CN113549606A - 具有改变特性的α-淀粉酶变异体 - Google Patents

具有改变特性的α-淀粉酶变异体 Download PDF

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CN113549606A
CN113549606A CN202110696049.5A CN202110696049A CN113549606A CN 113549606 A CN113549606 A CN 113549606A CN 202110696049 A CN202110696049 A CN 202110696049A CN 113549606 A CN113549606 A CN 113549606A
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卡斯滕.安德森
亨里克.奥斯特达尔
彼得.斯卡格林德
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Abstract

本发明涉及具有改变特性的α‑淀粉酶变异体，具体地本发明涉及多肽，特别是Termamyl‑样α‑淀粉酶的变异体(突变体)，该变异体具有α‑淀粉酶活性且相对于所述亲本α‑淀粉酶表现出至少一种下列特性改变：底物特异性，底物结合，底物裂解方式，热稳定性，pH/活性谱，pH/稳定性谱，氧化稳定性，Ca2+依赖性，比活，特别是洗衣和洗碗的应用。

Description

具有改变特性的α-淀粉酶变异体
本申请是申请日为2005年07月05日，申请号为“201610851835.7”、名称为“具有改变特性的α-淀粉酶变异体”的发明专利申请的分案申请。
技术领域
本发明涉及亲本Termamyl-样α-淀粉酶的变异体(突变体)，该变异体具有α-淀粉酶活性且相对于所述亲本α-淀粉酶表现出至少一种下列特性改变：底物特异性，底物结合，底物裂解方式，热稳定性，pH活性谱(profile)，pH稳定性谱，氧化稳定性，Ca2+依赖性，pI减少和增加以及改进的洗涤效果，比活，在例如，高温和/或低/高pH条件下，特别是在低钙浓度下的稳定性，和在洗涤剂(detergent)存在下的稳定性，例如在洗涤剂中的贮存稳定性。本发明的变异体适合于淀粉转化，乙醇生产，衣物洗涤，盘子洗涤，硬表面清洁，纺织品脱浆(desizing)，和/或甜味剂生产。
背景技术
α-淀粉酶(α-1,4-葡聚糖-4-葡聚糖水解酶，E.C.3.2.1.1)由一组酶构成，它催化淀粉和其它直链和支链1,4-糖苷键寡糖和多糖的水解。
本发明的目的是提供特别适用于洗涤剂用途的改进α-淀粉酶。
本发明的概述
本发明的目的是提供Termamyl-样淀粉酶，与相应的亲本α-淀粉酶，即，未突变的α-淀粉酶相比该变异体具有α-淀粉酶活性且相对于所述亲本α-淀粉酶表现出至少一种上述特性改变。
具体地，本发明涉及如下各项：
1.亲本Termamyl-样α-淀粉酶的变异体，其包含在选自如下组中的一个或多个位置改变：
26,30,33,82,37,106,118,128,133,149,150,160,178,182,186,193,203,214,231,256,257,258,269,270,272,283,295,296,298,299,303,304,305,311,314,315,318,319,339,345,361,378,383,419,421,437,441,444,445,446,447,450,461,471,482,484,
其中
(a)改变是独立地
(i)在占据该位置的氨基酸下游插入氨基酸，
(ii)缺失占据该位置的氨基酸，或
(iii)用不同的氨基酸取代占据该位置的氨基酸，
(b)该变异体具有α-淀粉酶活性，和
(c)各位置相应于具有亲本Termamyl-样α-淀粉酶的氨基酸序列的亲本α-淀粉酶的氨基酸序列位置，该亲本Termamyl-样α-淀粉酶具有SEQ ID NO:12所示的AA560的氨基酸序列。
2.项1的变异体，其中该突变是：
R26S,D30N,N33D,R82H,K37T,N106D,K118Q,N128Y,G133E,A,G149A,N,N150H,Q,Y160F,Y178F,G182T,G186A,T193S,N,D,E,Q,Y203L,V214I,T,D231N,G256K,T257I,G258D,K269S,Q,N270F,Y,D,L272I,V,A,N283D,Y295F,N,D,Q,E,N296K,Q,E,Y298F,H,N299F,Y,Q,T,S303Q,K,Y304F,R,K,G305D,Q311N,Q,K,R,T,S,Y,F,N314D,S,T,Q,G315N,S,T,V318L,Q319E,K,S,T,A339S,T,E345N,R,Q361E,G378K,K383R,T419N,H421Y,N437H,F441L,R444E,Y,N445Q,K446R,A447Y,V450T,T461P,N471E,W482Y,N484Q。
3.项1-2的变异体，其中该变异体在一个或多个甲硫氨酸残基上具有额外的突变。
4.项3的变异体，其中该甲硫氨酸残基是：
M9,M10,M116,M202,M208,M261,M309,M323,M382,M410,M430,和M440。
5.项4的变异体，其中该突变是：
M9L,I,M10L,M105L,I,F,M116N,D,L,I,F,W,R,K,M202I,L,V,T,M208F,Y,L,I,M261L,I,M309L,I,M323L,I,S,T,A,Q,E,N,D,M382L,I,Y,F,K,M410L,I,V,M430L,I,和M440L,I,F,Y。
6.项1-5的变异体，其中该变异体具有一个或多个下列突变：
M9L+M202I,
M9L+M202I+M323T,
M9L+M202I+323T+M382Y,
M9L+M202I+Y295F+A339S,
M9L+M202I+Y295F,
M9L+M202I+A339S,
M9L+M202I+Y295F+A339S,
M9L+M202I+Y295F+A339S+E345R,
M9L+G149A+M202I+Y295F+A339S+E345R,
M9L+M202L,
M9L+M202L+M323T,
M9L+M202L+M232T+M382Y,
M9L+M202L+Y295F+A339S,
M9L+M202L+Y295F,
M9L+M202L+A339S,
M9L+M202L+Y295F+A339S,
M9L+M202L+Y295F+A339S,E345R,
M9L+G149A+M202L+Y295F+A339S+E345R,
M9L+M202T,
M9L+M202T+M323T,
M9L+M202T+M323T+M382Y,
M9L+M202T+Y295F+A339S,
M9L+M202T+Y295F,
M9L+M202T+A339S,
M9L+M202T+Y295F+A339S,
M9L+M202T+Y295F+A339S+E345R,
M9L+G149A+M202T+Y295F+A339S+E345R,
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y,
M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S,
M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202L+V214T+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202I+V214I+Y295F+M323T+A339S+E345R+M382Y,
M9L+G149A+G182T+G186A+M202L+V214I+Y295F+N299Y+M323T+A339S,
M9L+G149A+G182T+G186A+M202I+T257I+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R+N471E,
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y+N471E,
M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S+N471E,
M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R+N471E,
M202L+M105F+M208F,
G133E+M202L+Q361E,
G133E+M202L+R444E,
M202L+Y295F,
M202L+A339S,
M202L+M323T,
M202L+M323T+M309L,
M202L+M323T+M430I,
M202L+V214T+R444Y,
M202L+N283D+Q361E,
M202L+M382Y+K383R,
M202L+K446R+N484Q,
M202I+Y295F,
M202I+A339S,
M202I+M105F+M208F,
G133E+M202I+Q361E,
G133E+M202I+R444E,
M202I+M202I+M323T,
M202I+M202I+M323T+M309L,
M202I+M323T+M430I,
M202I+V214T+R444Y,
M202I+N283D+Q361E,
M202I+M382Y+K383R,
M202I+K446R+N484Q,
M202V+M105F+M208F,
G133E+M202V+Q361E,
G133E+M202V+R444E,
M202V+M323T,
M202V+M323T+M309L,
M202V+M323T+M430I,
M202V+M323T+M9L,
M202V+V214T+R444Y,
M202V+N283D+Q361E,
M202V+M382Y+K383R,
M202V+K446R+N484Q,
M202T+M105F+M208F,
G133E+M202T+Q361E,
G133E+M202T+R444E,
M202T+Y295F,
M202T+A339S,
M202T+M323T,
M202T+M323T+M309L,
M202T+M323T+M430I,
M202T+M323T+M9L,
M202T+V214T+R444Y,
M202T+N283D+Q361E,
M202T+A339S,
M202T+Y295F
M202T+N299F,Y,
M202T+M382Y+K383R,
M202T+K446R+N484Q。
7.项1-6的变异体，其中该变异体还含有突变D183*+G184*。
8.项1-7的变异体，其中该变异体还含有在R118的突变，特别是R118K。
9.项1-8的变异体，其中该变异体还含有在N195的突变，特别是N195F。
10.项1-9的变异体，其中该变异体还含有在R320的突变，特别是R320K。
11.项1-10的变异体，其中该变异体还含有在R458的突变，特别是R458K。
12.项1-11的变异体，其中该变异体含有突变D183*+G184*+R118K+N195F+R320K+R458K与一个或多个下列突变的组合：
K118Q,
K37T,
H421Y,
V450T,
K383R,
N445Q,
Y178F,
V318L,
W482Y,
N283D+Q361E,
M105F+M208F,
M202L+M323T+M430I,
K446R+N484Q,
R444Y,
N106D,
Y203L,
G133E+Q361E,
M323E,
V214T,
M202L+M323T+M309L,
M202L,
M202L+M323T,
M202L+M323T+M9L+M382Y+K383R,
M202L+M323T+M9L+M382Y,
M202L+M323T+M9L。
13.项12的变异体，其中该变异体含有突变D183*+G184*+R118K+N195F+R320K+R458K+M202L+M323T+M9L。
14.项13的变异体，其中该变异体还含有一个或多个下列突变：
T461P,
Y298H,
G133E+R444E,
Y298F,
M202T,
M202I,
M202V,
V214T+M323E+M382Y+K383R+N471E
Y178F+G258D+T419N+N437H
G149N+N150Q+M382Y+K383R
Y160F+V214T+M382Y
N128Y+G149A+V214T+D231N+M382Y+F441L
R82H+N128Y+G149A+V214T+M382Y
N150H+V214T
V214T+E345N
V214T+G305D+M382Y+R444E
V214T+M382Y+A447Y
M202I+V214T+M382Y+K383R+R444Y
V214T+G378K
V214T+A256K
R26S+D30N+N33D+V214T+M382Y。
15.项1-14中任一项的变异体，其中该亲本Termamyl-样α-淀粉酶来源于地衣芽孢杆菌，解淀粉芽孢杆菌，嗜热脂肪芽孢杆菌，芽孢杆菌属菌种NCIB 12289，NCIB 12512，NCIB12513或DSM 9375，或DSMZ no.12649，KSM AP1378，或KSM K36或KSM K38的菌株。
16.项1-15的变异体，其中该亲本Termamyl-样α-淀粉酶是选自SEQ ID NOS:2,4,6,8,10,12,13,14,15 16,17,和18所述的组中的任何α-淀粉酶。
17.根据项1-16任一项的变异体，其中该亲本Termamyl-样α-淀粉酶具有与SEQ IDNO:4有至少60％，优选70％，更优选至少80％，甚至更优选至少大约90％，甚至更优选至少95％，甚至更优选至少97％，和甚至更优选至少99％同一性程度的氨基酸序列。
18.项1-17中任一项的变异体，其中该亲本Termamyl-样α-淀粉酶由核酸序列编码，该核酸序列在低等，优选中等，优选高度严格条件下与SEQ ID NO:11的核酸序列杂交。
19.项1-18的变异体，该变异体相对于所述亲本α-淀粉酶具有至少一种下列特性改变：底物特异性，底物结合，底物裂解模式，热稳定性，pH活性谱，pH稳定性谱，氧化稳定性，Ca2+依赖性，pI减少和增加以及改进的洗涤效果，比活，在例如，高温和/或低/高pH条件下，特别是在低钙浓度下的稳定性，和在去污剂存在下的稳定性，例如在去污剂中的贮存稳定性。
20.一种DNA构建体，它含有编码根据项1至19任一项的α-淀粉酶变异体的DNA序列。
21.一种重组表达载体，它携带根据项20的DNA构建体。
22.用根据项20的DNA构建体或根据项21的载体转化的细胞。
23.根据项22的细胞，它是微生物，优选细菌或真菌，特别是革兰氏阳性细菌，例如枯草芽孢杆菌，地衣芽孢杆菌，迟缓芽孢杆菌,短芽孢杆菌，嗜热脂肪芽孢杆菌，嗜碱芽孢杆菌,解淀粉芽孢杆菌,凝固芽孢杆菌，环状芽孢杆菌，灿烂芽孢杆菌或苏芸金芽孢杆菌。
24.一种组合物，它含有项1－19的α-淀粉酶变异体。
25.一种去污剂添加剂，它含有根据项1至19任一项的α-淀粉酶变异体，任选以无尘粒状，稳定液体或受保护酶的形式。
26.根据项25的去污剂添加剂，它含有0.02-200mg酶蛋白/g添加剂。
27.根据项25或26的去污剂添加剂，它还含有另一酶，例如蛋白酶，脂肪酶，过氧化物酶，甘露聚糖酶，产麦芽糖淀粉酶，CGTase，淀粉酶或另一淀粉分解酶，例如葡糖淀粉酶，和/或纤维素酶。
28.一种去污剂组合物，它含有根据项1到19任一项的α-淀粉酶变异体。
29.根据项28的去污剂组合物，它还含有另一酶，例如蛋白酶，脂肪酶，过氧化物酶，甘露聚糖酶，产麦芽糖淀粉酶，CGTase，另一种淀粉分解酶和/或纤维素酶。
30.含有根据项1到19任一项的α-淀粉酶变异体的手工或自动餐具去污剂组合物。
31.根据项30的餐具去污剂组合物，它还含有另一酶，例如蛋白酶，脂肪酶，过氧化物酶，甘露聚糖酶，产麦芽糖淀粉酶，CGTase，淀粉酶或另一种淀粉分解酶，例如葡糖淀粉酶，和/或纤维素酶。
32.含有根据项1到19任一项的α-淀粉酶变异体的手工或自动洗衣洗涤组合物。
33.根据项32的洗衣洗涤组合物，它还含有另一种酶，例如蛋白酶，脂肪酶，过氧化物酶，甘露聚糖酶，产麦芽糖淀粉酶，CGTase，淀粉酶或另一种淀粉分解酶，例如葡糖淀粉酶和/或纤维素酶。
34.根据项1到19任一项的α-淀粉酶变异体或根据项24到34的组合物在洗涤和/或餐具中的用途。
35.根据项1到19任一项的α-淀粉酶变异体或根据项24到34的组合物在纺织品脱浆中的用途。
36.根据项1到19任一项的α-淀粉酶变异体或根据项24到34的组合物在淀粉液化中的用途。
37.生产根据项1-19任一项的变异体的方法，其中在有助于生产该变异体的条件下培养根据项22至23任一项的细胞，随后从培养物中回收该变异体。
命名法
在本说明书和权利要求书中，使用氨基酸残基的常规单字母和3字母代码。为了便于参照，本发明的α-淀粉酶变异体使用如下命名法描述：原始氨基酸:位置:取代氨基酸
根据该命名法，例如在第30位丙氨酸取代天冬酰胺表示为：Ala30Asn或A30N
在相同位置缺失丙氨酸表示为：Ala30*或A30*
插入另一氨基酸残基，例如赖氨酸，表示为：Ala30AlaLys或A30AK
缺失连续的一段氨基酸残基，例如氨基酸残基30-33，表示为(30-33)*或Δ(A30-N33)。
如果与其它α-淀粉酶相比特定α-淀粉酶含有“缺失”且在该位置含有插入，则表示为：
*36Asp或*36D，表示在第36位插入天冬氨酸。
多个突变用加号分开，即：Ala30Asn+Glu34Ser或A30N+E34S
表示在第30位和34位分别用丙氨酸和谷氨酸取代天冬酰胺和丝氨酸的突变。
当在给定位置可插入一个或多个可选择的氨基酸残基时，表示为：
A30N,E或
A30N或A30E
另外，当本文鉴定了适合于修饰的位置而没有建议任何具体的修饰时，应理解为任意氨基酸残基都可取代该位置的氨基酸残基。因此，例如，当提及修饰第30位的丙氨酸，但没有指定时，应理解为该丙氨酸可缺失或用任何其它氨基酸，即R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V中的任一个取代
此外，“A30X”是指下列取代中的任一个：
A30R,A30N,A30D,A30C,A30Q,A30E,A30G,A30H,A30I,A30L,A30K,A30M,A30F,A30P,A30S,A30T,A30W,A30Y,或A30V；或简写为：A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V。
如果用于编号的亲本酶在该位置已经具有建议取代的所述氨基酸残基，则使用如下命名法：
例如，如果野生型中存在N或V之一则表示为“X30N”或“X30N,V”。
因此，这意味着其它相应的亲本酶在第30位取代成“Asn”或“Val”。
氨基酸残基的特性
带电荷的氨基酸:
Asp,Glu,Arg,Lys,His
带负电荷的氨基酸(前面为带最大负电荷的残基):
Asp,Glu
带正电荷的氨基酸(前面为带最大正电荷的残基):
Arg,Lys,His
中性氨基酸:
Gly,Ala,Val,Leu,Ile,Phe,Tyr,Trp,Met,Cys,Asn,Gln,Ser,Thr,Pro
疏水氨基酸残基(疏水性最大的残基在后面列出):
Gly,Ala,Val,Pro,Met,Leu,Ile,Tyr,Phe,Trp,
亲水氨基酸(亲水性最大的残基在后面列出):
Thr,Ser,Cys,Gln,Asn
附图的简要描述
图1是13个亲本Termamyl-样α-淀粉酶的氨基酸序列的序列对比。
本发明的详细描述
本发明的目的是提供多肽，例如酶，特别是α-淀粉酶，它相对于所述亲本多肽至少一种下列特性改变：底物特异性，底物结合，底物裂解方式，热稳定性，pH/活性谱(profile)，氧化稳定性，Ca2+依赖性，和特别是在衣物和盘子洗涤应用中的比活。该特性将在下文中进一步限定。
多肽
根据本发明的多肽包括具有生物学活性，抗微生物活性，和酶活性的蛋白质。
涉及的酶活性包括蛋白酶，淀粉酶，CGTases，甘露聚糖酶，产麦芽糖淀粉酶(maltogenic amylases)，葡糖淀粉酶，糖酶，转移酶，裂合酶，氧化还原酶，脂肪酶。
在一个优选的实施方案中，该酶是α-淀粉酶，特别是芽孢杆菌属(Bacillus)或曲霉属(Aspergillus)α-淀粉酶。在优选的实施方案中，芽孢杆菌属α-淀粉酶是Termamyl-样淀粉酶。
具有生物学活性的多肽包括，EPO，TPO，生长激素，调节肽，凝血因子，抗体等。
SP722的三级结构和模拟(modelling)另一Termamyl-样α-淀粉酶的三级结构
根据WO 01/66712附件1所示的SP722的三级结构发现了本发明的α-淀粉酶突变体。根据其它三级结构可发现其它多肽的突变体。
根据WO 01/66712附件1公开的SP722三级结构建立了另一碱性Termamyl-样淀粉酶，即AA560的模型。AA560α-淀粉酶与模板淀粉酶(SP722)大约87％相同且序列对比不含插入或缺失。
本发明的发现可应用到与SEQ ID NO:12所示的Termamyl-样α-淀粉酶具有至少60％同一性，优选至少70％同一性，更优选80％同一性，甚至更优选85％同一性，甚至更优选90％同一性，甚至更优选95％同一性，甚至更优选97％同一性，甚至更优选99％同一性的Termamyl-样淀粉酶上。优选的是该发现可用于碱性Termamyl-样α-淀粉酶，特别是与SP722(SEQ ID NO:4，在图1的序列对比中编号为7)相比在一级结构的序列对比中具有相同长度而没有添加氨基酸残基或间隙(gaps)的碱性α-淀粉酶。具体地说，该发现可用于下列碱性Termamyl-样α-淀粉酶：SP690(SEQ ID NO:2)，SP722(SEQ ID NO:4)，AA560(SEQ ID NO:12)，#707α-淀粉酶(SEQ ID NO:13)，在WO 97/00324中公开的KSM AP 1378α-淀粉酶，在WO02/10356中公开的#SP7-7α-淀粉酶，或其片断或截短形式。后面所述的碱性α-淀粉酶在上述相互作用区周围具有非常相似的三级晶体结构，且具有相同的一级结构长度，即485个氨基酸。
与此相反，例如，当与SP722进行序列对比时，Termamyl(表示为图1序列对比中的序列号1)缺少两个氨基酸残基(位置1和2)；在位置174和181-182有间隙；在位置378-381有3个额外的氨基酸残基。
与SP722进行序列对比时，BAN(表示为图1序列对比中的序列号4)缺少5个氨基酸残基(位置1-4和488)；在位置174和181-182有间隙；在位置378-381有3个额外的氨基酸残基。
与SP722进行序列对比时，BSG(表示为图1序列对比中的序列号3)缺少1个氨基酸残基(位置1)；且在位置489-519有31个额外的氨基酸残基。
与SP722进行序列对比时，KSM-K36和KSM-K38(EP 1,022,334-A)缺少5个氨基酸残基(位置1和2)且在位置174和181-182有间隙。
与SP722进行序列对比时，AA180，AA20和Amrk385(丹麦专利申请号PA 2000 00347或PCT/DK01/00133)在位置261有一个额外的氨基酸。
下面描述了如何从另一α-淀粉酶模拟Termamyl-样α-淀粉酶。该方法可按例如上文所述推广(exprepolated)到其它多肽。
Termamyl-样α-淀粉酶的模拟
WO 96/23874提供了Termamyl-样α-淀粉酶的三级结构(3D结构)，X-射线晶体结构数据，它由解淀粉芽孢杆菌(B.amyloliquefaciens)α-淀粉酶(BANTM)的300个N-末端氨基酸残基和地衣芽孢杆菌(B.licheniformis)α-淀粉酶的C-末端氨基酸301-483组成(SEQ IDNO:8)。WO 96/23874还描述了根据对亲本Termamyl-样α-淀粉酶的结构分析设计(模拟)相对于该亲本表现出特性改变的该亲本Termamyl-样α-淀粉酶的变异体的方法。
根据在此作为参考文献引用的WO 96/23874可模拟其它Termamyl-样结构。
为了获得本发明的变异体，可按实施例1所述根据SP722的三级结构(在附件1中公开)设计(模拟)AA560的三级结构。同样可构建其它Termamyl-样α-淀粉酶的结构(例如，本文所述的那些结构)。
Termamyl-样α-淀粉酶
由芽孢杆菌属菌种产生的许多α-淀粉酶在氨基酸水平上具有高度同源性(同一性)。
许多芽孢杆菌属α-淀粉酶的同一性可参见下表1(ClustalW)：
表1
Figure BDA0003128425700000131
例如，发现包含SEQ ID NO:8所示氨基酸序列的地衣芽孢杆菌α-淀粉酶(可作为TermamylTM购买)与包含SEQ ID NO:10所示氨基酸序列的解淀粉芽孢杆菌α-淀粉酶具有大约81％的同源性且与包含SEQ ID NO:6所示氨基酸序列的嗜热脂肪芽孢杆菌(B.stearothermophilus)α-淀粉酶有大约65％的同源性。其它同源性α-淀粉酶包括WO 95/26397中公开且分别在本文中以SEQ ID NO:2和SEQ ID NO:4所示的SP690和SP722。其它淀粉酶是来自芽孢杆菌属菌种且以SEQ ID NO:12所示的AA560α-淀粉酶，和Tsukamoto等，Biochemical and Biophysical Research Communications,151(1988),第25-31页所述的来自芽孢杆菌属菌种的#707α-淀粉酶。
KSM AP1378α-淀粉酶在WO 97/00324(来自KAO Corporation)中公开。根据本发明还包含在EP 1,022,334中公开的K38和K38α-淀粉酶。
其它α-淀粉酶以SEQ ID NOS:13,14,15,16,17,和18显示。
还有其它的同源性α-淀粉酶包括在EP 0252666中描述的地衣芽孢杆菌菌株(ATCC27811)产生的α-淀粉酶，和在WO 91/00353和WO 94/18314中鉴定的α-淀粉酶。其它商品Termamyl-样α-淀粉酶包含在以下列商品名销售的产品中：OptithermTM和TakathermTM(来自Solvay)；MaxamylTM(来自Gist-brocades/Genencor)，Spezym AATM和Spezyme Delta AATM(来自Genencor)，和KeistaseTM(来自Daiwa)，PurastarTMST 5000E，PURASTRATMHPAM L(来自Genencor Int.).
由于在这些α-淀粉酶之间发现的基本同源性，因此可认为它们属于同类α-淀粉酶，即“Termamyl-样α-淀粉酶”类型。
因此，在本文中，术语“Termamyl-样α-淀粉酶”试图表示这样的α-淀粉酶，即，它在氨基酸水平上表现出与TermamylTM，即，具有本文SEQ ID NO:8所示氨基酸序列的地衣芽孢杆菌α-淀粉酶具有基本同一性。
换句话说，所有下列具有本文SEQ ID NOS:2,4,6,8,10,12,13,14,15,16,17,和18所示氨基酸序列的α-淀粉酶都认为是“Termamyl-样α-淀粉酶”。其它Termamyl-样α-淀粉酶是如下α-淀粉酶：i)与SEQ ID NOS:2,4,6,8,10,12,13,14,15,16,17和18所示的氨基酸序列中的至少一个显示出至少60％，例如至少70％，例如至少75％，或至少80％，至少85％，至少90％，至少95％，至少97％，至少99％同源性和/或ii)由这样的DNA序列编码，该DNA序列与明显来自本说明书SEQ ID NOS:1,3,5,7,9,11的编码上述α-淀粉酶的DNA序列(其编码序列分别编码本文SEQ ID NOS:2,4,6,8,10和12所示的氨基酸序列)杂交。
应理解在本申请中还包括由1)与Termamyl具有高度同源性的催化域和2)糖结合域(CBM)组成的Termamyl淀粉酶。该结合域位于相对于催化域序列的N-末端或相对于催化域的C-末端，可能存在超过1个的CBM，它位于N-或C-末端或两者都有。具有CBM的淀粉酶可来自天然来源或者是遗传工程融合编码淀粉酶的基因与编码CBM的基因的结果。
同源性(同一性)
同源性可测定为两个序列之间的同一性程度，它表明了第一个序列从第二个序列衍生。同源性可借助于本领域已知的计算机程序合适地测定，例如GCG程序包中提供的GAP(上述)。因此，Gap GCGv8可使用同一性的默认记分矩阵和下列默认参数：分别用于核酸序列比较的GAP产生罚分5.0和GAP延长罚分(extension penalty)0.3，和分别用于蛋白质序列比较的GAP产生罚分3.0和GAP延长罚分0.1。GAP使用Needleman和Wunsch,(1970),J.Mol.Biol.48,p.443-453的方法进行序列对比并计算同一性。
Termamyl(SEQ ID NO:8)与例如，另一α-淀粉酶之间的结构对比可用于鉴定在其它Termamyl-样α-淀粉酶中的相当/相应位置。获得所述结构对比的一个方法是使用GCG程序包的Pile Up程序，使用间隙罚分的默认值，即间隙产生罚分3.0和间隙延长罚分0.1。其它结构对比方法包括疏水簇分析(Gaboriaud等，(1987),FEBS LETTERS 224,第149-155页)和反向穿线(threading)(Huber,T；Torda,AE,PROTEIN SCIENCE Vol.7,No.1第142-149页(1998))。
杂交
用于按照上述特性ii)鉴定多肽，例如Termamyl-样α-淀粉酶的寡核苷酸探针可根据所述α-淀粉酶的全长或部分核苷酸或氨基酸序列合适地制备。
检测杂交的合适条件包括在5xSSC中预浸泡和在20％甲酰胺，5xDen-hardt's溶液，50mM磷酸钠，pH 6.8，和50mg变性超声处理小牛胸腺DNA的溶液中～40℃下预杂交1小时，接着在添加了100mM ATP的相同溶液中～40℃下杂交18小时，接着在2xSSC,0.2％SDS中在40℃下(低度严格)，优选在50℃下(中等严格)，更优选在65℃下(高度严格)，甚至更优选在～75℃下(极高严格条件)洗涤滤膜30分钟3次。关于杂交方法的更多细节可参见Sambrook等，Molecular Cloning:A Laboratory Manual,第二版,Cold Spring Harbor,1989。
在本文中，“来自于”不仅用于表示由所述生物菌株生产或可生产的α-淀粉酶，而且还表示由该菌株分离的DNA序列编码且在用所述DNA序列转化的宿主生物中产生的α-淀粉酶。最后，该术语用于表示这样的α-淀粉酶，它由合成和/或cDNA来源的DNA序列编码且具有所述α-淀粉酶的鉴定特征。该术语还用于表示该亲本α-淀粉酶是天然存在的α-淀粉酶的变异体，即该变异体是对天然存在的α-淀粉酶的一个或多个氨基酸残基进行修饰(插入，取代，缺失)的结果。
亲本Termamyl-样α-淀粉酶
根据本发明，上述所有的Termamy-样α-淀粉酶可用作亲本(即，主链)α-淀粉酶。在本发明优选的实施方案中，亲本α-淀粉酶来源于地衣芽孢杆菌，例如，上述之一，例如具有SEQ ID NO:10所示氨基酸序列的地衣芽孢杆菌α-淀粉酶。
在一个优选的实施方案中，亲本Termamyl-样α-淀粉酶是SP722或BSG或AA560，包括SP722+R181*+G182*,SP722+D183*+G184*；SP722+D183*+G184*+N195F；SP722+D183*+G184*+M202L；SP722+D183*+G184*+N195F+M202L；SP722+D183*+G184*+R181Q；SP722+D183*+G184*+R118K+N195F+R320K+R458K；BSG+I181*+G182*；BSG+I181*+G182*+N193F；BSG+I181*+G182*+M200L；BSG+I181*+G182*+N193F+M200L；
AA560+D183*+G184*；AA560+D183*+G184*+N195F；AA560+D183*+G184*+M202L；AA560+D183*+G184*+N195F+M202L；AA560+D183*+G184*+R118K+N195F+R320K+R458K中任一种。
“BSG+I181*+G182*+N193F”是指具有如下突变的嗜热脂肪芽孢杆菌(B.stearothermophilus)α-淀粉酶：在位置I181和G182缺失和在位置193由Asn(N)取代成Phe(F)。
亲本杂合Termamyl-样α-淀粉酶
亲本α-淀粉酶(即，主链α-淀粉酶)也可以是杂合α-淀粉酶，即这样的一种α-淀粉酶，它包含来自至少一种α-淀粉酶的部分氨基酸序列的组合。
亲本杂合α-淀粉酶可以是根据氨基酸同源性(同一性)和/或DNA杂交(如上所述)测定属于Termamyl-样α-淀粉酶家族的α-淀粉酶。在这种情况下，杂合α-淀粉酶一般由至少一部分Termamyl-样α-淀粉酶和选自微生物(细菌或真菌)和/或哺乳动物来源的Termamyl-样α-淀粉酶或非-Termamyl-样α-淀粉酶的一种或多种其它α-淀粉酶的部分组成。
因此，亲本杂合α-淀粉酶可包含来自至少两种Termamyl-样α-淀粉酶，或来自至少一种Termamyl-样和至少一种非-Termamyl-样细菌α-淀粉酶，或来自至少一种Termamyl-样和至少一种真菌α-淀粉酶的部分氨基酸序列的组合。提供部分氨基酸序列的该Termamyl-样α-淀粉酶可以是任何本文提及的特异性Termamyl-样α-淀粉酶。
例如，亲本α-淀粉酶可包含来源于地衣芽孢杆菌菌株的α-淀粉酶的C-末端部分，和来源于解淀粉芽孢杆菌菌株或来源于嗜热脂肪芽孢杆菌菌株的α-淀粉酶的N-末端部分。例如，亲本α-淀粉酶可包含地衣芽孢杆菌α-淀粉酶的C-末端部分的至少430个氨基酸残基，且例如，可包含a)相应于具有SEQ ID NO:10所示氨基酸序列的解淀粉芽孢杆菌α-淀粉酶的37个N-末端氨基酸残基的氨基酸片断和相应于具有SEQ ID NO:8所示氨基酸序列的地衣芽孢杆菌α-淀粉酶的445个C-末端氨基酸残基的氨基酸片断，或除了N-末端35个氨基酸残基(成熟蛋白质中)被BAN(成熟蛋白质)，即，SEQ ID NO:10所示的解淀粉芽孢杆菌α-淀粉酶的N-末端33个残基取代外，与Termamyl序列，即，SEQ ID NO:8所示的Bacilluslicheniformisα-淀粉酶相同的杂合Termamyl-样α-淀粉酶；或b)相应于具有SEQ ID NO:6所示氨基酸序列的嗜热脂肪芽孢杆菌α-淀粉酶的68个N-末端氨基酸残基的氨基酸片断和相应于具有SEQ ID NO:8所示氨基酸序列的地衣芽孢杆菌α-淀粉酶的415个C-末端氨基酸残基的氨基酸片断。
另一合适的亲本杂合α-淀粉酶是由BAN，解淀粉芽孢杆菌α-淀粉酶N-末端(成熟蛋白质的氨基酸1-300)和Termamyl的C-末端(成熟蛋白质的氨基酸301-483)组成的以前在WO96/23874(来自Novo Nordisk)中描述的α-淀粉酶。
另一合适的亲本杂合α-淀粉酶由SEQ ID NOS:2,4,6,8,10,12,13,14,15,16,17,或18的序列，和SEQ ID NO:13(AMY1048)的最后99个氨基酸组成。
在本发明的一个优选的实施方案中，亲本Termamyl-样α-淀粉酶是SEQ ID NO:8和SEQ ID NO:10的杂合α-淀粉酶。具体地说，亲本杂合Termamyl-样α-淀粉酶可以是包含SEQID NO:8所示的地衣芽孢杆菌α-淀粉酶的445个C-末端氨基酸残基和SEQ ID NO:10所示的来源于解淀粉芽孢杆菌α-淀粉酶的33个N-末端氨基酸残基的杂合α-淀粉酶，它还可合适地具有下列突变：H156Y+A181T+N190F+A209V+Q264S(使用SEQ ID NO:8中的编号)。后一提及的杂合体用于下面的实施例且称为LE174。
其它具体涉及的亲本α-淀粉酶包括具有少数突变的LE174，即，具有下列突变的刚才上文提及的杂合体：A181T+N190F+A209V+Q264S；N190F+A209V+Q264S；A209V+Q264S；Q264S；H156Y+N190F+A209V+Q264S；H156Y+A209V+Q264S；H156Y+Q264S；H156Y+A181T+A209V+Q264S；H156Y+A181T+Q264S；H156Y+Q264S；H156Y+A181T+N190F+Q264S；H156Y+A181T+N190F；H156Y+A181T+N190F+A209V。这些杂合体也认为是本发明的部分。
在一个优选的实施方案中，亲本Termamyl-样α-淀粉酶是LE174，包括LE174+G48A+T49I+G107A+I201F；LE174+M197L；或LE174+G48A+T49I+G107A+M197L+I201F中任一个。
其它涉及的亲本α-淀粉酶包括LE429，它是具有另一取代I201F的LE174。根据本发明，LE335是α-淀粉酶，它与LE429相比，具有另外的取代T49I+G107A；LE399是LE335+G48A，即，具有G48A+T49I+G107A+I201F的LE174。
构建本发明的变异体
构建感兴趣的变异体可通过在有助于产生该变异体的条件下培养包含编码该变异体的DNA序列的微生物来实现。然后可从所得的培养液中回收该变异体。这点在下面进行详细地描述。
改变的特性
下面讨论存在于本发明变异体中的突变与可能由其引起的所需特性改变(相对于亲本Termamyl-样α-淀粉酶)之间的关系。
如上所述，本发明涉及特别是在高温和/或低pH，特别是在低钙浓度下具有改变特性的Termamyl-样α-淀粉酶。
在本发明上下文中，“高温”是指从70-120℃的温度，优选80-100℃，特别是85-95℃。
在本发明上下文中，术语“低pH”是指从4-6范围内的pH，优选4.2-5.5，特别是4.5-5。
在本发明上下文中，术语“高pH”是指从8-11范围内的pH，特别是8.5-10.6。
在本发明上下文中，术语“低钙浓度”是指低于60ppm的游离钙水平，优选40ppm，更优选25ppm，特别是5ppm钙。
与具体涉及的改变特性相联系的具体涉及的亲本Termamyl-样α-淀粉酶是上述亲本Termamyl-样α-淀粉酶和亲本杂合Termamyl-样α-淀粉酶。SP722α-淀粉酶用作起点，但在例如，Termamyl,BSG,BAN,AA560,SP690,AA180,KSM AP1378,SP7-7和#707,K38,和K36的相应位置应理解为也被公开。
设计改进氧化稳定性的淀粉酶变异体：
已表明SEQ ID NO:8中的M197或SEQ ID NO:12中相当的M202在漂白剂，例如，洗涤剂中的过硼酸盐等存在的条件下增加稳定性。另外SEQ ID NO:8中的M15突变也显示出相同效果，但是对于SEQ ID NO:2,4,6,10,和12和在相当于M15位置没有相应甲硫氨酸的其它淀粉酶，发现其它残基，特别是其它甲硫氨酸增加的稳定性超过对于M202所观察到的情况。这些残基包括但不限于SEQ ID NO:12,17,和18中的M9,M10,M105,M116(在SP690,SP722,AMRK385中不存在)M202,M208,M261,M309,M323(仅存在于AA560,SP722中),M382,M410(SP.7-7),M430,M440，而在SEQ ID NO:16(AAI-10)中，最感兴趣的位置是：M10,M105,M202,M208,M246,M286,M309,M430,M440,M454，在SEQ ID NO:14(Amrk385)中最感兴趣的位置是：M9,M10,M105,M202,M208,M262,M310,M383,M431,M441，在SEQ ID NO:15(K38)中最感兴趣的位置是：M7,M8,M103,M107,M277,M281,M304,M318,M380,M425,M435。最优选的取代是：M9L,I,M10L,M105L,I,F,M116N,D,L,I,F,W,R,K,M202L,I,T,V,M208F,Y,L,I,M261L,I,M309L,I,M323L,I,S,T,A,Q,E,N,D,M382L,I,Y,F,K,M410L,I,V,M430L,I,M440L,I,F,Y。
如上所述，在漂白剂存在的条件下M202显示出对于稳定性是重要的。然而将M202突变成对于稳定性优选的取代时，降低该淀粉酶的活性。为了再活化该淀粉酶，已表明沿推定的底物结合裂口进行的取代对该活性有益。它们包括：T193,K269,N270,L272,Y295,N296,N299,S303,Y304,Q311,N314,G315,Q319,和A339等。优选的突变是：T193S,N,D,E,Q,K269S,Q,N270F,Y,D,L272I,V,A,Y295F,N,D,Q,E,N296K,Q,E,N299F,Y,Q,T,S303Q,K,Y304F,R,K,Q311N,Q,K,R,T,S,Y,F,N314D,S,T,Q,G315N,S,T,Q319E,K,S,T,A339S,T。
洗涤应用的最佳酶为能最佳工作必须满足几个标准。它在使用前在洗涤剂基质中应当稳定，它在洗涤期间应当稳定且它在洗涤期间应当有高度活性。已报道了优化各标准的几个例子，但是由于氧化稳定性淀粉酶活性低且有活性的淀粉酶稳定性差，因此，本发明的范围是鉴定满足所有三个要求的最佳取代组合。优选的组合是：
M9L+M202I
M9L+M202I+M323T
M9L+M202I+M323T+M382Y
M202I+Y295F
M202I+A339S
M9L+M202I+Y295F+A339S
M9L+M202I+Y295F
M9L+M202I+A339S
M9L+M202I+Y295F+A339S
M9L+M202I+Y295F+A339S+E345R
M9L+G149A+M202I+Y295F+A339S+E345R
M9L+M202L
M9L+M202L+M323T
M9L+M202L+M323T+M382Y
M202L+Y295F
M202L+A339S
M9L+M202L+Y295F+A339S
M9L+M202L+Y295F
M9L+M202L+A339S
M9L+M202L+Y295F+A339S
M9L+M202L+Y295F+A339S+E345R
M9L+G149A+M202L+Y295F+A339S+E345R
M9L+M202T
M9L+M202T+M323T
M9L+M202T+M323T+M382Y
M202T+Y295F
M202T+A339S
M9L+M202T+Y295F+A339S
M9L+M202T+Y295F
M9L+M202T+A339S
M9L+M202T+Y295F+A339S
M9L+M202T+Y295F+A339S+E345R
M9L+G149A+M202T+Y295F+A339S+E345R
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y
M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339SM9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R
M9L+G149A+M202L+V214T+Y295F+N299Y+M323T+A339S+E345R
M9L+G149A+M202I+V214I+Y295F+M323T+A339S+E345R+M382Y
M9L+G149A+G182T+G186A+M202L+V214I+Y295F+N299Y+M323T+A339S
M9L+G149A+G182T+G186A+M202I+T257I+Y295F+N299Y+M323T+A339S+E345R
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R+N471E
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y+N471E
M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S+N471E
M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R+N471E
在第一个方面，亲本Termamyl-样α-淀粉酶的变异体包含在选自如下组中一个或多个位置有改变：
26,30,33,82,37,106,118,128,133,149,150,160,178,182,186,193,203,214,231,256,257,258,269,270,272,283,295,296,298,299,303,304,305,311,314,315,318,319,339,345,361,378,383,419,421,437,441,444,445,446,447,450,461,471,482,484,
其中
(a)改变是独立地
(i)在占据该位置的氨基酸下游插入一个氨基酸，
(ii)缺失占据该位置的氨基酸，或
(iii)用不同的氨基酸取代占据该位置的氨基酸，
(b)该变异体具有α-淀粉酶活性，和
(c)各位置相应于具有亲本Termamyl-样α-淀粉酶的氨基酸序列的亲本α-淀粉酶的氨基酸序列位置，该亲本Termamyl-样α-淀粉酶具有SEQ ID NO:12所示的AA560的氨基酸序列。
在优选的实施方案中，本发明的变异体(使用SEQ ID NO:12的编号)具有一个或多个下列突变/取代：
R26S,D30N,N33D,R82H,K37T,N106D,K118Q,N128Y,G133E,A,G149A,N,N150H,Q,Y160F,Y178F,G182T,G186A,T193S,N,D,E,Q,Y203L,V214I,T,D231N,G256K,T257I,G258D,K269S,Q,N270F,Y,D,L272I,V,A,N283D,Y295F,N,D,Q,E,N296K,Q,E,Y298F,H,N299F,Y,Q,T,S303Q,K,Y304F,R,K,G305D,Q311N,Q,K,R,T,S,Y,F,N314D,S,T,Q,G315N,S,T,V318L,Q319E,K,S,T,A339S,T,E345N,R,Q361E,G378K,K383R,T419N,H421Y,N437H,F441L,R444E,Y,N445Q,K446R,A447Y,V450T,T461P,N471E,W482Y,N484Q。
使用SEQ ID NO:12作为编号基础，优选的双重，三重和多重突变包括：
M9L+M202I,
M9L+M202I+M323T,
M9L+M202I+323T+M382Y,
M9L+M202I+Y295F+A339S,
M9L+M202I+Y295F,
M9L+M202I+A339S,
M9L+M202I+Y295F+A339S,
M9L+M202I+Y295F+A339S+E345R,
M9L+G149A+M202I+Y295F+A339S+E345R,
M9L+M202L,
M9L+M202L+M323T,
M9L+M202L+M232T+M382Y,
M9L+M202L+Y295F+A339S,
M9L+M202L+Y295F,
M9L+M202L+A339S,
M9L+M202L+Y295F+A339S,
M9L+M202L+Y295F+A339S,E345R,
M9L+G149A+M202L+Y295F+A339S+E345R,
M9L+M202T,
M9L+M202T+M323T,
M9L+M202T+M323T+M382Y,
M9L+M202T+Y295F+A339S,
M9L+M202T+Y295F,
M9L+M202T+A339S,
M9L+M202T+Y295F+A339S,
M9L+M202T+Y295F+A339S+E345R,
M9L+G149A+M202T+Y295F+A339S+E345R,
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y,
M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S,
M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202L+V214T+Y295F+N299Y+M323T+A339S+E345R,M9L+G149A+M202I+V214I+Y295F+M323T+A339S+E345R+M382Y,
M9L+G149A+G182T+G186A+M202L+V214I+Y295F+N299Y+M323T+A339S,
M9L+G149A+G182T+G186A+M202I+T257I+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R+N471E,
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y+N471E,
M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S+N471E,
M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R+N471E,
M202L+M105F+M208F,
G133E+M202L+Q361E,
G133E+M202L+R444E,
M202L+Y295F,
M202L+A339S,
M202L+M323T,
M202L+M323T+M309L,
M202L+M323T+M430I,
M202L+V214T+R444Y,
M202L+N283D+Q361E,
M202L+M382Y+K383R,
M202L+K446R+N484Q,
M202I+Y295F,
M202I+A339S,
M202I+M105F+M208F,
G133E+M202I+Q361E,
G133E+M202I+R444E,
M202I+M202I+M323T,
M202I+M202I+M323T+M309L,
M202I+M323T+M430I,
M202I+V214T+R444Y,
M202I+N283D+Q361E,
M202I+M382Y+K383R,
M202I+K446R+N484Q,
M202V+M105F+M208F,
G133E+M202V+Q361E,
G133E+M202V+R444E,
M202V+M323T,
M202V+M323T+M309L,
M202V+M323T+M430I,
M202V+M323T+M9L,
M202V+V214T+R444Y,
M202V+N283D+Q361E,
M202V+M382Y+K383R,
M202V+K446R+N484Q,
M202T+M105F+M208F,
G133E+M202T+Q361E,
G133E+M202T+R444E,
M202T+Y295F,
M202T+A339S,
M202T+M323T,
M202T+M323T+M309L,
M202T+M323T+M430I,
M202T+M323T+M9L,
M202T+V214T+R444Y,
M202T+N283D+Q361E,
M202T+A339S,
M202T+Y295F
M202T+N299F,Y,
M202T+M382Y+K383R,
M202T+K446R+N484Q
稳定性
在本发明上下文中，对于实现改变的稳定性，特别是改良的稳定性(即，更高或更低)，特别是在高温(即，70-120℃)和/或极端pH(即，低或高pH，即分别为pH 4-6或pH 8-11下)，特别是在低于60ppm的游离(即，未结合，因此在溶液中)钙浓度下改良的稳定性重要的突变(包括氨基酸取代和缺失)包括“改变的特性”部分所列的任何突变。该稳定性可按下面“材料和方法”部分所述来测定。
Ca2+稳定性
改变的Ca2+稳定性是指在Ca2+排除(depletion)的条件下酶稳定性改良，即稳定性更高或更低。在本发明上下文中，对于实现改变的Ca2+稳定性，特别是改良的Ca2+稳定性，即，更高或更低的稳定性，特别是在高pH(即，pH 8-10.5)下改良的Ca2+稳定性重要的突变(包括氨基酸取代和缺失)包括“改变的特性”部分所列的任何突变。
比活
在本发明的另一方面，对于获得表现出改变的比活(specific activity)，特别是增加或降低比活，特别是在从10-60℃，优选在20-50℃，特别是在30-40℃下改变的比活的变异体重要的突变(包括氨基酸取代和缺失)包括“改变的特性”部分所列的任何突变。该比活可按下面“材料和方法”部分所述来测定。
氧化稳定性
本发明的变异体可具有改变的氧化稳定性，特别是与亲本α-淀粉酶相比更高的氧化稳定性。增强的氧化稳定性在例如，洗涤剂组合物中具有优势而降低的氧化稳定性在淀粉液化组合物中具有优势。氧化稳定性可按下面“材料和方法”部分所述来测定。
改变的pH活性谱
对于获得具有改变的pH活性谱(profile)，特别是在高pH(即，pH 8-10.5)或低pH(即，pH 4-6)下活性提高的变异体重要的位置和突变包括位于靠近活性位点残基的氨基酸残基的突变。
优选的特异性突变/取代是对所述位置进行在上面“改变的特性”部分所列的突变/取代。合适的测定法在下面“材料和方法”部分描述。
洗涤效果
对于获得特别是在中性到高pH，即，pH 6-11，优选pH 8.5-11下具有改进的洗涤效果的变异体重要的位置和突变包括在所述位置进行的上文“改变的特性”部分所列的具体突变/取代。洗涤效果可按下面“材料和方法”部分所述检测。
制备本发明的α-淀粉酶变异体的方法
将突变导入基因的一些方法是本领域已知的。在简要讨论编码α-淀粉酶的DNA序列的克隆后，将讨论在α-淀粉酶编码序列内特定位点产生突变的方法。
克隆编码α-淀粉酶的DNA序列
编码亲本α-淀粉酶的DNA序列可使用本领域熟知的各种方法从产生所述α-淀粉酶的任何细胞或微生物中分离。首先，使用来自产生所需研究的α-淀粉酶的生物的染色体DNA或信使RNA构建基因组DNA和/或cDNA文库。然后，如果已知该α-淀粉酶的氨基酸序列，可合成同源的标记寡核苷酸探针并用于从所述生物制备的基因组文库中鉴定编码α-淀粉酶的克隆。另外，含有与已知α-淀粉酶基因同源的序列的标记寡核苷酸探针可用作探针以便使用低度严格的杂交和洗涤条件鉴定编码α-淀粉酶的克隆。
鉴定编码α-淀粉酶的克隆的另一方法包括将基因组DNA片断插入表达载体，例如质粒中，用所得的基因组DNA文库转化α-淀粉酶阴性细菌，然后将转化的细菌涂布到含有α-淀粉酶底物的琼脂上，从而允许鉴定表达α-淀粉酶的克隆。
作为选择，可用已建立的标准方法，例如，S.L.Beaucage和M.H.Caruthers,Tetrahedron Letters 22,1981,第1859-1869页所述的亚磷酸胺方法或Matthes等，The EMBO J.3,1984,第801-805页所述的方法通过合成制备编码该酶的DNA序列。在亚磷酸胺方法中，在例如，自动DNA合成仪上合成寡核苷酸，纯化，退火，连接并在合适的载体中克隆。
最后，该DNA序列可以是混合的基因组和合成来源，混合的合成和cDNA来源或混合的基因组和cDNA来源，按照标准技术通过连接合成的，基因组或cDNA来源的片断(视情况，该片断相应于完整DNA序列的各个部分)制备。该DNA序列也可通过聚合酶链式反应(PCR)使用特异性引物来制备，例如按US 4,683,202或R.K.Saiki等，Science 239,1988,第487-491页所述。
定点诱变
一旦分离了编码α-淀粉酶的DNA序列，鉴定了突变所需的位点，可使用合成寡核苷酸导入突变。这些寡核苷酸含有所需突变位点侧翼的核苷酸序列；在寡核苷酸合成期间插入突变核苷酸。在一个具体方法中，在携带α-淀粉酶基因的载体中产生桥接编码α-淀粉酶序列的DNA单链缺口。然后将携带所需突变的合成核苷酸与单链DNA的同源部分退火。然后用DNA聚合酶I(Klenow片断)填充剩下的缺口并使用T4连接酶连接该构建体。该方法的具体实施例在Morinaga等，(1984)中描述。US 4,760,025公开了通过对序列盒进行较小改变导入编码多个突变的寡核苷酸。然而，通过Morinaga方法可在任何时间导入甚至更大数目的各种突变，因为可导入各种长度的多个寡核苷酸。
将突变导入编码α-淀粉酶的DNA序列中的另一方法在Nelson和Long(1989)中描述。它包含通过使用化学合成的DNA链作为PCR反应的一个引物导入含有所需突变的PCR片断的3-步生产法。通过用限制性内切核酸酶裂解并再插入表达质粒中可从PCR-产生的片断中分离携带该突变的DNA片断。
提供本发明的变异体的另一方法包括基因改组，例如在WO 95/22625(AffymaxTechnologies N.V.)或WO 96/00343(Novo Nordisk A/S)中所述，或产生包含所述突变，例如，取代和/或缺失的杂合酶的其它相应技术。
随机诱变
随机诱变适合于作为定位(localised)或区域特异性随机诱变在翻译成所示氨基酸序列的所述基因的至少三个部分中，或在整个基因内进行。
对编码亲本α-淀粉酶的DNA序列的随机诱变可通过使用本领域已知的任何方法按常规进行。
与上述内容相关，本发明的另一方面涉及产生亲本α-淀粉酶变异体的方法，例如，其中该变异体表现出在靠近分支点裂解寡糖底物的能力降低，且还表现出相对于亲本底物特异性提高和/或比活提高，该方法为：
(a)对编码亲本α-淀粉酶的DNA序列进行随机诱变，
(b)在宿主细胞中表达步骤(a)获得的突变DNA序列，和
(c)筛选表达相对于亲本α-淀粉酶具有改变特性(即，热稳定性)的α-淀粉酶变异体的宿主细胞。
本发明上述方法的步骤(a)优选使用掺杂(doped)引物进行。例如，随机诱变可通过使用合适的物理或化学诱变剂，通过使用合适的寡核苷酸，或者通过对DNA序列进行PCR产生的诱变来进行。另外，随机诱变可通过使用这些诱变剂的任意组合进行。诱变剂可以是，例如，诱导转换，易位，倒位，拼凑，缺失和/或插入的试剂。
适合于该目的的物理或化学诱变剂的例子包括紫外(UV)辐射，羟胺，N-甲基-N'-硝基-N-亚硝基胍(MNNG)，O-甲基羟胺，亚硝酸，乙基甲磺酸(EMS)，亚硫酸氢钠，甲酸，和核苷酸类似物。当使用该试剂时，一般通过在适合于发生诱变的条件下在选定诱变剂存在下温育待诱变的编码亲本酶的DNA序列并选择具有所需特性的突变DNA来进行诱变。当使用寡核苷酸进行诱变时，可在寡核苷酸合成期间在需改变的位置用三个非亲本核苷酸掺杂或锲入(spiked)该寡核苷酸。进行掺杂或锲入使得可以避免不需要的氨基酸密码子。掺杂或锲入的寡核苷酸可通过任何公开的技术，使用例如，PCR，LCR或认为合适的任何DNA聚合酶和连接酶掺入编码α-淀粉酶的DNA中。优选的是，掺杂使用“固定随机掺杂”进行，其中预先确定各位置中野生型和突变的百分数。另外，掺杂可定向优先导入某些核苷酸，从而优先导入一个或多个特定氨基酸残基。可进行掺杂以便例如，允许在各位置导入90％野生型和10％的突变。选择掺杂方案的另一考虑是以遗传和蛋白质结构约束为基础。掺杂方案可通过使用DOPE程序进行，该程序特别是确保避免导入终止密码子。当使用PCR-产生的诱变时，在增加核苷酸错误掺入的条件下对化学处理或未处理的编码亲本α-淀粉酶的基因进行PCR(Deshler 1992；Leung等，Technique,Vol.1,1989,第11-15页)。大肠杆菌的增变株(Fowler等，Molec.Gen.Genet.,133,1974,第179-191页)，啤酒糖酵母(S.cereviseae)或任何其它微生物可用于随机诱变编码α-淀粉酶的DNA，通过例如，将含有亲本糖基化酶的质粒转化进增变株，繁殖含有该质粒的增变株和从该增变株分离突变的质粒。随后可将突变质粒转化进表达生物中。待诱变的DNA序列可方便地存在于从表达亲本α-淀粉酶的生物制备的基因组或cDNA文库中。另外，该DNA序列可存在于诸如质粒或噬菌体的合适载体中，这样用诱变剂温育或与其接触。待诱变的DNA也可通过整合在所述细胞的基因组中或者存在于该细胞携带的载体中而存在于宿主细胞中。最后，待诱变的DNA可以是分离的形式，应明白进行随机诱变的DNA序列优选是cDNA或基因组DNA序列。在某些情况下，在进行表达步骤b)或筛选步骤c)之前扩增突变的DNA序列是合适的。该扩增可按照本领域已知的方法进行，目前优选的方法是使用根据亲本酶的DNA或氨基酸序列制备的寡核苷酸引物的PCR产生的扩增。用诱变剂温育或与其接触后，通过在允许发生表达的条件下培养携带该DNA序列的合适宿主细胞可表达突变的DNA。用于该目的的宿主细胞是用任选存在于载体中的突变DNA序列转化的细胞，或在诱变处理期间携带编码亲本酶的DNA序列的细胞。合适宿主细胞的例子如下：革兰氏阳性细菌，例如枯草芽孢杆菌，地衣芽孢杆菌(Bacillus licheniformis)，迟缓芽孢杆菌(Bacillus lentus),短芽孢杆菌(Bacillus brevis)，嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)，嗜碱芽孢杆菌(Bacillus alkalophilus),解淀粉芽孢杆菌(Bacillus amyloliquefaciens),凝固芽孢杆菌(Bacillus coagulans)，环状芽孢杆菌(Bacillus circulans)，灿烂芽孢杆菌(Bacillus lautus),巨大芽孢杆菌(Bacillusmegaterium)，苏芸金芽孢杆菌(Bacillus thuringiensis)，Streptomyces lividans或Streptomyces murinus；和革兰氏阴性细菌，例如大肠杆菌。突变的DNA序列还可包含编码允许表达突变的DNA序列的功能的DNA序列。
定位(Localised)随机诱变
可将随机诱变有利地定位于所述亲本α-淀粉酶的一部分上。例如，当该酶的某些区域被鉴定为对于该酶的给定特性特别重要且当修饰时预期将产生具有改良特性的变异体时这是有利的。当亲本酶的三级结构被阐明且与该酶的功能相关联时一般可鉴定该区域。
通过使用上述PCR产生的诱变技术或本领域已知的任何其它合适的技术可方便地进行定位的，或区域特异性的随机诱变。作为选择，通过例如，插入合适的载体中可分离编码待修饰的部分DNA序列的DNA序列，且通过使用任何上述诱变方法随后对所述的部分进行诱变。
提供α-淀粉酶变异体的另一方法
提供本发明的变异体的另一方法包括本领域已知的基因改组方法，包括例如在WO95/22625(Affymax Technologies N.V.)和WO 96/00343(Novo Nordisk A/S)中所述的方法。
表达α-淀粉酶变异体
根据本发明，通过上述方法，或者通过本领域已知的任何可供选择的方法产生的编码变异体的DNA可使用一般包括编码启动子，操纵子，核糖体结合位点，翻译起始信号，和任选阻遏基因或各种激活基因的调控序列的表达载体以酶的形式表达。
携带编码本发明的α-淀粉酶变异体的DNA序列的重组表达载体可以是可方便地对其进行重组DNA操作的任何载体，且载体的选择通常依赖于它将要导入的宿主细胞。因此，该载体可以是自主复制载体，即，作为染色体外实体存在，其复制独立于染色体复制的载体，例如，质粒，噬菌体或染色体外元件，微型染色体或人工染色体。作为选择，该载体可以是导入宿主细胞时整合进宿主细胞基因组并与整合进的染色体一起复制的载体。
在载体中，该DNA序列应与合适的启动子序列可操作地连接。该启动子可以是在选定的宿主细胞中表现出转录活性的任何DNA序列且可来自于与该宿主细胞同源或异源的编码蛋白质的基因。指导编码本发明的α-淀粉酶变异体的DNA序列在特别是细菌宿主中转录的合适启动子的例子是大肠杆菌lac操纵子的启动子，天蓝色链霉菌(Streptomycescoelicolor)琼脂糖酶基因dagA启动子，地衣芽孢杆菌α-淀粉酶基因(amyL)的启动子，嗜热脂肪芽孢杆菌产麦芽糖淀粉酶基因(amyM)的启动子，解淀粉芽孢杆菌α-淀粉酶(amyQ)的启动子，枯草芽孢杆菌xylA和xylB基因的启动子等。为了在真菌宿主中转录，有用的启动子的例子是来自编码米曲霉TAKA淀粉酶，Rhizomucor miehei天冬氨酸蛋白酶，黑曲霉中性α-淀粉酶，黑曲霉酸稳定性α-淀粉酶，黑曲霉葡糖淀粉酶，Rhizomucor miehei脂肪酶，米曲霉碱性蛋白酶，米曲霉丙糖磷酸异构酶或构巢曲霉(A.nidulans)乙酰胺酶的基因的那些启动子。
本发明的表达载体也可包含与编码本发明的α-淀粉酶变异体的DNA序列可操作地连接的合适的转录终止子和在真核细胞中的多聚腺苷化序列。终止和多聚腺苷化序列可合适地来源于与启动子相同的来源。
该载体还可包含使得该载体能够在所述宿主细胞中复制的DNA序列。该序列的例子是质粒pUC19,pACYC177,pUB110,pE194,pAMB1和pIJ702的复制起点。
该载体也可包含选择性标记，例如，其产物弥补宿主细胞缺陷的基因，例如，枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或赋予抗生素抗性，例如氨苄青霉素，卡那霉素，氯霉素或四环素抗性的基因。另外，该载体可包含曲霉属选择标记，例如amdS,argB,niaD和sC，产生潮霉素抗性的标记，或该选择可通过共转化实现，例如在WO 91/17243中所述。
尽管细胞内表达在某些方面，例如，当使用某些细菌作为宿主细胞时具有优势，但是一般优选在细胞外表达。一般来说，本文提及的芽孢杆菌属α-淀粉酶包含允许表达的蛋白酶分泌进培养基的前肽区(preregion)。如果需要，该前肽区可用不同前肽区或信号序列取代，它可通过取代编码各前肽区的DNA序列按常规完成。
用于分别连接编码α-淀粉酶变异体，启动子，终止子和其它元件的本发明的DNA构建体，并将它们插入含有复制必需信息的合适载体的方法是本领域的技术人员熟知的(参见，例如，Sambrook等，Molecular Cloning:A Laboratory Manual，第二版，Cold SpringHarbor,1989)。
包含上述本发明的DNA构建体或表达载体的本发明的细胞用作重组生产本发明的α-淀粉酶变异体的宿主细胞是有利的。可通过将该DNA构建体(一个或多个拷贝)方便地整合进宿主染色体中用编码变异体的本发明的DNA构建体转化该细胞。一般认为该整合是具有优势的，因为该DNA序列在细胞中稳定维持的可能性更大。将DNA构建体整合进宿主染色体可按照常规方法进行，例如，通过同源或异源重组。作为选择，该细胞可用与不同类型的宿主细胞相联系的上述表达载体转化。
本发明的细胞可以是高等生物的细胞，例如哺乳动物或昆虫，但是优选微生物细胞，例如细菌或真菌(包括酵母)细胞。
合适的细菌的例子是革兰氏阳性细菌，例如枯草芽孢杆菌，地衣芽孢杆菌，迟缓芽孢杆菌,短芽孢杆菌，嗜热脂肪芽孢杆菌，嗜碱芽孢杆菌,解淀粉芽孢杆菌,凝固芽孢杆菌，环状芽孢杆菌，灿烂芽孢杆菌,巨大芽孢杆菌，苏芸金芽孢杆菌，或Streptomyces lividans或Streptomyces murinus，或革兰氏阴性细菌，例如大肠杆菌。细菌的转化可通过例如，原生质体转化或通过使用感受态细胞以本身已知的方式实现。
酵母生物合适地选自糖酵母属(Saccharomyces)或裂殖糖酵母属(Schizosaccharomyces)的菌种，例如啤酒糖酵母(Saccharomyces cerevisiae)。有利的丝状真菌属于曲霉属的菌种，例如米曲霉或黑曲霉。真菌细胞可通过包含原生质体形成和原生质体转化的方法进行转化，接着以本身已知的方式再生细胞壁。转化曲霉属宿主细胞的合适方法在EP 238 023中描述。
在另一方面，本发明涉及产生本发明的α-淀粉酶变异体的方法，该方法包括在有助于生产该变异体的条件下培养上述宿主细胞并从细胞和/或培养基中回收该变异体。
用于培养该细胞的培养基可以是适合于所述宿主细胞生长并获得本发明的α-淀粉酶变异体表达的任何常规培养基。合适的培养基可从供应商获得或者可按照公开的配方(例如，在美国典型培养物保藏中心的产品目录中所述)制备。
从宿主细胞分泌的α-淀粉酶变异体可按常规通过熟知的方法从培养基中回收，包括通过离心或过滤分离细胞与培养基，借助于诸如硫酸铵的盐沉淀培养基中的蛋白质成份，接着使用色谱法，例如离子交换色谱法，亲和色谱法，或类似方法。
工业应用
本发明的α-淀粉酶变异体具有允许各种工业应用的宝贵特性。具体地说，本发明的酶变异体适用于用作洗涤，洗碗，和硬表面清洁去污剂组合物的成份。
具有改变特性的本发明的变异体可用于淀粉加工，特别是淀粉转化，特别是淀粉的液化(参见，例如，US 3,912,590,EP专利申请号252 730和63 909，WO 99/19467，和WO96/28567，所有文献在此引用以供参考)。本发明还涉及用于淀粉转化用途的组合物，它除了本发明的变异体外，还包含葡糖淀粉酶，支链淀粉酶，和其它α-淀粉酶。
另外，本发明的变异体还特别用于从淀粉或完整谷粒生产增甜剂和乙醇(参见，例如，在此作为参考文献引用的美国专利号5,231,017)，例如燃料，饮料和工业乙醇。
本发明的变异体也可用于纺织品，织物和服装的脱浆(参见，例如，WO 95/21247,US专利4,643,736，EP 119,920，在此引用以供参考)，啤酒制备或酿造，纸浆和纸生产，和饲料和食品生产。
淀粉转化
常规淀粉转化方法，例如液化和糖化方法在例如，美国专利号3,912,590和EP专利公开号252,730和63,909中描述，在此引用以供参考。
在一个实施方案中，将淀粉降解成诸如糖或脂肪替代品的低分子量糖成份的淀粉转化方法包括去分支步骤。
淀粉转化成糖
为了将淀粉转化成糖，应解聚淀粉。该解聚过程由预处理步骤和两个或三个连续的加工步骤组成，即，液化过程，糖化过程和依赖于所需最终产物任选的异构化过程。
天然淀粉的预处理
天然淀粉由显微颗粒组成，它在室温下不溶于水。当加热含水淀粉浆料时，该颗粒膨胀并最终破裂，将淀粉分子分散进溶液中。在该“糊化”过程中，粘度显著增加。由于在典型的工业过程中固形物水平是30-40％，因此淀粉要变稀或“液化”(liquefied)使其能够处理。这种粘度的降低目前主要通过酶降解获得。
液化(liquefaction)
液化步骤期间，长链淀粉由α-淀粉酶降解为分支和线性较短单位(麦芽糖糊精)。液化步骤在105-110℃下进行5到10分钟，接着在95℃进行1－2小时。pH位于5.5到6.2之间。为了保证在这些条件下的最佳酶稳定性，加入1mM钙(40ppm游离钙离子)。该处理后，液化的淀粉具有10-15的“葡萄糖当量”(DE)。
糖化
经过液化过程后，通过加入葡糖淀粉酶(例如，AMG)和去分支酶，例如异淀粉酶(美国专利号4,335,208)或支链淀粉酶(例如，PromozymeTM)(美国专利号4,560,651)将麦芽糖糊精转化为葡萄糖。在该步骤前，将pH值降低到低于4.5，维持高温(高于95℃)以便灭活液化α-淀粉酶以减少称为“潘糖前体”的短寡糖形成，该寡糖不能被去分支酶正确水解。
将温度降低到60℃，加入葡糖淀粉酶和去分支酶。该糖化过程进行24-72小时。
一般来说，在液化步骤后变性α-淀粉酶时，大约0.2-0.5％的糖化产物是不能被支链淀粉酶降解的分支的三糖62-α-葡糖基麦芽糖(潘糖)。如果在糖化期间存在来自液化步骤的活性淀粉酶(即，不变性)，该水平可高达1-2％，这是非常不希望的，因为它明显降低了糖化产量。
异构化
当所需的最终糖产物是例如，高果糖糖浆时，可将葡萄糖糖浆转变成果糖。在糖化过程后，将pH值增加到6-8的范围内，优选7.5，且通过离子交换去掉钙。然后使用例如，固定化葡萄糖异构酶(例如，SweetzymeTM IT)将葡萄糖糖浆转化成高果糖糖浆。
乙醇生产
一般来说，从完整谷粒生产醇(乙醇)可分成4个主要步骤：
-磨碎
-液化
-糖化
-发酵
磨碎
磨碎谷粒以便打开该结构并允许进一步加工。使用湿磨或干磨两个步骤。在干磨中，磨碎完整种仁并用于该方法的剩下部分。湿磨可产生极好的胚芽(germ)和粗粉(meal)(淀粉颗粒和蛋白质)分离且除少数例外情况外一般用于平行生产糖浆的情况。
液化
在液化过程中，淀粉颗粒通过水解溶解成大部分DP高于4的麦芽糖糊精。水解可通过酸处理或通过α-淀粉酶的酶处理进行。酸水解用于有限的情况中。粗制材料可以是磨细的完整谷粒或来自淀粉加工的副产物(side stream)。
酶液化一般作为三步加热浆料法进行。将浆料加热到60-95℃之间，优选80-85℃，加入酶。然后在95-140℃之间，优选105-125℃下喷射蒸煮该浆料，冷却到60-95℃并加入更多酶以获得最终水解。液化过程在pH 4.5-6.5，一般在5到6之间的pH下进行。磨碎并液化的谷粒也称为麦芽汁。
糖化
为了生产可被酵母代谢的低分子量糖DP1-3，可进一步水解液化产生的麦芽糖糊精。水解一般通过葡糖淀粉酶进行酶处理，作为选择，可使用α-葡糖苷酶或酸性α-淀粉酶。全部糖化步骤可持续72小时，然而，通常仅进行一般40-90分钟的预糖化和随后在发酵期间完成糖化(SSF)。糖化一般在从30-65℃，一般大约60℃的温度下和在pH 4.5下进行。
发酵
一般来自糖酵母属(Saccharomyces)菌种的酵母加入麦芽汁中且发酵进行24-96小时，例如一般35-60小时。温度在26-34℃之间，一般在大约32℃下，pH为pH 3-6，优选大约pH 4-5。
注意最广泛使用的方法是同时糖化和发酵(SSF)方法，其中没有糖化保持期，这意味着酵母和酶一起加入。当进行SSF时，通常在发酵前立即引入在超过50℃温度下的预糖化步骤。
蒸馏
发酵后，蒸馏麦芽汁提取乙醇。
根据本发明方法获得的乙醇可用作例如，燃料乙醇；饮料乙醇，即可饮用的精制酒精(neutral ethanol)；或工业乙醇。
副产品
发酵剩余物是一般以液体形式或干燥形式用于动物饲料的谷粒。
如何进行液化，糖化，发酵，蒸馏，和回收乙醇的进一步细节是技术人员熟知的。
根据本发明的方法，糖化和发酵可同时或分开进行。
纸浆和纸生产
本发明的碱性α-淀粉酶也可用于从淀粉强化的废纸或薄纸板生产木质素纤维素材料，例如纸浆，纸和薄纸板，特别是在pH超过7下进行再制浆和淀粉酶通过降解强化淀粉促进废料分解的情况下。本发明的α-淀粉酶特别用于从淀粉覆盖的打印纸生产造纸纸浆的方法中。该方法可按WO 95/14807所述进行，包括如下步骤：
a)分解纸以便生产纸浆，
b)在步骤a)之前，期间或之后用淀粉降解酶处理，和
c)在步骤a)和b)之后从纸浆分离墨汁颗粒
本发明的α-淀粉酶在酶修饰的淀粉与诸如碳酸钙，高岭土(kaolin)和粘土的碱性填料一起用于造纸的情况下对于修饰淀粉也非常有用。使用本发明的碱性α-淀粉酶，使得在该填料存在下修饰淀粉成为可能，从而使得整体方法更简单。
纺织品，织物和衣服的脱浆
本发明的α-淀粉酶在纺织品，织物或衣服脱浆中也非常有用。在纺织品加工工业中，α-淀粉酶在脱浆过程中按传统用作辅助剂以促进含淀粉浆料的去除，该浆料在纺织期间用作覆盖织物纱线的保护剂。纺织后完全去掉浆料涂层对于确保清洗，漂白和染色织物的随后加工中的最佳效果是重要的。优选酶促淀粉分解，因为它不会对纤维材料产生任何有害影响。为了降低加工成本和提高工厂产量(mill throughput)，有时将脱浆加工与清洗(scouring)和漂白步骤合并。在这种情况下，一般使用诸如碱或氧化剂的非酶辅助剂分解淀粉，因为传统的α-淀粉酶与高pH水平和漂白剂非常不相容。非酶分解淀粉浆料导致一些纤维损坏，因为使用了相当烈性的化学品。因此，需要使用本发明的α-淀粉酶，因为它们在碱性溶液中具有改良的性能。该α-淀粉酶可单独使用或者在对含纤维素的织物或纺织品进行脱浆时可与纤维素酶联合使用。
脱浆和漂白方法是本领域熟知。例如，该方法在本文作为参考文献引用的WO 95/21247，美国专利4,643,736，EP 119,920中描述。
商业上可获得的用于脱浆的产品包括来自Novozymes A/S的
Figure BDA0003128425700000371
和
Figure BDA0003128425700000372
ULTRA。
啤酒生产
本发明的α-淀粉酶在啤酒生产方法中也非常有用；一般在麦芽汁处理期间添加该α-淀粉酶。
去污剂组合物
本发明的α-淀粉酶可添加到去污剂组合物中并因此成为去污剂组合物的一种成份。
本发明的去污剂组合物可配制成例如，手工或机械洗衣的去污剂组合物，包括适合于染色织物预处理的洗衣添加剂组合物和漂洗添加的织物软化剂组合物，或配制成用于常规家庭硬表面清洁操作的去污剂组合物，或配制成用于手工或机械的洗碗操作。
在一个具体方面，本发明提供了包含本发明酶的去污剂添加剂。该去污剂添加剂以及去污剂组合物可包含一个或多个其它酶，例如蛋白酶，脂肪酶，过氧化物酶，另一淀粉分解酶，例如，另一α-淀粉酶，葡糖淀粉酶，产麦芽糖淀粉酶，CGTase和/或纤维素酶，甘露聚糖酶(例如MANNAWAYTM，来自Novozymes,Denmark)，果胶酶，果胶裂解酶，角质酶，和/或漆酶。
一般来说，选定酶的特性应与选择的去污剂相容，(即，pH最适，与其它酶和非酶成份等相容)，且该酶应当以有效量存在。
蛋白酶：合适的蛋白酶包括动物，植物或微生物来源的蛋白酶。优选微生物来源。包括化学修饰或蛋白质改造的突变体。该蛋白酶可以是丝氨酸蛋白酶或金属蛋白酶，优选碱性微生物蛋白酶或胰蛋白酶样蛋白酶。碱性蛋白酶的例子有枯草杆菌蛋白酶，特别是来源于芽孢杆菌属的那些枯草杆菌蛋白酶，例如，枯草杆菌蛋白酶Novo，枯草杆菌蛋白酶Carlsberg，枯草杆菌蛋白酶309，枯草杆菌蛋白酶147和枯草杆菌蛋白酶168(在WO 89/06279中描述)。胰蛋白酶样蛋白酶的例子是胰蛋白酶(例如，猪或牛来源的胰蛋白酶)和在WO 89/06270和WO 94/25583中所述的镰孢属(Fusarium)蛋白酶。
有用的蛋白酶的例子是在WO 92/19729，WO 98/20115，WO 98/20116，和WO 98/34946中所述的变异体，特别是在一个或多个下列位置：27,36,57,76,87,97,101,104,120,123,167,170,194,206,218,222,224,235和274中具有取代的变异体。
优选的商业上可获得的蛋白酶包括
Figure BDA0003128425700000381
Figure BDA0003128425700000382
和
Figure BDA0003128425700000383
(来自Novozymes A/S),
Figure BDA0003128425700000384
MAXACAL,
Figure BDA0003128425700000385
Figure BDA0003128425700000386
PURAFECT
Figure BDA0003128425700000387
(Genencor International Inc.)。
脂肪酶：合适的脂肪酶包括细菌或真菌来源的那些脂肪酶。包括化学修饰的或蛋白质改造的突变体。有用的脂肪酶的例子包括来自腐质酶属(Humicola)(同义词Thermomyces)，例如，来自EP 258 068和EP 305 216中所述的H.lanuginosa(T.lanuginosus)或来自WO 96/13580中所述的H.insolens的脂肪酶，假单胞菌属(Pseudomonas)脂肪酶，例如，来自P.alcaligenes或类产碱假单胞菌(P.pseudoalcaligenes)(EP 218 272)，葱头假单胞菌(P.cepacia)(EP 331 376)，司徒茨氏假单胞菌(P.stutzeri)(GB 1,372,034)，荧光假单胞菌(P.fluorescens)，假单胞菌属菌株SD 705(WO 95/06720和WO 96/27002)，P.wisconsinensis(WO 96/12012)的脂肪酶，芽孢杆菌属脂肪酶，例如，来自枯草芽孢杆菌(Dartois等，(1993),Biochemica et BiophysicaActa,1131,253-360)，嗜热脂肪芽孢杆菌(JP 64/744992)或短小芽孢杆菌(B.pumilus)(WO91/16422)的脂肪酶。
其它例子是脂肪酶变异体，例如在WO 92/05249,WO 94/01541,EP 407225,EP 260105,WO 95/35381,WO 96/00292,WO 95/30744,WO 94/25578,WO 95/14783,WO 95/22615,WO 97/04079和WO 97/07202中所述的那些变异体。
优选的商业上可获得的脂肪酶包括LIPOLASETM和LIPOLASE ULTRATM(NovozymesA/S)。
淀粉酶：合适的淀粉酶(α和/或β)包括细菌或真菌来源的那些淀粉酶。包括化学修饰的或蛋白质改造的变异体。淀粉酶包括例如，从芽孢杆菌属，例如地衣芽孢杆菌的特定菌株获得的α-淀粉酶，它在GB 1,296,839中进行了更详细的描述。有用的α-淀粉酶的例子是在WO 94/02597，WO 94/18314，WO 96/23873，和WO 97/43424中所述的变异体，特别是在一个或多个下列位置：15,23,105,106,124,128,133,154,156,181,188,190,197,202,208,209,243,264,304,305,391,408,和444中具有取代的变异体。
商业上可获得的α-淀粉酶是DURAMYLTM,LIQUEZYMETMTERMAMYLTM,NATALASETM,SUPRAMYLTM,STAINZYMETM,FUNGAMYLTM和BANTM(Novozymes A/S),RAPIDASETM,PURASTARTM和PURASTAR OXAMTM(来自Genencor International Inc.)。
纤维素酶：合适的纤维素酶包括细菌或真菌来源的那些酶。包括化学修饰或蛋白质改造的变异体。合适的纤维素酶包括来自芽孢杆菌属，假单胞菌属，腐质酶属,镰孢属(Fusarium),草根霉属(Thielavia),支顶孢属(Acremonium)的纤维素酶，例如，在US 4,435,307,US 5,648,263,US 5,691,178,US 5,776,757和WO 89/09259中公开的由Humicolainsolens,Myceliophthora thermophila和尖孢镰孢(Fusarium oxysporum)产生的真菌纤维素酶。
特别合适的纤维素酶是具有颜色喜好优点(colour care benefit)的碱性或中性纤维素酶。该纤维素酶的例子是在EP 0 495 257，EP 0 531 372，WO 96/11262，WO 96/29397，WO 98/08940中所述的纤维素酶。其它例子是纤维素酶变异体，例如在WO 94/07998,EP 0 531 315,US 5,457,046,US 5,686,593,US 5,763,254,WO 95/24471,WO 98/12307和PCT/DK98/00299中所述的那些变异体。
商业上可获得的纤维素酶包括
Figure BDA0003128425700000391
和
Figure BDA0003128425700000392
(Novozymes A/S),
Figure BDA0003128425700000393
和PURADAX
Figure BDA0003128425700000394
(Genencor International Inc.)，和
Figure BDA0003128425700000395
(Kao Corporation)。
过氧化物酶/氧化酶：合适的过氧化物酶/氧化酶包括植物，细菌或真菌来源的那些酶。包括化学修饰或蛋白质改造的突变体。有用的过氧化物酶的例子包括来自Coprinus，例如，来自C.cinereus的过氧化物酶及其变异体，例如在WO 93/24618,WO 95/10602,和WO98/15257中所述的那些酶。
商业上可获得的过氧化物酶包括
Figure BDA0003128425700000396
(Novozymes A/S)。
通过加入含有一种或多种酶的分开的(separate)添加剂，或者通过加入含有所有这些酶的组合添加剂可在去污剂组合物中包含去污剂酶。本发明的去污剂添加剂，即，分开的添加剂或组合添加剂可配制成例如，粒状，液体，浆液等。优选的去污剂添加剂配制品是粒状，特别是无尘粒状，液体，特别是稳定液体，或浆液。
无尘粒状可按例如，在US 4,106,991和4,661,452中所述生产且任选通过本领域已知的方法包衣。蜡状包衣材料的例子是具有1000至20000平均分子量的聚环氧乙烷产物(聚乙二醇，PEG)；具有16到50个环氧乙烷单元的乙氧基化壬烷基酚；其中醇含有12到20个碳原子且有15到80个环氧乙烷单元的乙氧基化脂肪醇；脂肪醇；脂肪酸；和脂肪酸的单-和二-和三酸甘油酯。适合于通过流化床技术应用的成膜包衣材料的例子在GB 1483591中提供。液体酶制品可通过例如，按照已有方法加入诸如丙二醇的多元醇，糖或糖醇，乳酸或硼酸来稳定。根据EP 238,216中公开的方法可制备保护的酶。
本发明的去污剂组合物可以是任何方便的形式，例如，棒状，片剂，粉末，颗粒，糊剂或液体。液体去污剂可以是含水的，一般含高达70％的水和0-30％有机溶剂，或可不含水。
去污剂组合物含有一种或多种表面活性剂，它可以是包括半极性的非离子，和/或阴离子和/或阳离子和/或两性离子的表面活性剂。表面活性剂一般以0.1％到60％的重量水平存在。
当其中包含时，去污剂通常含有从大约1％到大约40％的阴离子表面活性剂，例如，线性烷基苯磺酸盐，α-链烯磺酸盐，烷基硫酸盐(脂肪醇硫酸盐)，脂肪醇乙氧硫酸盐，仲链烷磺酸盐，α-硫代脂肪酸甲酯，烷基或链烯基琥珀酸或肥皂。
当其中包含时，去污剂通常含有从大约0.2％到大约40％的非离子表面活性剂，例如，乙氧基化醇，乙氧基化壬烷基酚，烷基多聚糖苷，烷基二甲基氧化胺，乙氧基化脂肪酸一乙醇酰胺，脂肪酸一乙醇酰胺，聚羟基烷基脂肪酸酰胺，或葡糖胺的N-酰基N-烷基衍生物("葡糖酰胺")。
去污剂可含有0-65％的去污剂组份或络合剂，例如沸石，二磷酸盐，三磷酸盐，膦酸(phosphonate)，碳酸盐，柠檬酸盐，氮三乙酸，乙二胺四乙酸，二亚乙基三胺五乙酸，烷基或烯基琥珀酸，可溶性硅酸盐或分层硅酸盐(例如，SKS-6，来自Hoechst)。
该去污剂(detergent)可包含一个或多个聚合物。例子有羧甲基纤维素，聚乙烯吡咯烷酮，聚乙二醇，聚乙烯醇，聚乙烯啶-N-氧化物，聚乙烯咪唑，聚羧酸酯，例如聚丙烯酸酯，马来酸/丙烯酸共聚物和月桂基异丁烯酸/丙烯酸共聚物。
去污剂可含有漂白系统，它可包含H2O2源，例如可与诸如四乙酰乙二胺或壬酰羟苯磺酸的过酸形成漂白活化剂结合的过硼酸盐或过碳酸盐。作为选择，漂白系统可含有例如酰胺，亚胺，或砜形式的过氧酸。
本发明的去污剂组合物的酶可使用常规稳定剂稳定，例如，诸如丙二醇或甘油的多元醇，糖或糖醇，乳酸，硼酸，或硼酸衍生物，例如，芳香硼酸酯，或苯基硼酸衍生物，例如4-甲酰基苯基硼酸，且该组合物可按例如，WO 92/19709和WO 92/19708所述配制。
该去污剂也可含有其它常规去污剂成份，例如，织物调节剂，包括粘土，泡沫激发剂，泡沫抑制剂，抗腐蚀剂，尘土悬浮剂，抗尘土再沉积剂，染料，杀菌剂，光亮剂，水溶助剂(hydrotropes)，玷污抑制剂，或香料。
本发明还涉及在去污剂组合物中以相应于每升洗涤溶液0.001-100mg酶蛋白，优选每升洗涤溶液0.005-5mg酶蛋白，更优选每升洗涤溶液0.01-1mg酶蛋白且特别是每升洗涤溶液0.1-1mg酶蛋白的量加入任何酶，特别是本发明的酶。
本发明的酶还可掺入在此作为参考文献引用的WO 97/07202中公开的去污剂配制品中。
洗碗去污剂组合物
本发明的酶也可用于洗碗去污剂组合物中，包括下列组合物：
1)粉剂自动洗碗组合物
非离子表面活性剂 0.4 -2.5％
硅酸钠 0 -20％
二硅酸钠 3 -20％
三磷酸钠 20 -40％
碳酸钠 0 -20％
过硼酸钠 2 -9％
四乙酰乙二胺(TAED) 1 -4％
硫酸钠 5 -33％
酶 0.0001 -0.1％
2)粉剂自动洗碗组合物
Figure BDA0003128425700000411
Figure BDA0003128425700000421
3)粉剂自动洗碗组合物
非离子表面活性剂 0.5 -2.0％
二硅酸钠 25 -40％
柠檬酸钠 30 -55％
碳酸钠 0 -29％
碳酸氢钠 0 -20％
一水过硼酸钠 0 -15％
四乙酰乙二胺(TAED) 0 -6％
马来酸/丙烯酸共聚物 0 -5％
粘土 1 -3％
聚氨基酸 0 -20％
聚丙烯酸钠 0 -8％
酶 0.0001 -0.1％
4)粉剂自动洗碗组合物
Figure BDA0003128425700000422
Figure BDA0003128425700000431
5)粉剂自动洗碗组合物
非离子表面活性剂 1 -7％
二硅酸钠 18 -30％
柠檬酸三钠 10 -24％
碳酸钠 12 -20％
单过硫酸(2KHSO5.KHSO4.K2SO4) 15 -21％
漂白稳定剂 0.1 -2％
马来酸/丙烯酸共聚物 0 -6％
二亚乙基三胺五乙酸酯，五钠盐 0 -2.5％
酶 0.0001 -0.1％
硫酸钠，水 平衡
6)具有清洁表面活性剂系统的粉剂和液体洗碗组合物
Figure BDA0003128425700000432
Figure BDA0003128425700000441
7)非水液体自动洗碗组合物
Figure BDA0003128425700000442
8)无水液体洗碗组合物
Figure BDA0003128425700000443
9)触变液体自动洗碗组合物
Figure BDA0003128425700000444
Figure BDA0003128425700000451
10)液体自动洗碗组合物
乙氧基化醇 0 -20％
磺酸脂肪酸酯 0 -30％
十二烷基硫酸钠 0 -20％
烷基聚糖苷酯(Alkyl polyglycoside) 0 -21％
油酸 0 -10％
一水二硅酸钠 18 -33％
脱水柠檬酸钠 18 -33％
硬脂酸钠 0 -2.5％
一水过硼酸钠 0 -13％
四乙酰乙二胺(TAED) 0 -8％
马来酸/丙烯酸共聚物 4 -8％
酶 0.0001 -0.1％
11)含保护性漂白颗粒的液体自动洗碗组合物
Figure BDA0003128425700000452
Figure BDA0003128425700000461
12)1),2),3),4),6)和10)所述的自动洗碗组合物，其中过硼酸盐被过碳酸盐取代。
13)1)-6)所述的自动洗碗组合物，它还含有锰催化剂。锰催化剂可以是，例如，“用于低温漂白的有效锰催化剂”，Nature 369,1994,第637-639页中所述的化合物之一。
材料和方法
酶:
SP722:SEQ ID NO:4，可从Novozymes获得，且在WO 95/26397中描述。
AA560:SEQ ID NO:12；在WO 00/60060中公开且可从Novozymes A/S获得；在丹麦专利申请号PA 1999 00490中公开；于1999年1月25日在DSMZ保藏且保藏号为DSMZno.12649。
AA560按布达佩斯条约在用于专利程序的微生物国际保藏中心DeutsheSammmlung von Microorganismen und Zellkulturen GmbH(DSMZ),Mascheroder Weg 1b,D-38124Braunschweig DE保藏。
AX379:可从Novozymes获得。
枯草芽孢杆菌(Bacillus subtilis)SHA273：参见WO 95/10603。
质粒
pJE1含有编码SP722α-淀粉酶变异体的基因(SEQ ID NO:4)：即相应于成熟蛋白氨基酸D183-G184的6个核苷酸缺失。JE1基因的转录由amyL启动子指导。该质粒还含有复制起点和从质粒pUB110获得的赋予氯霉素抗性的cat-基因(Gryczan,TJ等，(1978),J.Bact.134:318-329)。
pDN1528含有编码Termamyl,amyL的完整基因，其表达由其自身的启动子指导。另外，该质粒含有来自质粒pUB110的复制起点，ori,和来自质粒pC194的赋予氯霉素抗性的cat基因。pDN1528在WO 96/23874的图9中显示。
方法：
常规分子生物学方法：
除非另有说明，DNA操作和转化使用标准分子生物学方法进行(Sambrook等，(1989)；Ausubel等，(1995)；Harwood和Cutting(1990)。
模型构建
检索蛋白质结构数据库，例如“The Protein Data Bank(PDB)(<http:// www.pdb.bnl.gov/>)”或“The Brookhaven databank at Brookhaven NationalLaboratory,US”中与需要建立模型的所述分子相似的蛋白质。对氨基酸序列进行序列对比，考虑结构保守区并将坐标(coordinates)从对照蛋白复制到目的蛋白。从具有相似氨基酸序列的其它蛋白质，或者通过使用大多数3D软件包，例如Biosym,MSI的Homology中建立的随机结构产生功能设定具有插入和缺失的坐标区域。
当设定了目的蛋白的所有氨基酸的坐标并通过例如，Biosym,MSI的Discover程序的命令END REPAIR和SPLICE REPAIR将该片断连接在一起时，需要精选该模型。该模型的能量首先通过松弛该分子来最小化(Discover程序中的RELAX命令)且随后通过分子动力学来最小化。
参考文献可参见同源性构建软件，例如Biosym,MSI的Homology的操作手册。
获得感兴趣区域的方法：
有3个已知的细菌α-淀粉酶3D结构。两个地衣芽孢杆菌α-淀粉酶，Brookhavendatabase 1BPL(Machius等，(1995),J.Mol.Biol.246,第545-559页)和1VJS(Song等，(1996),Enzymes for Carbohydrate 163Engineering(Prog.Biotechnol.V 12)。这两个结构在两个钙离子和一个钠离子结合位点周围的区域缺少所谓B-结构域的重要结构片断。还有一个α-淀粉酶BA2的3D结构(WO 96/23874，它是公开的BANTM(SEQ ID NO.5)和地衣芽孢杆菌α-淀粉酶(SEQ ID NO.4)之间的杂合体，它含有全部B-结构域且因此含有A和B结构域之间的金属离子。另外，来自B.stearothermophilus的α-淀粉酶主要部分的结构也被Sued[？]公开且碱性α-淀粉酶SP722的结构在WO 01/66712中提供。
为了构建给定α-淀粉酶的最佳模型，选择非常同源的结构，即地衣芽孢杆菌α-淀粉酶的良好模型最好根据BA2的结构来构建，因此它是B.amyloliquefacienceα-淀粉酶的良好模型，而类似AA560,SP707,SP7-7和KSM-AP1378的碱性α-淀粉酶最好根据SP722α-淀粉酶的结构来构建。
从SP722的三级结构同源性构建AA560
如上所述，AA560α-淀粉酶(SEQ ID NO:12)与SP722(SEQ ID NO:4)的总体同源性是大约87％。AA560与SP722的序列对比表明其中没有插入或缺失，也可参见图1。
AA560α-淀粉酶的三级结构是使用“材料和方法”部分所述的方法“模型构建”在附件1所公开的结构上建立的模型。
在UNIX工作站运行Insight和BIOSYM,MSI的同源性软件可显示SP722的结构。对氨基酸序列进行序列对比并将Sp722的坐标(coordinated)设定到AA560的氨基酸上。在SP722结构中缺失的AA560的前4个氨基酸的坐标(coordinates)由“END REPAIR”功能指定。
AA560的模型通过首先使用“RELAX”命令松弛氨基酸侧向改变，然后运行分子动力学以最小化该3D模型的能量。选择Insight 95,MSI的默认参数用于两种松弛分子动力学。
α-淀粉酶变异体的发酵和纯化
发酵和纯化可按本领域熟知的方法进行。
α-淀粉酶和变异体的发酵
发酵可按本领域熟知的或者如下的方法进行。
将含有相关表达质粒的枯草芽孢杆菌划线在具有相关抗生素的LB-琼脂糖平板上，并在37℃下生长过夜。
将菌落转移到500ml摇瓶中的添加了相关抗生素(例如，10mg/l氯霉素)的100mlBPX培养基中。
BPX培养基的组成成份：
Figure BDA0003128425700000491
培养物在37℃下以270rpm摇动4到5天。
通过以4500rpm离心20-25分钟从发酵肉汤中去掉细胞和细胞碎片。然后过滤上清获得完全澄清的溶液。浓缩滤液并在UF-滤膜(10000截留膜)上洗涤且将缓冲液更换成20mM乙酸盐pH 5.5。将UF-滤液加样到S-琼脂糖F.F.上并在相同缓冲液中用0.2M NaCl通过洗脱步骤进行洗脱。洗脱物用10mM Tris,pH 9.0进行透析并加样到Q-琼脂糖F.F.上，用在6倍柱体积内从0-0.3MNaCl的线性梯度进行洗脱。合并含有活性的馏分(通过Phadebas测定法测量)，将pH调节到pH 7.5并通过用0.5％W/vol.活性炭处理5分钟去掉剩余的颜色。
稳定性测定
使用如下方法测量淀粉酶的稳定性：
将酶在相应条件下温育。在各时间点，例如0,5,10,15和30分钟后取样并在测定缓冲液(0.1M 50mM Britton缓冲液，pH 7.3)中稀释25倍(相同稀释度用于所有的取样)并在标准条件pH 7.3,37℃下使用Phadebas测定法(Pharmacia)测量活性。
温育前(0分钟)测定的活性用作对照(100％)。计算下降百分数作为温育时间的函数。表中表示了在例如，温育30分钟后的剩余活性。
测量钙-和pH-依赖性稳定性
执行正常的工业溶解程序，使用pH 6.0-6.2作为溶解pH并添加40ppm游离钙以提高在95℃-105℃下的稳定性。进行本文提出的一些取代以提高在如下条件下的稳定性：
1.比pH 6.2更低的pH和/或
2.比40ppm游离钙更低的游离钙水平。
可使用两个不同的方法测量在所述α-淀粉酶中通过不同取代获得的稳定性改变：
方法 1.在降低的pH,pH 5.0下，在存在5ppm游离钙时测量稳定性的一种测定法。
将10微克变异体在如下条件：0.1M乙酸盐溶液，pH调节到pH 5.0，含有5ppm钙和5％w/w普通玉米淀粉(不含钙)中温育。温育在95℃水浴中进行30分钟。
方法2.在不含游离钙且其中pH维持在pH 6.0时测量稳定性的一种测定法。该测定法可测量钙灵敏度的下降：
将10微克变异体在如下条件下温育：0.1M乙酸钠，pH调节到pH 6.0，含有5％w/w普通玉米淀粉(不含钙)。温育在95℃的水浴中进行30分钟。
α-淀粉酶活性的测定法
1.Phadebas测定法
α-淀粉酶活性通过采用
Figure BDA0003128425700000502
片作为底物的方法来测定。Phadebas片(
Figure BDA0003128425700000501
Amylase Test，Pharmacia Diagnostic提供)含有交联不溶性蓝色淀粉聚合物，它与牛血清白蛋白和缓冲剂物质和成片剂混合。
对于每次测量，将一片悬浮于含有5ml 50mM Britton-Robinson缓冲液(50mM乙酸，50mM磷酸，50mM硼酸，0.1mM CaCl2，用NaOH将pH调节到目的值)的试管中。在感兴趣的温度下在水浴中进行检测。待测α-淀粉酶在x ml的50mM Britton-Robinson缓冲液中稀释。将1ml该α-淀粉酶溶液加入到5ml 50mM Britton-Robinson缓冲液中。淀粉被α-淀粉酶水解产生可溶的蓝色片段。在620nm下以分光光度法测定所得蓝色溶液的吸光度是α-淀粉酶活性的函数。
重要的是在温育10或15分钟后(检测时间)测量的620nm下吸光度在0.2到2.0的620nm下吸光度单位范围内。在该吸光度范围内，活性与吸光度之间呈线性(Lambert-Beer定律)。因此必须调节酶的稀释度以符合该准则。在具体设定的条件(温度，pH，反应时间，缓冲液条件)下，1mg给定的α-淀粉酶将水解一定量的底物并产生蓝色。在620nm下测定颜色强度。测定的吸光度与所述α-淀粉酶在给定条件下的比活(活性/mg纯α-淀粉酶蛋白质)成正比。
2.可供选择的方法
通过采用PNP-G7底物的方法测定α-淀粉酶活性。PNP-G7是对-硝基苯-α,D-麦芽七糖苷的缩写，它是封闭(blocked)的寡糖，可被内切淀粉酶裂解。裂解后，试剂盒中包含的α-葡萄糖苷酶消化底物以释放游离的PNP分子，该分子为黄色，因此可通过可见分光光度法在λ＝405nm(400-420nm)下测量。含有PNP-G7底物和α-葡萄糖苷酶的试剂盒由Boehringer-Mannheim生产(产品目录号：1054635)。
为了制备底物，将1瓶底物(BM 1442309)加入5ml缓冲液(BM1442309)中。为了制备α-葡萄糖苷酶，将1瓶α-葡萄糖苷酶(BM 1462309)加入45ml缓冲液(BM1442309)中。通过将5mlα-葡萄糖苷酶溶液与1ml底物混合制备工作液。
通过将20μl酶溶液转移到96孔微量滴定板中并在25℃下温育进行测定。加入25℃的200μl工作液。混合溶液并预温育1分钟，在3分钟内每隔15秒测量OD 405nm下的吸光度。
时间依赖型吸光度曲线的斜率与所述α-淀粉酶在给定条件下的比活(每mg酶的活性)成正比。
比活测定
使用上述方法之一以活性/mg酶测定比活。按产品说明书进行(也可参见下文“α-淀粉酶活性测定法”)。
氧化稳定性测定
将含有本发明不同变异体的粗滤培养液在pH 9.0的50mM Britton-Robinson缓冲液中稀释到淀粉酶活性为100KNU/ml(上文定义)并在40℃下温育。随后加入H2O2至200mM的浓度，将pH值再调节到9.0。在15秒后和5，15，和30分钟后通过取样测量活性，将它们用冰冷的水稀释5倍并将它们贮存在冰上。使用上述Phadebas方法测量剩余的活性，其中在620nm下以分光光度法测量所得蓝色溶液的吸光度是α-淀粉酶活性的函数。相对于15秒后的活性计算在5,15和30分钟后的活性以测定稳定性。
洗涤效果
分别在25℃和40℃下；在从9-10.5范围内的pH下；在从6到15OdH范围内的水硬度；Ca:Mg比为从2:1到4:1的条件下在剂量为依赖于去污剂含有从3到5g/l所述α-淀粉酶变异体的不同去污剂溶液(参见上文材料部分所述)中通过洗涤弄脏的试验样品15和30分钟评估洗涤效果。
向去污剂溶液中以例如，0.01-5mg/l的浓度加入重组α-淀粉酶变异体。试验样品用橙色稻米淀粉弄脏(CS-28样品可从CFT,Center for Test Material,Holland获得)。
洗涤后，通过使用Elrepho Remission分光光度计测量460nm下的褪色(remission)来评估样品。结果表示为ΔR＝用α-淀粉酶洗涤样品的褪色减去在相同条件下不含α-淀粉酶洗涤的样品褪色。
实施例
实施例1
构建AA560 SEQ ID NO:12的变异体
SEQ ID NO:12所示的编码AA560α-淀粉酶的基因位于质粒pTVB223中。该淀粉酶在枯草芽孢杆菌中从该构建体上的amyL启动子表达。
按Sarkar和Sommer,(1990),BioTechniques 8:404-407所述的大引物方法构建具有M202L突变的本发明的变异体。所得的编码含M202L的AX379淀粉酶的质粒命名为pCA202-AX379。
本发明其它变异体的构建以相同的方式进行。
实施例2
测定洗涤活性
使用在活性检测中分别用作对照(表中第1栏)的淀粉酶AX379或AX413所述的常规方法构建淀粉酶变异体。AX413变异体通过导入下表所示的突变由AX379产生。
在模拟欧洲的40℃洗涤条件下在去污剂溶液中测量活性。制备每5ml4g/L去污剂溶液1片Phadebas片剂的悬液并在Eppendorf管中适当搅拌。在40℃下预热5分钟后，加入淀粉酶且混合物在剧烈搅拌下温育20分钟。通过加入10％1M NaOH终止反应并将试管以最小10000xg旋转3分钟。最后测量上清在650nm下的吸光度，使用无酶样品作为空白对照。
表3:
突变 活性提高
AX379 1
K118Q 1.1
K37T 1.1
H421Y 1.1
V450T 1.1
K383R 1.1
N445Q 1.1
Y178F 1.2
V318L 1.2
W482Y 1.2
N283D+Q361E 1.2
M105F+M208F 1.2
M202L+M323T+M430I 1.3
K446R+N484Q 1.4
R444Y 1.5
N106D 1.8
Y203L 2.0
G133E+Q361E 2.9
M323E 4.1
V214T 6.8
M202L+M323T+M309L 13
M202L 16
M202L+M323T 23
M202L+M323T+M9L+M382Y+K383R 25
M202L+M323T+M9L+M382Y 26
M202L+M323T+M9L(AX413) 27
表4:
Figure BDA0003128425700000531
Figure BDA0003128425700000541
实施例3
测定洗碗期间的稳定性
使用在活性检测中分别用作对照(表中第1栏)的淀粉酶AX379或AX413所述的常规方法构建淀粉酶变异体。AX413变异体通过导入下表所示的突变由AX379产生。
通过在40℃下温育含大约0.1mg/ml淀粉酶的用于自动洗碗的4g/l去污剂18小时测量淀粉酶稳定性。通过加入9倍体积的冷水(<5℃)终止温育并贮存在冰上。在1小时内使用Phadebas淀粉酶试剂盒测量活性且将去污剂样品的活性与在去污剂中在但在冰上温育相同时间的样品进行比较。
表5:
突变 剩余活性提高
AX413 1
V214T+M323E+M382Y+K383R+N471E 1.1
Y178F+G258D+T419N+N437H 1.1
G149N+N150Q+M382Y+K383R 1.1
Y160F+V214T+M382Y 1.2
N128Y+G149A+V214T+D231N+M382Y+F441L 1.2
R82H+N128Y+G149A+V214T+M382Y 1.2
N150H+V214T 1.2
V214T+E345N 1.2
V214T+G305D+M382Y+R444E 1.2
V214T+M382Y+A447Y 1.2
M202I+V214T+M382Y+K383R+R444Y 1.2
V214T+G378K 1.3
V214T+A256K 1.3
R26S+D30N+N33D+V214T+M382Y 1.5
实施例4
按实施例1所述构建seq.ID no.12的淀粉酶变异体并使用丰富培养基在摇瓶中发酵。使用标准纯化方法从上清纯化淀粉酶变异体蛋白至超过90％的纯度。从A280吸光度计算蛋白质浓度，理论消光系数为2.9ml/mg/cm。
按“方法”部分所述使用G7-pNP活性测定法测量淀粉酶样品的活性，从而计算比活(SA)，即，每mg淀粉酶蛋白的G7-pNP活性并与同源对照淀粉酶进行比较。
Figure BDA0003128425700000551
Figure BDA0003128425700000552
Figure BDA0003128425700000553
Figure BDA0003128425700000554
Figure BDA0003128425700000561
实施例5
使用含有漂白剂和每升0.2mg淀粉酶蛋白的9g/l(Henkel)HDD传统去污剂在按比例缩减洗衣机中使用在20分钟内达到40℃的常规欧洲加热程序进行洗涤试验。用Ca,Mg和NaHCO3将水硬度调节到16°dH。
在含有色水稻淀粉的棉花样品(CS-28，来自CFT)上通过相对于无淀粉酶洗涤的样品白度测量在存在淀粉酶时洗涤后的样品白度评估洗涤效果。样品在机内过夜干燥后使用褪色分光光度计(Macbeth Color-Eye)测量白度。
Figure BDA0003128425700000562
实施例6
使用6g/l Persil Megaperls(Henkel)去污剂和每升0.2mg淀粉酶蛋白在按比例缩减洗衣机中使用在20分钟内达到40℃的常规欧洲加热程序进行洗涤试验。用Ca,Mg和NaHCO3将水硬度调节到16°dH。
在含有色稻米淀粉的棉花样品(CS-28，来自CFT)上通过相对于无淀粉酶洗涤的样品白度测量在淀粉酶存在时洗涤后的样品白度来评估洗涤效果。样品在机内过夜干燥后使用褪色分光光度计(Macbeth Color-Eye)测量白度。
序列表
<110> 诺维信公司(Novozymes A/S)
<120> 具有改变特性的α-淀粉酶变异体
<130> 10504-CN-PCD DIV.2
<160> 18
<170> PatentIn version 3.2
<210> 1
<211> 1455
<212> DNA
<213> 芽孢杆菌属SP690（Bacillus SP690）
<220>
<221> CDS
<222> (1)..(1455)
<400> 1
cat cat aat gga aca aat ggt act atg atg caa tat ttc gaa tgg tat 48
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
ttg cca aat gac ggg aat cat tgg aac agg ttg agg gat gac gca gct 96
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala
20 25 30
aac tta aag agt aaa ggg ata aca gct gta tgg atc cca cct gca tgg 144
Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
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aag ggg act tcc cag aat gat gta ggt tat gga gcc tat gat tta tat 192
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
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gat ctt gga gag ttt aac cag aag ggg acg gtt cgt aca aaa tat gga 240
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
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aca cgc aac cag cta cag gct gcg gtg acc tct tta aaa aat aac ggc 288
Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly
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att cag gta tat ggt gat gtc gtc atg aat cat aaa ggt gga gca gat 336
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
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ggt acg gaa att gta aat gcg gta gaa gtg aat cgg agc aac cga aac 384
Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
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cag gaa acc tca gga gag tat gca ata gaa gcg tgg aca aag ttt gat 432
Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp
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Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr
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cat ttt gat ggg aca gat tgg gat cag tca cgc cag ctt caa aac aaa 528
His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys
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ata tat aaa ttc agg gga aca ggc aag gcc tgg gac tgg gaa gtc gat 576
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
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aca gag aat ggc aac tat gac tat ctt atg tat gca gac gtg gat atg 624
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
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gat cac cca gaa gta ata cat gaa ctt aga aac tgg gga gtg tgg tat 672
Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr
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acg aat aca ctg aac ctt gat gga ttt aga ata gat gca gtg aaa cat 720
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
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ata aaa tat agc ttt acg aga gat tgg ctt aca cat gtg cgt aac acc 768
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
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aca ggt aaa cca atg ttt gca gtg gct gag ttt tgg aaa aat gac ctt 816
Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
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ggt gca att gaa aac tat ttg aat aaa aca agt tgg aat cac tcg gtg 864
Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val
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ttt gat gtt cct ctc cac tat aat ttg tac aat gca tct aat agc ggt 912
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
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ggt tat tat gat atg aga aat att tta aat ggt tct gtg gtg caa aaa 960
Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys
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cat cca aca cat gcc gtt act ttt gtt gat aac cat gat tct cag ccc 1008
His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
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Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala
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tat gca ttg gtt ctg aca agg gaa caa ggt tat cct tcc gta ttt tat 1104
Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
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ggg gat tac tac ggt atc cca acc cat ggt gtt ccg gct atg aaa tct 1152
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
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Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr
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cag cat gat tac ttt gat cat cat gat att atc ggt tgg aca aga gag 1248
Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu
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Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
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ggt cca ggt ggt aac aaa tgg atg tat gtg ggg aaa aat aaa gcg gga 1344
Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly
435 440 445
caa gtt tgg aga gat att acc gga aat agg aca ggc acc gtc aca att 1392
Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
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aat gca gac gga tgg ggt aat ttc tct gtt aat gga ggg tcc gtt tcg 1440
Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
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gtt tgg gtg aag caa 1455
Val Trp Val Lys Gln
485
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<213> 芽孢杆菌属SP690
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His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
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Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala
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Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
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Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
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Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
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Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly
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Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
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Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
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Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp
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Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr
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His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys
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Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
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Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
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Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr
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Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
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Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
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Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
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Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val
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Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
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Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys
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His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
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Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala
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Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
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Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
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Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr
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Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu
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Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
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Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly
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Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
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Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
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Val Trp Val Lys Gln
485
<210> 3
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<213> 芽孢杆菌属SP722
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His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
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Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser
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Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp
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aaa ggg act tcg caa aat gat gtg ggg tat gga gcc tat gat ctt tat 192
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
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Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
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Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly
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Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
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Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
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Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
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Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr
145 150 155 160
cat ttc gat ggt gta gat tgg gat caa tca cga caa ttc caa aat cgt 528
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg
165 170 175
atc tac aaa ttc cga ggt gat ggt aag gca tgg gat tgg gaa gta gat 576
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
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Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
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gat cat ccg gag gta gta aat gag ctt aga aga tgg gga gaa tgg tat 672
Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr
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Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
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Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala
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Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
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Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
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Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
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ggc aac tat gac atg gca aaa ctt ctt aat gga acg gtt gtt caa aag 960
Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys
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His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
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Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala
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tat gcg ctt att tta aca aga gaa caa ggc tat ccc tct gtc ttc tat 1104
Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
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ggt gac tac tat gga att cca aca cat agt gtc cca gca atg aaa gcc 1152
Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala
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aag att gat cca atc tta gag gcg cgt caa aat ttt gca tat gga aca 1200
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr
385 390 395 400
caa cat gat tat ttt gac cat cat aat ata atc gga tgg aca cgt gaa 1248
Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
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Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
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Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly
435 440 445
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Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile
450 455 460
aat gca gat gga tgg gct aat ttt tca gta aat gga gga tct gtt tcc 1440
Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser
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Ile Trp Val Lys Arg
485
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<211> 485
<212> PRT
<213> 芽孢杆菌属SP722
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His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
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Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser
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Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp
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Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
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Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
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Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly
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Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
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Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn
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Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr
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His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala
245 250 255
Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
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Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
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Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys
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His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
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Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala
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Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
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Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala
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Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr
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Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
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Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
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Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly
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Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile
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Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser
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Ile Trp Val Lys Arg
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Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu
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Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn
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Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
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Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
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Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
65 70 75 80
aaa gct caa tat ctt caa gcc att caa gcc gcc cac gcc gct gga atg 288
Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met
85 90 95
caa gtg tac gcc gat gtc gtg ttc gac cat aaa ggc ggc gct gac ggc 336
Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
100 105 110
acg gaa tgg gtg gac gcc gtc gaa gtc aat ccg tcc gac cgc aac caa 384
Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln
115 120 125
gaa atc tcg ggc acc tat caa atc caa gca tgg acg aaa ttt gat ttt 432
Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe
130 135 140
ccc ggg cgg ggc aac acc tac tcc agc ttt aag tgg cgc tgg tac cat 480
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His
145 150 155 160
ttt gac ggc gtt gat tgg gac gaa agc cga aaa ttg agc cgc att tac 528
Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr
165 170 175
aaa ttc cgc ggc atc ggc aaa gcg tgg gat tgg gaa gta gac acg gaa 576
Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu
180 185 190
aac gga aac tat gac tac tta atg tat gcc gac ctt gat atg gat cat 624
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205
ccc gaa gtc gtg acc gag ctg aaa aac tgg ggg aaa tgg tat gtc aac 672
Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn
210 215 220
aca acg aac att gat ggg ttc cgg ctt gat gcc gtc aag cat att aag 720
Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
225 230 235 240
ttc agt ttt ttt cct gat tgg ttg tcg tat gtg cgt tct cag act ggc 768
Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly
245 250 255
aag ccg cta ttt acc gtc ggg gaa tat tgg agc tat gac atc aac aag 816
Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys
260 265 270
ttg cac aat tac att acg aaa aca gac gga acg atg tct ttg ttt gat 864
Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp
275 280 285
gcc ccg tta cac aac aaa ttt tat acc gct tcc aaa tca ggg ggc gca 912
Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala
290 295 300
ttt gat atg cgc acg tta atg acc aat act ctc atg aaa gat caa ccg 960
Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro
305 310 315 320
aca ttg gcc gtc acc ttc gtt gat aat cat gac acc gaa ccc ggc caa 1008
Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln
325 330 335
gcg ctg cag tca tgg gtc gac cca tgg ttc aaa ccg ttg gct tac gcc 1056
Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala
340 345 350
ttt att cta act cgg cag gaa gga tac ccg tgc gtc ttt tat ggt gac 1104
Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp
355 360 365
tat tat ggc att cca caa tat aac att cct tcg ctg aaa agc aaa atc 1152
Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile
370 375 380
gat ccg ctc ctc atc gcg cgc agg gat tat gct tac gga acg caa cat 1200
Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His
385 390 395 400
gat tat ctt gat cac tcc gac atc atc ggg tgg aca agg gaa ggg ggc 1248
Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly
405 410 415
act gaa aaa cca gga tcc gga ctg gcc gca ctg atc acc gat ggg ccg 1296
Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
gga gga agc aaa tgg atg tac gtt ggc aaa caa cac gct gga aaa gtg 1344
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445
ttc tat gac ctt acc ggc aac cgg agt gac acc gtc acc atc aac agt 1392
Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser
450 455 460
gat gga tgg ggg gaa ttc aaa gtc aat ggc ggt tcg gtt tcg gtt tgg 1440
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp
465 470 475 480
gtt cct aga aaa acg acc gtt tct acc atc gct cgg ccg atc aca acc 1488
Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr
485 490 495
cga ccg tgg act ggt gaa ttc gtc cgt tgg acc gaa cca cgg ttg gtg 1536
Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val
500 505 510
gca tgg cct tga 1548
Ala Trp Pro
515
<210> 6
<211> 515
<212> PRT
<213> 芽孢杆菌属BSG
<400> 6
Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu
1 5 10 15
Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn
20 25 30
Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
35 40 45
Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
65 70 75 80
Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met
85 90 95
Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
100 105 110
Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln
115 120 125
Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe
130 135 140
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His
145 150 155 160
Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr
165 170 175
Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu
180 185 190
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205
Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn
210 215 220
Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
225 230 235 240
Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly
245 250 255
Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys
260 265 270
Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp
275 280 285
Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala
290 295 300
Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro
305 310 315 320
Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln
325 330 335
Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala
340 345 350
Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp
355 360 365
Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile
370 375 380
Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His
385 390 395 400
Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly
405 410 415
Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445
Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser
450 455 460
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp
465 470 475 480
Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr
485 490 495
Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val
500 505 510
Ala Trp Pro
515
<210> 7
<211> 1920
<212> DNA
<213> 地衣芽孢杆菌(Bacillus licheniformis)
<220>
<221> CDS
<222> (421)..(1872)
<400> 7
cggaagattg gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60
gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag 120
agattattaa aaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag 180
aagtgaagaa gcagagaggc tattgaataa atgagtagaa gcgccatatc ggcgcttttc 240
ttttggaaga aaatataggg aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300
tcatatgttt cacattgaaa ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360
cgattgctga cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420
gca aat ctt aat ggg acg ctg atg cag tat ttt gaa tgg tac atg ccc 468
Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15
aat gac ggc caa cat tgg agg cgt ttg caa aac gac tcg gca tat ttg 516
Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30
gct gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag gga 564
Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
acg agc caa gcg gat gtg ggc tac ggt gct tac gac ctt tat gat tta 612
Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
ggg gag ttt cat caa aaa ggg acg gtt cgg aca aag tac ggc aca aaa 660
Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
gga gag ctg caa tct gcg atc aaa agt ctt cat tcc cgc gac att aac 708
Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95
gtt tac ggg gat gtg gtc atc aac cac aaa ggc ggc gct gat gcg acc 756
Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110
gaa gat gta acc gcg gtt gaa gtc gat ccc gct gac cgc aac cgc gta 804
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125
att tca gga gaa cac cta att aaa gcc tgg aca cat ttt cat ttt ccg 852
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro
130 135 140
ggg cgc ggc agc aca tac agc gat ttt aaa tgg cat tgg tac cat ttt 900
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
gac gga acc gat tgg gac gag tcc cga aag ctg aac cgc atc tat aag 948
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
ttt caa gga aag gct tgg gat tgg gaa gtt tcc aat gaa aac ggc aac 996
Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190
tat gat tat ttg atg tat gcc gac atc gat tat gac cat cct gat gtc 1044
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205
gca gca gaa att aag aga tgg ggc act tgg tat gcc aat gaa ctg caa 1092
Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220
ttg gac ggt ttc cgt ctt gat gct gtc aaa cac att aaa ttt tct ttt 1140
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
ttg cgg gat tgg gtt aat cat gtc agg gaa aaa acg ggg aag gaa atg 1188
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
245 250 255
ttt acg gta gct gaa tat tgg cag aat gac ttg ggc gcg ctg gaa aac 1236
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn
260 265 270
tat ttg aac aaa aca aat ttt aat cat tca gtg ttt gac gtg ccg ctt 1284
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285
cat tat cag ttc cat gct gca tcg aca cag gga ggc ggc tat gat atg 1332
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300
agg aaa ttg ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg 1380
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser
305 310 315 320
gtt aca ttt gtc gat aac cat gat aca cag ccg ggg caa tcg ctt gag 1428
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
tcg act gtc caa aca tgg ttt aag ccg ctt gct tac gct ttt att ctc 1476
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
aca agg gaa tct gga tac cct cag gtt ttc tac ggg gat atg tac ggg 1524
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
acg aaa gga gac tcc cag cgc gaa att cct gcc ttg aaa cac aaa att 1572
Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile
370 375 380
gaa ccg atc tta aaa gcg aga aaa cag tat gcg tac gga gca cag cat 1620
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His
385 390 395 400
gat tat ttc gac cac cat gac att gtc ggc tgg aca agg gaa ggc gac 1668
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
405 410 415
agc tcg gtt gca aat tca ggt ttg gcg gca tta ata aca gac gga ccc 1716
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
ggt ggg gca aag cga atg tat gtc ggc cgg caa aac gcc ggt gag aca 1764
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445
tgg cat gac att acc gga aac cgt tcg gag ccg gtt gtc atc aat tcg 1812
Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460
gaa ggc tgg gga gag ttt cac gta aac ggc ggg tcg gtt tca att tat 1860
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480
gtt caa aga tag aagagcagag aggacggatt tcctgaagga aatccgtttt 1912
Val Gln Arg
tttatttt 1920
<210> 8
<211> 483
<212> PRT
<213> 地衣芽孢杆菌
<400> 8
Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15
Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30
Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95
Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro
130 135 140
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205
Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser
305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His
385 390 395 400
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445
Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Arg
<210> 9
<211> 2084
<212> DNA
<213> 芽孢杆菌属BAN
<220>
<221> CDS
<222> (343)..(1794)
<400> 9
gccccgcaca tacgaaaaga ctggctgaaa acattgagcc tttgatgact gatgatttgg 60
ctgaagaagt ggatcgattg tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc 120
atcagacagg gtatttttta tgctgtccag actgtccgct gtgtaaaaat aaggaataaa 180
ggggggttgt tattatttta ctgatatgta aaatataatt tgtataagaa aatgagaggg 240
agaggaaaca tgattcaaaa acgaaagcgg acagtttcgt tcagacttgt gcttatgtgc 300
acgctgttat ttgtcagttt gccgattaca aaaacatcag cc gta aat ggc acg 354
Val Asn Gly Thr
1
ctg atg cag tat ttt gaa tgg tat acg ccg aac gac ggc cag cat tgg 402
Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly Gln His Trp
5 10 15 20
aaa cga ttg cag aat gat gcg gaa cat tta tcg gat atc gga atc act 450
Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile Gly Ile Thr
25 30 35
gcc gtc tgg att cct ccc gca tac aaa gga ttg agc caa tcc gat aac 498
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln Ser Asp Asn
40 45 50
gga tac gga cct tat gat ttg tat gat tta gga gaa ttc cag caa aaa 546
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Gln Gln Lys
55 60 65
ggg acg gtc aga acg aaa tac ggc aca aaa tca gag ctt caa gat gcg 594
Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu Gln Asp Ala
70 75 80
atc ggc tca ctg cat tcc cgg aac gtc caa gta tac gga gat gtg gtt 642
Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly Asp Val Val
85 90 95 100
ttg aat cat aag gct ggt gct gat gca aca gaa gat gta act gcc gtc 690
Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val Thr Ala Val
105 110 115
gaa gtc aat ccg gcc aat aga aat cag gaa act tcg gag gaa tat caa 738
Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu Glu Tyr Gln
120 125 130
atc aaa gcg tgg acg gat ttt cgt ttt ccg ggc cgt gga aac acg tac 786
Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly Asn Thr Tyr
135 140 145
agt gat ttt aaa tgg cat tgg tat cat ttc gac gga gcg gac tgg gat 834
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala Asp Trp Asp
150 155 160
gaa tcc cgg aag atc agc cgc atc ttt aag ttt cgt ggg gaa gga aaa 882
Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly Glu Gly Lys
165 170 175 180
gcg tgg gat tgg gaa gta tca agt gaa aac ggc aac tat gac tat tta 930
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
185 190 195
atg tat gct gat gtt gac tac gac cac cct gat gtc gtg gca gag aca 978
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Thr
200 205 210
aaa aaa tgg ggt atc tgg tat gcg aat gaa ctg tca tta gac ggc ttc 1026
Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu Asp Gly Phe
215 220 225
cgt att gat gcc gcc aaa cat att aaa ttt tca ttt ctg cgt gat tgg 1074
Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu Arg Asp Trp
230 235 240
gtt cag gcg gtc aga cag gcg acg gga aaa gaa atg ttt acg gtt gcg 1122
Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe Thr Val Ala
245 250 255 260
gag tat tgg cag aat aat gcc ggg aaa ctc gaa aac tac ttg aat aaa 1170
Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr Leu Asn Lys
265 270 275
aca agc ttt aat caa tcc gtg ttt gat gtt ccg ctt cat ttc aat tta 1218
Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
280 285 290
cag gcg gct tcc tca caa gga ggc gga tat gat atg agg cgt ttg ctg 1266
Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg Arg Leu Leu
295 300 305
gac ggt acc gtt gtg tcc agg cat ccg gaa aag gcg gtt aca ttt gtt 1314
Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val Thr Phe Val
310 315 320
gaa aat cat gac aca cag ccg gga cag tca ttg gaa tcg aca gtc caa 1362
Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
325 330 335 340
act tgg ttt aaa ccg ctt gca tac gcc ttt att ttg aca aga gaa tcc 1410
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser
345 350 355
ggt tat cct cag gtg ttc tat ggg gat atg tac ggg aca aaa ggg aca 1458
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Thr
360 365 370
tcg cca aag gaa att ccc tca ctg aaa gat aat ata gag ccg att tta 1506
Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu Pro Ile Leu
375 380 385
aaa gcg cgt aag gag tac gca tac ggg ccc cag cac gat tat att gac 1554
Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp Tyr Ile Asp
390 395 400
cac ccg gat gtg atc gga tgg acg agg gaa ggt gac agc tcc gcc gcc 1602
His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser Ser Ala Ala
405 410 415 420
aaa tca ggt ttg gcc gct tta atc acg gac gga ccc ggc gga tca aag 1650
Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
425 430 435
cgg atg tat gcc ggc ctg aaa aat gcc ggc gag aca tgg tat gac ata 1698
Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp Tyr Asp Ile
440 445 450
acg ggc aac cgt tca gat act gta aaa atc gga tct gac ggc tgg gga 1746
Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp Gly Trp Gly
455 460 465
gag ttt cat gta aac gat ggg tcc gtc tcc att tat gtt cag aaa taa 1794
Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val Gln Lys
470 475 480
ggtaataaaa aaacacctcc aagctgagtg cgggtatcag cttggaggtg cgtttatttt 1854
ttcagccgta tgacaaggtc ggcatcaggt gtgacaaata cggtatgctg gctgtcatag 1914
gtgacaaatc cgggttttgc gccgtttggc tttttcacat gtctgatttt tgtataatca 1974
acaggcacgg agccggaatc tttcgccttg gaaaaataag cggcgatcgt agctgcttcc 2034
aatatggatt gttcatcggg atcgctgctt ttaatcacaa cgtgggatcc 2084
<210> 10
<211> 483
<212> PRT
<213> 芽孢杆菌属BAN
<400> 10
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp
1 5 10 15
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp
20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser
35 40 45
Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu
50 55 60
Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu
65 70 75 80
Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr
85 90 95
Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp
100 105 110
Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser
115 120 125
Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg
130 135 140
Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly
145 150 155 160
Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg
165 170 175
Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val
195 200 205
Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser
210 215 220
Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu
275 280 285
His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala
305 310 315 320
Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His
385 390 395 400
Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr
435 440 445
Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser
450 455 460
Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Lys
<210> 11
<211> 1458
<212> DNA
<213> 芽孢杆菌属AA560
<220>
<221> CDS
<222> (1)..(1458)
<400> 11
cac cat aat ggt acg aac ggc aca atg atg cag tac ttt gaa tgg tat 48
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
cta cca aat gac gga aac cat tgg aat aga tta agg tct gat gca agt 96
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser
20 25 30
aac cta aaa gat aaa ggg atc tca gcg gtt tgg att cct cct gca tgg 144
Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
aag ggt gcc tct caa aat gat gtg ggg tat ggt gct tat gat ctg tat 192
Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
gat tta gga gaa ttc aat caa aaa gga acc att cgt aca aaa tat gga 240
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly
65 70 75 80
acg cgc aat cag tta caa gct gca gtt aac gcc ttg aaa agt aat gga 288
Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly
85 90 95
att caa gtg tat ggc gat gtt gta atg aat cat aaa ggg gga gca gac 336
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
gct acc gaa atg gtt agg gca gtt gaa gta aac ccg aat aat aga aat 384
Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
caa gaa gtg tcc ggt gaa tat aca att gag gct tgg aca aag ttt gac 432
Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
ttt cca gga cga ggt aat act cat tca aac ttc aaa tgg aga tgg tat 480
Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr
145 150 155 160
cac ttt gat gga gta gat tgg gat cag tca cgt aag ctg aac aat cga 528
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg
165 170 175
att tat aaa ttt aga ggt gat gga aaa ggg tgg gat tgg gaa gtc gat 576
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp
180 185 190
aca gaa aac ggt aac tat gat tac cta atg tat gca gat att gac atg 624
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205
gat cac cca gag gta gtg aat gag cta aga aat tgg ggt gtt tgg tat 672
Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
acg aat aca tta ggc ctt gat ggt ttt aga ata gat gca gta aaa cat 720
Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
ata aaa tac agc ttt act cgt gat tgg att aat cat gtt aga agt gca 768
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala
245 250 255
act ggc aaa aat atg ttt gcg gtt gcg gaa ttt tgg aaa aat gat tta 816
Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
ggt gct att gaa aac tat tta aac aaa aca aac tgg aac cat tca gtc 864
Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285
ttt gat gtt ccg ctg cac tat aac ctc tat aat gct tca aaa agc gga 912
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly
290 295 300
ggg aat tat gat atg agg caa ata ttt aat ggt aca gtc gtg caa aga 960
Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg
305 310 315 320
cat cca atg cat gct gtt aca ttt gtt gat aat cat gat tcg caa cct 1008
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
gaa gaa gct tta gag tct ttt gtt gaa gaa tgg ttc aaa cca tta gcg 1056
Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala
340 345 350
tat gct ttg aca tta aca cgt gaa caa ggc tac cct tct gta ttt tat 1104
Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
gga gat tat tat ggc att cca acg cat ggt gta cca gcg atg aaa tcg 1152
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380
aaa att gac ccg att cta gaa gcg cgt caa aag tat gca tat gga aga 1200
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg
385 390 395 400
caa aat gac tac tta gac cat cat aat atc atc ggt tgg aca cgt gaa 1248
Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
405 410 415
ggg aat aca gca cac ccc aac tcc ggt tta gct act atc atg tcc gat 1296
Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
ggg gca gga gga aat aag tgg atg ttt gtt ggg cgt aat aaa gct ggt 1344
Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly
435 440 445
caa gtt tgg acc gat atc act gga aat cgt gca ggt act gtt acg att 1392
Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile
450 455 460
aat gct gat gga tgg ggt aat ttt tct gta aat gga gga tca gtt tct 1440
Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
att tgg gta aac aaa taa 1458
Ile Trp Val Asn Lys
485
<210> 12
<211> 485
<212> PRT
<213> 芽孢杆菌属AA560
<400> 12
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser
20 25 30
Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala
245 250 255
Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly
290 295 300
Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg
305 310 315 320
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg
385 390 395 400
Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly
435 440 445
Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Asn Lys
485
<210> 13
<211> 586
<212> PRT
<213> 芽孢杆菌属AMY1048
<400> 13
Gly Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala Gln
20 25 30
Ser Leu Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr
35 40 45
Lys Gly Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr Ala Gly
85 90 95
Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp
100 105 110
Gly Thr Glu Leu Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn
115 120 125
Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile
165 170 175
Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr
180 185 190
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp
195 200 205
His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val
210 215 220
Thr Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile
225 230 235 240
Lys Tyr Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Thr Gln Thr
245 250 255
Gln Lys Pro Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Ile Ser
260 265 270
Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe
275 280 285
Asp Ala Pro Leu His Asn Asn Phe Tyr Ile Ala Ser Lys Ser Gly Gly
290 295 300
Tyr Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln
305 310 315 320
Pro Thr Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Glu Pro Gly
325 330 335
Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr
340 345 350
Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly
355 360 365
Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Ala Leu Lys Ser Lys
370 375 380
Leu Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
385 390 395 400
His Asp Tyr Ile Asp Ser Ala Asp Ile Ile Gly Trp Thr Arg Glu Gly
405 410 415
Val Ala Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
420 425 430
Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys
435 440 445
Thr Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn
450 455 460
Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile
465 470 475 480
Trp Val Pro Lys Ile Ser Thr Thr Ser Gln Ile Thr Phe Thr Val Asn
485 490 495
Asn Ala Thr Thr Val Trp Gly Gln Asn Val Tyr Val Val Gly Asn Ile
500 505 510
Ser Gln Leu Gly Asn Trp Asp Pro Val His Ala Val Gln Met Thr Pro
515 520 525
Ser Ser Tyr Pro Thr Trp Thr Val Thr Ile Pro Leu Leu Gln Gly Gln
530 535 540
Asn Ile Gln Phe Lys Phe Ile Lys Lys Asp Ser Ala Gly Asn Val Ile
545 550 555 560
Trp Glu Asp Ile Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala Ser
565 570 575
Gly Ala Tyr Thr Ala Ser Trp Asn Val Pro
580 585
<210> 14
<211> 486
<212> PRT
<213> 芽孢杆菌属AMRK385
<400> 14
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
1 5 10 15
Leu Pro Asn Asp Gly Gln His Trp Asn Arg Leu Arg Asn Asp Ala Ala
20 25 30
Asn Leu Lys Asn Leu Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Ser Gln Leu Gln Ser Ala Ile Ala Ser Leu Gln Asn Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Trp Val Gln Ala Val Glu Val Asn Pro Ser Asn Arg Asn
115 120 125
Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Asn Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Ile Asn Glu Leu Arg Arg Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Asn His Val Arg Ser Thr
245 250 255
Thr Gly Lys Asn Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp
260 265 270
Leu Gly Ala Ile Glu Asn Tyr Leu His Lys Thr Asn Trp Asn His Ser
275 280 285
Val Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser
290 295 300
Gly Gly Asn Tyr Asp Met Arg Gln Ile Leu Asn Gly Thr Val Val Ser
305 310 315 320
Lys His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln
325 330 335
Pro Gly Glu Ala Leu Glu Ser Phe Val Glu Ala Trp Phe Lys Pro Leu
340 345 350
Ala Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe
355 360 365
Tyr Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Ala Ala Met Lys
370 375 380
Gly Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly
385 390 395 400
Thr Gln His Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg
405 410 415
Glu Gly Asn Ser Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser
420 425 430
Asp Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg His Lys Ala
435 440 445
Gly Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr
450 455 460
Ile Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val
465 470 475 480
Ser Ile Trp Val Asn Lys
485
<210> 15
<211> 480
<212> PRT
<213> 芽孢杆菌属SP.K38
<400> 15
Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu
1 5 10 15
Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu
20 25 30
Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn
85 90 95
Val Tyr Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr
100 105 110
Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp
115 120 125
Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser
130 135 140
Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe
145 150 155 160
Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn His Ile Phe Arg
165 170 175
Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val
195 200 205
Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp
210 215 220
Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr
225 230 235 240
Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu
245 250 255
Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe
260 265 270
Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu
275 280 285
Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met
290 295 300
Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala
305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu
325 330 335
Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu
340 345 350
Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly
355 360 365
Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu
370 375 380
Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe
385 390 395 400
Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg
405 410 415
Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser
420 425 430
Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp
435 440 445
Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp
450 455 460
Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln
465 470 475 480
<210> 16
<211> 485
<212> PRT
<213> 芽孢杆菌属AAI-10
<400> 16
His His Asp Gly Thr Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Asn
1 5 10 15
Val Pro Asn Asp Gly Gln His Trp Asn Arg Leu His Asn Asn Ala Gln
20 25 30
Asn Leu Lys Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Lys Ala Glu Leu Glu Arg Ala Ile Arg Ser Leu Lys Ala Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Phe Thr Glu Arg Val Gln Ala Val Glu Val Asn Pro Gln Asn Arg Asn
115 120 125
Gln Glu Val Ser Gly Thr Tyr Gln Ile Glu Ala Trp Thr Gly Phe Asn
130 135 140
Phe Pro Gly Arg Gly Asn Gln His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Ala Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Ile Asn Glu Leu Asn Arg Trp Gly Val Trp Tyr
210 215 220
Ala Asn Thr Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His
225 230 235 240
Ile Lys Phe Ser Phe Met Arg Asp Trp Leu Gly His Val Arg Gly Gln
245 250 255
Thr Gly Lys Asn Leu Phe Ala Val Ala Glu Tyr Trp Lys Asn Asp Leu
260 265 270
Gly Ala Leu Glu Asn Tyr Leu Ser Lys Thr Asn Trp Thr Met Ser Ala
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Gln Ala Ser Asn Ser Ser
290 295 300
Gly Asn Tyr Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val Gln Arg
305 310 315 320
His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro
325 330 335
Gly Glu Ala Leu Glu Ser Phe Val Gln Gly Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Thr Ile Leu Thr Arg Glu Gln Gly Tyr Pro Gln Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Ser Asp Gly Val Pro Ser Tyr Arg Gln
370 375 380
Gln Ile Asp Pro Leu Leu Lys Ala Arg Gln Gln Tyr Ala Tyr Gly Arg
385 390 395 400
Gln His Asp Tyr Phe Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Ala Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Lys Ala Gly
435 440 445
Glu Val Trp His Asp Met Thr Gly Asn Arg Ser Gly Thr Val Thr Ile
450 455 460
Asn Gln Asp Gly Trp Gly His Phe Phe Val Asn Gly Gly Ser Val Ser
465 470 475 480
Val Trp Val Lys Arg
485
<210> 17
<211> 485
<212> PRT
<213> 芽孢杆菌属KSM-AP1378
<400> 17
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala
20 25 30
Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
115 120 125
Gln Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys
165 170 175
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Ile Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205
Asp His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255
Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Ala Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300
Gly Tyr Arg Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys
305 310 315 320
His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Gly Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser
370 375 380
Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr
385 390 395 400
Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asp Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly
435 440 445
Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser
465 470 475 480
Val Trp Val Lys Gln
485
<210> 18
<211> 485
<212> PRT
<213> 芽孢杆菌属SP.7-7
<400> 18
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser
20 25 30
Asn Leu Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn
115 120 125
Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Gly Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255
Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Ile
260 265 270
Gly Ala Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly
290 295 300
Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg
305 310 315 320
His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala
340 345 350
Cys Ala Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys
385 390 395 400
Gln Asn Asp Tyr Leu Asp His His Asn Met Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Arg Asn Lys Ala Gly
435 440 445
Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Asn Asn
485

Claims (37)

1.亲本Termamyl-样α-淀粉酶的变异体，其包含在选自如下组中的一个或多个位置改变：
26,30,33,82,37,106,118,128,133,149,150,160,178,182,186,193,203,214,231,256,257,258,269,270,272,283,295,296,298,299,303,304,305,311,314,315,318,319,339,345,361,378,383,419,421,437,441,444,445,446,447,450,461,471,482,484,
其中
(a)改变是独立地
(i)在占据该位置的氨基酸下游插入氨基酸，
(ii)缺失占据该位置的氨基酸，或
(iii)用不同的氨基酸取代占据该位置的氨基酸，
(b)该变异体具有α-淀粉酶活性，和
(c)各位置相应于具有亲本Termamyl-样α-淀粉酶的氨基酸序列的亲本α-淀粉酶的氨基酸序列位置，该亲本Termamyl-样α-淀粉酶具有SEQ ID NO:12所示的AA560的氨基酸序列。
2.权利要求1的变异体，其中该突变是：
R26S,D30N,N33D,R82H,K37T,N106D,K118Q,N128Y,G133E,A,G149A,N,N150H,Q,Y160F,Y178F,G182T,G186A,T193S,N,D,E,Q,Y203L,V214I,T,D231N,G256K,T257I,G258D,K269S,Q,N270F,Y,D,L272I,V,A,N283D,Y295F,N,D,Q,E,N296K,Q,E,Y298F,H,N299F,Y,Q,T,S303Q,K,Y304F,R,K,G305D,Q311N,Q,K,R,T,S,Y,F,N314D,S,T,Q,G315N,S,T,V318L,Q319E,K,S,T,A339S,T,E345N,R,Q361E,G378K,K383R,T419N,H421Y,N437H,F441L,R444E,Y,N445Q,K446R,A447Y,V450T,T461P,N471E,W482Y,N484Q。
3.权利要求1-2的变异体，其中该变异体在一个或多个甲硫氨酸残基上具有额外的突变。
4.权利要求3的变异体，其中该甲硫氨酸残基是：
M9,M10,M116,M202,M208,M261,M309,M323,M382,M410,M430,和M440。
5.权利要求4的变异体，其中该突变是：
M9L,I,M10L,M105L,I,F,M116N,D,L,I,F,W,R,K,M202I,L,V,T,M208F,Y,L,I,M261L,I,M309L,I,M323L,I,S,T,A,Q,E,N,D,M382L,I,Y,F,K,M410L,I,V,M430L,I,和M440L,I,F,Y。
6.权利要求1-5的变异体，其中该变异体具有一个或多个下列突变：
M9L+M202I,
M9L+M202I+M323T,
M9L+M202I+323T+M382Y,
M9L+M202I+Y295F+A339S,
M9L+M202I+Y295F,
M9L+M202I+A339S,
M9L+M202I+Y295F+A339S,
M9L+M202I+Y295F+A339S+E345R,
M9L+G149A+M202I+Y295F+A339S+E345R,
M9L+M202L,
M9L+M202L+M323T,
M9L+M202L+M232T+M382Y,
M9L+M202L+Y295F+A339S,
M9L+M202L+Y295F,
M9L+M202L+A339S,
M9L+M202L+Y295F+A339S,
M9L+M202L+Y295F+A339S,E345R,
M9L+G149A+M202L+Y295F+A339S+E345R,
M9L+M202T,
M9L+M202T+M323T,
M9L+M202T+M323T+M382Y,
M9L+M202T+Y295F+A339S,
M9L+M202T+Y295F,
M9L+M202T+A339S,
M9L+M202T+Y295F+A339S,
M9L+M202T+Y295F+A339S+E345R,
M9L+G149A+M202T+Y295F+A339S+E345R,
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y,
M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S,
M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202L+V214T+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202I+V214I+Y295F+M323T+A339S+E345R+M382Y,
M9L+G149A+G182T+G186A+M202L+V214I+Y295F+N299Y+M323T+A339S,
M9L+G149A+G182T+G186A+M202I+T257I+Y295F+N299Y+M323T+A339S+E345R,
M9L+G149A+M202I+V214T+Y295F+N299Y+M323T+A339S+E345R+N471E,
M9L+G149A+M202L+V214I+Y295F+M323T+A339S+E345R+M382Y+N471E,
M9L+G149A+G182T+G186A+M202I+V214I+Y295F+N299Y+M323T+A339S+N471E,
M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R+N471E,
M202L+M105F+M208F,
G133E+M202L+Q361E,
G133E+M202L+R444E,
M202L+Y295F,
M202L+A339S,
M202L+M323T,
M202L+M323T+M309L,
M202L+M323T+M430I,
M202L+V214T+R444Y,
M202L+N283D+Q361E,
M202L+M382Y+K383R,
M202L+K446R+N484Q,
M202I+Y295F,
M202I+A339S,
M202I+M105F+M208F,
G133E+M202I+Q361E,
G133E+M202I+R444E,
M202I+M202I+M323T,
M202I+M202I+M323T+M309L,
M202I+M323T+M430I,
M202I+V214T+R444Y,
M202I+N283D+Q361E,
M202I+M382Y+K383R,
M202I+K446R+N484Q,
M202V+M105F+M208F,
G133E+M202V+Q361E,
G133E+M202V+R444E,
M202V+M323T,
M202V+M323T+M309L,
M202V+M323T+M430I,
M202V+M323T+M9L,
M202V+V214T+R444Y,
M202V+N283D+Q361E,
M202V+M382Y+K383R,
M202V+K446R+N484Q,
M202T+M105F+M208F,
G133E+M202T+Q361E,
G133E+M202T+R444E,
M202T+Y295F,
M202T+A339S,
M202T+M323T,
M202T+M323T+M309L,
M202T+M323T+M430I,
M202T+M323T+M9L,
M202T+V214T+R444Y,
M202T+N283D+Q361E,
M202T+A339S,
M202T+Y295F
M202T+N299F,Y,
M202T+M382Y+K383R,
M202T+K446R+N484Q。
7.权利要求1-6的变异体，其中该变异体还含有突变D183*+G184*。
8.权利要求1-7的变异体，其中该变异体还含有在R118的突变，特别是R118K。
9.权利要求1-8的变异体，其中该变异体还含有在N195的突变，特别是N195F。
10.权利要求1-9的变异体，其中该变异体还含有在R320的突变，特别是R320K。
11.权利要求1-10的变异体，其中该变异体还含有在R458的突变，特别是R458K。
12.权利要求1-11的变异体，其中该变异体含有突变D183*+G184*+R118K+N195F+R320K+R458K与一个或多个下列突变的组合：
K118Q,
K37T,
H421Y,
V450T,
K383R,
N445Q,
Y178F,
V318L,
W482Y,
N283D+Q361E,
M105F+M208F,
M202L+M323T+M430I,
K446R+N484Q,
R444Y,
N106D,
Y203L,
G133E+Q361E,
M323E,
V214T,
M202L+M323T+M309L,
M202L,
M202L+M323T,
M202L+M323T+M9L+M382Y+K383R,
M202L+M323T+M9L+M382Y,
M202L+M323T+M9L。
13.权利要求12的变异体，其中该变异体含有突变D183*+G184*+R118K+N195F+R320K+R458K+M202L+M323T+M9L。
14.权利要求13的变异体，其中该变异体还含有一个或多个下列突变：
T461P,
Y298H,
G133E+R444E,
Y298F,
M202T,
M202I,
M202V,
V214T+M323E+M382Y+K383R+N471E
Y178F+G258D+T419N+N437H
G149N+N150Q+M382Y+K383R
Y160F+V214T+M382Y
N128Y+G149A+V214T+D231N+M382Y+F441L
R82H+N128Y+G149A+V214T+M382Y
N150H+V214T
V214T+E345N
V214T+G305D+M382Y+R444E
V214T+M382Y+A447Y
M202I+V214T+M382Y+K383R+R444Y
V214T+G378K
V214T+A256K
R26S+D30N+N33D+V214T+M382Y。
15.权利要求1-14中任一项的变异体，其中该亲本Termamyl-样α-淀粉酶来源于地衣芽孢杆菌，解淀粉芽孢杆菌，嗜热脂肪芽孢杆菌，芽孢杆菌属菌种NCIB 12289，NCIB 12512，NCIB 12513或DSM 9375，或DSMZ no.12649，KSM AP1378，或KSM K36或KSM K38的菌株。
16.权利要求1-15的变异体，其中该亲本Termamyl-样α-淀粉酶是选自SEQ ID NOs:2,4,6,8,10,12,13,14,15 16,17,和18所述的组中的任何α-淀粉酶。
17.根据权利要求1-16任一项的变异体，其中该亲本Termamyl-样α-淀粉酶具有与SEQID NO:4有至少60％，优选70％，更优选至少80％，甚至更优选至少大约90％，甚至更优选至少95％，甚至更优选至少97％，和甚至更优选至少99％同一性程度的氨基酸序列。
18.权利要求1-17中任一项的变异体，其中该亲本Termamyl-样α-淀粉酶由核酸序列编码，该核酸序列在低等，优选中等，优选高度严格条件下与SEQ ID NO:11的核酸序列杂交。
19.权利要求1-18的变异体，该变异体相对于所述亲本α-淀粉酶具有至少一种下列特性改变：底物特异性，底物结合，底物裂解模式，热稳定性，pH活性谱，pH稳定性谱，氧化稳定性，Ca2+依赖性，pI减少和增加以及改进的洗涤效果，比活，在例如，高温和/或低/高pH条件下，特别是在低钙浓度下的稳定性，和在去污剂存在下的稳定性，例如在去污剂中的贮存稳定性。
20.一种DNA构建体，它含有编码根据权利要求1至19任一项的α-淀粉酶变异体的DNA序列。
21.一种重组表达载体，它携带根据权利要求20的DNA构建体。
22.用根据权利要求20的DNA构建体或根据权利要求21的载体转化的细胞。
23.根据权利要求22的细胞，它是微生物，优选细菌或真菌，特别是革兰氏阳性细菌，例如枯草芽孢杆菌，地衣芽孢杆菌，迟缓芽孢杆菌,短芽孢杆菌，嗜热脂肪芽孢杆菌，嗜碱芽孢杆菌,解淀粉芽孢杆菌,凝固芽孢杆菌，环状芽孢杆菌，灿烂芽孢杆菌或苏芸金芽孢杆菌。
24.一种组合物，它含有权利要求1-19的α-淀粉酶变异体。
25.一种去污剂添加剂，它含有根据权利要求1至19任一项的α-淀粉酶变异体，任选以无尘粒状，稳定液体或受保护酶的形式。
26.根据权利要求25的去污剂添加剂，它含有0.02-200mg酶蛋白/g添加剂。
27.根据权利要求25或26的去污剂添加剂，它还含有另一酶，例如蛋白酶，脂肪酶，过氧化物酶，甘露聚糖酶，产麦芽糖淀粉酶，CGTase，淀粉酶或另一淀粉分解酶，例如葡糖淀粉酶，和/或纤维素酶。
28.一种去污剂组合物，它含有根据权利要求1到19任一项的α-淀粉酶变异体。
29.根据权利要求28的去污剂组合物，它还含有另一酶，例如蛋白酶，脂肪酶，过氧化物酶，甘露聚糖酶，产麦芽糖淀粉酶，CGTase，另一种淀粉分解酶和/或纤维素酶。
30.含有根据权利要求1到19任一项的α-淀粉酶变异体的手工或自动餐具去污剂组合物。
31.根据权利要求30的餐具去污剂组合物，它还含有另一酶，例如蛋白酶，脂肪酶，过氧化物酶，甘露聚糖酶，产麦芽糖淀粉酶，CGTase，淀粉酶或另一种淀粉分解酶，例如葡糖淀粉酶，和/或纤维素酶。
32.含有根据权利要求1到19任一项的α-淀粉酶变异体的手工或自动洗衣洗涤组合物。
33.根据权利要求32的洗衣洗涤组合物，它还含有另一种酶，例如蛋白酶，脂肪酶，过氧化物酶，甘露聚糖酶，产麦芽糖淀粉酶，CGTase，淀粉酶或另一种淀粉分解酶，例如葡糖淀粉酶和/或纤维素酶。
34.根据权利要求1到19任一项的α-淀粉酶变异体或根据权利要求24到34的组合物在洗涤和/或餐具中的用途。
35.根据权利要求1到19任一项的α-淀粉酶变异体或根据权利要求24到34的组合物在纺织品脱浆中的用途。
36.根据权利要求1到19任一项的α-淀粉酶变异体或根据权利要求24到34的组合物在淀粉液化中的用途。
37.生产根据权利要求1-19任一项的变异体的方法，其中在有助于生产该变异体的条件下培养根据权利要求22至23任一项的细胞，随后从培养物中回收该变异体。
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