ID NO:3所示氨基酸序列的嗜热脂肪芽孢杆菌α-淀粉酶约79%同源。其它同源的α-淀粉酶包括得自芽孢杆菌菌种NCIB 12289,NCIB 12512,NCIB 12513或DSM 9375的α-淀粉酶(皆详细描述于WO 95/26397),和描述于Tsukamoto等,生物化学和生物物理研究通讯,151(1988),p25-31中的α-淀粉酶。
其它同源的α-淀粉酶包括由EP 0252666所述的地衣芽孢杆菌菌株(ATCC 27811)所产生的α-淀粉酶,和WO 9I/00353和WO 94/18314中鉴定的α-淀粉酶。其它可商购的Termamyl-样地衣芽孢杆菌α-淀粉酶是OptithermTM和TakathermTM(可得自Solvay),MaxamylTM(可得自Gist-brocades/Genencor),Spezym AATM和Spezyme Delta AATM(可得自Genencor)和KeistaseTM(可得自Daiwa)。
由于这些α-淀粉酶基本上同源,因此可以认为它们属于同一类α-淀粉酶,即“Termamyl-样α-淀粉酶”。
因此,在本发明的上下文中,术语“Termamyl-样α-淀粉酶”欲指在氨基酸水平上与TermamylTM(即具有本文SEQ ID NO:4所示氨基酸序列的地衣芽孢杆菌α-淀粉酶)基本上同源的α-淀粉酶。换句话说,Termamyl-样α-淀粉酶是具有本文SEQ ID NO:1,2,3,4,5,6,7或8所示氨基酸序列,和WO95/26397的SEQ ID NO:1(与本文SEQ ID NO:7所示氨基酸序列相同)或WO95/26397的SEQ ID NO:2(与本文SEQ ID NO:8所示氨基酸序列相同)或Tsukamoto等,1988(其氨基酸序列示于本文SEQ ID NO:6)所示氨基酸序列的α-淀粉酶,或者,Termamyl-样α-淀粉酶是:i)与SEQ ID NO:1或2或3或4或5或6或7或8所示的至少一个所述氨基酸序列的同源性(同一性)至少为60%,优选至少为70%,更优选至少为75%,甚至更优选至少为80%,尤其是至少为85%,尤其优选至少为90%,尤其是至少为95%,甚至尤其更优选至少为97%,尤其是至少为99%的α-淀粉酶,和/或ii)能与针对一种或多种所述α-淀粉酶产生的抗体发生免疫交叉反应的α-淀粉酶,和/或iii)由能在低至很高严紧条件下(所述条件在下文中描述)与编码上述-特定α-淀粉酶的DNA序列杂交的DNA序列编码的α-淀粉酶,所述特定α-淀粉酶的DNA序列分别示于本申请的SEQ ID NO:9,10,11,12和32(分别编码本文SEQ IDNO:1,2,3,4和5所示的氨基酸序列),WO 95/26397的SEQ ID NO:4(该DNA序列与终止密码子TAA一起示于本文SEQ ID NO:13,它编码本文SEQ IDNO:8所示的氨基酸序列)和WO 95/26397的SEQ ID NO:5(示于本文SEQ IDNO:14)。
关于特性i),可利用任何常规的算法测定“同源性”(同一性),优选使用GCG程序包第8版(1994年8月)的缺口程序,该程序使用缺口得分的缺省值,即缺口产生得分为3.0,缺口延伸得分为0.1(Genetic ComputerGroup(1991) GCG程序包程序手册,第8版,575 Science Drive,Madison,Wisconsin,USA 53711)。
在一个实施方案中,亲代Termamyl-样α-淀粉酶骨架具有的氨基酸序列与SEQ ID NO:4的同一性程度(按上述方法测定)至少为65%,优选至少为70%,优选至少为75%,更优选至少为80%,更优选至少为85%,甚至更优选至少约为90%,甚至更优选至少为95%,甚至更优选至少为97%,甚至更优选至少为99%。
可使用Termamyl(SEQ ID NO:4)和Termamyl-样α-淀粉酶之间的结构对比鉴定其它Termamyl-样α-淀粉酶中等同的/相应的位置。得到所述结构对比的一个方法是使用GCG程序包中的Pile Up程序,该程序使用缺省的缺口得分值,即缺口产生得分为3.0,缺口延伸得分为0.1。其它结构对比方法包括疏水簇分析(Gaboriaud等,(1987),FEBS LETTERS 224,p.149-155)和反向穿梭(threading)法(Huber,T;Torda,AE,蛋白质科学,卷7,1期,142-149(1998))。
例如,在已提及的多种Termamyl-样α-淀粉酶的氨基酸序列中,地衣芽孢杆菌α-淀粉酶C-结构域中靶残基的相应位置如下:
Termamyl-样α-淀粉酶 |
地衣芽孢杆菌(SEQ ID NO:4) S356 Y358 E376 S417 A420解淀粉芽孢杆菌(SEQ ID NO:5) S356 Y358 E376 S417 A420嗜热脂肪芽孢杆菌(SEQ ID NO:3)......Y361.....................Bac.WO95/26397(SEQ ID NO:2) ......Y363......S419.........Bac.WO95/26397(SEQ ID NO:1) ......Y363...................... |
这些保守氨基酸残基的突变对于增加高温下酸性pH和/或低钙浓度时的稳定性非常重要,这一点将在下文中进一步描述。
使用针对相关Termamyl-样α-淀粉酶的至少一个表位的抗体或与所述表位反应的抗体,可以检测α-淀粉酶的特性ii)(见上文),即免疫交叉反应性。通过本领域已知的方法,如Hudson等,实用免疫学,第3版(1989),BlackwellScientific Publications可以产生抗体,所述抗体可以是单克隆或多克隆抗体。使用本领域已知的试验,例如Western印迹或放射状免疫扩散试验(如Hudson等,1989所述)可以测定免疫交叉-反应性。在此方面,已发现了分别具有氨基酸序列SEQ ID NO:1,2,3,4,5,6,7或8的α-淀粉酶之间的免疫交叉-反应性。
可以在所述α-淀粉酶的完整或部分核苷酸或氨基酸序列的基础上适当制备寡核苷酸探针,然后使用所述探针,根据上述特性iii)鉴定Termamyl-样α-淀粉酶。
检测杂交的适当条件包括:在5×SSC中预浸泡,于~40℃,在20%甲酰胺,5×Denhardt’s溶液,50mM磷酸钠,pH6.8和50mg变性的经超声处理的小牛胸腺DNA中预杂交1小时,接着于~40℃,在添加有100mM ATP的相同溶液中杂交18小时,接着于40℃,用2×SSC,0.2%SDS将滤膜洗涤3次,每次30分钟(低严紧性),优选在50℃(中度严紧性),更优选在65℃(高严紧性),甚至更优选在~75℃(很高的严紧性)进行洗涤。有关杂交方法的更多细节描述干Sambrook等,分子克隆:实验室手册,第2版,冷泉港,1989。
在本发明的上下文中,“得自”不仅仅表示由所述生物体产生或可由所述生物体产生的α-淀粉酶,还指由分离自所述菌株的DNA序列编码的α-淀粉酶,和由所述DNA序列转化的宿主生物体产生的α-淀粉酶。最后,该术语还表示由合成的和/或cDNA来源的DNA序列编码的,并具有所述α-淀粉酶的鉴定特征的α-淀粉酶。该术语还表示亲代α-淀粉酶可以是天然α-淀粉酶的变体,即修饰(插入,取代,缺失)天然α-淀粉酶的一个或多个氨基酸残基而得到的变体。
亲代杂合α-淀粉酶
亲代α-淀粉酶(骨架)可以是杂合α-淀粉酶,即含有得自至少两种α-淀粉酶的部分氨基酸序列组合的α-淀粉酶。
亲代杂合α-淀粉酶可以是在氨基酸同源性和/或免疫交叉-反应性和/或DNA杂交(如上所述)的基础上被确定为属于Termamyl-样α-淀粉酶家族的杂合α-淀粉酶。此时,杂合α-淀粉酶一般由Termamyl-样α-淀粉酶的至少一个部分和一种或多种其它α-淀粉酶的部分组成,所述其它α-淀粉酶选自微生物(细菌或真菌)和/或哺乳动物来源的Termamyl-样α-淀粉酶或非-Termamyl-样α-淀粉酶。
因此,亲代杂合α-淀粉酶可含有得自至少两种Termamyl-样α-淀粉酶,或得自至少一种Termamyl-样和至少一种非-Termamyl-样细菌α-淀粉酶,或得自至少一种Termamyl-样和至少一种真菌α-淀粉酶的部分氨基酸序列组合。衍生得到部分氨基酸序列的Termamyl-样α-淀粉酶可以是例如本文所述的任何特定的Termamyl-样α-淀粉酶。
例如,亲代α-淀粉酶可含有得自地衣芽孢杆菌菌株的α-淀粉酶的C-末端部分,和得自解淀粉芽孢杆菌菌株或嗜热脂肪芽孢杆菌菌株的α-淀粉酶的N-末端部分。例如,亲代α-淀粉酶可含有地衣芽孢杆菌α-淀粉酶C-末端部分的至少430个氨基酸残基。所述杂合Termamyl-样α-淀粉酶可以与SEQID NO:4所示的地衣芽孢杆菌α-淀粉酶相同,不同之处仅在于:(成熟蛋白质)N-末端的35个氨基酸残基被SEQ ID NO:5所示的解淀粉芽孢杆菌α-淀粉酶(BAN)成熟蛋白质N-末端的33个氨基酸残基所取代。该杂合体也可由对应于嗜热脂肪芽孢杆菌α-淀粉酶(具有SEQ ID NO:3所示氨基酸序列)的68个N-末端氨基酸残基的氨基酸区段,和对应于地衣芽孢杆菌α-淀粉酶(具有SEQID NO:4所示氨基酸序列)的415个C-末端氨基酸残基的氨基酸区段组成。
非-Termamyl-样α-淀粉酶可以是例如真菌α-淀粉酶,哺乳动物或植物α-淀粉酶或细菌α-淀粉酶(不同于Termamyl-样α-淀粉酶)。这种α-淀粉酶的具体例子包括米曲霉TAKAα-淀粉酶,黑曲霉酸性α-淀粉酶,枯草芽孢杆菌α-淀粉酶,猪胰腺α-淀粉酶和大麦α-淀粉酶。所有这些α-淀粉酶都具有已阐明的,与本文所述典型Termamyl-样α-淀粉酶的结构显著不同的结构。
上述真菌α-淀粉酶,即得自黑曲霉和米曲霉的α-淀粉酶在氨基酸水平上高度同源,一般认为它们属于相同的α-淀粉酶家族。得自米曲霉的真菌α-淀粉酶可以商购,其商品名为FungamylTM。
另外,当提及Termamyl-样α-淀粉酶的特定变体(本发明的变体)时,提到该变体是通过常规方法,修饰(例如缺失或取代)特定Termamyl-样α-淀粉酶氨基酸序列中的特定氨基酸残基而获得的,应理解在另一个Termamyl-样α-淀粉酶的等同位置(由各个氨基酸序列之间最有可能的氨基酸序列对比测定)修饰得到的变体也包括在本发明的范围内。
本发明变体的优选实施方案是得自地衣芽孢杆菌α-淀粉酶(作为亲代Termamyl-样α-淀粉酶)的变体,例如,上述α-淀粉酶中的一个,如具有SEQID NO:4所示氨基酸序列的地衣芽孢杆菌α-淀粉酶的变体。
本发明变体经改变的特性
下文将讨论本发明变体中可能存在的改变/突变与由此产生的合乎需要的特性改变(相对于亲代Termamyl-样α-淀粉酶而言)之间的关系。
在高温,酸性pH和/或低钙浓度下稳定性的增加
本发明涉及亲代Termamyl-样α-淀粉酶的变体,与亲代α-淀粉酶相比,所述变体α-淀粉酶表面的一个或多个暴露于溶剂的氨基酸残基发生变化,从而使α-淀粉酶的总体亲水性增加和/或表面上所述暴露于溶剂之氨基酸残基的侧链中的甲基总数增加。
在优选的实施方案中,向内弯曲的凹面上的一个或多个暴露于溶剂的氨基酸残基变为更具疏水性的氨基酸残基。
在另一个优选的实施方案中,凸面上的一个或多个暴露于溶剂的氨基酸残基经改变后增加了侧链中的甲基数目。
本发明涉及亲代Termamyl-样α-淀粉酶的α-淀粉酶变体,该变体在选自下列的一个或多个位置处具有变化:E376,S417,A420,S356,Y358;
其中:(a)所述变化各为:
(i)在占用上述位置的氨基酸的下游插入一个氨基酸,
(ii)缺失占用上述位置的氨基酸,或
(iii)用不同的氨基酸取代占用上述位置的氨基酸,
(b)所述变体具有α-淀粉酶活性,和(c)每个位置对应于具有SEQ IDNO:4氨基酸序列的亲代Termamyl-样α-淀粉酶的氨基酸序列位置。
在一个实施方案中,变化是下列取代之一:
E376A,R,D,C,Q,G,H,I,K,L,M,N,F,P,S,T,W,Y,V;
在优选的实施方案中,取代是:E376K。
在一个实施方案中,变化是下列取代之一:
S417A,R,D,C,E,Q,G,H,I,K,L,M,N,F,P,T,W,Y,V;
在优选的实施方案中,取代是:S417T。
在一个实施方案中,变化是下列取代之一:
A420R,D,C,E,Q,G,H,I,K,L,M,N,F,P,S,T,W,Y,V;
在优选的实施方案中,取代是:A420Q,R。
在一个实施方案中,变化是下列取代之一:
S356A,R,D,C,E,Q,G,H,I,K,L,M,N,F,P,T,W,Y,V。
在一个实施方案中,变化是下列取代之一:
Y3 58A,R,D,C,E,Q,G,H,I,K,L,M,N,F,P,S,T,W,V。
在优选的实施方案中,取代是:Y358F。
在本发明的一个实施方案中,变体含有一个或多个下列取代:E376K,S417T,A420Q,R,S356A,Y358F。
使用下文阐明本发明的实施例2中所述的方法,测定在高温,酸性pH和/或低钙浓度下稳定性的增加。
用作制备本发明变体所用骨架的亲代Termamyl-样α-淀粉酶可以是上文所述的任何Termamyl-样α-淀粉酶。
特别希望它们是选自下列的亲代Termamyl-样α-淀粉酶:得自地衣芽孢杆菌,如地衣芽孢杆菌菌株ATCC 27811,解淀粉芽孢杆菌,嗜热脂肪芽孢杆菌,芽孢杆菌菌种NCIB 12289,NCIB12512,NCIB 12513或DSM 9375的亲代Termamyl-样α-淀粉酶,和SEQ ID NO:1,2,3,4,5,6,7和8所示的亲代Termamyl-样α-淀粉酶。
在本发明的一个实施方案中,亲代Termamyl-样α-淀粉酶是与SEQ IDNO:4所示的地衣芽孢杆菌α-淀粉酶(Termamyl)相同的杂合α-淀粉酶,不同之处在于(成熟蛋白质)N-末端的35个氨基酸残基被SEQ ID NO:5所示解淀粉芽孢杆菌α-淀粉酶(BAN)成熟蛋白质N-末端的33个氨基酸残基所取代。亲代Termamyl-样杂合α-淀粉酶可以是上述的杂合Termamyl-样α-淀粉酶,其进一步具有下列突变:H156Y+181T+190F+209V+264S(使用SEQ ID NO:4中的编号)。所述骨架在下文中被称为“LE174”。
亲代α-淀粉酶进一步在下列一个或多个位置具有突变较为有利:K176,I201和H205(使用SEQ ID NO:4中的编号),尤其有利的是具有一个或多个下列取代:K176R,I201F和H205N(使用SEQ ID NO:4中的编号),例如特别是下列取代:K176R+I201F+H205N(使用SEQ ID NO:4中的编号)。
本发明人发现:相对于亲代Termamyl-样α-淀粉酶而言,上述变体在95-160℃的温度(即高温),低于7.0的pH(即酸性pH)和/或低于1mM(40ppm)的钙浓度(即低钙浓度)下具有增加的稳定性。
改变(如通过取代来改变)一个或多个暴露于溶剂的氨基酸残基可以1)增加酶的总体亲水性,或2)增加暴露于溶剂的氨基酸残基侧链中的甲基数目,从而改善温度稳定性。优选将向内弯曲的凹面上的氨基酸残基改变(如通过取代来改变)为更具疏水性的残基。优选改变(如通过取代来改变)凸面上的氨基酸残基以增加侧链中的甲基数目。
使用国际互联网站点http://sunrise.cbs.umn.edu/cast/版本1.0中的CAST程序(1998年2月发布)(参考文献:Jie Liang,Herbert Edelsbrunner和ClareWoodward,1998,分析蛋白质袋和沟槽:测定结合位点几何学并涉及配体设计,蛋白质科学,7,pp.1884-1897),可以鉴定出靠近表面的凹面区域。由于直径为1.4埃的探针可进可出,因此在该程序中确定了通向表面的路径。在CAST程序中使用缺省参数,使用Brookhaven数据库(1BPL)中的地衣芽孢杆菌钙-耗尽的α-淀粉酶结构可以发现凹面沟槽:
三种类型的相互作用可被合理化:
A.残基侧链和蛋白质之间的相互作用,
B.残基侧链和周围环境中的水之间的相互作用,
C.水和蛋白质之间的相互作用。
将SEQ ID NO:4所示的亲代Termamyl-样α-淀粉酶用作骨架,认为下列位置暴露于溶剂,并可对其进行适当改变:
E376,S417,A420,S356,Y358。
使用W.kabsch和C.Sander,生物聚合物22(1983)pp.2577-2637的dssp程序,可鉴定其它Termamyl-样α-淀粉酶表面相应的和其它的暴露于溶剂的位置。使用WHATIF程序包中的AACAVI程序部分(G.Vriend,Whatif和药物设计程序,J.Mol.Graph.8,pp.52-56(1990)版本19980317),可以鉴定凸起的表面。
在本发明的一个实施方案中,变体含有一个或多个下列取代:E376K,S417T,A420Q,R,S356A,Y358F。
本发明人发现:通过将上述位置,即E376,S417,A420,S356,Y358(使用SEQ ID NO:4的编号)的突变与下列一个或多个位置,即K176,I201和H205的突变联合,可以使高温,酸性pH和/或低钙浓度下的稳定性有更大的提高。
下列其它取代是优选的:
K176A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V;
I201A,R,D,C,E,Q,G,H,L,K,M,N,F,P,S,T,W,Y,V;
H205A,R,D,C,E,Q,G,I,L,K,M,N,F,P,S,T,W,Y,V;
如阐明本发明的实施例2所示,使用被称为LE174的杂合α-淀粉酶作为亲代Termamyl-样α-淀粉酶,联合下列突变可以使稳定性增加:
K176+I201F+H205N+E376K+A420R或
K176+I201F+H205N+S417T+A420Q或
K176+I201F+H205N+S356A+Y358F。
本发明变体中的一般突变
优选本发明的变体除了含有上述变化外,还含有一个或多个修饰。因此,经修饰的α-淀粉酶变体中一个或多个脯氨酸残基被非脯氨酸残基取代较为有利,所述非脯氨酸残基可以是任何可能的天然非脯氨酸残基,优选为丙氨酸,甘氨酸,丝氨酸,苏氨酸,缬氨酸或亮氨酸。
类似地,优选亲代α-淀粉酶被修饰的氨基酸残基中存在的一个或多个半胱氨酸残基被非半胱氨酸残基取代,所述非半胱氨酸残基可以是例如丝氨酸,丙氨酸,苏氨酸,甘氨酸,缬氨酸或亮氨酸。
另外,本发明的变体可以(作为唯一的修饰或与上述任何修饰联合)被修饰,以使对应于SEQ ID NO:4中185-209的氨基酸片段中存在的一个或多个Asp和/或Glu分别被Asn和/或Gln取代。另外,用Arg取代Termamyl-样α-淀粉酶中对应于SEQ ID NO:4中185-209的氨基酸片段中存在的一个或多个Lys残基也很有意义。
应理解本发明包含掺入了两个或多个上述修饰的变体。
另外,在本文所述的任何变体中导入点突变较为有利。
克隆编码本发明α-淀粉酶的DNA序列
可使用本领域众所周知的多种方法,从产生所述α-淀粉酶的任何细胞或微生物中分离出编码亲代α-淀粉酶的DNA序列。首先,应使用源自产生待研究之α-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α-淀粉酶的氨基酸序列是已知的,可合成同源的经标记的寡核苷酸探针,使用该探针从制备自所述生物体的基因组文库中鉴定编码α-淀粉酶的克隆。或者,将含有与已知α-淀粉酶基因同源之序列的经标记的寡核苷酸探针用作探针,使用较低严紧度的杂交和洗涤条件鉴定编码α-淀粉酶的克隆。
另一种鉴定编码α-淀粉酶的克隆的方法包括:将基因组DNA片段插入表达载体,如质粒,用所得基因组DNA文库转化α-淀粉酶-阴性细菌,然后将经转化的细菌铺于含有α-淀粉酶底物的琼脂上,从而鉴定出表达α-淀粉酶的克隆。
或者,可通过已建立的标准方法合成制备编码酶的DNA序列,所述方法例如S.L.Beaucage和M.H.Caruthers(1981)所述的膦酰胺法或Matthes等(1984)所述的方法。在膦酰胺法中,可在例如自动化的DNA合成仪中合成寡核苷酸,对其进行纯化,退火,再连接并克隆至适当的载体。
最终,DNA序列可以是根据标准技术,通过连接合成的,基因组的或cDNA来源的片段(适当时是对应于完整DNA序列的多个部分的片段)而制备的混合的基因组和合成来源,混合的合成和cDNA来源或混合的基因组和cDNA来源的DNA序列。也可以使用特定的引物,如US 4,683,202或R.K.Saiki等(1988)所述的引物经聚合酶链反应(PCR)制备DNA序列。
定点诱变
一旦分离出编码α-淀粉酶的DNA序列,并鉴定出合乎需要的突变位点,可使用合成的寡核苷酸导入突变。这些寡核苷酸含有侧翼于所需突变位点的核苷酸序列;在合成寡核苷酸的过程中插入突变的核苷酸。在特定的方法中,在携有α-淀粉酶基因的载体中产生桥连α-淀粉酶-编码序列的单链DNA缺口。然后,使携有所需突变的合成核苷酸与该单链DNA的同源部分退火。然后用DNA聚合酶I(Klenow片段)补平其余缺口,使用T4连接酶连接构建体。该方法的特例描述于Morinaga等(1984)。US 4,760,025公开了通过对盒进行微小的改变而导入编码多个突变的寡核苷酸。然而,由于Morinaga法可以导入多种长度的多个寡核苷酸,因此可在任何一次导入甚至更多个突变。
Nelson和Long(1989)描述了另一种将突变导入编码α-淀粉酶的DNA序列的方法。所述方法包括以3个步骤产生含有所需突变的PCR片段,所述突变是通过使用化学合成的DNA链作为PCR反应的一个引物而导入的。通过用限制性内切核酸酶裂解,可从PCR-产生的片段中分离出携有突变的DNA片段,并将该片段重新插入表达质粒。
随机诱变
可在翻译成本文所示氨基酸序列的基因的至少3个部分中,或在整个基因内适当地进行随机诱变,所述诱变可以是定域的或区域-特异性的随机诱变。
利用本领域已知的任何方法,可以方便地对编码亲代α-淀粉酶的DNA序列进行随机诱变。
关于上文,本发明的另一方面涉及产生亲代α-淀粉酶的变体的方法,例如,其中变体相对于亲代而言,表现出经改变的或增加的热稳定性,所述方法包括:
(a)对编码亲代α-淀粉酶的DNA序列进行随机诱变,
(b)在宿主细胞中表达步骤(a)中得到的突变DNA序列,和
(c)筛选表达α-淀粉酶变体的宿主细胞,所述变体相对于亲代α-淀粉酶而言具有经改变的特性(即热稳定性)。
优选使用添加引物进行本发明上述方法中的步骤(a)。
例如,可使用适当的物理或化学诱变剂,使用适当的寡核苷酸,或通过对DNA序列进行PCR以产生诱变来进行随机诱变。另外,可使用这些诱变剂的任何组合进行随机诱变。诱变剂可以是例如诱导碱基转换,颠换,倒位,倒频,缺失和/或插入的试剂。
适用于本发明目的的物理或化学诱变剂的例子包括紫外线(UV)照射,羟胺,N-甲基-N’-硝基-N-亚硝基胍(MNNG),邻甲基羟胺,亚硝酸,乙基甲磺酸(EMS),亚硫酸氢钠,甲酸和核苷酸类似物。当使用这些诱变剂时,一般通过在适于发生诱变的条件下,在选定诱变剂的存在下保温待诱变的编码亲代酶的DNA序列来进行诱变,然后筛选具有所需特性的突变DNA。
当利用寡核苷酸进行诱变时,在合成待改变位置处的寡核苷酸的过程中,可同时添加寡核苷酸和3个非-亲代核苷酸。添加的目的是避免不必要氨基酸的密码子。可通过任何公开的技术,例如使用PCR,LCR或适当时使用任何DNA聚合酶和连接酶,将添加的寡核苷酸掺入编码α-淀粉酶的DNA。
优选使用“恒随机的添加”进行添加,其中各个位置的野生型和突变的百分比是预先确定好的。另外,添加可以导致优先导入某些核苷酸,从而优先导入一个或多个特定的氨基酸残基。例如,进行添加后可在每个位置导入90%野生型和10%突变。选择添加方案的其它考虑基于遗传学以及蛋白质-结构的限制。可使用DOPE程序制订添加方案,所述程序可特别地确保不导入终止密码子。
当使用PCR-产生的诱变时,可在增加核苷酸错误掺入的条件下,对经化学处理或未经处理的编码亲代α-淀粉酶的基因进行PCR(Deshler 1992;Leung等,技术,Vol.1,1989,PP.11-15)。
可使用大肠杆菌(Fowler等,Molec.Gen.Genet.,133,1974,pp.179-191),酿酒酵母或任何其它微生物的增变株对编码α-淀粉酶的DNA进行随机诱变,诱变方法包括例如:将含有亲代糖基酶的质粒转化至增变株,培养含有质粒的增变株,从增变株中分离突变的质粒。随后,将突变的质粒转化至表达生物体。
待诱变的DNA序列可以方便地存在于基因组或cDNA文库中,所述文库制备自表达亲代α-淀粉酶的生物体。或者,DNA序列可以存在于适当的载体,如质粒或噬菌体上,再与诱变剂一起保温或要不然暴露于诱变剂中。待诱变的DNA也可存在于宿主细胞中,或者整合于所述细胞的基因组中,或者存在于细胞所携带的载体上。最后,待诱变的DNA也可以是分离的形式。应理解接受随机诱变的DNA序列优选为cDNA或基因组DNA序列。
在某些情况下,在进行表达步骤b)或筛选步骤c)前,可以方便地扩增突变的DNA序列。可根据本领域已知的方法进行扩增,本发明优选的方法是:使用根据亲代酶的DNA或氨基酸序列制备的寡核苷酸引物进行PCR扩增。
与诱变剂保温或暴露于诱变剂之后,通过在允许发生表达的条件下培养携有突变DNA序列的适当宿主细胞来表达突变的DNA。用于此目的的宿主细胞可以是被突变的DNA序列(任选其存在于载体上)转化的宿主细胞,或者是在诱变处理的过程中携有编码亲代酶的DNA序列的宿主细胞。适当宿主细胞的例子如下: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;和革兰氏阴性细菌,如大肠杆菌。
突变的DNA序列可进一步含有能使突变的DNA序列表达的DNA序列。
定域随机诱变
随机诱变可有利地定域于所述亲代α-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α-淀粉酶变体的其它方法
提供α-淀粉酶变体的其它方法包括本领域已知的基因改组方法,所述方法包括例如WO 95/22625(Affymax Technologies N.V.)和WO96/00343(Novo Nordisk A/S)所述的方法。
表达本发明的α-淀粉酶变体
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。
携有编码本发明α-淀粉酶变体的DNA序列的重组表达载体可以是能对其方便地进行重组DNA操作的任何载体,载体的选择经常取决于导入该载体的宿主细胞。因此,载体可以是自我复制的载体,即作为染色体外实体存在的载体,所述载体的复制独立于染色体的复制,所述载体包括例如质粒,噬菌体或染色体外元件,微型染色体或人工染色体。或者,载体可以是当导入宿主细胞时可以整合至宿主细胞基因组,并与整合了该载体的染色体一起复制的载体。
在载体中,DNA序列应该与适当的启动子序列可操作相连。启动子可以是在选定宿主细胞中显示出转录活性的任何DNA序列,启动子可以得自编码与宿主细胞同源或异源之蛋白质的基因。介导编码本发明α-淀粉酶变体之DNA序列转录,尤其是在细菌宿主中的转录的适当启动子的例子是:大肠杆菌lac操纵子的启动子,天蓝色链霉菌琼脂酶基因dagA启动子,地衣芽孢杆菌α-淀粉酶基因(amyL)启动子,嗜热脂肪芽孢杆菌产麦芽糖淀粉酶基因(amyM)启动子,解淀粉芽孢杆菌α-淀粉酶(amyQ)启动子,枯草芽孢杆菌xylA和xylB基因的启动子等。为了在真菌宿主中转录,有用的启动子的例子是得自编码下列酶的基因的启动子:米曲霉TAKA淀粉酶,米赫根毛霉(Rhizomucor miehei)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。
本发明的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。
尽管细胞内表达在某些方面(例如当使用某些细菌作为宿主细胞时)有利,但一般优选在细胞外进行表达。通常,本文所述的芽孢杆菌α-淀粉酶含有允许表达的蛋白酶分泌至培养基的前区。必要时,该前区可被不同的前区或信号序列取代,通过取代编码各个前区的DNA序列可以方便地实现此目的。
分别连接编码α-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)。
含有上文所定义的本发明DNA构建体或表达载体的本发明细胞可以有利地用作宿主细胞,以重组产生本发明的α-淀粉酶变体。可以方便地通过将编码变体的本发明DNA构建体(一个或多个拷贝)整合至宿主染色体,用所述DNA构建体转化细胞。一般认为整合是有利的,因为整合后DNA序列可以在细胞中更加稳定地维持。根据常规方法,例如通过同源或异源重组可以将DNA构建体整合至宿主染色体。或者,可用与不同类型的宿主细胞有关的上述表达载体转化细胞。
本发明的细胞可以是高等生物,如哺乳动物或昆虫的细胞,但优选其为微生物细胞,例如细菌或真菌(包括酵母)细胞。
适当细菌的例子是: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,或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。通过例如原生质体转化,或通过以本身已知的方式使用感受态细胞即可实现细菌的转化。
酵母生物体可有利地选自糖酵母属或裂殖酵母属的种,例如酿酒酵母。丝状真菌可有利地属于曲霉属的种,例如米曲霉或黑曲霉。通过以下方法可转化真菌细胞,所述方法包括按本身已知的方式形成原生质体,转化原生质体,接着再生细胞壁。转化曲霉属宿主细胞的适当方法描述于EP 238 023。
另一方面,本发明涉及产生本发明α-淀粉酶变体的方法,所述方法包括在有利于产生变体的条件下培养上述宿主细胞,并从细胞和/或培养基中回收变体。
用于培养细胞的培养基可以是适于培养所述宿主细胞,并表达本发明α-淀粉酶变体的任何常规培养基。适当的培养基可以商购,或者可根据公开的配方(例如美国典型培养物保藏中心目录中所述的配方)制备。
通过众所周知的方法,包括通过离心或过滤将培养基与细胞分开,利用诸如硫酸铵的盐沉淀培养基中的蛋白质成分,接着利用层析法,如离子交换层析,亲和层析等,可以从培养基中方便地回收宿主细胞分泌的α-淀粉酶变体。
工业应用
本发明的α-淀粉酶变体具有有价值的特性,所述特性有多种工业用途。本发明的酶变体可用作洗涤,洗碟和硬-表面清洗去污剂组合物中的组分。多种变体对由淀粉产生增甜剂和乙醇,和/或织物脱浆特别有用。常规淀粉-转变过程,包括淀粉液化和/或糖化过程的条件描述于例如US 3,912,590和EP专利公开号252,730和63,909。
由淀粉产生增甜剂:
“传统的”由淀粉转变为果糖浆的方法一般由三个连续的酶促过程组成,即液化过程,接着是糖化过程和异构化过程。在液化过程中,在温度为95-160℃,pH值为5.5-6.2时,用α-淀粉酶(如TermamylTM)将淀粉降解约2小时,使其成为糊精。为了确保这些条件下的最适酶稳定性,需加入1mM钙(40ppm游离的钙离子)。
液化过程之后,通过加入葡糖淀粉酶(如AMGTM)和脱支酶,如异淀粉酶或支链淀粉酶(如PromozymeTM),将糊精转变为葡萄糖。在此步骤之前,将pH值降低为4.5以下,维持高温(95℃以上),使液化α-淀粉酶活性变性。将温度降低为60℃,加入葡糖淀粉酶和脱支酶。将糖化过程进行24-72小时。
糖化过程之后,将pH增加至pH值范围为6-8,优选为pH7.5,通过离子交换除去钙。然后使用固定化的葡萄糖异构酶(如SweetzymeTM)将葡萄糖浆转变为高果糖浆。
此方法的至少1个酶促改良是可以想象到的。即降低了液化α-淀粉酶的钙依赖性。添加游离的钙是确保α-淀粉酶足够高的稳定性所必需的,但游离的钙强烈地抑制了葡萄糖异构酶的活性,必需通过操作昂贵装置以除去游离的钙,使其水平降至3-5ppm以下。如果这种操作可以避免,液化过程在不加入游离钙离子时也能进行的话,就可以节省开支。
为了达到此目的,需要一种对钙的依赖性较低的Termamyl-样α-淀粉酶,该α-淀粉酶在游离钙浓度低(<40ppm)时是稳定的,且具有高活性。这种Termamyl-样α-淀粉酶应该在pH范围为4.5-6.5时,优选在pH范围为4.5-5.5时具有最适pH。
去污剂组合物
如上所述,可将本发明的变体适当掺入去污剂组合物中。关于去污剂组合物(如洗衣或洗碟去污剂)的相关成分,将变体配制在所述去污剂组合物中的适当方法,和相关类型的去污剂组合物的例子的其它细节可参见例如WO 96/23874和WO 97/07202。
含有本发明变体的去污剂组合物还可含有一种或多种其它的酶,例如脂肪酶,角质酶,蛋白酶,纤维素酶,过氧化物酶或漆酶和/或另一种α-淀粉酶。
以常规使用的浓度将本发明的α-淀粉酶变体掺入去污剂。本发明希望使用常规的去污剂剂量水平,以相当于每升洗涤/洗碟液0.00001-1mg(以纯的活性酶蛋白质计)α-淀粉酶的量掺入本发明的变体。
材料和方法
酶:
LE174杂合α-淀粉酶变体:LE174是与Termamyl序列,即SEQ ID NO:4所示的地衣芽孢杆菌α-淀粉酶相同的杂合Termamyl-样α-淀粉酶,不同之处在于:(成熟蛋白质)N-末端的35个氨基酸残基被SEQ ID NO:5所示的解淀粉芽孢杆菌α-淀粉酶(BAN)成熟蛋白质N-末端的33个氨基酸残基所取代,其进一步具有下列突变:H156Y+A181T+N190F+A209V+Q264S(使用SEQID NO:4的编号)。
构建pSNK101
该大肠杆菌/芽孢杆菌穿梭载体可用于导入突变,而不用在大肠杆菌中表达α-淀粉酶,然后以α-淀粉酶在芽孢杆菌中具有活性的方式修饰该载体。按下述构建载体:通过将含有来源于大肠杆菌之片段的1.2kb片段插入α-淀粉酶基因5’编码区的PstI位点,来灭活pX载体(在amyL中具有下列改变的pDN1528:BAN(1-33),H156Y,A181T,N190F,A209V, Q264S,实施例1中将进一步描述质粒pDN1528)中的α-淀粉酶基因。使用正向引物1:5’-gacctgcagtcaggc aacta-3’(SEQ ID NO:28)和反向引物1:5’-tagagtcgacctgcaggcat-3’(SEQ ID NO:29),由pUC19(GenBank登记号:X02514)扩增此片段。于37℃,用PstI消化PCR扩增子和含有α-淀粉酶基因的pX质粒达2小时。室温下连接pX载体片段和来源于大肠杆菌的扩增子达1小时,通过电转化转化至大肠杆菌。所得载体被称为pSnK101。
该大肠杆菌/芽孢杆菌穿梭载体可用于导入突变,而不用在大肠杆菌中表达α-淀粉酶,然后以α-淀粉酶在芽孢杆菌中具有活性的方式修饰该载体。按下述构建载体:通过将含有来源于大肠杆菌之片段的1.2kb片段插入α-淀粉酶基因5’编码区的PstI位点,来灭活pX载体(在amyL中具有下列改变的pDN1528:BAN(1-33),H156Y+A181T+N190F+A209V+Q264S,实施例1中将进一步描述质粒pDN1528)中的α-淀粉酶基因。使用正向引物2:5’-gacctgcagtcaggcaacta-3’(SEQ ID NO:30)和反向引物2:5’-tagagtcgacctgcaggcat-3’(SEQ ID NO:31),由pUC19(GenBank登记号:X02514)扩增此片段。于37℃,用PstI消化PCR扩增子和含有α-淀粉酶基因的pX质粒达2小时。室温下连接pX载体片段和来源于大肠杆菌的扩增子达1小时,通过电转化转化至大肠杆菌。所得载体被称为pSnK101。
低pH滤膜试验
于37℃,将芽孢杆菌文库铺于含10μg/ml氯霉素的TY琼脂平板上的醋酸纤维素(OE67,Schleicher&Schuell,Dassel,德国)和硝酸纤维素滤膜(Protran-Ba 85,Schleicher & Schuell,Dassel,德国)夹层,至少放置21小时。醋酸纤维素层位于TY琼脂平板上。
在铺平板之后,但在保温之前用针头特异性地标记每个滤膜夹层,以使阳性变体定位于滤膜上,将结合有变体的硝酸纤维素滤膜转移至含柠檬酸盐缓冲液,pH4.5的容器,90℃保温15分钟。室温下,将含菌落的醋酸纤维素滤膜储存于TY-平板上待用。保温之后,在含有1%琼脂糖,0.2%淀粉(皆溶于柠檬酸盐缓冲液,pH6.0)的试验平板上检测残留的活性。按与滤膜夹层相同的方式标记含硝酸纤维素滤膜的试验平板,50℃保温2小时。取出滤膜之后,用10%Lugol溶液染色试验平板。降解淀粉的变体被测定为深蓝色背景上的白点,然后在储存平板上鉴定该变体。在与第一次筛选相同的条件下再将阳性变体筛选2次。
二级筛选
重新筛选之后,从储存平板上挑选阳性转化子,在二级平板试验中进行检测。于37℃,在5ml LB+氯霉素中将阳性转化子培养22小时。于90℃,在柠檬酸盐缓冲液,pH4.5中保温每个阳性转化子和对照LE174变体的芽孢杆菌培养物,在0,10,20,30,40,60和80分钟时取样。将3微升样品点在试验平板上。用10%Lugol溶液染色试验平板。将试验板上残留活性高于对照的晕圈定为改良变体。通过核苷酸测序测定这些改良的变体。
发酵和纯化α-淀粉酶变体
在含有15μg/ml氯霉素的LB-琼脂平板上,用-80℃原种划线携有相关表达质粒的枯草芽孢杆菌菌株,37℃培养过夜。将菌落转移至500ml摇瓶内的100ml添加有15μg/ml氯霉素的BPX培养基中。
BPX培养基的组成:
马铃薯淀粉 100g/l
大麦粉 50g/l
BAN 5000 SKB 0.1g/l
酪蛋白酸钠 10g/l
大豆粉 20g/l
Na2HP4,12H2O 9g/l
PluronicTM 0.1g/l
于37℃,以270rpm振荡培养5天。
通过以4500rpm离心20-25分钟,从发酵肉汤中除去细胞和细胞碎片。然后,过滤上清液,得到十分清亮的溶液。在UF-滤器(10000截留膜)上浓缩和冲洗滤液,将缓冲液变为20mM醋酸盐,pH5.5。将UF-滤液上样于S-Sepharose F.F.,用含0.2M NaCl的相同缓冲液逐步洗脱。洗脱液对10mMTris,pH9.0透析,然后上样于Q-sepharose F.F.,用超过6倍柱体积的0-0.3MNaCl线性梯度进行洗脱。集中含有活性(通过Phadebas试验测定)的级分,将pH调节至pH7.5,用0.5%W/vol的活性炭处理5分钟除去残留的颜色。
稳定性的测定
使用相同的设置进行所有的稳定性试验。方法是:
在相关条件(1-4)下保温酶。在0,5,10,15和30分钟时取样,用试验缓冲液(0.1M 50mM Britton缓冲液pH7.3)稀释25倍(对所有取出的样品都作相同的稀释),在标准条件下(pH7.3,37℃)使用Phadebas试验(Pharmacia)测定活性。
将保温前(0分钟)测定的活性用作参照(100%)。百分比的下降被计算为保温时间的函数。表中显示了保温30分钟之后残留的活性。
活性测定-(KNU)
在根据下列条件测定α-淀粉酶的Novo Nordisk’s标准方法中,1000个α-淀粉酶单位(1KNU)是每小时降解5.26g淀粉(Merck,Amylum Solubile,Erg.B6,批号9947275)所需的酶量:
底物 可溶性淀粉
溶剂中的钙含量 0.0043M
反应时间 7-20分钟
温度 37℃
pH 5.6
Novo Nordisk’s分析法(AF 9)的详细描述即要即得。
比活的测定
检测α-淀粉酶的活性
通过利用Phadebas片为底物的方法测定α-淀粉酶的活性。Phadebas片(PhadebasAmylase Test,由Pharmacia Diagnostic提供)含有交联的不可溶的蓝色淀粉聚合物,所述聚合物已与牛血清白蛋白和缓冲物质混合,并被制成片状。
对每一个单次测定而言,将1片Phadebas悬浮于含有5ml 50mMBritton-Robinson缓冲液(50mM醋酸,50mM磷酸,50mM硼酸,0.1mM CaCl2,用NaOH将pH调节至所需的值)的试管中。在所需温度的水浴中进行试验。用x ml 50mM Britton-Robinson缓冲液稀释待检测的α-淀粉酶。在5ml 50mMBritton-Robinson缓冲液中加入1ml这种α-淀粉酶溶液。通过α-淀粉酶水解淀粉,给出可溶性的蓝色片段。用分光光度计测定所得蓝色溶液在620nm处的光吸收值,该值是α-淀粉酶活性的函数。
重要的是,保温10或15分钟之后(检测时间)测定的620nm光吸收值范围是0.2至2.0个光吸收单位。在此光吸收值范围内,活性和光吸收值之间呈现线性关系(Lambert-Beer定律)。因此,必须调节酶的稀释度以符合此标准。在特定的一套条件(温度,pH,反应时间,缓冲液浓度)下,1mg给定的α-淀粉酶可水解一定量的底物,并产生蓝色。测定620nm处的颜色强度。在给定的一套条件下,测定的光吸收值与所述α-淀粉酶的比活(活性/mg纯α-淀粉酶蛋白质)直接成比例。
实施例
实施例1
通过随机诱变构建Termamyl-样LE174α-淀粉酶变体,与亲代酶相比,
所述变体在低pH下具有改善的稳定性,其稳定性对钙离子的依赖性降低
随机诱变
为了改善亲代LE174α-淀粉酶变体在低pH和低钙浓度下的稳定性,在预先选定的区域进行随机诱变:
所述区域是:
区域 残基
SERI A425-Y438
SERII W411-L424
SERIII G397-G410
SERV T369-H382
SERVII G310-F323
SERIX L346-P359
对6个区域中的每一个而言,在上述区域中的每一个核苷酸位置使用相同的突变率(主要成分97%,三种其余核苷酸中的每一种各1%共给出3%突变),例如对A425密码子的第1位而言:97%C,1%A,1%T,1%G来合成随机寡核苷酸。6个随机的寡核苷酸和必要时使用的互补SOE辅助引物示于具有下列4种核苷酸分布的表1-6:
表1
RSERI:5′-GC GTT TTG CCG GCC GAC ATA 312 234 322 243 333 133444 233 423 242 212 211 243 343 CAA ACC TGA ATT-3′(SEQ ID NO:15)
表2
RSERII:5′-GC GTT TTG CCG GCC GAC ATA CAT TCG CTT TGC CCC ACCGGG TCC GTC TGT TAT TAA TGC CGC 311 133 241 122 243 113 341 432423 433 223 332 242 331 GCC GAC AAT GTC ATG GTG-3′(SEQ ID NO:16)
表3
RSERIII:5′-GTC GCC TTC CCT TGT CCA 433 413 112 423 124 424 423411 121 123 124 324 243 233 GTA CGC ATA CTG TTT TCT-3′(SEQ IDNO:17)
辅助引物FSERIII:5’-TGG ACA AGG GAA GGC GAC AG-3’(SEQ IDNO:18)
表4
RSERV:5-TAA GAT CGG TTC AAT TTT 424 222 311 443 144 112 223434 324 441 423 233 222 342 CCC GTA CAT ATC CCC GTA GAA-3(SEQID NO:19)
辅助引物FSERV:5’-AAA ATT GAA CCG ATC TTA-3’(SEQ IDNO:20)
表5
FSERVII:5′-TT CCA TGC TGC ATC GAC ACA GGG AGG CGG CTA TGA TATGAG GAA ATT GCT GAA 344 213 442 342 223 311 431 233 422 411 123442 213 122 TGT CGA TAA CCA-3′(SEQ ID NO:21)
辅助引物RSERVII:5’-TGT CGA TGC AGC ATG GAA-3’(SEQ IDNO:22)
表6
FSERIX:5′-GT CCA AAC ATG GTT TAA GCC 432 243 221 343 222 212232 313 114 441 123 244 121 333 TCA GGT TTT CTA CGG GGA-3′(SEQID NO:23)
辅助引物RSERIX:5’-GGC TTA AAC CAT GTT TGG AC-3’(SEQ IDNO:24)
每个突变的核苷酸位置的核苷酸分布:
1:97%A,1%T,1%C,1%G
2:97%T,1%A,1%C,1%G
3:97%C,1%A,1%T,1%G
4:97%G,1%A,1%T,1%C
构建质粒文库
使用引物1B:5’-CGA TTG CTG ACG CTG TTA TTT GCG-3’和表1所示的随机寡核苷酸,或表2所示的随机寡核苷酸,经PCR扩增两个约1.4kb的片段。用EcoRV和EagI消化pSnK101和所述PCR片段达2小时。纯化约3.6kb的载体片段和约1.3kb的PCR片段,连接过夜,转化至大肠杆菌,然后进一步按下文所述转化至芽孢杆菌宿主菌株。使用表3-6所示的针对各个区域的随机寡核苷酸(在图2中通称为aSER和bSER),和覆盖了LE174序列中的EcoRV和EagI位点的特定的地衣芽孢杆菌引物1B(SEQ ID NO:26)和#63:5’-CTA TCT TTG AAC ATA AAT TGA AAC C-3’(SEQ ID NO:27),经重叠延伸法(Horton等,基因,77(1989),pp61-68)产生PCR-文库-片段。图2显示了PCR策略。将PCR片段克隆至大肠杆菌/芽孢杆菌穿梭载体pSNK101(见材料和方法),从而在大肠杆菌中诱变,并立即在枯草芽孢杆菌中表达,以防止α-淀粉酶在大肠杆菌中的致死积累。在大肠杆菌中建立了克隆的PCR片段之后,消化质粒,得到经修饰的pUC19片段,物理连接启动子和突变的Termamyl基因,在芽孢杆菌宿主中进行表达。
筛选
在上文“材料和方法”一节中所述的低pH滤膜试验中筛选6个文库。
按实施例1所述制备下文实施例2表中所列的所有变体。
实施例2
测定稳定性
通常,为了改善95℃-105℃时的稳定性,在pH6.0-6.2,加入约40ppm游离钙时进行工业液化过程。制备本发明的变体,使之在
1.pH低于pH6.2和/或
2.游离钙水平低于40ppm游离钙时,稳定性改善。
使用在酸性pH(pH5.0),5ppm游离钙的存在下测定稳定性的试验测定稳定性的增加。
在下列条件下保温10μg变体:0.1M醋酸盐溶液,pH调节至pH5.0,含有5ppm钙和5%w/w普通的玉米淀粉(不含钙)。在95℃水浴中保温30分钟。
结果:
在pH5.0,5ppm钙,95℃保温时增加的稳定性
保温时间 | LE174,具有K176R+I201F+H205N | LE174,具有K176R+I201F+H205N+E376K+A420R | LE174,具有K176R+I201F+H205N+S417T+A420Q | LE174,具有K176R+I201F+H205N+S356A+Y358F |
0 | 100 | 100 | 100 | 100 |
5 | 65 | 61 | 66 | 66 |
10 | 58 | 53 | 60 | 59 |
15 | 51 | 48 | 55 | 56 |
30 | 36 | 39 | 45 | 49 |
比活测定
使用Phadebas试验(Pharmacia)(上述)将比活测定为活性/mg酶。使用本文材料和方法一节中所述的α-淀粉酶试验测定活性。
具有下列取代的LE174:
K176R+I201F+H205N
所测比活为:13400NU/mg
具有下列取代的LE174:
K176R+I201F+H205N+E376K+A420R:
所测比活为:14770NU/mg
具有下列取代的LE174:
K176R+I201F+H205N+S417T+A420Q:
所测比活为:16670NU/mg
具有下列取代的LE174:
K176R+I201F+H205N+S356A+Y358F:
所测比活为:15300NU/mg
参考文献
Klein,C等,生物化学1992,31,8740-8746,Mizuno,H等,分子生物学杂志,(1993),234,1282-1283,Chang,C等,分子生物学杂志,(1993),229,235-238,Larson,S.B.,分子生物学杂志,(1994),235,1560-1584,Lawson,C.L.,分子生物学杂志,(1994),236,590-600,Qian,M等,分子生物学杂志,(1993),231,785-799,Brady,R.L等,Acta Crystallogr.sect.B.47,527-535,Swift,H.J等,Acta Crystallogr.sect.B.47,535-544,A.Kadziola博士论文:“通过X-射线晶体学研究的大麦α-淀粉酶及其与底物类似物抑制剂的复合物”,哥本哈根大学化学系,1993,MacGregor,E.A.,食品水胶体,1987,Vol.1,No.5-6,p.B.Diderichsen和L.Christiansen,从嗜热脂肪芽孢杆菌中克隆产麦芽糖的α-淀粉酶,FEMS Microbiol letters:56:pp.53-60(1988),Hudson等,实用免疫学,第3版(1989),Blackwell Scientific Publications,Sambrook等,分子克隆:实验室手册,第2版,冷泉港,1989,S.L.Beaucage和M.H.Caruthers,四面体通讯,22,1981,pp.1859-1869,Matthes等,The EMBO J.3,1984,pp.801-805,R.K.Saiki等,科学,239,1988,pp.487-491,Morinaga等,(1984,生物技术2:646-639),Nelson和Long,分析生物化学,180,1989,pp.147-151,Hunkapiller等,1984,自然,310:105-111,R.Higuchi,B.Krummel和R.K.Saiki(1988),体外制备和特异性诱变DNA片段的一般方法:研究蛋白质和DNA的相互作用,核酸研究,16:7351-7367,Dubnau等,1971,分子生物学杂志,56,pp.209-221,Gryczan等,1978,细菌学杂志,134,pp.318-329,S.D.Erlich,1977,Proc.Natl.Acad.Sci.74,pp.1680-1682,Boel等,1990,生物化学,29,pp.6244-6249。
序列表
(1)序列资料:
(i)申请人:
(A)姓名:NOVO NORDISK A/S
(B)街道:Novo Alle
(C)城市:DK-2880 Bagsvaerd
(E)国家:丹麦
(F)邮编:DK-2880
(G)电话:+45 4444 8888
(H)电传:+45 4449 3256
(ii)发明标题:α-淀粉酶变体
(iii)序列数:32
(iv)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(2)SEQ ID NO:1的资料:
(i)序列特征:
(A)长度:485个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:蛋白质
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:1
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala
20 25 30
Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn sn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
115 120 125
Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys
165 170 175
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255
Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300
Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys
305 310 315 320
His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380
Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr
385 390 395 400
Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly
435 440 445
Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Val Trp Val Lys Gln
485
(2)SEQ ID NO:2的资料:
(i)序列特征:
(A)长度:485个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:蛋白质
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:2
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser
20 25 30
Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly
85 90 95
Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala
245 250 255
Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300
Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys
305 310 315 320
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala
370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr
385 390 395 400
Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly
435 440 445
Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Lys Arg
485
(2)SEQ ID NO:3的资料:
(i)序列特征:
(A)长度:514个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:蛋白质
(iii)生物:嗜热脂肪芽孢杆菌
(xi)序列描述:SEQ ID NO:3
Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu
1 5 10 15
Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn
20 25 30
Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
35 40 45
Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60
Leu Gly Glu Phe Asn Gln Lys Gly Ala Val Arg Thr Lys Tyr Gly Thr
65 70 75 80
Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met
85 90 95
Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
100 105 110
Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln
115 120 125
Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe
130 135 140
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His
145 150 155 160
Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr
165 170 175
Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu
180 185 190
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205
Pro Glu Val Val Thr Glu Leu Lys Ser Trp Gly Lys Trp Tyr Val Asn
210 215 220
Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
225 230 235 240
Phe Ser Phe Phe Pro Asp Trio Leu Ser Asp Val Arg Ser Gln Thr Gly
245 250 255
Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys
260 265 270
Leu His Asn Tyr Ile Met Lys Thr Asn Gly Thr Met Ser Leu Phe Asp
275 280 285
Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Thr
290 295 300
Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met LysAsp Gln Pro
305 310 315 320
Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln
325 330 335
Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala
340 345 350
Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp
355 360 365
Tyr Tyr Gly Ile Pro Gln Tyr Ash Ile Pro Ser Leu Lys Ser Lys Ile
370 375 380
Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His
385 390 395 400
Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val
405 410 415
Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445
Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser
450 455 460
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp
465 470 475 480
Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Trp Ser Ile Thr Thr
485 490 495
Arg Pro Trp Thr Asp Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val
500 505 510
Ala Trp
(2)SEQ ID NO:4的资料:
(i)序列特征:
(A)长度:483个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:蛋白质
(iii)生物:地衣芽孢杆菌
(xi)序列描述:SEQ ID NO:4
Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15
Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30
Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95
Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro
130 135 140
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205
Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser
305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His
385 390 395 400
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445
Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Arg
(2)SEQ ID NO:5的资料:
(i)序列特征:
(A)长度:480个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:蛋白质
(iii)生物:解淀粉芽孢杆菌
(xi)序列描述:SEQ ID NO:5
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp
1 5 10 15
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp
20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser
35 40 45
Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu
50 55 60
Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu
65 70 75 80
Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr
85 90 95
Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp
100 105 110
Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser
115 120 125
Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg
130 135 140
Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly
145 150 155 160
Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg
165 170 175
Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val
195 200 205
Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser
210 215 220
Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Sar Phe
225 230 235 240
Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu
275 280 285
His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala
305 310 315 320
Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His
385 390 395 400
Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr
435 440 445
Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser
450 455 460
Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr
465 470 475 480
(2)SEQ ID NO:6的资料:
(i)序列特征:
(A)长度:485个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:蛋白质
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:6
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser
20 25 30
Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly His Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala
245 250 255
Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly
290 295 300
Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg
305 310 315 320
His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser
370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys
385 390 395 400
Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly
435 440 445
Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
450 455 460
Ash Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Asn Lys
485
(2)SEQ ID NO:7的资料:
(i)序列特征:
(A)长度:485个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:蛋白质
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:7
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala
20 25 30
Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
115 120 125
Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys
165 170 175
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255
Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300
Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys
305 310 315 320
His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Giy Val Pro Ala Met Lys Ser
370 375 380
Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr
385 390 395 400
Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly
435 440 445
Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Val Trp Val Lys Gln
485
(2)SEQ ID NO:8的资料:
(i)序列特征:
(A)长度:485个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:蛋白质
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:8
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser
20 25 30
Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly
85 90 95
Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala
245 250 255
Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300
Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys
305 310 315 320
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala
370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr
385 390 395 400
Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly
435 440 445
Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Lys Arg
485
(2)SEQ ID NO:9的资料:
(i)序列特征:
(A)长度:1455个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:DNA(基因组)
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:9
CATCATAATG GAACAAATGG TACTATGATG CAATATTTCG AATGGTATTT GCCAAATGAC 60
GGGAATCATT GGAACAGGTT GAGGGATGAC GCAGCTAACT TAAAGAGTAA AGGGATAACA 120
GCTGTATGGA TCCCACCTGC ATGGAAGGGG ACTTCCCAGA ATGATGTAGG TTATGGAGCC 180
TATGATTTAT ATGATCTTGG AGAGTTTAAC CAGAAGGGGA CGGTTCGTAC AAAATATGGA 240
ACACGCAACC AGCTACAGGC TGCGGTGACC TCTTTAAAAA ATAACGGCAT TCAGGTATAT 300
GGTGATGTCG TCATGAATCA TAAAGGTGGA GCAGATGGTA CGGAAATTGT AAATGCGGTA 360
GAAGTGAATC GGAGCAACCG AAACCAGGAA ACCTCAGGAG AGTATGCAAT AGAAGCGTGG 420
ACAAAGTTTG ATTTTCCTGG AAGAGGAAAT AACCATTCCA GCTTTAAGTG GCGCTGGTAT 480
CATTTTGATG GGACAGATTG GGATCAGTCA CGCCAGCTTC AAAACAAAAT ATATAAATTC 540
AGGGGAACAG GCAAGGCCTG GGACTGGGAA GTCGATACAG AGAATGGCAA CTATGACTAT 600
CTTATGTATG CAGACGTGGA TATGGATCAC CCAGAAGTAA TACATGAACT TAGAAACTGG 660
GGAGTGTGGT ATACGAATAC ACTGAACCTT GATGGATTTA GAATAGATGC AGTGAAACAT 720
ATAAAATATA GCTTTACGAG AGATTGGCTT ACACATGTGC GTAACACCAC AGGTAAACCA 780
ATGTTTGCAG TGGCTGAGTT TTGGAAAAAT GACCTTGGTG CAATTGAAAA CTATTTGAAT 840
AAAACAAGTT GGAATCACTC GGTGTTTGAT GTTCCTCTCC ACTATAATTT GTACAATGCA 900
TCTAATAGCG GTGGTTATTA TGATATGAGA AATATTTTAA ATGGTTCTGT GGTGCAAAAA 960
CATCCAACAC ATGCCGTTAC TTTTGTTGAT AACCATGATT CTCAGCCCGG GGAAGCATTG 1020
GAATCCTTTG TTCAACAATG GTTTAAACCA CTTGCATATG CATTGGTTCT GACAAGGGAA 1080
CAAGGTTATC CTTCCGTATT TTATGGGGAT TACTACGGTA TCCCAACCCA TGGTGTTCCG 1140
GCTATGAAAT CTAAAATAGA CCCTCTTCTG CAGGCACGTC AAACTTTTGC CTATGGTACG 1200
CAGCATGATT ACTTTGATCA TCATGATATT ATCGGTTGGA CAAGAGAGGG AAATAGCTCC 1260
CATCCAAATT CAGGCCTTGC CACCATTATG TCAGATGGTC CAGGTGGTAA CAAATGGATG 1320
TATGTGGGGA AAAATAAAGC GGGACAAGTT TGGAGAGATA TTACCGGAAA TAGGACAGGC 1380
ACCGTCACAA TTAATGCAGA CGGATGGGGT AATTTCTCTG TTAATGGAGG GTCCGTTTCG 1440
GTTTGGGTGA AGCAA 1455
(2)SEQ ID NO:10的资料:
(i)序列特征:
(A)长度:1455个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:DNA(基因组)
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:10
CATCATAATG GGACAAATGG GACGATGATG CAATACTTTG AATGGCACTT GCCTAATGAT 60
GGGAATCACT GGAATAGATT AAGAGATGAT GCTAGTAATC TAAGAAATAG AGGTATAACC 120
GCTATTTGGA TTCCGCCTGC CTGGAAAGGG ACTTCGCAAA ATGATGTGGG GTATGGAGCC 180
TATGATCTTT ATGATTTAGG GGAATTTAAT CAAAAGGGGA CGGTTCGTAC TAAGTATGGG 240
ACACGTAGTC AATTGGAGTC TGCCATCCAT GCTTTAAAGA ATAATGGCGT TCAAGTTTAT 300
GGGGATGTAG TGATGAACCA TAAAGGAGGA GCTGATGCTA CAGAAAACGT TCTTGCTGTC 360
GAGGTGAATC CAAATAACCG GAATCAAGAA ATATCTGGGG ACTACACAAT TGAGGCTTGG 420
ACTAAGTTTG ATTTTCCAGG GAGGGGTAAT ACATACTCAG ACTTTAAATG GCGTTGGTAT 480
CATTTCGATG GTGTAGATTG GGATCAATCA CGACAATTCC AAAATCGTAT CTACAAATTC 540
CGAGGTGATG GTAAGGCATG GGATTGGGAA GTAGATTCGG AAAATGGAAA TTATGATTAT 600
TTAATGTATG CAGATGTAGA TATGGATCAT CCGGAGGTAG TAAATGAGCT TAGAAGATGG 660
GGAGAATGGT ATACAAATAC ATTAAATCTT GATGGATTTA GGATCGATGC GGTGAAGCAT 720
ATTAAATATA GCTTTACACG TGATTGGTTG ACCCATGTAA GAAACGCAAC GGGAAAAGAA 780
ATGTTTGCTG TTGCTGAATT TTGGAAAAAT GATTTAGGTG CCTTGGAGAA CTATTTAAAT 840
AAAACAAACT GGAATCATTC TGTCTTTGAT GTCCCCCTTC ATTATAATCT TTATAACGCG 900
TCAAATAGTG GAGGCAACTA TGACATGGCA AAACTTCTTA ATGGAACGGT TGTTCAAAAG 960
CATCCAATGC ATGCCGTAAC TTTTGTGGAT AATCACGATT CTCAACCTGG GGAATCATTA 1020
GAATCATTTG TACAAGAATG GTTTAAGCCA CTTGCTTATG CGCTTATTTT AACAAGAGAA 1080
CAAGGCTATC CCTCTGTCTT CTATGGTGAC TACTATGGAA TTCCAACACA TAGTGTCCCA 1140
GCAATGAAAG CCAAGATTGA TCCAATCTTA GAGGCGCGTC AAAATTTTGC ATATGGAACA 1200
CAACATGATT ATTTTGACCA TCATAATATA ATCGGATGGA CACGTGAAGG AAATACCACG 1260
CATCCCAATT CAGGACTTGC GACTATCATG TCGGATGGGC CAGGGGGAGA GAAATGGATG 1320
TACGTAGGGC AAAATAAAGC AGGTCAAGTT TGGCATGACA TAACTGGAAA TAAACCAGGA 1380
ACAGTTACGA TCAATGCAGA TGGATGGGCT AATTTTTCAG TAAATGGAGG ATCTGTTTCC 1440
ATTTGGGTGA AACGA 1455
(2)SEQ ID NO:11的资料:
(i)序列特征:
(A)长度:1548个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:DNA(基因组)
(iii)生物:嗜热脂肪芽孢杆菌
(xi)序列描述:SEQ ID NO:11
GCCGCACCGT TTAACGGCAC CATGATGCAG TATTTTGAAT GGTACTTGCC GGATGATGGC 60
ACGTTATGGA CCAAAGTGGC CAATGAAGCC AACAACTTAT CCAGCCTTGG CATCACCGCT 120
CTTTGGCTGC CGCCCGCTTA CAAAGGAACA AGCCGCAGCG ACGTAGGGTA CGGAGTATAC 180
GACTTGTATG ACCTCGGCGA ATTCAATCAA AAAGGGACCG TCCGCACAAA ATACGGAACA 240
AAAGCTCAAT ATCTTCAAGC CATTCAAGCC GCCCACGCCG CTGGAATGCA AGTGTACGCC 300
GATGTCGTGT TCGACCATAA AGGCGGCGCT GACGGCACGG AATGGGTGGA CGCCGTCGAA 360
GTCAATCCGT CCGACCGCAA CCAAGAAATC TCGGGCACCT ATCAAATCCA AGCATGGACG 420
AAATTTGATT TTCCCGGGCG GGGCAACACC TACTCCAGCT TTAAGTGGCG CTGGTACCAT 480
TTTGACGGCG TTGATTGGGA CGAAAGCCGA AAATTGAGCC GCATTTACAA ATTCCGCGGC 540
ATCGGCAAAG CGTGGGATTG GGAAGTAGAC ACGGAAAACG GAAACTATGA CTACTTAATG 600
TATGCCGACC TTGATATGGA TCATCCCGAA GTCGTGACCG AGCTGAAAAA CTGGGGGAAA 660
TGGTATGTCA ACACAACGAA CATTGATGGG TTCCGGCTTG ATGCCGTCAA GCATATTAAG 720
TTCAGTTTTT TTCCTGATTG GTTGTCGTAT GTGCGTTCTC AGACTGGCAA GCCGCTATTT 780
ACCGTCGGGG AATATTGGAG CTATGACATC AACAAGTTGC ACAATTACAT TACGAAAACA 840
GACGGAACGA TGTCTTTGTT TGATGCCCCG TTACACAACA AATTTTATAC CGCTTCCAAA 900
TCAGGGGGCG CATTTGATAT GCGCACGTTA ATGACCAATA CTCTCATGAA AGATCAACCG 960
ACATTGGCCG TCACCTTCGT TGATAATCAT GACACCGAAC CCGGCCAAGC GCTGCAGTCA 1020
TGGGTCGACC CATGGTTCAA ACCGTTGGCT TACGCCTTTA TTCTAACTCG GCAGGAAGGA 1080
TACCCGTGCG TCTTTTATGG TGACTATTAT GGCATTCCAC AATATAACAT TCCTTCGCTG 1140
AAAAGCAAAA TCGATCCGCT CCTCATCGCG CGCAGGGATT ATGCTTACGG AACGCAACAT 1200
GATTATCTTG ATCACTCCGA CATCATCGGG TGGACAAGGG AAGGGGGCAC TGAAAAACCA 1260
GGATCCGGAC TGGCCGCACT GATCACCGAT GGGCCGGGAG GAAGCAAATG GATGTACGTT 1320
GGCAAACAAC ACGCTGGAAA AGTGTTCTAT GACCTTACCG GCAACCGGAG TGACACCGTC 1380
ACCATCAACA GTGATGGATG GGGGGAATTC AAAGTCAATG GCGGTTCGGT TTCGGTTTGG 1440
GTTCCTAGAA AAACGACCGT TTCTACCATC GCTCGGCCGA TCACAACCCG ACCGTGGACT 1500
GGTGAATTCG TCCGTTGGAC CGAACCACGG TTGGTGGCAT GGCCTTGA 1548
(2)SEQ ID NO:12的资料:
(i)序列特征:
(A)长度:1920个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:DNA(基因组)
(iii)生物:地衣芽孢杆菌
(xi)序列描述:SEQ ID NO:12
CGGAAGATTG GAAGTACAAA AATAAGCAAA AGATTGTCAA TCATGTCATG AGCCATGCGG 60
GAGACGGAAA AATCGTCTTA ATGCACGATA TTTATGCAAC GTTCGCAGAT GCTGCTGAAG 120
AGATTATTAA AAAGCTGAAA GCAAAAGGCT ATCAATTGGT AACTGTATCT CAGCTTGAAG 180
AAGTGAAGAA GCAGAGAGGC TATTGAATAA ATGAGTAGAA GCGCCATATC GGCGCTTTTC 240
TTTTGGAAGA AAATATAGGG AAAATGGTAC TTGTTAAAAA TTCGGAATAT TTATACAACA 300
TCATATGTTT CACATTGAAA GGGGAGGAGA ATCATGAAAC AACAAAAACG GCTTTACGCC 360
CGATTGCTGA CGCTGTTATT TGCGCTCATC TTCTTGCTGC CTCATTCTGC AGCAGCGGCG 420
GCA AAT CTT AAT GGG ACG CTG ATG CAG TAT TTT GAA TGG TAC ATG CCC 468
AAT GAC GGC CAA CAT TGG AGG CGT TTG CAA AAC GAC TCG GCA TAT TTG 516
GCT GAA CAC GGT ATT ACT GCC GTC TGG ATT CCC CCG GCA TAT AAG GGA 564
ACG AGC CAA GCG GAT GTG GGC TAC GGT GCT TAC GAC CTT TAT GAT TTA 612
GGG GAG TTT CAT CAA AAA GGG ACG GTT CGG ACA AAG TAC GGC ACA AAA 660
GGA GAG CTG CAA TCT GCG ATC AAA AGT CTT CAT TCC CGC GAC ATT AAC 708
GTT TAC GGG GAT GTG GTC ATC AAC CAC AAA GGC GGC GCT GAT GCG ACC 756
GAA GAT GTA ACC GCG GTT GAA GTC GAT CCC GCT GAC CGC AAC CGC GTA 804
ATT TCA GGA GAA CAC CTA ATT AAA GCC TGG ACA CAT TTT CAT TTT CCG 852
GGG CGC GGC AGC ACA TAC AGC GAT TTT AAA TGG CAT TGG TAC CAT TTT 900
GAC GGA ACC GAT TGG GAC GAG TCC CGA AAG CTG AAC CGC ATC TAT AAG 948
TTT CAA GGA AAG GCT TGG GAT TGG GAA GTT TCC AAT GAA AAC GGC AAC 996
TAT GAT TAT TTG ATG TAT GCC GAC ATC GAT TAT GAC CAT CCT GAT GTC 1044
GCA GCA GAA ATT AAG AGA TGG GGC ACT TGG TAT GCC AAT GAA CTG CAA 1092
TTG GAC GGT TTC CGT CTT GAT GCT GTC AAA CAC ATT AAA TTT TCT TTT 1140
TTG CGG GAT TGG GTT AAT CAT GTC AGG GAA AAA ACG GGG AAG GAA ATG 1188
TTT ACG GTA GCT GAA TAT TGG CAG AAT GAC TTG GGC GCG CTG GAA AAC 1236
TAT TTG AAC AAA ACA AAT TTT AAT CAT TCA GTG TTT GAC GTG CCG CTT 1284
CAT TAT CAG TTC CAT GCT GCA TCG ACA CAG GGA GGC GGC TAT GAT ATG 1332
AGG AAA TTG CTG AAC GGT ACG GTC GTT TCC AAG CAT CCG TTG AAA TCG 1380
GTT ACA TTT GTC GAT AAC CAT GAT ACA CAG CCG GGG CAA TCG CTT GAG 1428
TCG ACT GTC CAA ACA TGG TTT AAG CCG CTT GCT TAC GCT TTT ATT CTC 1476
ACA AGG GAA TCT GGA TAC CCT CAG GTT TTC TAC GGG GAT ATG TAC GGG 1524
ACG AAA GGA GAC TCC CAG CGC GAA ATT CCT GCC TTG AAA CAC AAA ATT 1572
GAA CCG ATC TTA AAA GCG AGA AAA CAG TAT GCG TAC GGA GCA CAG CAT 1620
GAT TAT TTC GAC CAC CAT GAC ATT GTC GGC TGG ACA AGG GAA GGC GAC 1668
AGC TCG GTT GCA AAT TCA GGT TTG GCG GCA TTA ATA ACA GAC GGA CCC 1716
GGT GGG GCA AAG CGA ATG TAT GTC GGC CGG CAA AAC GCC GGT GAG ACA 1764
TGG CAT GAC ATT ACC GGA AAC CGT TCG GAG CCG GTT GTC ATC AAT TCG 1812
GAA GGC TGG GGA GAG TTT CAC GTA AAC GGC GGG TCG GTT TCA ATT TAT 1860
GTT CAA AGA TAG AAGAGCAGAG AGGACGGATT TCCTGAAGGA AATCCGTTTT 1912
TTTATTTT 1920
(2)SEQ ID NO:13的资料:
(i)序列特征:
(A)长度:1455个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:DNA(基因组)
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:13
CATCATAATG GAACAAATGG TACTATGATG CAATATTTCG AATGGTATTT GCCAAATGAC 60
GGGAATCATT GGAACAGGTT GAGGGATGAC GCAGCTAACT TAAAGAGTAA AGGGATAACA 120
GCTGTATGGA TCCCACCTGC ATGGAAGGGG ACTTCCCAGA ATGATGTAGG TTATGGAGCC 180
TATGATTTAT ATGATCTTGG AGAGTTTAAC CAGAAGGGGA CGGTTCGTAC AAAATATGGA 240
ACACGCAACC AGCTACAGGC TGCGGTGACC TCTTTAAAAA ATAACGGCAT TCAGGTATAT 300
GGTGATGTCG TCATGAATCA TAAAGGTGGA GCAGATGGTA CGGAAATTGT AAATGCGGTA 360
GAAGTGAATC GGAGCAACCG AAACCAGGAA ACCTCAGGAG AGTATGCAAT AGAAGCGTGG 420
ACAAAGTTTG ATTTTCCTGG AAGAGGAAAT AACCATTCCA GCTTTAAGTG GCGCTGGTAT 480
CATTTTGATG GGACAGATTG GGATCAGTCA CGCCAGCTTC AAAACAAAATATATAAATTC 540
AGGGGAACAG GCAAGGCCTG GGACTGGGAA GTCGATACAG AGAATGGCAA CTATGACTAT 600
CTTATGTATG CAGACGTGGA TATGGATCAC CCAGAAGTAA TACATGAACT TAGAAACTGG 660
GGAGTGTGGT ATACGAATAC ACTGAACCTT GATGGATTTA GAATAGATGC AGTGAAACAT 720
ATAAAATATA GCTTTACGAG AGATTGGCTT ACACATGTGC GTAACACCAC AGGTAAACCA 780
ATGTTTGCAG TGGCTGAGTT TTGGAAAAAT GACCTTGGTG CAATTGAAAA CTATTTGAAT 840
AAAACAAGTT GGAATCACTC GGTGTTTGAT GTTCCTCTCC ACTATAATTT GTACAATGCA 900
TCTAATAGCG GTGGTTATTA TGATATGAGA AATATTTTAA ATGGTTCTGT GGTGCAAAAA 960
CATCCAACAC ATGCCGTTAC TTTTGTTGAT AACCATGATT CTCAGCCCGG GGAAGCATTG 1020
GAATCCTTTG TTCAACAATG GTTTAAACCA CTTGCATATG CATTGGTTCT GACAAGGGAA 1080
CAAGGTTATC CTTCCGTATT TTATGGGGAT TACTACGGTA TCCCAACCCA TGGTGTTCCG 1140
GCTATGAAAT CTAAAATAGA CCCTCTTCTG CAGGCACGTC AAACTTTTGC CTATGGTACG 1200
CAGCATGATT ACTTTGATCA TCATGATATT ATCGGTTGGA CAAGAGAGGG AAATAGCTCC 1260
CATCCAAATT CAGGCCTTGC CACCATTATG TCAGATGGTC CAGGTGGTAA CAAATGGATG 1320
TATGTGGGGA AAAATAAAGC GGGACAAGTT TGGAGAGATA TTACCGGAAA TAGGACAGGC 1380
ACCGTCACAA TTAATGCAGA CGGATGGGGT AATTTCTCTG TTAATGGAGG GTCCGTTTCG 1440
GTTTGGGTGA AGCAA 1455
(2)SEQ ID NO:14的资料:
(i)序列特征:
(A)长度:1455个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:DNA(基因组)
(iii)生物:芽孢杆菌属的种
(xi)序列描述:SEQ ID NO:14
CATCATAATG GGACAAATGG GACGATGATG CAATACTTTG AATGGCACTT GCCTAATGAT 60
GGGAATCACT GGAATAGATT AAGAGATGAT GCTAGTAATC TAAGAAATAG AGGTATAACC 120
GCTATTTGGA TTCCGCCTGC CTGGAAAGGG ACTTCGCAAA ATGATGTGGG GTATGGAGCC 180
TATGATCTTT ATGATTTAGG GGAATTTAAT CAAAAGGGGA CGGTTCGTAC TAAGTATGGG 240
ACACGTAGTC AATTGGAGTC TGCCATCCAT GCTTTAAAGA ATAATGGCGT TCAAGTTTAT 300
GGGGATGTAG TGATGAACCA TAAAGGAGGA GCTGATGCTA CAGAAAACGT TCTTGCTGTC 360
GAGGTGAATC CAAATAACCG GAATCAAGAA ATATCTGGGG ACTACACAAT TGAGGCTTGG 420
ACTAAGTTTG ATTTTCCAGG GAGGGGTAAT ACATACTCAG ACTTTAAATG GCGTTGGTAT 480
CATTTCGATG GTGTAGATTG GGATCAATCA CGACAATTCC AAAATCGTAT CTACAAATTC 540
CGAGGTGATG GTAAGGCATG GGATTGGGAA GTAGATTCGG AAAATGGAAA TTATGATTAT 600
TTAAT3TATG CAGATGTAGA TATGGATCAT CCGGAGGTAG TAAATGAGCT TAGAAGATGG 660
GGAGAATGGT ATACAAATAC ATTAAATCTT GATGGATTTA GGATCGATGC GGTGAAGCAT 720
ATTAAATATA GCTTTACACG TGATTGGTTG ACCCATGTAA GAAACGCAAC GGGAAAAGAA 780
ATGTTTGCTG TTGCTGAATT TTGGAAAAAT GATTTAGGTG CCTTGGAGAA CTATTTAAAT 840
AAAACAAACT GGAATCATTC TGTCTTTGAT GTCCCCCTTC ATTATAATCT TTATAACGCG 900
TCAAATAGTG GAGGCAACTA TGACATGGCA AAACTTCTTA ATGGAACGGT TGTTCAAAAG 960
CATCCAATGC ATGCCGTAAC TTTTGTGGAT AATCACGATT CTCAACCTGG GGAATCATTA 1020
GAATCATTTG TACAAGAATG GTTTAAGCCA CTTGCTTATG CGCTTATTTT AACAAGAGAA 1080
CAAGGCTATC CCTCTGTCTT CTATGGTGAC TACTATGGAA TTCCAACACA TAGTGTCCCA 1140
GCAATGAAAG CCAAGATTGA TCCAATCTTA GAGGCGCGTC AAAATTTTGC ATATGGAACA 1200
CAACATGATT ATTTTGACCA TCATAATATA ATCGGATGGA CACGTGAAGG AAATACCACG 1260
CATCCCAATT CAGGACTTGC GACTATCATG TCGGATGGGC CAGGGGGAGA GAAATGGATG 1320
TACGTAGGGC AAAATAAAGC AGGTCAAGTT TGGCATGACA TAACTGGAAA TAAACCAGGA 1380
ACAGTTACGA TCAATGCAGA TGGATGGGCT AATTTTTCAG TAAATGGAGG ATCTGTTTCC 1440
ATTTGGGTGA AACGA 1455
(2)SEQ ID NO:15的资料:
(i)序列特征:
(A)长度:74个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“RSERI”
(ix)特征:
(A)名称/关键词:misc-特征
(B)位置:21-62
(D)其它信息:/Note=1:97%A,1%T,1%C,1%G
2:97%T,1%A,1%C,1%G
3:97%C,1%A,1%T,1%G
4:97%G,1%A,1%T,1%C
(xi)序列描述:SEQ ID NO:15
GCGTTTTGCC GGCCGACATA 3122343222 4333313344
4233423242 2122112433 43CAAACCTG AATT 74
(2)SEQ ID NO:16的资料:
(i)序列特征:
(A)长度:122个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“RSERII”
(ix)特征:
(A)名称/关键词:misc-特征
(B)位置:63-104
(D)其它信息:/Note=1:97%A,1%T,1%C,1%G
2:97%T,1%A,1%C,1%G
3:97%C,1%A,1%T,1%G
4:97%G,1%A,1%T,1%C
(xi)序列描述:SEQ ID NO:16
GCGTTTTGCC GGCCGACATA CATTCGCTTT GCCCCACCGG GTCCGTCTGT
TATTAATGCC GC31113324 1122243113 3414324234 3322333224
2331GCCGAC AATGTCATGG TG 122
(2)SEQ ID NO:17的资料:
(i)序列特征:
(A)长度:78个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“RSERIII”
(ix)特征:
(A)名称/关键词:misc-特征
(B)位置:19-60
(D)其它信息:/Note=1:97%A,1%T,1%C,1%G
2:97%T,1%A,1%C,1%G
3:97%C,1%A,1%T,1%G
4:97%G,1%A,1%T,1%C
(xi)序列描述:SEQ ID NO:17
GTCGCCTTCC CTTGTCCA43 3413112423 1244244234 1112112312
4324243233 GTACGCATAC TGTTTTCT 78
(2)SEQ ID NO:18的资料:
(i)序列特征:
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“FSERIII”
(xi)序列描述:SEQ ID NO:18
TGGACAAGGG AAGGCGACAG 20
(2)SEQ ID NO:19的资料:
(i)序列特征:
(A)长度:81个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“RSERV”
(ix)特征:
(A)名称/关键词:misc-特征
(B)位置:19-60
(D)其它信息:/Note=1:97%A,1%T,1%C,1%G
2:97%T,1%A,1%C,1%G
3:97%C,1%A,1%T,1%G
4:97%G,1%A,1%T,1%C
(xi)序列描述:SEQ ID NO:19
TAAGATCGGT TCAATTTT42 4222311443 1441122234 3432444142
3233222342 CCCGTACATA TCCCCGTAGA A
(2)SEQ ID NO:20的资料:
(i)序列特征:
(A)长度:18个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“FSERV”
(xi)序列描述:SEQ ID NO:20
AAAATTGAAC CGATCTTA 18
(2)SEQ ID NO:21的资料:
(i)序列特征:
(A)长度:107个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“FSERVII”
(ix)特征:
(A)名称/关键词:misc-特征
(B)位置:54-95
(D)其它信息:/Note=1:97%A,1%T,1%C,1%G
2:97%T,1%A,1%C,1%G
3:97%C,1%A,1%T,1%G
4:97%G,1%A,1%T,1%C
(xi)序列描述:SEQ ID NO:21
TTCCATGCTG CATCGACACA GGGAGGCGGC TATGATATGA GGAAATTGCT
GAA3442134 4234222331 1431233422 4111234422 13122TGTCG
ATAACCA 108
(2)SEQ ID NO:22的资料:
(i)序列特征:
(A)长度:18个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“RSERVII”
(xi)序列描述:SEQ ID NO:22
TGTCGATGCA GCATGGAA 18
(2)SEQ ID NO:23的资料:
(i)序列特征:
(A)长度:80个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“FSERIX”
(ix)特征:
(A)名称/关键词:misc-特征
(B)位置:21-62
(D)其它信息:/Note=1:97%A,1%T,1%C,1%G
2:97%T,1%A,1%C,1%G
3:97%C,1%A,1%T,1%G
4:97%G,1%A,1%T,1%C
(xi)序列描述:SEQ ID NO:23
GTCCAAACAT GGTTTAAGCC 4322432213 4322221223 2313114441
1232441213 33TCAGGTTT TCTACGGGGA 80
(2)SEQ ID NO:24的资料:
(i)序列特征:
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“RSERIX”
(xi)序列描述:SEQ ID NO:25
GGCTTAAACC ATGTTTGGAC 20
(2)SEQ ID NO:26的资料:
(i)序列特征:
(A)长度:24个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“引物1B”
(xi)序列描述:SEQ ID NO:26
CGATTGCTGA CGCTGTTATT TGCG 24
(2)SEQ ID NO:27的资料:
(i)序列特征:
(A)长度:25个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“引物#63”
(xi)序列描述:SEQ ID NO:27
CTATCTTTGA ACATAAATTG AAACC 25
(2)SEQ ID NO:28的资料:
(i)序列特征:
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“正向引物1”
(xi)序列描述:SEQ ID NO:28
gacctgcagt caggcaacta 20
(2)SEQ ID NO:29的资料:
(i)序列特征:
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“反向引物1”
(xi)序列描述:SEQ ID NO:29
tagagtcgac ctgcaggcat 20
(2)SEQ ID NO:30的资料:
(i)序列特征:
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“正向引物2”
(xi)序列描述:SEQ ID NO:30
gacctgcagt caggcaacta 20
(2)SEQ ID NO:31的资料:
(i)序列特征:
(A)长度:25个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:其它核酸
(ix)特征:
(A)名称/关键词:misc-特征
(B)其它信息:/desc=“反向引物2”
(xi)序列描述:SEQ ID NO:31
tagagtcgac ctgcaggcat 20
(2)SEQ ID NO:32的资料:
(i)序列特征:
(A)长度:2084个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑构型:线性
(ii)分子类型:DNA(基因组)
(iii)生物:解淀粉芽孢杆菌
(ix)特征:
(A)名称/关键词:CDS
(B)位置:343…1794
(xi)序列描述:SEQ ID NO:32
GCCCCGCACA TACGAAAAGA CTGGCTGAAA ACATTGAGCC TTTGATGACT GATGATTTGG 60
CTGAAGAAGT GGATCGATTG TTTGAGAAAA GAAGAAGACC ATAAAAATAC CTTGTCTGTC 120
ATCAGACAGG GTATTTTTTA TGCTGTCCAG ACTGTCCGCT GTGTAAAAAT AAGGAATAAA 180
GGGGGGTTGT TATTATTTTA CTGATATGTA AAATATAATT TGTATAAGAA AATGAGAGGG 240
AGAGGAAACA TGATTCAAAA ACGAAAGCGG ACAGTTTCGT TCAGACTTGT GCTTATGTGC 300
ACGCTGTTAT TTGTCAGTTT GCCGATTACA AAAACATCAG CC GTA AAT GGC ACG 354
CTG ATG CAG TAT TTT GAA TGG TAT ACG CCG AAC GAC GGC CAG CAT TGG 402
AAA CGA TTG CAG AAT GAT GCG GAA CAT TTA TCG GAT ATC GGA ATC ACT 450
GCC GTC TGG ATT CCT CCC GCA TAC AAA GGA TTG AGC CAA TCC GAT AAC 498
GGA TAC GGA CCT TAT GAT TTG TAT GAT TTA GGA GAA TTC CAG CAA AAA 546
GGG ACG GTC AGA ACG AAA TAC GGC ACA AAA TCA GAG CTT CAA GAT GCG 594
ATC GGC TCA CTG CAT TCC CGG AAC GTC CAA GTA TAC GGA GAT GTG GTT 642
TTG AAT CAT AAG GCT GGT GCT GAT GCA ACA GAA GAT GTA ACT GCC GTC 690
GAA GTC AAT CCG GCC AAT AGA AAT CAG GAA ACT TCG GAG GAA TAT CAA 738
ATC AAA GCG TGG ACG GAT TTT CGT TTT CCG GGC CGT GGA AAC ACG TAC 786
AGT GAT TTT AAA TGG CAT TGG TAT CAT TTC GAC GGA GCG GAC TGG GAT 834
GAA TCC CGG AAG ATC AGC CGC ATC TTT AAG TTT CGT GGG GAA GGA AAA 882
GCG TGG GAT TGG GAA GTA TCA AGT GAA AAC GGC AAC TAT GAC TAT TTA 930
ATG TAT GCT GAT GTT GAC TAC GAC CAC CCT GAT GTC GTG GCA GAG ACA 978
AAA AAA TGG GGT ATC TGG TAT GCG AAT GAA CTG TCA TTA GAC GGC TTC 1026
CGT ATT GAT GCC GCC AAA CAT ATT AAA TTT TCA TTT CTG CGT GAT TGG 1074
GTT CAG GCG GTC AGA CAG GCG ACG GGA AAA GAA ATG TTT ACG GTT GCG 1122
GAG TAT TGG CAG AAT AAT GCC GGG AAA CTC GAA AAC TAC TTG AAT AAA 1170
ACA AGC TTT AAT CAA TCC GTG TTT GAT GTT CCG CTT CAT TTC AAT TTA 1218
CAG GCG GCT TCC TCA CAA GGA GGC GGA TAT GAT ATG AGG CGT TTG CTG 1266
GAC GGT ACC GTT GTG TCC AGG CAT CCG GAA AAG GCG GTT ACA TTT GTT 1314
GAA AAT CAT GAC ACA CAG CCG GGA CAG TCA TTG GAA TCG ACA GTC CAA 1362
ACT TGG TTT AAA CCG CTT GCA TAC GCC TTT ATT TTG ACA AGA GAA TCC 1410
GGT TAT CCT CAG GTG TTC TAT GGG GAT ATG TAC GGG ACA AAA GGG ACA 1458
TCG CCA AAG GAA ATT CCC TCA CTG AAA GAT AAT ATA GAG CCG ATT TTA 1506
AAA GCG CGT AAG GAG TAC GCA TAC GGG CCC CAG CAC GAT TAT ATT GAC 1554
CAC CCG GAT GTG ATC GGA TGG ACG AGG GAA GGT GAC AGC TCC GCC GCC 1602
AAA TCA GGT TTG GCC GCT TTA ATC ACG GAC GGA CCC GGC GGA TCA AAG 1650
CGG ATG TAT GCC GGC CTG AAA AAT GCC GGC GAG ACA TGG TAT GAC ATA 1698
ACG GGC AAC CGT TCA GAT ACT GTA AAA ATC GGA TCT GAC GGC TGG GGA 1746
GAG TTT CAT GTA AAC GAT GGG TCC GTC TCC ATT TAT GTT CAG AAA TAA 1794
GGTAATAAAA AAACACCTCC AAGCTGAGTG CGGGTATCAG CTTGGAGGTG CGTTTATTTT 1854
TTCAGCCGTA TGACAAGGTC GGCATCAGGT GTGACAAATA CGGTATGCTG GCTGTCATAG 1914
GTGACAAATC CGGGTTTTGC GCCGTTTGGC TTTTTCACAT GTCTGATTTT TGTATAATCA 1974
ACAGGCACGG AGCCGGAATC TTTCGCCTTG GAAAAATAAG CGGCGATCGT AGCTGCTTCC 2034
AATATGGATT GTTCATCGGG ATCGCTGCTT TTAATCACAA CGTGGGATCC 2084
Claims (10)
1.亲代Termamyl-样α-淀粉酶的变体,与亲代α-淀粉酶相比,所述α-淀粉酶变体中,表面有一个或多个暴露于溶剂的氨基酸残基发生变化以使该α-淀粉酶的总体亲水性增加和/或表面上所述暴露于溶剂的氨基酸残基的侧链中甲基总数增加。
2.亲代Termamyl-样α-淀粉酶的变体,该变体在选自下列的一个或多个位置处具有变化:E376,S417,A420,S356,Y358;
其中:(a)所述变化各为:
(i)在占用上述位置的氨基酸的下游插入一个氨基酸,
(ii)缺失占用上述位置的氨基酸,或
(iii)用不同的氨基酸取代占用上述位置的氨基酸,
(b)所述变体具有α-淀粉酶活性,和(c)每个位置对应于具有SEQ IDNO:4氨基酸序列的亲代Termamyl-样α-淀粉酶的氨基酸序列位置。
3.权利要求1-2中任一项的变体,其中亲代Termamyl-样α-淀粉酶得自地衣芽孢杆菌,解淀粉芽孢杆菌,嗜热脂肪芽孢杆菌的菌株,芽孢杆菌菌种NCIB 12289,NCIB 12512,NCIB 12513或DSM 9375。
4.DNA构建体,其含有编码权利要求1至3中任一项的α-淀粉酶变体的DNA序列。
5.重组表达载体,其携有权利要求4的DNA构建体。
6.被权利要求4的DNA构建体或权利要求5的载体转化的细胞。
7.去污剂添加剂,其含有权利要求1至3中任一项的α-淀粉酶变体,所述变体任选为不成粉末的颗粒,稳定化的液体或被保护的酶的形式。
8.组合物,其含有:
(i)具有SEQ ID NO:4所示序列的地衣芽孢杆菌α-淀粉酶与得自具有SEQ ID NO:3所示序列的嗜热脂肪芽孢杆菌α-淀粉酶(作为亲代Termamyl-样α-淀粉酶)的权利要求1至3中任一项的一种或多种变体的混合物;或
(ii)具有SEQ ID NO:3所示序列的嗜热脂肪芽孢杆菌α-淀粉酶与得自一种或多种其它亲代Termamyl-样α-淀粉酶的权利要求1至3中任一项的一种或多种变体的混合物;或
(iii)得自具有SEQ ID NO:3所示序列的嗜热脂肪芽孢杆菌α-淀粉酶(作为亲代Termamyl-样α-淀粉酶)的权利要求1至3中任一项的一种或多种变体与得自一种或多种其它亲代Termamyl-样α-淀粉酶的本发明的一种或多种变体的混合物。
9.权利要求1至3中任一项的α-淀粉酶变体或权利要求8的组合物用于洗涤和/或洗碟、织物脱浆或淀粉液化的用途。
10.产生亲代Termamyl-样α-淀粉酶的变体的方法,其中变体相对于亲代而言,在高温下表现出增加的稳定性,所述方法包括:
(a)对编码亲代Termamyl-样α-淀粉酶的DNA序列进行随机诱变,
(b)在宿主细胞中表达步骤(a)中得到的突变DNA序列,和
(c)筛选表达突变α-淀粉酶的宿主细胞,相对于亲代Termamyl-样α-淀粉酶而言,所述变体在高温下具有增加的稳定性。
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US09/193,068 US6197565B1 (en) | 1998-11-16 | 1998-11-16 | α-Amylase variants |
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AT (1) | ATE318305T1 (zh) |
AU (1) | AU1151200A (zh) |
CA (1) | CA2350837C (zh) |
DE (1) | DE69930016T2 (zh) |
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CN103890170A (zh) * | 2011-10-17 | 2014-06-25 | 诺维信公司 | α-淀粉酶变体以及对其进行编码的多核苷酸 |
CN103890170B (zh) * | 2011-10-17 | 2018-02-02 | 诺维信公司 | α‑淀粉酶变体以及对其进行编码的多核苷酸 |
CN103834606A (zh) * | 2014-01-16 | 2014-06-04 | 北京中科星冠生物技术有限责任公司 | 一种表达耐酸高温α-淀粉酶基因突变体的工程菌株 |
CN107312764A (zh) * | 2017-02-21 | 2017-11-03 | 南京百斯杰生物工程有限公司 | α淀粉酶变体 |
CN107312764B (zh) * | 2017-02-21 | 2020-05-12 | 南京百斯杰生物工程有限公司 | α淀粉酶变体 |
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JP2002530072A (ja) | 2002-09-17 |
CN1333821A (zh) | 2002-01-30 |
EP1676913B1 (en) | 2015-08-05 |
JP4550285B2 (ja) | 2010-09-22 |
US6197565B1 (en) | 2001-03-06 |
ES2259249T3 (es) | 2006-09-16 |
CA2350837C (en) | 2011-10-18 |
CA2350837A1 (en) | 2000-05-25 |
AU1151200A (en) | 2000-06-05 |
WO2000029560A1 (en) | 2000-05-25 |
EP1676913A3 (en) | 2007-08-29 |
DE69930016T2 (de) | 2006-10-05 |
ATE318305T1 (de) | 2006-03-15 |
DK1131418T3 (da) | 2006-06-26 |
DE69930016D1 (de) | 2006-04-27 |
EP1676913A2 (en) | 2006-07-05 |
EP1131418A1 (en) | 2001-09-12 |
CN100374557C (zh) | 2008-03-12 |
EP1131418B1 (en) | 2006-02-22 |
CN101240269B (zh) | 2012-04-18 |
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