CN100342016C - 多点置换的蛋白酶变体 - Google Patents
多点置换的蛋白酶变体 Download PDFInfo
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- CN100342016C CN100342016C CNB988114364A CN98811436A CN100342016C CN 100342016 C CN100342016 C CN 100342016C CN B988114364 A CNB988114364 A CN B988114364A CN 98811436 A CN98811436 A CN 98811436A CN 100342016 C CN100342016 C CN 100342016C
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Abstract
公开了由天然存在的或重组的非人类蛋白酶的DNA序列衍生的新型蛋白酶变体。变异蛋白酶的获得方法通常是,对编码天然存在的或重组的蛋白酶的前体DNA序列进行体外修饰,在前体蛋白酶的氨基酸序列中引起多处氨基酸残基的置换。这种变体蛋白酶具有与前体蛋白酶不同的性质,如改变的洗涤性能。置换的氨基酸残基相应于解淀粉芽孢杆菌枯草杆菌蛋白酶位置62、212、230、232、252和257。
Description
相关申请
本申请是1998年10月23日提交的美国专利申请08/956,323、1998年10月23日提交的美国专利申请08/956,564和1998年10月23日提交的美国专利申请08/956,324的部分继续申请,所有这些申请在此都完整引用。
发明背景
丝氨酸蛋白酶是羰基水解酶的一个亚组。其包括多种类别的具有广泛特性和生物功能的酶。Stroud,R.,科学美国人(Sci.Amer.)131:74-88。尽管其功能的多样性,但丝氨酸蛋白酶的催化机制至少由两种遗传分离的酶家族来完成:1)枯草杆菌蛋白酶和2)哺乳动物胰凝乳蛋白酶相关的丝氨酸蛋白酶及同源细菌丝氨酸蛋白酶(例如胰蛋白酶和灰色链霉菌(S.gresius)胰蛋白酶)。这两个丝氨酸蛋白酶家族显示非常相似的催化机理。Kraut,J.(1977),生物化学年述(Annu.Rev.Biochem.),46:331-358。此外,尽管一级结构不相关,但这两个酶家族的三级结构存在由丝氨酸、组氨酸和天冬氨酸组成的氨基酸保守催化三联体。
枯草杆菌蛋白酶是由多种芽孢杆菌属的种及其它微生物大量分泌的丝氨酸蛋白酶(分子量约为27,500)。已经测定了芽孢杆菌属至少九个不同种的枯草杆菌蛋白酶的蛋白质序列。Markland,F.S.等人(1983),Hooppe-Seyler’s Z.Physiol.Chem.,364:1537-1540。曾经报道了来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、地衣芽孢杆菌(Bacilluslicheniformis)和迟缓芽孢杆菌(B.Ientus)几种天然变体的枯草杆菌蛋白酶的三维晶体结构。这些研究表明,尽管枯草杆菌蛋白酶与哺乳动物丝氨酸蛋白酶在遗传上是不相关的,但它具有相似的活性部位结构。含有共价结合的肽抑制剂的枯草杆菌蛋白酶(Robertus,J.D.等人(1972),生物化学(Biochemistry),11:2439-2449)或产物复合物(Robertus,J.D.等人(1976),生物化学杂志(J.Biol.Chem.),251:1097-1103)的x-射线晶体结构也提供了关于枯草杆菌蛋白酶的活性部位和推断的底物结合裂隙的信息。另外,曾经报道了关于枯草杆菌蛋白酶的大量动力学和化学修饰研究(Svendsen,B.(1976),Carlsberg研究通讯(Carlsberg Res.Commun.),41:237-291;Markland,F.S.,同前),并且至少有一条报道,其中枯草杆菌蛋白酶残基222的甲硫氨酸的侧链被过氧化氢转化为甲硫氨酸-亚砜(Stauffer,D.C.等人(1965),生物化学杂志,244:5333-5338),并进行了广泛的位点专一诱变(Wells和Estell(1988)TIBS 13:291-297)。
发明概述
本发明的一个目的在于提供在一个或多个残基位置处含有氨基酸置换的蛋白酶变体,残基位置相应于选自解淀粉芽孢杆菌枯草杆菌蛋白酶位置62、212、230、232、252和257的残基。
尽管可利用任何上述所列氨基酸置换的任一组合,本发明优选的蛋白酶变体酶包括如下组合的氨基酸残基的置换。所有的残基位置相应于解淀粉芽孢杆菌枯草杆菌蛋白酶位置:
(1)一种蛋白酶变体,包括在位置62以及在一个或多个如下位置:103、104、109、159、213、232、236、245、248和252中的氨基酸残基的置换;
(2)一种蛋白酶变体,包括在位置212以及在一个或多个如下位置:12、98、102、103、104、159、232、236、245、248和252中的氨基酸残基的置换;
(3)一种蛋白酶变体,包括在位置230以及在一个或多个如下位置:68、103、104、159、232、236、和245中的氨基酸残基的置换;
(4)一种蛋白酶变体,包括在位置232以及在一个或多个如下位置:1、9、12、61、62、68、76、97、98、101、102、103、104、109、130、131、159、183、185、205、209、210、212、213、217、230、236、245、248、252、257、260、270和275中的氨基酸残基的置换;
(5)一种蛋白酶变体,包括在位置232以及在一个或多个如下位置:103、104、236和245中的氨基酸残基的置换;
(6)一种蛋白酶变体,包括在位置232和103以及在一个或多个如下位置:1、9、12、61、62、68、76、97、98、101、102、103、104、109、130、131、159、183、185、205、209、210、212、213、217、230、236、245、248、252、257、260、270和275中的氨基酸残基的置换;
(7)一种蛋白酶变体,包括在位置232和104以及在一个或多个如下位置:1、9、12、61、62、68、76、97、98、101、102、103、104、109、130、131、159、183、185、205、209、210、212、213、217、230、236、245、248、252、257、260、270和275中的氨基酸残基的置换;
(8)一种蛋白酶变体,包括在位置232和236以及在一个或多个如下位置:1、9、12、61、62、68、76、97、98、101、102、103、104、109、130、131、159、183、185、205、209、210、212、213、217、230、236、245、248、252、257、260、270和275中的氨基酸残基的置换;
(9)一种蛋白酶变体,包括在位置232和245以及在一个或多个如下位置:1、9、12、61、62、68、76、97、98、101、102、103、104、109、130、131、159、183、185、205、209、210、212、213、217、230、236、245、248、252、257、260、270和275中的氨基酸残基的置换;
(10)一种蛋白酶变体,包括在位置232、103、104、236和245以及在一个或多个如下位置:1、9、12、61、62、68、76、97、98、101、102、103、104、109、130、131、159、183、185、205、209、210、212、213、217、230、236、245、248、252、257、260、270和275中的氨基酸残基的置换;
(11)一种蛋白酶变体,包括在位置252以及在一个或多个如下位置:1、9、12、61、62、68、76、97、98、101、102、103、104、109、130、131、159、183、185、210、212、213、217、236、245、248、和270中的氨基酸残基的置换;
(12)一种蛋白酶变体,包括在位置252以及在一个或多个如下位置:103、104、236和245中的氨基酸残基的置换;
(13)一种蛋白酶变体,包括在位置252和103以及在一个或多个如下位置:1、9、12、61、62、68、97、98、101、102、103、104、109、130、131、159、183、185、210、212、213、217、232、236、245、248、和270中的氨基酸残基的置换;
(14)一种蛋白酶变体,包括在位置252和104以及在一个或多个如下位置:1、9、12、61、62、68、97、98、101、102、103、104、109、130、131、159、183、185、210、212、213、217、232、236、245、248和270中的氨基酸残基的置换;
(15)一种蛋白酶变体,包括在位置252和236以及在一个或多个如下位置:1、9、12、61、62、68、97、98、101、102、103、104、109、130、131、159、183、185、210、212、213、217、232、236、245、248、和270中的氨基酸残基的置换;
(16)一种蛋白酶变体,包括在位置252和245以及在一个或多个如下位置:1、9、12、61、62、68、97、98、101、102、103、104、109、130、131、159、183、185、210、212、213、217、232、236、245、248和270中的氨基酸残基的置换;
(17)一种蛋白酶变体,包括在位置252、103、104、236和245以及在一个或多个如下位置:1、9、12、61、62、68、97、98、101、102、103、104、109、130、131、159、183、185、210、212、213、217、232、236、245、248和270中的氨基酸残基的置换;和
(18)一种蛋白酶变体,包括在位置257以及在一个或多个如下位置:68、103、104、205、209、210、232、236、245、270和275中的氨基酸残基的置换。
更优选的蛋白酶变体的置换选自相应于解淀粉芽孢杆菌枯草杆菌蛋白酶的表1中位置的残基位置。
表1
76 | 103 | 104 | 212 | 271 | ||||||||
76 | 103 | 104 | 252 | 261 | ||||||||
76 | 103 | 104 | 212 | 258 | ||||||||
4 | 76 | 103 | 104 | 159 | 217 | 252 | ||||||
12 | 62 | 76 | 103 | 104 | 159 | |||||||
76 | 103 | 104 | 212 | 268 | 271 | |||||||
76 | 87 | 103 | 104 | 212 | 271 | |||||||
76 | 103 | 104 | 212 | 245 | 271 | |||||||
76 | 103 | 104 | 134 | 141 | 212 | 271 | ||||||
76 | 103 | 104 | 212 | 236 | 243 | 271 | ||||||
20 | 62 | 76 | 103 | 104 | ||||||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
76 | 103 | 104 | 232 | 245 | ||||||||
24 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | |||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | ||||||
68 | 103 | 104 | 140 | 159 | 232 | 236 | 245 | 252 |
表1(续)
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
68 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | 275 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 103 | 104 | 116 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | |||||
10 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 203 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 237 | 245 | |||||
68 | 76 | 79 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 183 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 174 | 206 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 188 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | |||||
68 | 98 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | |||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | ||||
76 | 103 | 104 | 232 | 236 | 245 | 257 | ||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 275 | ||||
76 | 103 | 104 | 257 | 275 | ||||||||
68 | 103 | 104 | 159 | 224 | 232 | 236 | 245 | 257 | ||||
76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 76 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 159 | 211 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 214 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
20 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 259 | |||
68 | 87 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 261 | ||||
76 | 103 | 104 | 232 | 236 | 242 | 245 | ||||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
12 | 48 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
76 | 103 | 104 | 232 | 236 | 245 |
表1(续)
76 | 103 | 104 | 159 | 192 | 232 | 236 | 245 | |||||
76 | 103 | 104 | 147 | 159 | 232 | 236 | 245 | 248 | 251 | |||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 272 | |||
68 | 76 | 103 | 104 | 159 | 183 | 206 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 256 | ||||
68 | 76 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | ||||
27 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 116 | 159 | 170 | 185 | 232 | 236 | 245 | ||
61 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 99 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | |||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
68 | 103 | 104 | 159 | 185 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 185 | 210 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 213 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 216 | 232 | 236 | 245 | 248 | 252 | |||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 173 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 251 | 252 | |||
68 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
55 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 255 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 256 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 257 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 258 | |||
8 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 269 |
表1(续)
68 | 103 | 104 | 116 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 232 | 236 | 245 | 248 | 252 | |||||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
18 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 76 | 101 | 103 | 104 | 159 | 213 | 218 | 232 | 236 | 245 | 260 | |
68 | 103 | 104 | 159 | 228 | 232 | 236 | 245 | 248 | 252 | |||
33 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
68 | 76 | 89 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | |
61 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
103 | 104 | 159 | 205 | 210 | 232 | 236 | 245 | |||||
61 | 68 | 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | ||
61 | 68 | 103 | 104 | 133 | 137 | 159 | 232 | 236 | 245 | 248 | 252 | |
61 | 103 | 104 | 133 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 218 | 232 | 236 | 245 | 248 | 252 | |||
61 | 68 | 103 | 104 | 159 | 160 | 232 | 236 | 245 | 248 | 252 | ||
3 | 61 | 68 | 76 | 103 | 104 | 232 | 236 | 245 | 248 | 252 | ||
61 | 68 | 103 | 104 | 159 | 167 | 232 | 236 | 245 | 248 | 252 | ||
97 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
98 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
99 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 106 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | ||||
62 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 166 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 217 | 232 | 236 | 245 | 248 | 252 | ||||
20 | 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 159 | 206 | 217 | 232 | 236 | 245 | 248 | 252 |
表1(续)
62 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
27 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
38 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
38 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | 271 | ||
68 | 76 | 103 | 104 | 159 | 209 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 205 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 260 | |||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||
76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 126 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 205 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | |||||
103 | 104 | 159 | 230 | 236 | 245 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||||
103 | 104 | 159 | 232 | 236 | 245 | |||||||
68 | 103 | 104 | 159 | 174 | 232 | 236 | 245 | 257 | ||||
68 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 257 | ||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 257 | ||||
103 | 104 | 159 | 232 | 236 | 245 | 257 | ||||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | 261 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 261 | ||||
103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||||
103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 209 | 232 | 236 | 245 | 257 | |||||
68 | 76 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | ||
12 | 103 | 104 | 159 | 209 | 213 | 232 | 236 | 245 | 260 | |||
103 | 104 | 209 | 232 | 236 | 245 | 257 | ||||||
103 | 104 | 159 | 205 | 210 | 213 | 232 | 236 | 245 | 260 | |||
103 | 104 | 159 | 205 | 209 | 232 | 236 | 245 | 260 | ||||
68 | 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | |||
103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | 257 |
表1(续)
103 | 104 | 159 | 205 | 209 | 232 | 236 | 245 | 257 | ||||
68 | 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | 260 | ||
103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | ||||
103 | 104 | 159 | 209 | 210 | 232 | 236 | 245 | |||||
103 | 104 | 159 | 205 | 210 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 128 | 159 | 232 | 236 | 245 | |||||
48 | 103 | 104 | 159 | 230 | 236 | 245 | ||||||
48 | 68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | ||||
48 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
48 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 261 | |||
102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
12 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
101 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
102 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 232 | 236 | 244 | 245 | 248 | 252 | ||||
62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | 256 | ||
12 | 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 159 | 185 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
98 | 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 248 | 252 | |||
62 | 103 | 104 | 109 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 159 | 212 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 101 | 103 | 104 | 159 | 212 | 213 | 232 | 236 | 245 | 248 | 252 | |
103 | 104 | 159 | 232 | 245 | 248 | 252 | ||||||
103 | 104 | 159 | 230 | 245 | ||||||||
62 | 103 | 104 | 130 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 128 | 159 | 232 | 236 | 245 | 248 | 252 | |||
62 | 101 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 128 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 128 | 159 | 213 | 232 | 236 | 245 | 248 | 252 |
表1(续)
101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
101 | 103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | |||
98 | 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
99 | 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 205 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 230 | 236 | 245 | ||||||
101 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 248 | 252 | |||
76 | 101 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | 248 | 252 | |||
62 | 103 | 104 | 159 | 185 | 206 | 213 | 232 | 236 | 245 | 248 | 252 | 271 |
更优选的蛋白酶变体的置换选自相应于解淀粉芽孢杆菌枯草杆菌蛋白酶的表2中位置的残基位置。
表2
N76D | S103A | V104I | S212P | E271V | ||||||||
N76D | S103A | V104I | N252K | N261Y | ||||||||
N76D | S103A | V104I | S212P | G258R | ||||||||
V4E | N76D | S103A | V104I | G159D | L217E | N252D | ||||||
Q12H | N62H | N76D | S103A | V104I | G159D | |||||||
N76D | S103A | V104I | S212P | V268F | E271V | |||||||
N76D | S87R | S103A | V104I | S212P | E271V | |||||||
N76D | S103A | V104I | S212P | Q245L | E271V | |||||||
N76D | S103A | V104I | T134S | S141N | S212P | E271V | ||||||
N76D | S103A | V104I | S212P | Q236L | N243S | E271V | ||||||
G20V | N62S | N76D | S103A | V104I | ||||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
N76D | S103A | V104I | A232V | Q245R | ||||||||
S24T | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | |||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | N140D | G159D | A232V | Q236H | Q245R | N252K |
表2(续)
N43S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||
N43K | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||
V68A | S87G | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | R275S | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||
V68A | S103A | V104I | N116D | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | |||||
R10C | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | V203E | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | K237E | Q245R | |||||
V68A | N76D | 179N | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | S103A | V104I | G159D | N183D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A174V | Q206L | A232V | Q236H | Q245R | ||||
V68A | S103A | V104I | G159D | S188C | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A230T | A232V | Q236H | Q245R | |||||
V68A | A98T | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A215T | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248S | |||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R |
表2(续)
V68A | N76D | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | ||||
N76D | S103A | V104I | A232V | Q236H | Q245R | L257V | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | R275H | ||||
N76D | S103A | V104I | L257V | R275H | ||||||||
V68A | S103A | V104I | G159D | T224A | A232V | Q236H | Q245R | L257V | ||||
N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||
V68A | N76D | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | G211R | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | G211V | A232V | Q236H | Q245R | ||||
Q12R | V68A | N76D | S103A | V104I | G159D | Y214L | A232V | Q236H | Q245R | |||
V68A | N76D | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | ||||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
G20R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S259G | |||
V68A | S87R | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | T260V | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261G | ||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261W | ||||
N76D | S103A | V104I | A232V | Q236H | S242P | Q245R | ||||||
V68A | N76D | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R |
表2(续)
Q12R | A48V | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||
N76D | S103A | V104I | A232V | Q236H | Q245R | |||||||
N76D | S103A | V104I | G159D | Y192F | A232V | Q236H | Q245R | |||||
N76D | S103A | V104I | V147I | G159D | A232V | Q236H | Q245R | N248S | K251R | |||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | A272S | |||
V68A | N76D | S103A | V104I | G159D | N183K | Q206L | A232V | Q236H | Q245R | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S256R | ||||
V68A | N76D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | ||||
K27R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | N116T | G159D | R170S | N185S | A232V | Q236H | Q245R | ||
G61E | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | S99N | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | Y209F | A232V | Q236H | Q245R | N248D | N252K |
表2(续)
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D | |||
V68A | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210T | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210S | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | N185D | P210L | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212A | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | T213S | A232V | Q236H | Q245R | N248D | N252K | ||||
V68A | A103V | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213G | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A215V | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216T | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216V | A232V | Q236H | Q245R | N248D | N252K |
表2(续)
V68A | S103A | V104I | G159D | S216C | A232V | Q236H | Q245R | N248D | N252K | |||
G20A | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | N173D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | K251V | N252K | |||
V68A | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252L | ||||
P55S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T255V | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256N | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256E | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | L257R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | G258D | |||
18V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N269D | ||
V68A | S103A | V104I | N116S | G159D | A232V | Q236H | Q245R | N248D | N252K | T260E | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D |
表2(续)
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | A232S | Q236H | Q245R | N248D | N252K | |||||
V68A | S103A | V104I | G159D | A232V | Q236R | Q245R | N248D | N252K | ||||
N18S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245V | N248D | N252K | ||||
V68A | N76D | S101T | S103A | V104I | G159D | T213R | N218S | A232V | Q236H | Q245R | T260A | |
V68A | S103A | V104I | G159D | A228V | A232V | Q236H | Q245R | N248D | N252K | |||
T33S | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | N76D | E89D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | |
G61E | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | V205I | P210I | A232V | Q236H | Q245R | |||||
G61E | V68A | S103A | V104I | S130A | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
G61E | V68A | S103A | V104I | A133S | Q137R | G159D | A232V | Q236H | Q245R | N248D | N252K | |
G61E | S103A | V104I | A133V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248G | N252K | ||||
V68A | S103A | V104I | G159D | N218S | A232V | Q236H | Q245R | N248D | N252K | |||
G61E | V68A | S103A | V104I | G159D | S160V | A232V | Q236H | Q245R | N248D | N252K | ||
S3L | G61E | V68A | N76D | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K |
表2(续)
G61E | V68A | S103A | V104I | G159D | S167F | A232V | Q236H | Q245R | N248D | N252K | ||
G97E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
A98D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S99E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | S106E | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | Q109E | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | ||||
S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
N62D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | S166D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | L217E | A232V | Q236H | Q245R | N248D | N252K | ||||
G20R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
S103A | V104I | G159D | Q206R | L217E | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K |
表2(续)
S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
K27N | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
T38G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
T38A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | E271G | ||
V68A | N76D | S103A | V104I | G159D | Y209W | r213R | A232V | Q236H | Q245R | T260A | ||
V68A | N76D | S103A | V104I | G159D | P210I | T213R | A232V | Q236H | Q245R | T260A | ||
V68A | N76D | S103A | V104I | G159D | V205I | T213R | A232V | Q236H | Q245R | T260A | ||
V68A | N76D | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | T260A | |||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | ||||
N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | ||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A230V | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | L126F | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | |||||
S103A | V104I | G159D | A230V | Q236H | Q245R |
表2(续)
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | T260A | |||||
S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||
V68A | S103A | V104I | G159D | A174V | A232V | Q236H | Q245R | L257V | ||||
V68A | S103A | V104I | G159D | A194S | A232V | Q236H | Q245R | L257V | ||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | ||||
S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | ||||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | N261W | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | N261W | ||||
S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||
S103A | V104I | G1590 | P210I | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | |||||
V68A | N76D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | ||
Q12R | S103A | V104I | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | |||
S103A | V104I | Y209W | A232V | Q236H | Q245R | L257V | ||||||
S103A | V104I | G159D | V205I | P210I | T213R | A232V | Q236H | Q245R | T260A | |||
S103A | V104I | G159D | V205I | Y209W | A232V | Q236H | Q245R | T260A | ||||
V68A | S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | |||
S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | L257V | |||
S103A | V104I | G159D | V205I | Y209W | A232V | Q236H | Q245R | L257V |
表2(续)
IV68A | S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | T260A | ||
S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | ||||
S103A | V104I | G159D | Y209W | P210I | A232V | Q236H | Q245R | |||||
S103A | V104I | G159D | V205I | P210I | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | |||||
A48V | S103A | V104I | G159D | A230V | Q236H | Q245R | ||||||
A48V | V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | ||||
A48V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
A48V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | N261W | |||
G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
Q12R | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
G102A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | N184S | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | N184G | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | A232V | Q236H | V244T | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | A232V | Q236H | V244A | Q245R | N248D | N252K |
表2(续)
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | S256R | ||
Q12R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | Q206E | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | T213Q | A232V | Q236H | Q245R | N248D | N252K | |||
A98L | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | N248D | N252K | |||
N62D | S103A | V104I | Q109R | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S103A | V104I | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S101G | S103A | V104I | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | |
S103A | V104I | G159D | A232V | Q245R | N248D | N252K | ||||||
S103A | V104I | G159D | A230V | Q245R | ||||||||
N62D | S103A | V104I | S130G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | S128G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S101G | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K |
N62D | S103A | V104I | S128G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S103A | V104I | S128L | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260A | |||
S101G | S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
A98V | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S99G | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | A230V | Q236H | Q245R | ||||||
S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | |||
N76D | S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | G159D | A230V | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S103A | V104I | G159D | N185D | Q206E | T213R | A232V | Q236H | Q245R | N248D | N252K | E271Q |
本发明另外一个目的在于提供编码这些蛋白酶变体的DNA序列,以及含有这些变异体DNA序列的表达载体。
更进一步,本发明的另一目的在于提供用这些载体转化的宿主细胞,以及能够表达这种DNA而在细胞内或细胞外产生蛋白酶变体的宿主细胞。
进一步提供一种含有本发明的蛋白酶变体的清洁用组合物。
另外,也提供一种含有本发明的蛋白酶变体的动物饲料。
还提供了一种用于处理纺织品的含有本发明的蛋白酶变体之组合物。
附图简述
图1A-C描述解淀粉芽孢杆菌枯草杆菌蛋白酶的DNA及氨基酸序列,和该基因的部分限制酶切图谱。
图2描述解淀粉芽孢杆菌(BPN)’和迟缓芽孢杆菌(野生型)枯草杆菌蛋白酶之间的保守氨基酸残基。
图3A和3B描述四种枯草杆菌蛋白酶的氨基酸序列。最上一行代表来源于解淀粉芽孢杆菌枯草杆菌蛋白酶(有时也称为枯草杆菌蛋白酶BPN’)的枯草杆菌蛋白酶的氨基酸序列。第二行描述来源于枯草芽孢杆菌的枯草杆菌蛋白酶的氨基酸序列。第三行描述来源于地衣芽孢杆菌的枯草杆菌蛋白酶的氨基酸序列。第四行描述来源于迟缓芽孢杆菌的枯草杆菌蛋白酶(在PCT WO89/06276中也称为枯草杆菌蛋白酶309)的氨基酸序列。符号*表示与枯草杆菌蛋白酶BPN’相比特定氨基酸残基缺如。
发明详述
蛋白酶是通常用来切割蛋白质或肽的肽键的羰基水解酶。在此使用时,“蛋白酶”意思是天然存在的蛋白酶或重组蛋白酶。天然存在的蛋白酶包括α-氨酰肽水解酶、肽酰氨基酸水解酶、酰基氨基水解酶、丝氨酸羧基肽酶、金属羧基肽酶、巯基蛋白酶、羧基蛋白酶和金属蛋白酶。包括丝氨酸、金属、巯基和酸性蛋白酶,以及内切蛋白酶和外切蛋白酶。
本发明包括非天然存在的羰基水解酶变体的蛋白酶(蛋白酶变体),与衍生出该变体氨基酸序列的前体羰基水解酶相比,其具有不同的蛋白水解活性、稳定性、底物特异性、pH分布曲线和/或性能特征。特别是,这些蛋白酶变体具有自然界中未知的氨基酸序列,它是由于前体蛋白酶的大多数氨基酸残基被替换为不同氨基酸而产生的。前体蛋白酶可以是天然存在的蛋白酶或重组蛋白酶。
此处有用的蛋白酶变体包含19种天然存在的L-氨基酸中任一种在指定氨基酸残基位置处的置换。这些置换能在任何前体枯草杆菌蛋白酶(原核、真核、哺乳动物等)中进行。在整个申请中,以常用的单字母及三字母密码的形式提及不同的氨基酸。这些密码由Dale,M.W.(1989),《细菌分子遗传学》(Molecular Genetics of Bacteria),John Wiley & Sons,Ltd.,附录B确定。
此处有用的蛋白酶变体优选地来源于芽孢杆菌属的枯草杆菌蛋白酶。更优选地,蛋白酶变体来源于迟缓芽孢杆菌枯草杆菌蛋白酶和/或枯草杆菌蛋白酶309。
枯草杆菌蛋白酶是一般用来切割蛋白质或肽的肽键的细菌或真菌蛋白酶。在此使用时,“枯草杆菌蛋白酶”意思是天然存在的枯草杆菌蛋白酶或重组枯草杆菌蛋白酶。已知一系列天然存在的枯草杆菌蛋白酶是由不同微生物种产生且常常为分泌的。该系列成员的氨基酸序列不是完全同源的。然而,该系列中的枯草杆菌蛋白酶显示相同或相似类型的蛋白水解活性。该类丝氨酸蛋白酶具有一种共同的氨基酸序列,该序列定义了有别于胰凝乳蛋白酶相关种类丝氨酸蛋白酶的催化三联体。枯草杆菌蛋白酶和胰凝乳蛋白酶相关的丝氨酸蛋白酶都含有包括天冬氨酸、组氨酸和丝氨酸的催化三联体。在枯草杆菌蛋白酶相关蛋白酶中,这些氨基酸从氨基端向羧基端阅读的相对顺序是天冬氨酸-组氨酸-丝氨酸。但是,在胰凝乳蛋白酶相关蛋白酶中相对顺序为组氨酸-天冬氨酸-丝氨酸。因此,枯草杆菌蛋白酶在此是指含有枯草杆菌蛋白酶相关蛋白酶的催化三联体的丝氨酸蛋白酶。实例包括但不限于此处图3中所确定的枯草杆菌蛋白酶。一般而言,并且对于本发明,蛋白酶中氨基酸的编号对应于分配给图1所示成熟解淀粉芽孢杆菌枯草杆菌蛋白酶序列的编号。
“重组枯草杆菌蛋白酶”或“重组蛋白酶”是指一种枯草杆菌蛋白酶或蛋白酶,其中编码枯草杆菌蛋白酶或蛋白酶的DNA序列受到修饰,产生一种变异(或突变)DNA序列,该序列编码天然存在的氨基酸序列中一种或多种氨基酸的置换、缺失或插入。产生这种修饰、并且可与此处公开的方法结合的适当方法包括美国专利RE34,606、美国专利5,204,015和美国专利5,185,258、美国专利5,700,676、美国专利5,801,038和美国专利5,763,257中公开的方法。
“非人类的枯草杆菌蛋白酶”及其编码DNA可以从多种原核及真核生物中获得。原核生物的合适实例包括革兰氏阴性生物如大肠杆菌或假单胞菌(Pseudomonas)、革兰氏阳性菌如微球菌(Micrococcus)或芽孢杆菌。可从中获得枯草杆菌蛋白酶及其基因的真核生物的实例包括酵母如酿酒酵母(Saccharomyces cerevisiae)、真菌如曲霉(Aspergillus)属的种。
“蛋白酶变体”具有由“前体蛋白酶”的氨基酸序列衍生的氨基酸序列。前体蛋白酶包括天然存在的蛋白酶和重组蛋白酶。蛋白酶变体的氨基酸序列通过前体氨基酸序列中一种或多种氨基酸的置换、缺失或插入而由前体蛋白酶氨基酸序列“衍生”。这种修饰是编码前体蛋白酶氨基酸序列的“前体DNA序列”的修饰,而不是前体蛋白酶自身的操作。对前体DNA序列进行这种操作的适当方法包括此处公开的方法,以及本领域技术人员公知的方法(参见,例如,EP 0 328299、WO89/06279和已经在此引用的美国专利及申请)。
此处鉴定了在相应于选自解淀粉芽孢杆菌枯草杆菌蛋白酶残基位置62、212、230、232、252和257处的一个和多个残基位置的定点置换。
优选的变体是具有在相应于表1中解淀粉芽孢杆菌枯草杆菌蛋白酶位置的残基位置上置换的组合的那些变体。
更优选的变体是具有在相应于表2中解淀粉芽孢杆菌枯草杆菌蛋白酶位置的残基位置上置换的组合的那些变体。
更为优选的变体是具有在相应于表3中解淀粉芽孢杆菌枯草杆菌蛋白酶位置的残基位置上置换的组合的那些变体。
表3
68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | |||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | ||||||
68 | 103 | 104 | 140 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 224 | 232 | 236 | 245 | 257 | ||||
76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 76 | 103 | 104 | 159 | 211 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 214 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
20 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 259 | |||
68 | 76 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 261 | ||||
12 | 48 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
76 | 103 | 104 | 159 | 192 | 232 | 236 | 245 | |||||
76 | 103 | 104 | 147 | 159 | 232 | 236 | 245 | 248 | 251 | |||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 272 | |||
68 | 76 | 103 | 104 | 159 | 183 | 206 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 256 | ||||
68 | 76 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | ||||
27 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | |||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 |
表3(续)
68 | 103 | 104 | 213 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 216 | 232 | 236 | 245 | 248 | 252 | |||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 255 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 256 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
68 | 103 | 104 | 159 | 228 | 232 | 236 | 245 | 248 | 252 | |||
68 | 76 | 89 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | |
68 | 103 | 104 | 159 | 218 | 232 | 236 | 245 | 248 | 252 |
这些氨基酸位置编号是指那些分配给图1所示成熟解淀粉芽孢杆菌枯草杆菌蛋白酶序列的编号。然而,本发明不限于该特定枯草杆菌蛋白酶的突变,而是扩展到在一定位置处含有这样的氨基酸残基的前体蛋白酶,该残基“等同”于解淀粉芽孢杆菌枯草杆菌蛋白酶中特别确定的残基。在本发明的一个优选实施方案中,前体蛋白酶是迟缓芽孢杆菌枯草杆菌蛋白酶,并在迟缓芽孢杆菌中对应于以上所列位点的等同氨基酸残基位置处进行置换。
如果前体蛋白酶的残基(氨基酸)位置与解淀粉芽孢杆菌枯草杆菌蛋白酶中的特定残基或该残基的一部分同源(即,在一级或三级结构中位置相对应)或类似(即,具有相同或相似的化学结合、反应或相互作用的功能能力),则它相当于解淀粉芽孢杆菌枯草杆菌蛋白酶的残基。
为了建立与一级结构的同源性,将前体蛋白酶的氨基酸序列直接与解淀粉芽孢杆菌枯草杆菌蛋白酶一级序列相比较,特别与公知在序列已知的枯草杆菌蛋白酶中不变的一组残基相比较。例如,在此图2显示解淀粉芽孢杆菌枯草杆菌蛋白酶与迟缓芽孢杆菌枯草杆菌蛋白酶之间的保守残基。对比保守残基,为维持序列对比进行必要的插入和缺失(即,避免保守残基由于任意缺失和插入而消除)之后,确定等同于解淀粉芽孢杆菌枯草杆菌蛋白酶一级序列中特定氨基酸的残基。保守残基的对比优选地应当保持100%的这些残基。然而,超过75%或低至50%的保守残基的对比也足以确定等同的残基。催化三联体Asp32/His64/Ser221的保守性应当保持。Siezen等人(1991)蛋白质工程(Protein Eng.)4(7):719-737显示了大量丝氨酸蛋白酶的序列对比。Siezen等人将此分组称为枯草杆菌酶(Subtilase)或枯草杆菌蛋白酶样丝氨酸蛋白酶。
例如,图3中,排列对比了解淀粉芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌(carlsbergensis)和迟缓芽孢杆菌的枯草杆菌蛋白酶之氨基酸序列,以提供氨基酸序列之间最大量的同源性。这些序列的比较显示,在每种序列中含有许多保守残基。图2中确定了这些保守残基(BPN’与迟缓芽孢杆菌之间)。
因此,可用这些保守残基确定其它枯草杆菌蛋白酶,诸如迟缓芽孢杆菌枯草杆菌蛋白酶(1989年7月13日公布的PCT公开文本WO89/06279)、此处优选的蛋白酶前体酶或被称为PB92的枯草杆菌蛋白酶(EP 0 328 299,它与优选的迟缓芽孢杆菌枯草杆菌蛋白酶高度同源)中与解淀粉芽孢杆菌枯草杆菌蛋白酶相应的等同氨基酸残基。在图3A和3B中将这些枯草杆菌蛋白酶中某些氨基酸序列与解淀粉芽孢杆菌枯草杆菌蛋白酶的序列相对比,产生保守残基的最大同源性。如所能看到的,与解淀粉芽孢杆菌枯草杆菌蛋白酶相比,迟缓芽孢杆菌序列中存在大量缺失。因此,例如,在其它枯草杆菌蛋白酶中与解淀粉芽孢杆菌枯草杆菌蛋白酶中的Val165等同的氨基酸对于迟缓芽孢杆菌和地衣芽孢杆菌而言是异亮氨酸。
也可以通过测定已经通过x-射线晶体分析法确定三级结构的前体蛋白酶在三级结构水平上的同源性,来确定“等同的残基”。等同的残基被定义为前体蛋白酶和解淀粉芽孢杆菌枯草杆菌蛋白酶特定氨基酸残基的两个或多个主链原子的原子坐标(N对N、CA对CA、C对C和O对O)经对比后在0.13nm优选地0.1nm之内的残基。在定向并定位最佳模型之后完成对比,得到所述蛋白酶与解淀粉芽孢杆菌枯草杆菌蛋白酶的非氢蛋白质原子的原子坐标的最大重叠。最佳模型是在可获得的最高分辨力时对于实验衍射数据可得到最低R因子的晶体模型。
与解淀粉芽孢杆菌枯草杆菌蛋白酶特定残基功能类似的等同残基被定义为前体蛋白酶的如下氨基酸,它们可采取一种构象,使得它们以一种确定的方式改变、修饰或贡献于蛋白质结构、底物结合或催化,且其归因于解淀粉芽孢杆菌枯草杆菌蛋白酶的特定残基。此外,它们是前体蛋白酶的那类残基(通过x-射线晶体分析法已获得其三级结构),其占据了类似位点,使得尽管基于占据同源位点,特定残基的主链原子也许不能满足等同的标准,但该残基的至少两个侧链原子之原子坐标位于解淀粉芽孢杆菌枯草杆菌蛋白酶相应侧链原子的0.13nm内。在EPO公开文本0 251 446(相当于美国专利5,182,204,其公开内容在此引用作为参考)中阐述了解淀粉芽孢杆菌枯草杆菌蛋白酶的三维结构的坐标,并能如上所述用来在三级结构水平上确定等同的残基。
为了置换所确定的某些残基是保守残基,而其它的则不是。对于不保守的残基而言,一种或多种氨基酸的置换限于产生这样一种变体的置换,该变体具有一种与自然中所发现的氨基酸序列不对应的序列。对于保守残基而言,这些置换不能产生天然存在的序列。本发明的蛋白酶变体包括蛋白酶变体的成熟形式,以及这些蛋白酶变体的原-形式和前原-形式。前原-形式是优选的结构,因为其有利于蛋白酶变体的表达、分泌和成熟。
“原序列”是指与蛋白酶成熟形式的N端部分结合的氨基酸序列,当它被去除后,导致该蛋白酶“成熟”形式的出现。实质上发现许多蛋白水解酶为翻译酶原产物,在不存在翻译后加工的情况下以该方式表达。用于产生蛋白酶变体的一种优选原序列是解淀粉芽孢杆菌枯草杆菌蛋白酶的推定的原序列,尽管也可使用其它蛋白酶原序列。
“信号序列”或“前序列”是指与蛋白酶N端部分结合或与原蛋白酶N端部分结合的任何氨基酸序列,它们可能参与蛋白酶成熟形式或原形式的分泌。信号序列的这种定义是一种功能性定义,意为包括蛋白酶基因N端部分所编码的所有氨基酸序列,其在天然条件下参与蛋白酶分泌的实现。本发明利用这些序列实现此处所述蛋白酶变体的分泌。一种可能的信号序列包含枯草芽孢杆菌枯草杆菌蛋白酶信号序列的前7个氨基酸残基,这些残基与迟缓芽孢杆菌枯草杆菌蛋白酶信号序列(ATCC21536)的剩余部分相融合。
蛋白酶变体的“前原”形式由含有与蛋白酶氨基端有效连接的原序列的蛋白酶成熟形式和与原序列氨基端有效连接的“前”序列或“信号”序列组成。
“表达载体”是指一种DNA构建体,其含有与能实现该DNA在适当宿主中表达的适当控制序列有效连接的DNA序列。这类控制序列包括实现转录的启动子、控制这种转录的任意操纵子序列、编码适当mRNA核糖体结合位点的序列和控制转录与翻译终止的序列。载体可以是质粒、噬菌体颗粒,或者仅仅是有效的基因组插入片段。一旦转化到适当宿主中,载体可不依赖于宿主基因组复制并起作用,或者在某些情况中可能整合到基因组自身中。在本说明书中,“质粒”和“载体”有时交换使用,因为质粒是目前最常用的载体形式。然而,本发明意在包括表达载体的其它形式,它们具有等同的功能,并且是或者可成为本领域所公知的。
本发明中所用的“宿主细胞”通常是原核或真核宿主,它们优选地用美国专利RE 34,606中公开的方法操作,以使其不能分泌酶促活性的内切蛋白酶。表达蛋白酶的一种优选宿主细胞是芽孢杆菌株BG2036,它缺乏具有有酶活性的中性蛋白酶和碱性蛋白酶(枯草杆菌蛋白酶)。美国专利5,264,366中详述了菌株BG2036的构建。表达蛋白酶的其它宿主细胞包括枯草芽孢杆菌1168(在美国专利RE 34,606和美国专利5,264,366中也有描述,其公开内容在此引用作为参考),以及任何合适的芽孢杆菌属的菌株,如地衣芽孢杆菌、迟缓芽孢杆菌等。
用以重组DNA技术构建的载体转化或转染宿主细胞。这些转化的宿主细胞能复制编码蛋白酶变体的载体或表达希望的蛋白酶变体。对于编码蛋白酶变体前-形式或前原-形式的载体而言,这些变体当表达时一般由宿主细胞向宿主细胞培养基中分泌。
当描述两个DNA区域之间的关系时,“有效连接”仅意味着它们在功能上彼此相关。例如,如果一种前序列作为信号序列参与蛋白质成熟形式的分泌,最可能的是参与信号序列的切割,则该前序列与肽有效连接。如果一种启动子控制一种编码序列的转录,则该启动子与该序列有效连接;如果一种核糖体结合位点位于一定位置使得翻译成为可能,则它与编码序列有效连接。
编码天然存在的前体蛋白酶的基因可依照本领域技术人员公知的通用方法获得。这些方法一般包括:合成含有编码目标蛋白酶区推定序列的标记探针、由表达该蛋白酶的生物制备基因组文库,以及通过与该探针杂交为了获得目标基因筛选该文库。然后对阳性杂交克隆作图并测定。
然后用克隆的蛋白酶转化宿主细胞,以表达该蛋白酶。然后将蛋白酶基因连接于高拷贝数质粒中。该质粒在宿主中复制,在此意义上它含有质粒复制所必需的众所周知的元件:与所述基因有效连接的启动子(如果它可被宿主识别,即转录,则可作为基因本身的同源启动子)、转录终止区和聚腺苷酸化区(在某些真核宿主细胞中,它对于由宿主从蛋白酶基因转录的mRNA的稳定性是必需的),它是外源的或由蛋白酶基因的内源终止区所提供的,质粒中还期望含有选择性基因,如抗生素抗性基因,它使得质粒感染的宿主细胞通过在含有抗生素的培养基中生长以维持连续培养成为可能。高拷贝数质粒也含有宿主的复制起点,从而能在细胞质中不受染色体限制地产生大量质粒。然而,宿主基因组中整合多拷贝的蛋白酶基因也处于本发明之内。利用对同源重组特别敏感的原核和真核生物利于此过程。
该基因可能是天然迟缓芽孢杆菌基因。此外,也可以产生编码天然存在的或突变的前体蛋白酶的合成基因。这种方法中,测定前体蛋白酶的DNA和/或氨基酸序列。之后合成多个、重叠的合成单链DNA片段,杂交并连接后产生编码前体蛋白酶的合成DNA。在美国专利5,204,015的实施例3中描述了合成的基因构建体的一个实例,其公开内容在此引用作为参考。
一旦克隆了天然存在的或合成的前体蛋白酶基因,即进行大量修饰,以增强天然存在的前体蛋白酶合成之外的基因用途。这些修饰包括如美国专利RE 34,606和EPO公布号0 251 446所公开的重组蛋白酶的产生和此处所述的蛋白酶变体的产生。
可用下列盒式诱变法促进本发明的蛋白酶变体的构建,尽管也可使用其它方法。首先,获得天然存在的编码蛋白酶的基因,并全部或部分测序。然后为了寻找希望在编码的酶中进行一个或多个氨基酸突变(缺失、插入或置换)的位点,扫描该序列。评价侧翼于该位点的序列是否存在估计限制酶切位点的存在,用于将基因的短片段替换为表达时编码不同突变体的寡核苷酸集合体。这些限制酶切位点优选地是蛋白酶基因内的唯一位点,以便于基因片段的置换。然而,可使用在蛋白酶基因中不过度丰余的任何适宜的限制酶切位点,只要限制酶切消化产生的基因片段能够在适当的序列中重新装配即可。如果在与所选位点适当距离内(10-15个核苷酸)的位置处不存在限制酶切位点,则可通过以在最终构建中不改变读框也不改变所编码氨基酸的方式置换基因中的核苷酸来产生这些位点。为了改变其序列以符合目标序列,基因突变依照公知的方法通过M13引物延伸来实现。根据遗传密码的丰余性、基因的限制酶图谱和大量不同的限制酶,常规进行定位适当侧翼区并估计所需改变以得到两种适宜的限制酶切位点序列的工作。注意到如果可获得适宜的侧翼限制酶切位点,则上述方法需要仅仅与不含该位点的侧翼区结合应用。
一旦克隆了天然存在的DNA或合成的DNA,即用同源的限制酶消化侧翼于待突变位点的限制酶切位点,并将多数末端互补的寡核苷酸盒连接于该基因中。该方法简化了诱变,因为能合成所有寡核苷酸使其具有相同的限制酶切位点,且合成的接头不是产生限制酶切位点所必需的。
在此使用时,蛋白水解活性被定义为每毫克活性酶的肽键水解率。测定蛋白水解活性有许多众所周知的方法(K.M.Kalisz,“微生物蛋白酶”《生物化学工程/生物技术进展》(Advances in BiochemicalEngineering/Biotechnology),A.Fiechter编,1988)。除了改进的蛋白水解活性之外,或者作为一种备择,本发明的变异蛋白酶可能具有其它改进的性质,如Km、kcat或Kcat/Km比值和/或改进的底物特异性和/或改进的pH活性分布曲线。这些酶能量身定做般地用于例如预计在肽制剂中存在的特定底物,或适于水解过程如洗衣用途。
本发明的一个方面,目的在于获得一种与前体蛋白酶相比,在至少一种去污剂配方中和、或在至少一种洗涤条件下具有改变的,优选的改善的洗涤性能的蛋白酶变体。
洗涤条件多种多样,包括不同的去污剂配方、洗涤用水体积、洗涤用水温度和可接触蛋白酶变体的洗涤时间。例如,在不同领域中使用的去污剂配方在洗涤用水(wash water)中含有不同浓度的有关成分。例如,欧洲去污剂体系一般在洗涤用水中含有大约4500-5000ppm的去污剂成分,而日本去污剂体系一般在洗涤用水中含有大约667ppm的去污剂成分。在北美洲,尤其在美国,去污剂体系一般在洗涤用水中含有约975ppm的去污剂成分。
低去污剂浓度体系包括在洗涤用水中存在低于大约800ppm的去污剂成分的去污剂。日本去污剂一般被认为是低去污剂浓度体系,因为它们在洗涤用水中存在大约667ppm的去污剂成分。
中去污剂浓度包括在洗涤用水中存在大约800ppm到约2000ppm的去污剂成分的去污剂。北美去污剂一般被认为是中去污剂浓度体系,因为它们在洗涤用水中存在大约975ppm的去污剂成分。巴西的体系一般在洗涤用水中存在大约1500ppm的去污剂成分。
高去污剂浓度体系包括在洗涤用水中存在高于大约2000ppm的去污剂成分的去污剂。欧洲去污剂一般被认为是高去污剂浓度体系,因为它们在洗涤用水中存在大约4500-5000ppm的去污剂成分。
拉丁美洲去污剂一般是高泡沫磷酸盐助洗去污剂,在拉丁美洲使用的去污剂范围可能处于中及高去污剂浓度,因为在洗涤用水中它们有1500ppm到6000ppm的去污剂成分。如上所述,巴西的体系一般在洗涤用水中含有大约1500ppm的去污剂成分。然而,其它高泡沫磷酸盐助洗去污剂地区,不限于其它拉丁美洲国家,可能有在洗涤用水中存在可达大约6000ppm的去污剂成分的高去污剂浓度体系。
根据前述,显然典型洗涤溶液中去污剂组合物的浓度在全世界范围内各不相同,从低于大约800ppm的去污剂组合物(“低去污剂浓度地区”),例如在日本为大约667ppm,到约800ppm-约2000ppm(“中去污剂浓度地区”),例如在美国约为975ppm、在巴西约为1500ppm,到高于大约2000ppm(“高去污剂浓度地区”),例如在欧洲为大约4500ppm-约5000ppm,在高泡沫助洗剂地区约为6000ppm。
典型洗涤溶液的浓度由经验确定。例如,在美国,一种典型的洗涤机可容纳大约64.4升体积的洗涤溶液。因此,为了获得在洗涤溶液中大约975ppm去污剂的浓度,必须向64.4升洗涤溶液中加入大约62.79克去污剂组合物。这种量是由消费者用去污剂所带量杯测量的加入洗涤用水中的典型量。
作为又一实例,不同地理区域使用不同洗涤温度。在日本的洗涤用水温度一般低于在欧洲使用的温度。
根据本发明的一个方面,包括了在至少一种洗涤条件下显示具有改善的洗涤性能的蛋白酶变体。
本发明的另一方面中,已确定在选自相应于解淀粉芽孢杆菌枯草杆菌蛋白酶之62、212、230、232、252和257残基位置的氨基酸置换对于酶之洗涤性能的改善十分重要。
这些置换优选地在迟缓芽孢杆菌(重组的或天然型)枯草杆菌蛋白酶中进行,尽管该置换也可在任何芽孢杆菌蛋白酶,优选地在芽孢杆菌属的枯草杆菌蛋白酶中进行。
根据用变异蛋白酶获得的筛选结果,解淀粉芽孢杆菌枯草杆菌蛋白酶中的显著突变对于这些酶的蛋白水解活性、性能和/或稳定性以及这些变异酶的清洁或洗涤能力至关重要。
本发明的许多蛋白酶变体可用于配制不同的去污剂组合物或个人护理制剂如洗发剂或洗涤剂。许多公知的化合物是在含有本发明之蛋白酶突变体的组合物中有用的适当表面活性剂。其包括非离子、阴离子、阳离子或两性离子去污剂,如Barry J.Anderson的US 4,404,128和Jiri Flora等人的US 4,261,868中所公开的。一种合适的去污剂制剂是美国专利5,204,015(前文引用作为参考)的实施例7中所述的制剂。本领域技术人员熟悉能用作清洁用组合物的不同制剂。除了典型的清洁用组合物之外,易于理解本发明的蛋白酶变体也可用于使用天然或野生型蛋白酶的任何目的。因此,例如,这些变体能用于固体肥皂或液体肥皂的用途、餐具清洁(dishcare)剂、隐形眼镜清洁液或产品、肽水解、废物处理、纺织品用途、用作蛋白质生产中的融合-切割酶,等等。本发明的变体可在去污剂组合物中具有增强的性能(与前体相比)。在此使用时,在去污剂中的增强性能定义为某些酶敏感性污迹如草或血液的清除加强,如在标准洗涤循环后通过通用评估所测定的。
本发明的蛋白酶能以大约0.01%到大约5%(优选地0.1%-0.5%)的重量百分比水平配制于已知的pH6.5-12.0的粉末及液体去污剂中。这些去污剂清洁用组合物也能包含其它的酶如已知的蛋白酶、淀粉酶、纤维素酶、脂肪酶或糖苷内切酶,以及助洗剂和稳定剂。
本发明的蛋白酶向常规清洁用组合物中的添加不产生任何特殊的应用限制。换句话说,适合于去污剂的任何温度和pH也适合于该组合物,只要pH位于上述范围之内,而温度低于所述蛋白酶的变性温度。另外,本发明的蛋白酶也能在不含去污剂的清洁用组合物中使用,单独或与助洗剂和稳定剂结合使用。
本发明也涉及含有本发明的蛋白酶变体的清洁用组合物。清洁用组合物另外也可含有常用于清洁用组合物的添加剂。其能选自但不限于:漂白剂、表面活性剂、助洗剂、酶和漂白催化剂。本领域的一名普通技术人员可容易地理解何种添加剂适合包含于组合物中。此处提供的列表绝不是完全的,而只是作为适当添加剂的实例。本领域的一名普通技术人员可容易地理解,应当只使用那些与该组合物中的酶及其它成分如表面活性剂相适合的添加剂。
制成后,清洁用组合物中存在的添加剂的量为大约0.01%-约99.9%,优选地约1%-约95%,更优选地约1%-约80%。
本发明的变异蛋白酶能包含于动物饲料中,如作为动物饲料添加剂部分,例如,如US 5,612,055、US 5,314,692和US 5,147,642所述。
本发明的一个方面是含有本发明的变异蛋白酶的用于纺织品处理的组合物。如出版物如RD 216,034、EP 134,267、US 4,533,359和EP 344,259中所述,能用该组合物处理如丝绸或毛。
以下以实施例的方式描述,而不应理解为对权利要求书范围的限制。
此处引用的所有出版物和专利在此都完全引入作为参考。
实施例1
用本领域众所周知的方法生产并纯化大量蛋白酶变体。所有突变都在迟缓芽孢杆菌GG36枯草杆菌蛋白酶中进行。变体示于表4。
表4
N76D | S103A | V104I | M222S | |||||||||
N76D | A98E | S103A | V104I | |||||||||
N76D | S78T | S103A | V104I | |||||||||
N76D | S103A | V104I | I107V | |||||||||
V4E | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | I246V | |||||||||
N76D | N77D | S103A | V104I | |||||||||
N76D | S103A | V104I | N183D | N218I | ||||||||
A16T | N76D | S103A | V104I | N248D | ||||||||
A1E | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | N261D | |||||||||
N76D | S103A | V104I | S160T | |||||||||
N76D | S103A | V104I | S216C | |||||||||
H17Q | N76D | S103A | V104I | |||||||||
S37T | N76D | S103A | V104I | |||||||||
N76D | N77D | S103A | V104I | A174V |
表4(续)
T38S | N76D | S103A | V104I | |||||||||
T38S | N76D | S103A | V104I | K237Q | ||||||||
I8V | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | N183D | |||||||||
R19L | N76D | S103A | V104I | |||||||||
A13V | N76D | S103A | V104I | |||||||||
R19C | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | N184D | |||||||||
N76D | S103A | V104I | N252D | |||||||||
N76D | S103A | V104I | S259C | |||||||||
N76D | S103A | V104I | K251T | |||||||||
N76D | P86S | S103A | V104I | |||||||||
I72V | N76D | S103A | V104I | N185D | ||||||||
N76D | S103A | V104I | K237E | T274A | ||||||||
N76D | S103A | V104I | S160L | |||||||||
N76D | S103A | V104I | A228V | |||||||||
P55S | N76D | S103A | V104I | S240T | ||||||||
N76D | S103A | V104I | A254T |
表4(续)
N76D | S103A | I104N | N204T | |||||||||
N76D | S103A | V104I | N204D | |||||||||
N43S | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | G159D | |||||||||
R10H | N76D | S103A | V104I | V177A | ||||||||
T58S | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | A270V | |||||||||
N76D | S103A | V104I | N185D | |||||||||
K27N | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | L262M | |||||||||
N76D | S78P | S103A | V104I | |||||||||
S24P | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | S166G | Q236R | K251R | |||||||
H17L | N76D | S103A | V104I | K237E | ||||||||
N76D | S103A | V104I | S130L | |||||||||
N76D | S103A | V104I | Q109R | |||||||||
N76D | S99R | S103A | V104I | N204T | ||||||||
N76D | S103A | V104I | D181N |
表4(续)
Q12R | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | S212P | E271V | ||||||||
N76D | S103A | V104I | N252K | N261Y | ||||||||
N76D | S103A | V104I | S242T | |||||||||
N76D | S103A | V104I | E271Q | |||||||||
Q12R | N76D | S103A | V104I | S242T | ||||||||
N43S | N76D | S103A | V104I | N116K | N183I | |||||||
N76D | S103A | V104I | G258R | |||||||||
N76D | S103A | V104I | E271G | |||||||||
G61R | N76D | S103A | V104I | |||||||||
T38S | N76D | S103A | V104I | Q182R | Y263H | |||||||
N76D | S103A | V104I | Q182R | A272S | ||||||||
N76D | S103A | V104I | Q109R | I246V | ||||||||
N76D | S87G | S103A | V104I | Q206R | H249Q | S265G | ||||||
N76D | S103A | V104I | Q137R | N238Y | E271V | |||||||
S103A | V104I | A228T | ||||||||||
N76D | S103A | V104I | Q182R | I198V | ||||||||
L21M | N76D | S103A | V104I | Q182R |
表4(续)
N76D | S103A | V104I | M119I | Q137R | ||||||||
N76D | S103A | V104I | Q137R | N248S | ||||||||
A13T | N76D | S103A | V104I | Q206R | ||||||||
N76D | S103A | V104I | Q206R | |||||||||
N76D | S103A | V104I | S212P | G258R | ||||||||
T58S | N76D | S103A | V104I | E271G | ||||||||
N76D | S103A | V104I | Q206E | N261D | ||||||||
V4E | N76D | S103A | V104I | Q206E | ||||||||
N76D | N77D | S103A | V104I | Q206E | ||||||||
N76D | S103A | V104I | A158E | |||||||||
N76D | S103A | V104I | Q206E | |||||||||
V4E | N76D | S103A | V104I | G159D | L217E | K251Q | ||||||
V4E | N76D | S103A | V104I | G159D | L217E | N252D | ||||||
N76D | N77D | S103A | V104I | A133T | N185D | K251T | ||||||
N76D | S103A | V104I | G159D | Q206E | V244A | |||||||
v4E | N76D | S103A | V104I | S188E | ||||||||
V4E | N76D | S103A | V104I | A158E | ||||||||
N76D | N77D | S103A | V104I | N185D |
表4(续)
N76D | S103A | V104I | Q206E | K251T | ||||||||
A48T | N76D | S103A | V104I | L111M | G159D | |||||||
V68A | N76D | S103A | V104I | G159D | Q236H | |||||||
L42V | N76D | S103A | V104I | G159D | ||||||||
Q12H | N62H | N76D | S103A | V104I | G159D | |||||||
L42I | N76D | S103A | V104I | G159D | ||||||||
N76D | S103A | V104I | G146S | G159D | ||||||||
N76D | S103A | V104I | G159D | N238S | ||||||||
N76D | S103A | V104I | G159D | T224A | ||||||||
N76D | S103A | V104I | S212P | V268F | E271V | |||||||
N76D | E89A | S103A | V104I | |||||||||
N76D | S87R | S103A | V104I | S212P | E271V | |||||||
N76D | S103A | V104I | S212P | Q245L | E271V | |||||||
N76D | S103A | V104I | T134S | S141N | S212P | E271V | ||||||
N76D | S103A | V104I | S212P | Q236L | N243S | E271V | ||||||
N76D | S103A | V104I | Q109R | Q245R | ||||||||
N76D | S103A | V104I | Q109R | P210L | ||||||||
G20V | N62S | N76D | S103A | V104I |
表4(续)
V68A | N76D | S103A | V104I | Q236H | ||||||||
V68A | N76D | S103A | V104I | G159D | Q236H | E271V | ||||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245R | ||||||
V68A | N76D | S103A | V104I | G159D | L217I | Q236H | E271V | |||||
H17Q | V68A | N76D | S103A | V104I | ||||||||
V68A | N76D | S103A | V104I | |||||||||
V68A | N76D | S103A | V104I | G159D | Q236R | |||||||
V68A | L75R | N76D | S103A | V104I | G159D | Q236H | ||||||
V68A | N76D | N76D | S103A | A114V | V121I | G159D | Q236H | Q245R | ||||
Q12R | V68A | N76D | S103A | V104I | G159D | Q236H | ||||||
V68A | N76D | S103A | V104I | G159D | Y209S | Q236H | T253K | |||||
V68A | N76D | S103A | V104I | N117K | G159D | N184S | Q236H | |||||
V68A | N76D | S103A | V104I | G159D | Q236H | N243I | ||||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245L | ||||||
V68A | N76D | S103A | V104I | A142V | G159D | |||||||
V68A | N76D | S103A | V104I | N123S | G159D | Q236H | H249Y | |||||
V68A | N76D | S103A | V104I | G159D | Q236H | H249Q | ||||||
N76D | S103A | V104I | M222S | Q245R |
表4(续)
N76D | S103A | V104I | Q12R | M222S | H249R | |||||||
N76D | S103A | V104I | N173R | M222S | ||||||||
N76D | S103A | V104I | M222S | Y263F | ||||||||
L21M | N76D | S103A | V104I | M222S | K237R | Y263F | ||||||
N76D | S103A | V104I | Q109R | M222S | ||||||||
N76D | S103A | V104I | Q109R | M222S | E271D | |||||||
G61R | N76D | S103A | V104I | M222S | ||||||||
N76D | S103A | V104I | Q137R | M222S | ||||||||
N76D | S103A | V104I | Q109R | M222S | N248S | |||||||
N76D | S103A | V104I | M222S | H249R | ||||||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245R | N261D | |||||
V68A | N76D | S103A | V104I | S141N | G159D | Q236H | Q245R | T255S | ||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245R | R247H | |||||
V68A | N76D | S103A | V104I | G159D | A174V | N204D | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | N204D | Q236H | Q245R | |||||
V68A | N76D | S103A | V104I | A133V | G159D | N218D | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
V68A | N76D | S103A | V104I | G159D | A194I | V203A | Q236H | Q245R |
表4(续)
Q12R | N76D | S103A | V104I | M222S | Q245R | |||||||
N76D | S103A | V104I | A232V | Q245R | ||||||||
S24T | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | |||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||
Q12R | N76D | S103A | I104T | M222S | V244I | Q245R | ||||||
Q12R | N76D | S103A | M222S | P210T | Q245R | |||||||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | ||||||
T22K | V68A | N76D | S103A | V104I | ||||||||
V68A | N76D | S103A | N184D | |||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | N140D | G159D | A232V | Q236H | Q245R | N252K | ||||
N43S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||
N43K | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||
V68A | S87G | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | R275S | |||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N248S | L262M |
表4(续)
Q12R | N76D | S103A | I104T | S130T | A215V | M222S | Q245R | |||||
Q12R | N76D | S103A | I104T | S130T | M222S | V227A | Q245R | L262S | ||||
Q12R | N76D | S103A | I104T | S130T | A215T | M222S | Q245R | |||||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N261D | |||||
N76D | S103A | I104T | S130T | M222S | Q245R | |||||||
Q12R | N76D | S103A | I104T | S130T | N218D | M222S | Q245R | L262S | N269D | |||
Q12R | S57P | N76D | S103A | I104T | S130T | M222S | Q245R | K251Q | ||||
Q12R | N76D | S103A | I104T | S130T | R170S | N185D | M222S | N243D | Q245R | |||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | V268A | |||||
Q12R | N76D | S103A | I104T | S130T | M222S | P210S | Q245R | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||
V68A | S103A | V104I | N116D | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | |||||
R10C | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | V203E | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | K237E | Q245R | |||||
V68A | N76D | I79N | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | S103A | V104I | G159D | N183D | A232V | Q236H | Q245R |
表4(续)
V68A | S103A | V104I | G159D | A174V | Q206L | A232V | Q236H | Q245R | ||||
V68A | S103A | V104I | G159D | S188C | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A230T | A232V | Q236H | Q245R | |||||
V68A | A98T | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A215T | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248S | |||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
V68A | N76D | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | ||||
N76D | S103A | V104I | A232V | Q236H | Q245R | L257V | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | R275H | ||||
N76D | S103A | V104I | L257V | R275H | ||||||||
V68A | S103A | V104I | G159D | T224A | A232V | Q236H | Q245R | L257V | ||||
N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||
V68A | N76D | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | G211R | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | G211V | A232V | Q236H | Q245R | ||||
Q12R | V68A | N76D | S103A | V104I | G159D | Y214L | A232V | Q236H | Q245R |
表4(续)
V68A | N76D | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | ||||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
G20R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S259G | |||
V68A | S87R | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | T260V | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261G | ||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261W | ||||
N76D | S103A | V104I | A232V | Q236H | S242P | Q245R | ||||||
V68A | N76D | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | ||||
Q12R | A48V | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||
N76D | S103A | V104I | A232V | Q236H | Q245R | |||||||
N76D | S103A | V104I | G159D | Y192F | A232V | Q236H | Q245R | |||||
N76D | S103A | V104I | V147I | G159D | A232V | Q236H | Q245R | N248S | K251R | |||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | A272S | |||
V68A | N76D | S103A | V104I | G159D | N183K | Q206L | A232V | Q236H | Q245R | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S256R | ||||
V68A | N76D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | ||||
K27R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | N116T | G159D | R170S | N185S | A232V | Q236H | Q245R |
表4(续)
G61E | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | S99N | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | Y209F | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D | |||
V68A | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210T | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210S | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | N185D | P210L | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212A | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K |
表4(续)
V68A | S103A | V104I | G159D | S212E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | T213S | A232V | Q236H | Q245R | N248D | N252K | ||||
V68A | A103V | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213G | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A215V | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216T | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216V | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216C | A232V | Q236H | Q245R | N248D | N252K | |||
G20A | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | N173D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | K251V | N252K | |||
V68A | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252L | ||||
P55S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F |
表4(续)
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T255V | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256N | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256E | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | L257R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | G258D | |||
I8V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N269D | ||
V68A | S103A | V104I | N116S | G159D | A232V | Q236H | Q245R | N248D | N252K | T260E | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | A232S | Q236H | Q245R | N248D | N252K | |||||
V68A | S103A | V104I | G159D | A232V | Q236R | Q245R | N248D | N252K | ||||
N18S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245V | N248D | N252K | ||||
V68A | N76D | S101T | S103A | V104I | G159D | T213R | N218S | A232V | Q236H | Q245R | T260A |
表4(续)
V68A | S103A | V104I | G159D | A228V | A232V | Q236H | Q245R | N248D | N252K | |||
T33S | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | N76D | E89D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | |
G61E | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | V205I | P210I | A232V | Q236H | Q245R | |||||
G61E | V68A | S103A | V104I | S130A | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
G61E | V68A | S103A | V104I | A133S | Q137R | G159D | A232V | Q236H | Q245R | N248D | N252K | |
G61E | S103A | V104I | A133V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248G | N252K | ||||
V68A | S103A | V104I | G159D | N218S | A232V | Q236H | Q245R | N248D | N252K | |||
G61E | V68A | S103A | V104I | G159D | S160V | A232V | Q236H | Q245R | N248D | N252K | ||
S3L | G61E | V68A | N76D | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | ||
G61E | V68A | S103A | V104I | G159D | S167F | A232V | Q236H | Q245R | N248D | N252K | ||
G97E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
A98D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S99E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K |
表4(续)
G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | S106E | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | Q109E | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | ||||
S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
N62D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | S166D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | L217E | A232V | Q236H | Q245R | N248D | N252K | ||||
G20R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
S103A | V104I | G159D | Q206R | L217E | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | |||
S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
K27N | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
T38G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
T38A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A |
表4(续)
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | E271G | ||
V68A | N76D | S103A | V104I | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | ||
V68A | N76D | S103A | V104I | G159D | P210I | T213R | A232V | Q236H | Q245R | T260A | ||
V68A | N76D | S103A | V104I | G159D | V205I | T213R | A232V | Q236H | Q245R | T260A | ||
V68A | N76D | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | T260A | |||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | ||||
N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | ||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A230V | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | L126F | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | |||||
S103A | V104I | G159D | A230V | Q236H | Q245R | |||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | T260A | |||||
S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||
V68A | S103A | V104I | G159D | A174V | A232V | Q236H | Q245R | L257V | ||||
V68A | S103A | V104I | G159D | A194S | A232V | Q236H | Q245R | L257V |
表4(续)
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | ||||
S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | ||||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | N261W | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | N261W | ||||
S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||
S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | |||||
V68A | N76D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | ||
Q12R | S103A | V104I | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | |||
S103A | V104I | Y209W | A232V | Q236H | Q245R | L257V | ||||||
S103A | V104I | G159D | V205I | P210I | T213R | A232V | Q236H | Q245R | T260A | |||
S103A | V104I | G159D | V205I | Y209W | A232V | Q236H | Q245R | T260A | ||||
V68A | S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | |||
S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | L257V | |||
S103A | V104I | G159D | V205I | Y209W | A232V | Q236H | Q245R | L257V | ||||
V68A | S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | T260A | ||
S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | ||||
S103A | V104I | G159D | Y209W | P210I | A232V | Q236H | Q245R |
表4(续)
S103A | V104I | G159D | V205I | P210I | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | |||||
A48V | S103A | V104I | G159D | A230V | Q236H | Q245R | ||||||
A48V | V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | ||||
A48V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
A48V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | N261W | |||
G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
Q12R | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
G102A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | N184S | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | N184G | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | A232V | Q236H | V244T | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | A232V | Q236H | V244A | Q245R | N248D | N252K | ||||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | S256R | ||
Q12R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K |
表4(续)
S101G | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | Q206E | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | T213Q | A232V | Q236H | Q245R | N248D | N252K | |||
A98L | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | N248D | N252K | |||
N62D | S103A | V104I | Q109R | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S103A | V104I | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S101G | S103A | V104I | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | |
S103A | V104I | G159D | A232V | Q245R | N248D | N252K | ||||||
S103A | V104I | G159D | A230V | Q245R | ||||||||
N62D | S103A | V104I | S130G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | S128G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S101G | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S103A | V104I | S128G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K |
表4(续)
N62D | S103A | V104I | S128L | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260A | |||
S101G | S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
A98V | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S99G | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | A230V | Q236H | Q245R | ||||||
S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | |||
N76D | S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | G159D | A230V | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S103A | V104I | G159D | N185D | Q206E | T213R | A232V | Q236H | Q245R | N248D | N252K | E271Q |
实施例2
用美国系列号60/068,796“一种测定优选的酶和/或优选的去污剂组合物的改进方法”中所述的微观(microswatch)测定法检测如实施例1所述制备的大量蛋白酶变体在两种类型的去污剂及洗涤条件中的表现。
表5中列出检测的变体以及在两种不同去污剂中测试的结果。A栏;0.67g/l过滤的Ariel Ultra(Procter & Gamble,Cincinnati,OH,美国),溶于每加仑含有3格令混合Ca2+/Mg2+硬度的溶液中,20℃下每孔使用0.3ppm的酶。B栏;3.38g/l过滤的Ariel Futur(Procter & Gamble,Cincinnati,OH,美国),溶于每加仑含有15格令混合Ca2+/Mg2+硬度的溶液中,40℃下每孔使用0.3ppm的酶。
表5
A | B | |||||||||||
N76D | S103A | V104I | 1 | 1 | ||||||||
S103A | V104I | A228T | 0.56 | 1.11 | ||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | 1.4 | 1.85 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.77 | 1.20 | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | 2.26 | 1.67 | ||||
V68A | S103A | V104I | N140D | G159D | A232V | Q236H | Q245R | N252K | 2.96 | 1.42 | ||
N43S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | 1.91 | 1.80 | ||
N43K | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | 2.05 | 1.78 | |||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | 2.00 | 1.34 | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | 2.38 | 1.67 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | 2.83 | 0.53 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | K237E | Q245R | 2.87 | 0.20 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252S | 2.56 | 1.41 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | R275H | 3.97 | 0.47 | ||
V68A | S103A | V104I | G159D | T224A | A232V | Q236H | Q245R | L257V | 3.35 | 1.28 | ||
G61E | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 3.77 | 0.09 |
表5(续)
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 3.50 | 0.47 | |
V68A | S103A | V104I | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | 2.8 | 1.46 | |
N76D | A98E | S103A | V104I | 1.56 | 0.28 | |||||||
V4E | N76D | S103A | V104I | 1.22 | 0.33 | |||||||
N76D | N77D | S103A | V104I | 1.13 | 0.36 | |||||||
A16T | N76D | S103A | V104I | N248D | 1.22 | 0.43 | ||||||
A1E | N76D | S103A | V104I | 1.12 | 0.32 | |||||||
N76D | S103A | V104I | N261D | 1.54 | 0.33 | |||||||
N76D | S103A | V104I | S216C | 1.04 | 0.13 | |||||||
N76D | N77D | S103A | V104I | A174V | 1.09 | 0.35 | ||||||
T38S | N76D | S103A | V104I | K237Q | 1.11 | 0.55 | ||||||
N76D | S103A | V104I | N183D | 1.50 | 0.25 | |||||||
R19L | N76D | S103A | V104I | 1.11 | 0.48 | |||||||
R19C | N76D | S103A | V104I | 1.05 | 0.19 | |||||||
N76D | S103A | V104I | N184D | 1.32 | 0.29 | |||||||
N76D | S103A | V104I | N252D | 1.19 | 0.53 | |||||||
N76D | S103A | V104I | S259C | 0.92 | 0.12 | |||||||
N76D | S103A | V104I | K251T | 1.31 | 0.43 |
表5(续)
N76D | P86S | S103A | V104I | 1.00 | 0.98 | |||||||
I72V | N76D | S103A | V104I | N185D | 1.70 | 0.37 | ||||||
N76D | S103A | V104I | K237E | T274A | 1.12 | 0.16 | ||||||
N76D | S103A | V104I | A228V | 1.13 | 0.99 | |||||||
N76D | S103A | V104I | G159D | 1.88 | 0.23 | |||||||
H17L | N76D | S103A | V104I | K237E | 1.29 | 0.28 | ||||||
N76D | S103A | V104I | S130L | 0.52 | 0.71 | |||||||
N76D | S103A | V104I | Q109R | 0.23 | 1.26 | |||||||
N76D | S99R | S103A | V104I | N204T | 0.21 | 0.87 | ||||||
N76D | S103A | V104I | D181N | 0.24 | 1.07 | |||||||
Q12R | N76D | S103A | V104I | 0.61 | 1.31 | |||||||
N76D | S103A | V104I | S212P | E271V | 0.69 | 1.35 | ||||||
N76D | S103A | V104I | N252K | N261Y | 0.37 | 1.02 | ||||||
N76D | S103A | V104I | S242T | 0.98 | 0.92 | |||||||
N76D | S103A | V104I | E271Q | 0.63 | 1.25 | |||||||
Q12R | N76D | S103A | V104I | S242T | 0.49 | 1.32 | ||||||
N43S | N76D | S103A | V104I | N116K | N183I | 0.39 | 1.10 | |||||
N76D | S103A | V104I | G258R | 0.34 | 1.17 |
表5(续)
N76D | S103A | V104I | E271G | 0.57 | 1.25 | |||||||
N76D | S103A | V104I | Q182R | I198V | 0.22 | 0.95 | ||||||
L21M | N76D | S103A | V104I | Q182R | 0.24 | 0.98 | ||||||
N76D | S103A | V104I | M119I | Q137R | 0.13 | 0.91 | ||||||
N76D | S103A | V104I | Q137R | N248S | 0.16 | 1.02 | ||||||
A13T | N76D | S103A | V104I | Q206R | 0.31 | 1.01 | ||||||
N76D | S103A | V104I | Q206R | 0.33 | 1.02 | |||||||
N76D | S103A | V104I | S212P | G258R | 0.38 | 1.06 | ||||||
T58S | N76D | S103A | V104I | E271G | 0.84 | 1.26 | ||||||
N76D | S103A | V104I | Q206E | N261D | 1.97 | 0.04 | ||||||
V4E | N76D | S103A | V104I | Q206E | 1.51 | 0.05 | ||||||
N76D | N77D | S103A | V104I | Q206E | 1.40 | 0.04 | ||||||
N76D | S103A | V104I | A158E | 1.95 | 0.16 | |||||||
N76D | S103A | V104I | Q206E | 2.41 | 0.88 | |||||||
N76D | N77D | S103A | V104I | A133T | N185D | K251T | 1.34 | 0.03 | ||||
N76D | S103A | V104I | Q206E | N261D | 1.78 | 0.04 | ||||||
N76D | S103A | V104I | G159D | Q206E | V244A | 2.16 | 0.04 | |||||
V4E | N76D | S103A | V104I | S188E | 1.91 | 0.04 |
表5(续)
V4E | N76D | S103A | V104I | A158E | 2.06 | 0.04 | ||||||
N76D | S103A | V104I | Q206E | K251T | 1.73 | 0.06 | ||||||
A48T | N76D | S103A | V104I | L111M | G159D | 2.04 | 0.16 | |||||
V68A | N76D | S103A | V104I | G159D | Q236H | 3.20 | 0.09 | |||||
L42V | N76D | S103A | V104I | G159D | 1.83 | 0.17 | ||||||
Q12H | N62H | N76D | S103A | V104I | G159D | 1.42 | 0.14 | |||||
L42I | N76D | S103A | V104I | G159D | 1.86 | 0.18 | ||||||
N76D | S103A | V104I | G146S | G159D | 1.87 | 0.19 | ||||||
N76D | S103A | V104I | G159D | N238S | 1.90 | 0.15 | ||||||
N76D | S103A | V104I | G159D | T224A | 1.61 | 0.07 | ||||||
N76D | S103A | V104I | S212P | V268F | E271V | 0.44 | 1.42 | |||||
N76D | S87R | S103A | V104I | S212P | E271V | 0.39 | 2.03 | |||||
N76D | S103A | V104I | S212P | Q245L | E271V | 0.62 | 1.79 | |||||
N76D | S103A | V104I | Q109R | Q245R | 0.11 | 1.78 | ||||||
N76D | S103A | V104I | Q109R | P210L | 0.12 | 1.21 | ||||||
G20V | N62S | N76D | S103A | V104I | 1.63 | 0.78 | ||||||
V68A | N76D | S103A | V104I | Q236H | 2.37 | 0.44 | ||||||
V68A | N76D | S103A | V104I | G159D | Q236H | E271V | 2.97 | 0.45 |
表5(续)
V68A | N76D | S103A | V104I | G159D | Q236H | Q245R | 3.00 | 0.61 | ||||
V68A | N76D | S103A | V104I | G159D | L217I | Q236H | E271V | 2.71 | 0.12 | |||
H17Q | V68A | N76D | S103A | V104I | 2.46 | 0.38 | ||||||
V68A | N76D | S103A | V104I | 2.46 | 0.61 | |||||||
V68A | N76D | S103A | V104I | G159D | Q236R | 3.31 | 0.11 | |||||
V68A | L75R | N76D | S103A | V104I | G159D | Q236H | 3.06 | 0.14 | ||||
V68A | N76D | S103A | V104I | A114V | V121I | G159D | Q236H | Q245R | 3.11 | 0.40 | ||
Q12R | V68A | N76D | S103A | V104I | G159D | Q236H | 3.12 | 0.34 | ||||
V68A | N76D | S103A | V104I | G159D | Y209S | Q236H | T253K | 3.18 | 0.03 | |||
V68A | N76D | S103A | V104I | N117K | G159D | N184S | Q236H | 2.78 | 0.06 | |||
V68A | N76D | S103A | V104I | Q236H | 2.49 | 0.57 | ||||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245L | 3.37 | 0.03 | ||||
V68A | N76D | S103A | V104I | N123S | G159D | Q236H | H249Y | 3.11 | 0.03 | |||
V68A | N76D | S103A | V104I | G159D | Q236H | H249Q | 3.15 | 0.04 | ||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245R | N261D | 3.31 | 0.03 | |||
V68A | N76D | S103A | V104I | S141N | G159D | Q236H | Q245R | T255S | 3.26 | 0.62 | ||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245R | R247H | 2.78 | 0.03 | |||
V68A | N76D | S103A | V104I | G159D | A174V | N204D | Q236H | Q245R | 3.28 | 0.02 |
表5(续)
V68A | N76D | S103A | V104I | G159D | N204D | Q236H | Q245R | 3.34 | 0.02 | |||
V68A | N76D | S103A | V104I | A133V | G159D | N218D | Q236H | Q245R | 3.28 | 0.03 | ||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | 2.91 | 0.58 | |||
V68A | N76D | S103A | V104I | G159D | A194I | V203A | Q236H | Q245R | 2.86 | 0.13 | ||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | 1.30 | 1.73 | |
T22K | V68A | N76D | S103A | V104I | 1.83 | 1.13 | ||||||
V68A | N76D | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | 1.28 | 1.54 | ||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | 3.72 | 0.8 | ||
N76D | S103A | V104I | A232V | Q236H | Q245R | L257V | 0.6 | 1.5 | ||||
N76D | S103A | V104I | L257V | R275H | 1.91 | 0.15 | ||||||
N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | 1.92 | 1.09 | |||
V68A | N76D | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | 3.57 | 0.99 | ||
V68A | N76D | S103A | V104I | G159D | G211R | A232V | Q236H | Q245R | 1.74 | 1.76 | ||
V68A | N76D | S103A | V104I | G159D | G211V | A232V | Q236H | Q245R | 3.15 | 1.06 | ||
Q12R | V68A | N76D | S103A | V104I | G159D | Y214L | A232V | Q236H | Q245R | 2.33 | 1.92 | |
V68A | N76D | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | 1.67 | 1.45 | ||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | 2.16 | 1.72 | ||
G20R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S259G | 2.77 | 1.59 |
表5(续)
V68A | N76D | S87R | S103A | V104I | G159D | A232V | Q236H | Q245R | T260V | 2.62 | 1.49 | |
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261G | 2.92 | 0.68 | ||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261W | 2.17 | 1.37 | ||
N76D | S103A | V104I | A232V | Q236H | S242P | Q245R | 0.48 | 1.2 | ||||
V68A | N76D | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | 2.92 | 0.76 | ||
Q12R | A48V | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | 2.09 | 1.86 | |
N76D | S103A | V104I | A232V | Q236H | Q245R | 0.51 | 1.44 | |||||
N76D | S103A | V104I | G159D | Y192F | A232V | Q236H | Q245R | 1.60 | 1.14 | |||
N76D | S103A | V104I | V147I | G159D | A232V | Q236H | Q245R | N248S | K251R | 1.35 | 1.29 | |
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | A272S | 1.92 | 1.81 | |
V68A | N76D | S103A | V104I | G159D | N183K | Q206L | A232V | Q236H | Q245R | 1.17 | 1.53 | |
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S256R | 2.01 | 1.72 | ||
V68A | N76D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | 2.09 | 1.62 | ||
K27R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | 3.00 | 1.08 | ||
V68A | N76D | S103A | V104I | N116T | G159D | R170S | N185S | A232V | Q236H | Q245R | ND | ND |
N76D | S103A | V104I | M222S | Q245R | 1.01 | 1.23 | ||||||
Q12R | N76D | S103A | V104I | M222S | H249R | 0.57 | 1.65 | |||||
N76D | S103A | V104I | N173R | M222S | 0.86 | 0.46 |
表5(续)
N76D | S103A | V104I | M222S | Y263F | 1.24 | 0.77 | ||||||
L21M | N76D | S103A | V104I | M222S | K237R | Y263F | 1.18 | 0.76 | ||||
N76D | S103A | V104I | Q109R | M222S | 0.52 | 1.16 | ||||||
N76D | S103A | V104I | Q109R | M222S | E271D | 0.56 | 1.12 | |||||
G61R | N76D | S103A | V104I | M222S | 0.43 | 0.96 | ||||||
N76D | S103A | V104I | Q137R | M222S | 0.42 | 1.25 | ||||||
N76D | S103A | V104I | M222S | H249R | 1.15 | 1.01 | ||||||
Q12R | N76D | S103A | V104I | M222S | Q245R | 0.53 | 1.46 | |||||
N76D | S103A | V104I | A232V | Q245R | 0.69 | 1.56 | ||||||
Q12R | N76D | S103A | I104T | M222S | V244I | Q245R | 0.66 | 1.74 | ||||
Q12R | N76D | S103A | V104I | M222S | P210T | Q245R | 0.52 | 1.56 | ||||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | 0.70 | 1.61 | ||||
Q12R | N76D | S103A | I104T | S130T | A215V | M222S | Q245R | 0.79 | 1.85 | |||
Q12R | N76D | S103A | I104T | S130T | M222S | V227A | Q245R | L262S | 0.78 | 1.56 | ||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N261D | 1.25 | 1.30 | |||
N76D | S103A | I104T | S130T | M222S | Q245R | 1.29 | 1.30 | |||||
Q12R | S57P | N76D | S103A | I104T | S130T | M222S | Q245R | K251Q | 1.44 | 0.16 | ||
Q12R | N76D | S103A | I104T | S130T | R170S | N185D | M222S | N243D | Q245R | 2.01 | 0.04 |
表5(续)
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | V268A | 0.77 | 1.60 | |||
Q12R | N76D | S103A | I104T | S130T | M222S | P210S | Q245R | 0.73 | 1.66 | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | 2.09 | 0.86 |
实施例3
表6中列出检测的来自实施例1中的变体以及在四种不同去污剂中测试的结果。进行如实施例2中的相同操作考察了变体蛋白酶在下列去污剂中的洗涤性能。A栏;0.67g/l过滤的Ariel Ultra(Procter & Gamble,Cincinnati,OH,美国),溶于每加仑含有3格令混合Ca2+/Mg2+硬度的溶液中,20℃下每孔使用0.3ppm的酶。B栏;3.38g/l过滤的Ariel Futur(Procter & Gamble,Cincinnati,OH,美国),溶于每加仑含有15格令混合Ca2+/Mg2+硬度的溶液中,40℃下每孔使用0.3ppm的酶。C栏,3.5g/LHSP1去污剂(Procter & Gamble,Cincinnati,OH,美国),溶于每加仑含有8格令混合Ca2+/Mg2+硬度的溶液中,20℃下每孔使用0.3ppm的酶。D栏,1.5ml/l Tide KT去污剂(Procter & Gamble,Cincinnati,OH,美国),溶于每加仑含有3格令混合Ca2+/Mg2+硬度的溶液中,20℃下每孔使用0.3ppm的酶。
表6
A | B | C | D | |||||||||||||
N76D | S103A | V104I | 1 | 1 | 1 | 1 | ||||||||||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 1.44 | 1.41 | 1.39 | 1.26 | |||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | 2.34 | 1.49 | 1.65 | 2.35 | |||
V68A | S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | 1.05 | 1.41 | 1.20 | 1.19 | |||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 1.81 | 1.72 | 1.66 | 1.31 | |||
V68A | S103A | V104I | G159D | Y209F | A232V | Q236H | Q245R | N248D | N252K | 2.19 | 1.38 | 1.60 | 2.02 | |||
V68A | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | 2.91 | 0.91 | 1.48 | 2.70 | |||
V68A | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | N248D | N252K | 0.93 | 1.39 | 1.23 | 0.80 | |||
V68A | S103A | V104I | G159D | N185D | P210L | A232V | Q236H | Q245R | N248D | N252K | 2.67 | 0.86 | 1.41 | 2.88 | ||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | N248D | N252K | 2.22 | 1.43 | 1.55 | 1.78 | |||
V68A | S103A | V104I | G159D | S212C | A232V | Q236H | Q245R | N248D | N252K | 2.30 | 1.43 | 1.63 | 2.07 | |||
V68A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 2.31 | 1.47 | 1.62 | 2.01 | |||
V68A | S103A | V104I | G159D | S212E | A232V | Q236H | Q245R | N248D | N252K | 2.63 | 0.56 | 1.36 | 2.66 | |||
V68A | S103A | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | 2.75 | 0.50 | 1.27 | 2.78 | |||
V68A | S103A | V104I | T213S | A232V | Q236H | Q245R | N248D | N252K | 1.11 | 1.38 | 1.31 | 0.75 |
表6(续)
V68A | A103V | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | 2.27 | 0.15 | 1.12 | 2.01 | |||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 1.37 | 1.42 | 1.37 | 1.06 | |||
V68A | S103A | V104I | G159D | A215V | A232V | Q236H | Q245R | N248D | N252K | 2.14 | 1.40 | 1.53 | 1.54 | |||
V68A | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | N248D | N252K | 1.22 | 1.58 | 1.47 | 1.20 | |||
V68A | S103A | V104I | G159D | S216T | A232V | Q236H | Q245R | N248D | N252K | 2.12 | 1.36 | 1.56 | 1.56 | |||
V68A | S103A | V104I | G159D | S216V | A232V | Q236H | Q245R | N248D | N252K | 1.88 | 1.36 | 1.47 | 1.87 | |||
V68A | S103A | V104I | G159D | S216C | A232V | Q236H | Q245R | N248D | N252K | 2.24 | 0.33 | 1.07 | 2.89 | |||
V68A | S103A | V104I | G159D | N173D | A232V | Q236H | Q245R | N248D | N252K | 2.43 | 0.46 | 1.29 | 2.42 | |||
V68A | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | 0.98 | 1.46 | 1.24 | 0.95 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F | 2.52 | 1.00 | 1.42 | 2.42 | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252L | 2.05 | 1.13 | 1.30 | 1.85 | ||||
P55S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F | 2.61 | 0.91 | 1.43 | 3.22 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T255V | 2.18 | 1.36 | 1.58 | 1.72 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256N | 2.14 | 1.46 | 1.59 | 1.65 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256E | 2.46 | 0.77 | 1.33 | 2.58 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256R | 1.31 | 1.52 | 1.46 | 0.94 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260R | 1.21 | 1.41 | 1.31 | 1.05 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | L257R | 1.51 | 1.41 | 0.85 | 1.18 |
表6(续)
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | G258D | 2.56 | 0.59 | 1.30 | 2.64 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | 1.02 | 1.47 | 1.37 | 0.84 | |||
V68A | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | 1.04 | 1.50 | 1.32 | 0.73 | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245V | N248D | N252K | 2.60 | 0.93 | 1.41 | 2.67 | ||||
V68A | S103A | V104I | G159D | A228V | A232V | Q236H | Q245R | N248D | N252K | 2.31 | 1.38 | 1.53 | 1.57 | |||
G61E | V68A | S103A | V104I | S130A | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.83 | 0.25 | 133 | 2.44 | ||
G61E | S103A | V104I | A133V | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.10 | 0.97 | 1.36 | 2.29 | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248G | N252K | 1.37 | 1.54 | 0.89 | 1.27 | ||||
V68A | S103A | V104I | G159D | N218S | A232V | Q236H | Q245R | N248D | N252K | 2.30 | 1.50 | 1.62 | 1.56 | |||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 1.72 | 1.72 | 1.67 | 1.15 | |||
V68A | N76D | E89D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | 1.32 | 1.30 | 1.11 | 1.28 | |
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.50 | 0.83 | 1.43 | 2.25 | |||
G61E | V68A | S103A | V104I | G159D | S160V | A232V | Q236H | Q245R | N248D | N252K | 4.20 | 0.07 | ND | 1.28 | ||
S3L | G61E | V68A | N76D | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | 3.47 | 0.60 | ND | 1.45 | ||
G61E | V68A | S103A | V104I | G159D | Y167F | A232V | Q236H | Q245R | N248D | N252K | 4.32 | 0.79 | ND | 1.55 | ||
G97E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 3.14 | 0.41 | ND | 1.40 | ||||
A98D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.71 | 0.68 | ND | 1.72 | ||||
S99E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.97 | 0.68 | ND | 1.71 |
表6(续)
S101E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 3.50 | 0.27 | ND | 1.90 | ||||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.24 | 1.80 | ND | 1.33 | ||||
G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 3.35 | 1.33 | ND | 1.69 | ||||
S103A | V104I | S106E | G159D | A232V | Q236H | Q245R | N248D | N252K | 4.88 | 0.55 | ND | 2.71 | ||||
S103A | V104I | Q109E | G159D | A232V | Q236H | Q245R | N248D | N252K | 4.22 | 1.05 | ND | 2.40 | ||||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | 5.45 | 2.19 | ND | 2.58 | ||||
S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | 3.76 | 2.16 | ND | 1.82 | ||||
N62D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 7.42 | 0.13 | ND | 2.46 | ||||
S103A | V104I | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | 5.43 | 1.36 | ND | 2.84 | ||||
S103A | V104I | G159D | S166D | A232V | Q236H | Q245R | N248D | N252K | 5.12 | 1.21 | ND | 3.97 | ||||
S103A | V104I | G159D | L217E | A232V | Q236H | Q245R | N248D | N252K | 6.38 | 0.95 | ND | 3.09 | ||||
G20R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 3.17 | 2.83 | ND | 2.60 | ||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 4.38 | 1.92 | ND | 2.54 | |||
S103A | V104I | G159D | Q206R | L217E | A232V | Q236H | Q245R | N248D | N252K | 3.05 | 2.61 | ND | 1.10 | |||
N62D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | 4.09 | 2.46 | ND | 2.55 | |||
S103A | V104I | G159D | N184G | A232V | Q236H | Q245R | N248D | N252K | 2.32 | 2.08 | ND | 2.40 | ||||
S103A | V104I | G159D | A232V | Q236H | V244T | Q245R | N248D | N252K | 2.34 | 2.04 | ND | 1.86 | ||||
S103A | V104I | G159D | A232V | Q236H | V244A | Q245R | N248D | N252K | 2.24 | 2.11 | ND | 1.95 |
表6(续)
K27N | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.81 | 1.56 | ND | 2.47 | ||||
T38G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.30 | 2.09 | ND | 1.82 | ||||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | S256R | 2.63 | 2.66 | ND | 1.44 | ||
Q12R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 2.01 | 2.78 | ND | 1.99 | ||
N62D | S103A | V104I | G159D | N185D | Q206E | T213R | A232V | Q236H | Q245R | N248D | N252K | E271Q | 7.74 | 0.94 | ND | 5.39 |
S101G | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | 5.14 | 1.41 | ND | 1.92 | |||
S101G | S103A | V104I | G159D | Q206E | A232V | Q236H | Q245R | N248D | N252K | 4.97 | 0.57 | ND | 1.36 | |||
S101G | S103A | V104I | G159D | T213Q | A232V | Q236H | Q245R | N248D | N252K | 2.41 | 1.86 | ND | 1.01 | |||
A98L | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 4.42 | 0.50 | ND | 2.88 | |||
S101G | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 5.86 | 1.20 | ND | 3.84 | |||
G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 5.87 | 2.10 | ND | 3.19 | |||
Q12R | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 2.98 | 2.67 | ND | 2.17 | ||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 4.02 | 0.41 | ND | 2.25 | ||
S101G | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 6.63 | 2.07 | ND | 2.08 | ||
G102A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 2.03 | 2.48 | ND | 2.25 | |||
N62D | S103A | V104I | Q109R | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 2.96 | 2.76 | ND | 2.34 | ||
S103A | V104I | G159D | A232V | Q245R | N248D | N252K | 2.74 | 2.10 | ND | 1.86 | ||||||
S103A | V104I | G159D | A230V | Q245R | 2.11 | 2.35 | ND | 1.49 |
表6(续)
N62D | S103A | V104I | S130G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 3.42 | 0.71 | ND | 2.58 | ||
S101G | S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.59 | 1.32 | ND | 1.61 | |||
S101G | S103A | V104I | S128G | G159D | A232V | Q236H | Q245R | N248D | N252K | 1.30 | 1.23 | ND | 9.0 | |||
S101G | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.94 | 0.71 | ND | 1.08 | |||
N62D | S101G | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 3.17 | 0.83 | ND | 2.35 | ||
N62D | S103A | V104I | S128G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 2.15 | 1.38 | ND | 1.77 | ||
N62D | S103A | V104I | S128L | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 3.07 | 0.07 | ND | 1.45 | ||
S101G | S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.26 | 1.16 | ND | 3.05 | |||
A98V | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 1.82 | 1.34 | ND | 1.08 | |||
S99G | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 2.16 | 1.47 | ND | 1.20 | |||
S101G | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 1.79 | 1.38 | ND | 1.01 | |||
S101G | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | 1.15 | 1.18 | ND | 8.7 | |||
S101G | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | 1.47 | 1.23 | ND | 1.03 | |||
S101G | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | N248D | N252K | 1.90 | 1.38 | ND | 1.05 | |||
S101G | S103A | V104I | G159D | A230V | Q236H | Q245R | 1.55 | 1.51 | ND | 1.23 | ||||||
S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | 1.96 | 1.30 | ND | 1.10 | |||
N76D | S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | 2.49 | 0.80 | ND | 1.25 | ||
G61E | V68A | S103A | V104I | G159D | S160V | A232V | Q236H | Q245R | N248D | N252K | 420 | 7 | 128 | ND |
表6(续)
S3L | G61E | V68A | N76D | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | 347 | 60 | 145 | ND | ||
G61E | V68A | S103A | V104I | G159D | Y167F | A232V | Q236H | Q245R | N248D | N252K | 432 | 79 | 155 | ND | ||
G97E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 314 | 41 | 140 | ND | ||||
A98D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 271 | 68 | 172 | ND | ||||
S99E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 297 | 68 | 171 | ND | ||||
S101E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 350 | 27 | 190 | ND | ||||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 224 | 180 | 133 | ND | ||||
G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 335 | 133 | 169 | ND | ||||
S103A | V104I | S106E | G159D | A232V | Q236H | Q245R | N248D | N252K | 488 | 55 | 271 | ND | ||||
S103A | V104I | Q109E | G159D | A232V | Q236H | Q245R | N248D | N252K | 422 | 105 | 240 | ND | ||||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | 545 | 219 | 258 | ND | ||||
S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | 376 | 216 | 182 | ND | ||||
N62D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 742 | 13 | 246 | ND | ||||
S103A | V104I | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | 543 | 136 | 284 | ND | ||||
S103A | V104I | G159D | S166D | A232V | Q236H | Q245R | N248D | N252K | 512 | 121 | 397 | ND | ||||
S103A | V104I | G159D | L217E | A232V | Q236H | Q245R | N248D | N252K | 638 | 95 | 309 | ND | ||||
G20R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 317 | 283 | 260 | ND | ||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 438 | 192 | 254 | ND |
表6(续)
S103A | V104I | G159D | Q206R | L217E | A232V | Q236H | Q245R | N248D | N252K | 305 | 261 | 110 | ND | |||
N62D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | 409 | 246 | 255 | ND | |||
S103A | V104I | G159D | N184G | A232V | Q236H | Q245R | N248D | N252K | 232 | 208 | 240 | ND | ||||
S103A | V104I | G159D | A232V | Q236H | V244T | Q245R | N248D | N252K | 234 | 204 | 186 | ND | ||||
S103A | V104I | G159D | A232V | Q236H | V244A | Q245R | N248D | N252K | 224 | 211 | 195 | ND | ||||
K27N | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 281 | 156 | 247 | ND | ||||
T38G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 230 | 209 | 182 | ND | ||||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | S256R | 263 | 266 | 144 | ND | ||
Q12R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 201 | 278 | 199 | |||
N62D | S103A | V104I | G159D | N185D | Q206E | T213R | A232V | Q236H | Q245R | N248D | N252K | E271Q | 774 | 94 | 539 | ND |
S101G | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | 514 | 141 | 192 | ND | |||
S101G | S103A | V104I | G159D | Q206E | A232V | Q236H | Q245R | N248D | N252K | 497 | 57 | 136 | ND | |||
S101G | S103A | V104I | G159D | T213Q | A232V | Q236H | Q245R | N248D | N252K | 241 | 186 | 101 | ND | |||
A98L | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 442 | 50 | 288 | ND | |||
S101G | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 586 | 120 | 384 | ND | |||
G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 587 | 210 | 319 | ND | |||
Q12R | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 298 | 267 | 217 | ND | ||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 402 | 41 | 225 | ND |
表6(续)
S101G | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 663 | 207 | 208 | ND | ||
G102A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 203 | 248 | 225 | ND | |||
N62D | S103A | V104I | Q109R | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 296 | 276 | 234 | ND | ||
S103A | V104I | G159D | A232V | Q245R | N248D | N252K | 274 | 210 | 186 | ND | ||||||
S103A | V104I | G159D | A230V | Q245R | 211 | 235 | 149 | ND | ||||||||
N62D | S103A | V104I | S130G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 342 | 71 | 258 | ND | ||
S101G | S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | 259 | 132 | 161 | ND | |||
S101G | S103A | V104I | S128G | G159D | A232V | Q236H | Q245R | N248D | N252K | 130 | 123 | 90 | ND | |||
S101G | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | N248D | N252K | 294 | 71 | 108 | ND | |||
N62D | S101G | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 317 | 83 | 235 | ND | ||
N62D | S103A | V104I | S128G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 215 | 138 | 177 | ND | ||
N62D | S103A | V104I | S128L | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | 307 | 7 | 145 | ND | ||
S101G | S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | 226 | 116 | 305 | ND | |||
A98V | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 182 | 134 | 108 | ND | |||
S99G | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 216 | 147 | 120 | ND | |||
S101G | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | 179 | 138 | 101 | ND | |||
S101G | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | 115 | 118 | 87 | ND | |||
S101G | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | 147 | 123 | 103 | ND |
表6(续)
S101G | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | N248D | N252K | 190 | 138 | 105 | ND | |||
S101G | S103A | V104I | G159D | A230V | Q236H | Q245R | 155 | 151 | 123 | ND | ||||||
S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | 196 | 130 | 110 | ND | |||
N76D | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | 249 | 80 | 125 | ND |
Claims (10)
1.一种蛋白酶变体,其为在相应于选自解淀粉芽孢杆菌枯草杆菌蛋白酶232残基位置的氨基酸置换的如下蛋白酶变体:
V68A N76D S103A V104I G159D A232V Q236H Q245R,
N76D S103A V104I A232V Q245R,
S24T V68A N76D S103A V104I G159D A232V Q236H Q245R,
V68A S103A V104I G159D A232V Q236H Q245R N252K,
V68A N76D S103A V104I G159D T213R A232V Q236H Q245R T260A,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D A232V Q236H Q245R,
V68A S103A V104I N140D G159D A232V Q236H Q245R N252K,
N43S V68A S103A V104I G159D A232V Q236H Q245R N252K,
N43K V68A S103A V104I G159D A232V Q236H Q245R,
N43D V68A S103A V104I G159D A232V Q236H Q245R N252K,
V68A S87G S103A V104I G159D A232V Q236H Q245R N252K R275S,
V68A S103A V104I G159D A232V Q236H Q245R L257V,
V68A S103A V104I N116D G159D A232V Q236H Q245R,
V68A S103A V104I G159D A232V Q236H Q245R N248D,
R10C V68A S103A V104I G159D A232V Q236H Q245R,
V68A S103A V104I G159D V203E A232V Q236H Q245R,
V68A S103A V104I G159D A232V Q236H K237E Q245R,
V68A N76D I79N S103A V104I G159D A232V Q236H Q245R,
V68A S103A V104I G159D N183D A232V Q236H Q245R,
V68A S103A V104I G159D A174V Q206L A232V Q236H Q245R,
V68A S103A V104I G159D S188C A232V Q236H Q245R,
V68A S103A V104I G159D A230T A232V Q236H Q245R,
V68A A98T S103A V104I G159D A232V Q236H Q245R,
V68A S103A V104I G159D A215T A232V Q236H Q245R,
V88A S103A V104I G159D A232V Q236H Q245R N248S,
V68A N76D S103A V104I G159D A232V Q236H Q245R,
V68A N76D S103A V104I G159D P210R A232V Q236H Q245R,
V68A N76D S103A V104I G159D A232V Q236H Q245R L257V,
N76D S103A V104I A232V Q236H Q245R L257V,
V68A S103A V104I G159D A232V Q236H Q245R L257V R275H,
V68A S103A V104I G159D T224A A232V Q236H Q245R L257V,
N76D S103A V104I G159D A232V Q236H Q245R L257V,
V68A N76D S103A V104I G159D Y209W A232V Q236H Q245R,
V68A N76D S103A V104I G159D G211R A232V Q236H Q245R,
V68A N76D S103A V104I G159D G211V A232V Q236H Q245R,
Q12R V68A N76D S103A V104I G159D Y214L A232V Q236H Q245R,
V68A N76D S103A V104I G159D A215R A232V Q236H Q245R,
Q12R V68A N76D S103A V104I G159D A232V Q236H Q245R,
G20R V68A N76D S103A V104I G159D A232V Q236H Q245R S259G,
V68A S87R N76D S103A V104I G159D A232V Q236H Q245R T260V,
V68A N76D S103A V104I G159D A232V Q236H Q245R N261G,
V68A N76D S103A V104I G159D A232V Q236H Q245R N261W,
N76D S103A V104I A232V Q236H S242P Q245R,
V68A N76D S103A V104I G159D P210L A232V Q236H Q245R,
Q12R A48V V68A N76D S103A V104I G159D A232V Q236H Q245R,
N76D S103A V104I A232V Q236H Q245R,
N76D S103A V104I G159D Y192F A232V Q236H Q245R,
N76D S103A V104I V147I G159D A232V Q236H Q245R N248S K251R,
Q12R V68A N76D S103A V104I G159D A232V Q236H Q245R A272S,
V68A N76D S103A V104I G159D N183K Q206L A232V Q236H Q245R,
V68A N76D S103A V104I G159D A232V Q236H Q245R S256R,
V68A N76D S103A V104I G159D Q206R A232V Q236H Q245R,
K27R V68A N76D S103A V104I G159D A232V Q236H Q245R,
V68A N76D S103A V104I N116T G159D R170S N185S A232V Q236H Q245R,
G61E V68A S103A V104I G159D A232V Q236H Q245R N248D N252K,
N43D V68A S103A V104I G159D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D S212P A232V Q236H Q245R N248D N252K,
V68A S103A V104I S99N G159D N184D A232V Q236H Q245R N248D N252K,
S103A V104I G159D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D Y209W A232V Q236H Q245R N248D N252K,
V68A S103A V104I Q109R G159D A232V Q236H Q245R N248D N252K,
G20R V68A S103A V104I G159D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D Y209F A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K N261D,
V68A S103A V104I G159D N185D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D P210R A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D P210T A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D P210S A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D N185D P210L A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D P210L A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D S212A A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D S212G A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D S212E A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D T213E A232V Q236H Q245R N248U N252K,
V68A S103A V104I T213S A232V Q236H Q245R N248D N252K,
V68A A103V V104I G159D T213E A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D T213R A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D T213G A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D A215V A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D A215R A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D S216T A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D S216V A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D S216C A232V Q236H Q245R N248D N252K,
G20A V68A S103A V104I G159D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D N173D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D A232V Q236H Q245R N248D K251V N252K,
V68A S103A V104I G159D Q206R A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252F,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252L,
P55S V68A S103A V104I G159D A232V Q236H Q245R N248D N252F,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K T255V,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K S256N,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K S256E,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K S256R,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K T260R,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K L257R,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K G258D,
I8V V68A S103A V104I G159D A232V Q236H Q245R N248D N252K N269D,
V68A S103A V104I N116S G159D A232V Q236H Q245R N248D N252K T260E,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K N261R,
V68A S103A V104I G159D A232V Q236H Q245R N248D N252K N261D,
V68A N76D S103A V104I G159D A232V Q236H Q245R N248D N252K,
V68A S103A V104I A232V Q236H Q245R N248D N252K,
S103A V104I G159D A232S Q236H Q245R N248D N252K,
V68A S103A V104I G159D A232V Q236R Q245R N248D N252K,
N18S V68A S103A V104I G159D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D A232V Q236H Q245V N248D N252K,
V68A N76D S101T S103 AV104I G159D T213R N218S A232V Q236H Q245R T260A,
V68A S103A V104I G159D A228V A232V Q236H Q245R N248D N252K,
T33S V68A N76D S103A V104I G159D A232V Q236H Q245R N248D N252K,
V68A N76D E89D S103A V104I G159D P210L T213R A232V Q236H Q245R T260A,
G61E V68A N76D S103A V104I G159D A232V Q236H Q245R N248D N252K,
S103A V104I G159D V205I P210I A232V Q236H Q245R,
G61E V68A S103A V104I S130A G159D A232V Q236H Q245R N248D N252K,
G61E V68A S103A V104I A133S Q137R G159D A232V Q236H Q245R N248D N252K,
G61E S103A V104I A133V G159D A232V Q236H Q245R N248D N252K,
V68A S103A V104I G159D A232V Q236H Q245R N248G N252K,
V68A S103A V104I G159D N218S A232V Q236H Q245R N248D N252K,
G61E V68A S103A V104I G159D S160V A232V Q236H Q245R N248D N252K,
S3L G61E V68A N76D S103A V104I A232V Q236H Q245R N248D N252K,
G61E V68A S103A V104I G159D S167F A232V Q236H Q245R N248D N252K,
G97E S103A V104I G159D A232V Q236H Q245R N248D N252K,
A98D S103A V104I G159D A232V Q236H Q245R N248D N252K,
S99E S103A V104I G159D A232V Q236H Q245R N248D N252K,
S101E S103A V104I G159D A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D A232V Q236H Q245R N248D N252K,
G102A S103A V104I G159D A232V Q236H Q245R N248D N252K,
S103A V104I S106E G159D A232V Q236H Q245R N248D N252K,
S103A V104I Q109E G159D A232V Q236H Q245R N248D N252K,
S103A V104I G159D A232V Q236H Q245R N248D N252K N261R,
S103A V104I Q109R G159D A232V Q236H Q245R N248D N252K,
N62D S103A V104I G159D A232V Q236H Q245R N248D N252K,
S03A V104I G159D N184D A232V Q236H Q245R N248D N252K,
S103A V104I G159D S166D A232V Q236H Q245R N248D N252K,
S103A V104I G159D L217E A232V Q236H Q245R N248D N252K,
G20R N62D S103A V104I G1590 T213R A232V Q236H Q245R N248D N252K,
N62D S103A V104I G159D T213R A232V Q236H Q245R N248D N252K,
S103A V104I G159D Q206R L217E A232V Q236H Q245R N248D N252K,
N62D S103A V104I G159D Q206R A232V Q236H Q245R N248D N252K,
S103A V104I S130G G159D A232V Q236H Q245R N248D N252K,
S103A V104I P131V G159D A232V Q236H Q245R N248D N252K,
K27N S103A V104I G159D A232V Q236H Q245R N248D N252K,
T38G S103A V104I G159D A232V Q236H Q245R N248D N252K,
T38A N76D S103A V104I G159D T213R A232V Q236H Q245R T260A,
V68A N76D S103A V104I G159D T213R A232V Q236H Q245R T260A E271G,
V68A N76D S103A V104I G159D Y209W T213R A232V Q236H Q245R T260A,
V68A N76D S103A V104I G159D P210I T213R A232V Q236H Q245R T260A,
V68A N76D S103A V104I G159D V205I T213R A232V Q236H Q245R T260A,
V68A N76D S103A V104I G159D P210I A232V Q236H Q245R T260A,
V68A S103A V104I G159D T213R A232V Q236H Q245R T280A,
N76D S103A V104I G159D T213R A232V Q236H Q245R T260A,
V68A S103A V104I G159D Y209W A232V Q236H Q245R,
V68A S103A V104I G159D P210I A232V Q236H Q245R,
V68A S103A V104I G159D A230V A232V Q236H Q245R,
V68A S103A V104I G159D L126F A232V Q236H Q245R,
V68A S103A V104I G159D V205I A232V Q236H Q245R,
V68A S103A V104I G159D P210L A232V Q236H Q245R,
V68A S103A V104I G159D A232V Q236H Q245R T260A,
S103A V104I G159D A232V Q236H Q245R,
V68A S103A V104I G159D A174V A232V Q236H Q245R L257V,
V68A S103A V104I G159D A194S A232V Q236H Q245R L257V,
V68A S103A V104I G159D Y209W A232V Q236H Q245R L257V,
S103A V104I G159D A232V Q236H Q245R L257V,
V68A N76D S103A V104I G159D T213R A232V Q236H Q245R T260A N261W,
V68A S103A V104I G159D A232V Q236H Q245R L257V N261W,
S103A V104I G159D T213R A232V Q236H Q245R T260A,
S103A V104I G159D P210I A232V Q236H Q245R N248D N252K,
S103A V104I G159D Y209W A232V Q236H Q245R L257V,
V68A N76D S103A V104I G159D P210L T213R A232V Q236H Q245R T260A,
Q12R S103A V104I G159D Y209W T213R A232V Q236H Q245R T260A,
S103A V104I Y209W A232V Q236H Q245R L257V,
S103A V104I G159D V205I P210I T213R A232V Q236H Q245R T260A,
S103A V104I G159D V205I Y209W A232V Q236H Q245R T260A,
V68A S103A V104I G159D V205I Y209W P210I A232V Q236H Q245R,
S103A V104I G159D V205I Y209W P210I A232V Q236H Q245R L257V,
S103A V104I G159D V205I Y209W A232V Q236H Q245R L257V,
V68A S103A V104I G159D V205I Y209W P210I A232V Q236H Q245R T260A,
S103A V104I G159D V205I Y209W P210I A232V Q236H Q245R,
S103A V104I G159D Y209W P210I A232V Q236H Q245R,
S103A V104I G159D V205I P210I A232V Q236H Q245R,
V68A S103A V104I S128L G159D A232V Q236H Q245R,
A48V V68A S103A V104I G159D Y209W A232V Q236H Q245R,
A48V V68A S103A V104I G159D A232V Q236H Q245R N248D N252K,
A48V V68A S103A V104I G159D A232V Q236H Q245R L257V N261W,
G102A S103A V104I G159D S212G A232V Q236H Q245R N248D N252K,
Q12R G102A S103A V104I G159D S212G A232V Q236H Q245R N248D N252K,
S101G G102A S103A V104I G159D S212G A232V Q236H Q245R N248D N252K,
A98L G102A S103A V104I G159D S212G A232V Q236H Q245R N248D N252K,
G102A S103A V104I G159D T213R A232V Q236H Q245R N248D N252K,
S103A V104I P131V G159D A232V Q236H Q245R N248D N252K,
S103A V104I G159D N184S A232V Q236H Q245R N248D N252K,
S103A V104I G159D N184G A232V Q236H Q245R N248D N252K,
S103A V104I G159D A232V Q236H V244T Q245R N248D N252K,
S103A V104I G159D A232V Q236H V244A Q245R N248D N252K,
N62D S103A V104I G159D T213R A232V Q236H Q245R N248D N252K S256R,
Q12R N62D S103A V104I G159D T213R A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D N185D A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D Q206E A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D T213Q A232V Q236H Q245R N248D N252K,
A98L G102A S103A V104I G159D A232V Q236H Q245R N248D N252K,
S101G G102A S103A V104I G159D A232V Q236H Q245R N248D N252K,
A98L G102A S103A V104I G159D S212G A232V Q236H Q245R N248D N252K,
A98L G102A S103A V104I G159D S212G A232V Q236H N248D N252K,
N62D S103A V104I Q109R G159D T213R A232V Q236H Q245R N248D N252K,
N62D S103A V104I G159D S212G T213R A232V Q236H Q245R N248D N252K,
N62D S101G S103A V104I G159D S212G T213R A232V Q236H Q245R N248D N252K,
S103A V104I G159D A232V Q245R N248D N252K,
N62D S103A V104I S130G G159D T213R A232V Q236H Q245R N248D N252K,
S101G S103A V104I S130G G159D A232V Q236H Q245R N248D N252K,
S101G S103A V104I S128G G159D A232V Q236H Q245R N248D N252K,
S101G S103A V104I S128L G159D A232V Q236H Q245R N248D N252K,
N62D S101G S103A V104I G159D T213R A232V Q236H Q245R N248D N252K,
N62D S103A V104I S128G G159D T213R A232V Q236H Q245R N248D N252K,
N62D S103A V104I S128L G159D T213R A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D A232V Q236H Q245R N248D N252K T260A,
S101G S103A V104I P131V G159D A232V Q236H Q245R N248D N252K,
A98V S101G S103A V104I G159D A232V Q236H Q245F N248D N252K,
S99G S101G S103A V104I G159D A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D S212G A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D Y209W A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D P210I A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D V205I A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D A194P A232V Q236H Q245R N248D N252K,
N76D S101G S103A V104I G159D A194P A232V Q236H Q245R N248D N252K,
S101G S103A V104I G159D A230V A232V Q236H Q245R N248D N252K,
N62D S103A V104I G159D N185D Q206E A213R A232V Q245R Q245R N248D N252K E27Q。
2.权利要求1的蛋白酶变体,其还包含在位置62和230的一处或两处氨基酸残基的置换。
3.根据权利要求1的蛋白酶变体,其来源于芽孢杆菌属的枯草杆菌蛋白酶。
4.根据权利要求3的蛋白酶变体,其来源于迟缓芽孢杆菌枯草杆菌蛋白酶。
5.一种编码权利要求1的蛋白酶变体的DNA。
6.一种编码权利要求5的DNA的表达载体。
7.一种用权利要求6的表达载体转化的宿主细胞。
8.一种含有权利要求1的蛋白酶变体的清洁用组合物。
9.一种含有权利要求1的蛋白酶变体的动物饲料。
10.一种含有权利要求1的蛋白酶变体的用于处理纺织品的组合物。
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US08/956,564 | 1997-10-23 |
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