CN1537163A - 枯草杆菌酶变体 - Google Patents
枯草杆菌酶变体 Download PDFInfo
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- CN1537163A CN1537163A CNA028137477A CN02813747A CN1537163A CN 1537163 A CN1537163 A CN 1537163A CN A028137477 A CNA028137477 A CN A028137477A CN 02813747 A CN02813747 A CN 02813747A CN 1537163 A CN1537163 A CN 1537163A
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- variant
- deleted
- insert
- subtilase
- amino acid
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38609—Protease or amylase in solid compositions only
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38618—Protease or amylase in liquid compositions only
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
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- C—CHEMISTRY; METALLURGY
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D3/38654—Preparations containing enzymes, e.g. protease or amylase containing oxidase or reductase
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C12Y—ENZYMES
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- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
- C11D2111/10—Objects to be cleaned
- C11D2111/12—Soft surfaces, e.g. textile
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
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- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
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- Biomedical Technology (AREA)
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- Enzymes And Modification Thereof (AREA)
- Detergent Compositions (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及在一种或多种特性上与亲本枯草杆菌酶不同的新枯草杆菌酶变体,所述特性包括:洗涤性能、热稳定性、储藏稳定性或催化活性。本发明的变体适用于例如清洁或洗涤剂组合物,如洗衣洗涤剂组合物和洗盘组合物,包括自动洗盘组合物中。所述变体在氨基酸序列的52个指定位置之至少一个位置处包含缺失、替换或插入。
Description
技术领域
本发明涉及在一种或多种特性上与亲本枯草杆菌酶不同的新枯草杆菌酶(subtilase)变体,所述特性包括:洗涤性能、热稳定性、储藏稳定性或催化活性。本发明的变体适用于,例如,清洁或洗涤剂组合物,如洗衣洗涤剂组合物和洗盘组合物,包括自动洗盘组合物中。本发明还涉及编码该变体的分离的DNA序列、表达载体、宿主细胞,以及产生和使用本发明变体的方法。而且,本发明涉及包含本发明变体的清洁和洗涤剂组合物。
发明背景
在洗涤剂工业中,酶已在洗涤制剂中使用了30年以上。用于这些制剂的酶包括蛋白酶、脂肪酶、淀粉酶、纤维素酶和其它酶或它们的混合物。商业上最为重要的酶是蛋白酶。
数量不断增长的商用蛋白酶是天然存在的野生型蛋白酶的蛋白质工程化变体,如DURAZYM(Novo Nordisk A/S)、RELASE(Novo NordiskA/S)、MAXAPEM(Gist-Brocades N.V.)、PURAFECT(GenencorInternational,Inc.)。
而且,本领域描述了许多蛋白酶变体。WO 99/27082中给出了现有技术中蛋白酶变体的完整列表。
虽然已描述了大量有用的蛋白酶变体,但是对于许多工业应用,例如洗衣或硬表面清洁而言,仍需要新的改进的蛋白酶或蛋白酶变体。
因此,本发明的一个目的是提供改进的枯草杆菌酶变体用于此类目的。
发明概述
因此,在第一方面,本发明涉及包含至少一个或多个如下改变的枯草杆菌酶变体:
X11{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失,插入;
X30{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X31{R,N,D,Q,E,H,K,M,P,W,Y},缺失,插入;
X32{A,R,D,C,E,G,H,I,L,K,M,F,P,T,W,Y,V},插入;
X34{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失;
X65{A,R,C,G,H,I,L,K,M,F,T,W,Y,V},缺失,插入;
X66{A,R,C,H,I,L,K,M,F,T,W,Y,V},缺失,插入;
X67{A,R,N,Q,G,H,I,K,M,F,P,S,T,W,Y,V};
X68{R,N,D,Q,E,G,H,I,L,K,F,P,S,T,W,Y,V},缺失,插入;
X69{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X70G;
X71T;
X77N;
X83G,插入;
X84V;
X85{A,R,N,D,Q,E,G,H,I,L,M,F,S,T,W,Y,V};
X90{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X95{R,K,F,W,Y,V},缺失;
X107I
X110G,插入;
X121V,插入;
X122插入;
X123N;
X125S;
X150{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X152{A,R,N,D,Q,E,H,K,F,P,W,Y,V};
X153{R,N,D,Q,E,G,H,I,L,K,M,F,T,W,Y,V};
X154{A,R,G,H,I,L,M,F,W,Y,V};
X164{A,R,H,T,W};
X165{R,L,F,W,Y};
X166{W,Y},插入;
X175{R,N,D,Q,E,G,H,K,M,F,P,T,W,Y,V};
X177{R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X178{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X180{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X199{R,L,K,F,W,Y,V},缺失,插入;
X200{A,R,I,L,K,M,F,W,Y,V},缺失,插入;
X201{A,R,H,I,L,K,M,F,P,T,W,Y,V},缺失,插入;
X202{A,R,C,G,H,I,L,K,M,F,T,W,Y,V};
X207{A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V};
X220T,插入;
X223A,插入;
X225P,插入;
X226H,插入;
X227V,插入;
X228A,插入;
X229G,插入;
X230插入;
X231A,插入;
X253{A,R,N,D,Q,E,G,H,I,L,M,F,S,T,W,Y,V},缺失,插入;
X264{A,R,N,Q,G,H,I,L,M,F,S,T,W,Y,V},缺失,插入;
X266{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失,插入;
其中,
(a)所述改变各自独立地为
(i)以不同的氨基酸替换占据所述位置的氨基酸,
(ii)缺失占据所述位置的氨基酸,或
(iii)在占据所述位置的氨基酸的下游插入至少一个额外氨基酸,
(b)所述变体具有蛋白酶活性,且
(c)每个位置都对应于图1所示枯草杆菌蛋白酶BPN’氨基酸序列的位置。
第二方面,本发明涉及编码本发明枯草杆菌酶变体的分离的多核苷酸。
第三方面,本发明涉及包含本发明的分离多核苷酸的表达载体。
第四方面,本发明涉及用本发明的表达载体转化的微生物宿主细胞。
第五方面,本发明涉及用于产生本发明枯草杆菌酶变体的方法,其中在有利于该变体表达和分泌的条件下培养本发明的宿主,并回收该变体。
第六方面,本发明涉及包含本发明变体的清洁或洗涤剂组合物,优选地洗衣或洗盘组合物。
关于序列比对和编号参考图1,该图显示在枯草杆菌蛋白酶BPN′(a)(BASBPN)和枯草杆菌蛋白酶309(BLSAVI)(b)之间的序列比对。
在本专利申请中此序列比对(alignment)用作对残基进行编号的参照。
定义
在更详细地讨论本发明之前,首先定义以下的术语和约定。
氨基酸命名法
A = Ala = 丙氨酸
V = Val = 缬氨酸
L = Leu = 亮氨酸
I = Ile = 异亮氨酸
P = Pro = 脯氨酸
F = Phe = 苯丙氨酸
W = Trp = 色氨酸
M = Met = 甲硫氨酸
G = Gly = 甘氨酸
S = Ser = 丝氨酸
T = Thr = 苏氨酸
C = Cys = 半胱氨酸
Y = Tyr = 酪氨酸
N = Asn = 天冬酰胺
Q = Gln = 谷氨酰胺
D = Asp = 天冬氨酸
E = Glu = 谷氨酸
K = Lys = 赖氨酸
R = Arg = 精氨酸
H = His = 组氨酸
X = Xaa = 任何氨基酸
核酸命名法
A = 腺嘌呤
G = 鸟嘌呤
C = 胞嘧啶
T = 胸腺嘧啶(仅在DNA中)
U = 尿嘧啶(仅在RNA中)
变体名称的命名法和约定
在描述本发明产生的或考虑的多种枯草杆菌酶变体时,采用以下的命名法和约定以易于参照:
首先通过将分离的酶或亲本酶与枯草杆菌蛋白酶BPN′(BASBPN)比对来定义参考框。
可以由GCG软件包9.1版的GAP程序对变体进行编号而获得序列比对,其间使用了以下参数:缺口(gap)创建罚分=8,缺口延伸罚分=8,且所有的其它参数保持为它们的默认值。
另一种方法是使用枯草杆菌酶之间的已知公认比对,如在WO91/00345中所示的序列比对。在大部分情况下,这些差异并不重要。
由此,可以相对BASBPN(SEQ ID NO:3)定义许多缺失和插入。在图1中,枯草杆菌蛋白酶309(SEQ ID NO:4)与BASBPN相比,在位置36、58、158、162、163和164位有6处缺失。这些缺失在图1中用星号(*)表示。
一般采用以下的三个部分来表示对亲本酶所进行的多种修饰:
原始氨基酸
位置
替换的氨基酸
因此,符号G195E意指195位的甘氨酸用谷氨酸替换。
在原始氨基酸残基可以是任意氨基酸残基的情况下,有时可以用简略表达方式仅表示位置和替换的氨基酸:
位置
替换的氨基酸
这种符号尤其与同源枯草杆菌酶中的修饰相关(见下文)。
类似地,当替换氨基酸残基的身份不重要时,可以使用:
原始氨基酸
位置
当原始氨基酸和替换氨基酸均可以包含任意氨基酸时,则只指出位置,如:170。
当原始氨基酸和/或替换的氨基酸可以包含一种以上但不是所有的氨基酸时,所选择的氨基酸表示在括号内:
原始氨基酸
位置
{替换的氨基酸1,...,替换的氨基酸n}
对于具体的变体,使用具体的三个或一个字母代码,包括代码Xaa和X用来表示任何氨基酸残基。
替换:
用谷氨酸替换195位的甘氨酸表示为:
Gly195Glu或G195E
或者用任意氨基酸残基替换195位的甘氨酸表示为:
Gly195Xaa或G195X
或
Gly195或G195
因此用丝氨酸替换170位的任意氨基酸残基将表示为:
Xaa170Ser或X170S
或
170Ser或170S
这种符号尤其与同源枯草杆菌酶的修饰相关(见下文)。因此170Ser意在包括例如,BASBPN中的Lys170Ser修饰和BLSAVI中的Arg170Ser修饰(参见图1)。
对于其中的原始氨基酸和/或替换的氨基酸可以包含一种以上但不是所有的氨基酸的修饰而言,由甘氨酸、丙氨酸、丝氨酸或苏氨酸替换170位的精氨酸表示为
Arg170{Gly,Ala,Ser,Thr}或R170{G,A,S,T}
以表示变体
R170G、R170A、R170S和R170T。
缺失:
195位甘氨酸的缺失表示为:
Gly195*或G195*
相应地,一个以上氨基酸残基的缺失,如195和196位甘氨酸和亮氨酸的缺失表示为
Gly195*+Leu196*或G195*+L196*
插入:
额外的氨基酸残基的插入,如在G195后插入赖氨酸表示为:
Gly195GlyLys或G195GK;
或者,当插入一个以上的氨基酸残基,如在G195后插入Lys和Ala时,这种插入表示为:
Gly195GlyLysAla或G195GKA
在这些情况下,通过将小写字母加到所插入氨基酸残基之前的氨基酸残基位置号中来对所插入氨基酸残基进行编号。在以上的实例中,序列194至196因此为:
194 195 196
BLSAVI A-G-L
194 195 195a 195b 196
变体 A-G-K-A-L(SEQ ID NO:1)
当插入与所存在的氨基酸残基相同的氨基酸残基时,显然在命名法中出现了简并。例如,如果在以上的实例中,在甘氨酸之后插入甘氨酸,则将它表示为G195GG。对从
194 195 196
BLSAVI A-G-L
194 195 195a 196
至变体 A-G-G-L (SEQ ID NO:2)
194 194a 195 196的事实上同样的变化还可以表示为A194AG。
这些例子对本领域技术人员来说是显而易见的,因此表示法G195GG和用于此类插入的相应表示法意在包含这些等同的简并表示法。
填补缺口:
当与用于编号的枯草杆菌蛋白酶BPN′序列进行参照比较,在酶中存在缺失时,则对于在36位插入天冬氨酸而言,此位置处的插入表示为:
*36Asp或*36D
多重修饰:
用加号分隔含多重修饰的变体,如:
Arg170Tyr+Gly195Glu或R170Y+G195E代表在170和195位分别用酪氨酸和谷氨酸替换精氨酸和甘氨酸的修饰。
因此,Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr}表示以下变体:
Tyr167Gly+Arg170Gly,Tyr167Gly+Arg170Ala,
Tyr167Gly+Arg170Ser,Tyr167Gly+Arg170Thr,
Tyr167Ala+Arg170Gly,Tyr167Ala+Arg170Ala,
Tyr167Ala+Arg170Ser,Tyr167Ala+Arg170Thr,
Tyr167Ser+Arg170Gly,Tyr167Ser+Arg170Ala,
Tyr167Ser+Arg170Ser,Tyr167Ser+Arg170Thr,
Tyr167Thr+Arg170Gly,Tyr167Thr+Arg170Ala,
Tyr167Thr+Arg170Ser,和Tyr167Thr+Arg170Thr.
这种命名法与针对替换、代替、插入或缺失具有特定共同性质的氨基酸残基,如带正电荷(K、R、H)、带负电荷(D、E)残基的修饰,或意味着用一种小氨基酸替换另一种小氨基酸的诸如Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr}的保守氨基酸修饰特别相关。进一步的详细描述参见“发明详述”部分。
蛋白酶
断裂蛋白质底物中的酰胺键的酶归类为蛋白酶,或(可互换地)肽酶(见Walsh,1979,酶反应机理(Enzymatic Reaction Mechanisms).W.H.Freeman and Company,San Francisco,第3章)。
氨基酸位置/残基的编号
如果没有另外指出,本文所用的氨基酸编号对应于枯草杆菌酶BPN′(BASBPN)序列的氨基酸编号。对BPN′序列的详细描述,参见图1或Siezen等人,蛋白质工程(Protein Engng.)4(1991)719-737。
丝氨酸蛋白酶
丝氨酸蛋白酶是一种催化肽键水解的酶,其中在活性位点存在必需的丝氨酸残基(White,Handler和Smith,1973“生物化学原理(Principles of Biochemistry),”第五版,McGraw-Hill Book公司,纽约,第271-272页)。
细菌丝氨酸蛋白酶的分子量在20,000-45,000道尔顿的范围内。它们被二异丙基氟磷酸酯抑制。它们水解简单末端脂类,且其活性与真核生物糜蛋白酶(也是一种丝氨酸蛋白酶)相似。一个更为狭义的术语,碱性蛋白酶(包括一个亚组)反映某些丝氨酸蛋白酶的高pH最适值,为pH 9.0-11.0(综述参见Priest(1977)细菌学评论(BacteriologicalRev.)41:711-753)。
枯草杆菌酶
Siezen等人,蛋白质工程,4(1991)719-737和Siezen等人,蛋白质科学(Protein Science)6(1997)501-523提出丝氨酸蛋白酶的一个亚组,其暂称为枯草杆菌酶(subtilase)。它们是通过对170多个先前称为枯草杆菌蛋白酶样蛋白酶的丝氨酸蛋白酶的氨基酸序列进行同源性分析而定义的。枯草杆菌蛋白酶以前通常被定义为由革兰氏阳性细菌或真菌产生的丝氨酸蛋白酶,而现在根据Siezen等人则为枯草杆菌酶的一个亚组。已鉴定了大量的枯草杆菌酶,并已测定了许多枯草杆菌酶的氨基酸序列。对此类枯草杆菌酶及其氨基酸序列的更详细描述参见Siezen等人(1997)。
枯草杆菌酶的一个亚组,I-S1或″真″枯草杆菌蛋白酶,包含“经典的”枯草杆菌蛋白酶,如枯草杆菌蛋白酶168(BSS168)、枯草杆菌蛋白酶BPN′、枯草杆菌蛋白酶Carlsberg(ALCALASE,NOVOZYMES A/S)和枯草杆菌蛋白酶DY(BSSDY)。
Siezen等人(引文同上)识别了枯草杆菌酶的另一亚组,I-S2或强碱性枯草杆菌蛋白酶。亚组I-S2蛋白酶被描述为强碱性枯草杆菌蛋白酶并包括例如:枯草杆菌蛋白酶PB92(BAALKP)(MAXACAL,Gist-Brocades NV)、枯草杆菌蛋白酶309(SAVINASE,NOVOZYMESA/S)、枯草杆菌蛋白酶147(BLS147)(ESPERASE,NOVOZYMES A/S)和碱性弹性蛋白酶YaB(BSEYAB)。
″SAVINASE
″
SAVINASE由NOVOZYMES A/S销售。它是来自迟缓芽孢杆菌(B.Lentus)的枯草杆菌蛋白酶309,并且只在一个位置处不同于BAALKP(N87S,见此处的图1)。SAVINASE具有在图1中称为b)的氨基酸序列。
亲本枯草杆菌酶
术语“亲本枯草杆菌酶”描述根据Siezen等人(1991和1997)定义的枯草杆菌酶。关于进一步的细节,参见以上紧挨着的对“枯草杆菌酶”的描述。亲本枯草杆菌酶也可以是从天然来源分离的、并随后在保留枯草杆菌酶特征的情况下经过修饰的枯草杆菌酶。而且,亲本枯草杆菌酶还可以是通过如以下文献所述的DNA改组技术制备的枯草杆菌酶:J.E.Ness等人,自然生物技术(Nature Biotechnology),17,893-896(1999)。术语“亲本枯草杆菌酶”可另外称为“野生型枯草杆菌酶”。
作为参考下表提供了此处所述多种枯草杆菌酶的简称,对于其它简称,参见Siezen等,蛋白质工程(Protein Engng.)4(1991)719-737和Siezen等,蛋白质科学(Protein Science)6(1997)501-523。
表I
生物体 | 酶 | 简称 |
细菌:革兰氏阳性 | ||
枯草杆菌(Bacillus subtilis)168 | 枯草杆菌蛋白酶I168,apr | BSS168 |
解淀粉芽孢杆菌(Bacillus amyloliquefaciens) | 枯草杆菌蛋白酶BPN′(NOVO) | BASBPN |
枯草杆菌DY | 枯草杆菌蛋白酶DY | BSSDY |
地衣芽孢杆菌(Bacillus licheniformis) | 枯草杆菌蛋白酶Carlsberg | BLSCAR |
迟缓芽孢杆菌 | 枯草杆菌蛋白酶309 | BLSAVI |
迟缓芽孢杆菌 | 枯草杆菌蛋白酶147 | BLS147 |
嗜碱芽孢杆菌(Bacillus alcalophilus)PB92 | 枯草杆菌蛋白酶PB92 | BAPB92 |
芽孢杆菌YaB | 碱性弹性蛋白酶YaB | BYSYAB |
普通高温放线菌(Thermoactinomyces vulgaris) | thermitase | TVTHER |
枯草杆菌酶变体的修饰
本文所用的术语“修饰”定义为包括枯草杆菌酶的化学修饰以及对编码枯草杆菌酶的DNA所进行的遗传操作。修饰可以是氨基酸侧链的置换、目标氨基酸处的替换、缺失和/或插入。
枯草杆菌酶变体
在本发明的上下文中,术语枯草杆菌酶变体或突变的枯草杆菌酶指由表达突变基因的生物体产生的枯草杆菌酶,该突变基因来源于含原始或亲本基因并产生相应亲本酶的亲本微生物,其中为产生在适宜的宿主中表达时能产生所述的突变枯草杆菌酶的此突变基因,已经对所述亲本基因进行了突变。
同源枯草杆菌酶序列
在本上下文中,两种氨基酸序列之间的同源性用参数“同一性”描述。
为了确定两种枯草杆菌酶之间的同一性程度,可以应用(下文)GCG软件包9.1版的GAP程序以及相同的设定值。此程序的计算结果除了氨基酸序列比对外,还有两个序列之间的“同一性百分率”的计算结果。
根据此说明书,本领域技术人员可常规地鉴定可以根据本发明进行修饰的适宜同源性枯草杆菌酶。
分离的多核苷酸
术语“分离的”当应用于多核苷酸时是指该多核苷酸已经从其天然遗传环境中移出并因此不含其他外来或不需要的编码序列,并且该多核苷酸的存在形式适于在遗传工程化蛋白质生产系统中应用。此分离分子为那些从其天然环境中分离的分子并包括cDNA和基因组克隆。本发明的分离的DNA分子不含通常与之关联的其他基因,但可包含天然存在的5′和3′非翻译区如启动子和终止子。关联区域的鉴定对本领域内的普通技术人员而言是显而易见的(参见例如,Dynan和Tijan,自然(Nature)316:774-78,1985)。术语“分离的多核苷酸”另外可称为“克隆的多核苷酸”。
分离的蛋白质
术语“分离的”在应用于蛋白质时指此蛋白质已从其自然环境中移出。
在优选的形式中,分离的蛋白质基本上不含其它蛋白质,特别是不含其它同源蛋白质(即“同源杂质”(参见以下))。
通过SDS-PAGE测定,分离的蛋白质的纯度大于10%,优选大于20%,更优选大于30%。还优选提供高度纯化形式的蛋白质,即通过SDS-PAGE测定纯度大于40%,大于60%,大于80%,更优选大于95%,并最优选大于99%。
术语“分离的蛋白质”另外可称为“纯化的蛋白质”。
同源杂质
术语“同源杂质”意指任何来源于最初获得本发明枯草杆菌酶的同源细胞的杂质(如非本发明枯草杆菌酶的另一多肽)。
自……获得
本文所用与特定的微生物来源相关的术语“自……获得”意指该多核苷酸和/或枯草杆菌酶是由此特定来源或由已插入了来自于该来源的基因的细胞产生的。
底物
与蛋白酶的底物相关的术语“底物”应以其最广义的形式解释为包括含有至少一个易于被枯草杆菌蛋白酶水解的肽键(酰胺键)的化合物。
产物
与来自蛋白酶酶促反应的产物有关的术语“产物”在本发明的上下文中应解释为包括涉及蛋白酶枯草杆菌酶的水解反应的产物。产物可以是随后的水解反应的底物。
洗涤性能
在本发明上下文中,术语“洗涤性能”用以表示酶在例如洗涤或硬表面清洁过程中除去存在于待清洁物体上的蛋白质性污渍或有机污渍的能力。还参见本文实施例3中的“模型洗涤剂洗涤性能试验”。
性能因子
用下式定义术语“性能因子”:
P=R变体-R亲本
其中,P为性能因子,R变体为按“模型洗涤剂洗涤性能试验”中所述采用枯草杆菌酶变体处理之后的试验材料的反射能力(reflectance)(于460nm处测定),而R亲本为按“模型洗涤剂洗涤性能试验”中所述采用对应的亲本枯草杆菌酶处理之后的试验材料的反射能力(于460nm处测定)。进一步的详情参见本文实施例3中的“模型洗涤剂洗涤性能试验”。
附图简述
图1显示使用上述的GAP程序进行的枯草杆菌蛋白酶BPN′(a)和Savinase(b)之间的序列比对。
发明详述
本发明涉及在一种或多种特性上与亲本枯草杆菌酶不同的新枯草杆菌酶变体,所述特性包括:洗涤性能、热稳定性、储藏稳定性或催化活性。
作为本发明的一部分考虑的变体是与野生型枯草杆菌酶相比在至少一个或多个如下位置处具有一个或多个氨基酸残基的替换、缺失或插入的变体:11、30、31、32、34、65、66、67、68、69、70、71、77、83、84、85、90、95、107、110、121、122、123、125、150、152、153、154、164、165、166、175、177、178、180、199、200、201、202、207、220、223、225、226、227、228、229、230、231、253、264或266(BASBPN编号)。
在图1所示Savinase序列中,可突变的位置为一个或多个如下位置:V11,V30,L31,D32,G34,G65,T66,H67,V68,A69,G70,T71,N77,G83,V84,A85,L90,V95,I107,G110,V121,A122,N123,S125,V150,A152,S153,G154,*164,*165,*166,M175,V177,G178,T180,V199,A200,P201,G202,S207,T220,A223,P225,H226,V227,A228,G229,A230,A231,T253,G264或G266。
所考虑的具体替换/缺失/插入为:
X11{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失,插入;
X30{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X31{R,N,D,Q,E,H,K,M,P,W,Y},缺失,插入;
X32{A,R,D,C,E,G,H,I,L,K,M,F,P,T,W,Y,V},插入;
X34{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失;
X65{A,R,C,G,H,I,L,K,M,F,T,W,Y,V},缺失,插入;
X66{A,R,C,H,I,L,K,M,F,T,W,Y,V},缺失,插入;
X67{A,R,N,Q,G,H,I,K,M,F,P,S,T,W,Y,V};
X68{R,N,D,Q,E,G,H,I,L,K,F,P,S,T,W,Y,V},缺失,插入;
X69{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X70G;
X71T;
X77N;
X83G,插入;
X84V;
X85{A,R,N,D,Q,E,G,H,I,L,M,F,S,T,W,Y,V};
X90{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X95{R,K,F,W,Y,V},缺失;
X107I
X110G,插入;
X121V,插入;
X122插入;
X123N;
X125S;
X150{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X152{A,R,N,D,Q,E,H,K,F,P,W,Y,V};
X153{R,N,D,Q,E,G,H,I,L,K,M,F,T,W,Y,V};
X154{A,R,G,H,I,L,M,F,W,Y,V};
X164{A,R,H,T,W};X165{R,L,F,W,Y};
X166{W,Y},插入;
X175{R,N,D,Q,E,G,H,K,M,F,P,T,W,Y,V};
X177{R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X178{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X180{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X199{R,L,K,F,W,Y,V},缺失,插入;
X200{A,R,I,L,K,M,F,W,Y,V},缺失,插入;
X201(A,R,H,I,L,K,M,F,P,T,W,Y,V),缺失,插入;
X202{A,R,C,G,H,I,L,K,M,F,T,W,Y,V};
X207{A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V};
X220T,插入;
X223A,插入;
X225P,插入;
X226H,插入;
X227V,插入;
X228A,插入;
X229G,插入;
X230插入;
X231A,插入;
X253{A,R,N,D,Q,E,G,H,I,L,M,F,S,T,W,Y,V},缺失,插入;
X264{A,R,N,Q,G,H,I,L,M,F,S,T,W,Y,V},缺失,插入;
X266{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失,插入;
其中,每个位置都对应于图1所示枯草杆菌蛋白酶BPN’中的位置。
本发明第一方面的枯草杆菌酶变体可以是从自然界中鉴定和分离的亲本或野生型枯草杆菌酶。
此亲本野生型枯草杆菌酶可以通过本领域已知的标准技术特异地筛选。
进行此筛选的一个优选方式可以是从多种不同微生物,优选从不同芽孢杆菌菌株的枯草杆菌酶,特异地PCR扩增目的保守DNA区域。
由于枯草杆菌酶的DNA和氨基酸序列具有同源性,故在此意义上该酶是一组保守酶。因此,可以构建位于目的多核苷酸序列侧翼的相对特异引物。
使用此PCR引物从多种不同的微生物(优选从不同的芽孢杆菌菌株)扩增DNA,然后对所述的扩增PCR片段进行DNA测序,可以鉴定出产生本发明枯草杆菌酶变体的菌株。在鉴定到此菌株和目的枯草杆菌酶的部分DNA序列后,本领域技术人员可以常规地完成该枯草杆菌酶的克隆、表达和纯化。
然而,可以想到的是,本发明枯草杆菌酶变体主要是亲本枯草杆菌酶的变体。
适于本文所述用途的枯草杆菌酶变体可以通过本领域已知标准技术,例如定点/随机诱变或不同枯草杆菌酶序列的DNA改组来构建。参见此处“材料和方法”部分(见下文)的更详细描述。
本领域技术人员知道,此处描述的变体可以包含一个或多个其它的改变,尤其是一个或多个其它的替换。
而且,此处描述的变体可以在不仅一个位置上包含突变。例如,本发明的变体可以在一个位置、两个位置或两个以上位置,如3个、4个或5个位置上含有突变。
现有技术中已知,用类似的氨基酸残基对某氨基酸残基进行的所谓保守替代预期将仅造成酶特征的微小变化。
下列表II列出了保守氨基酸替换的组。
表II
保守氨基酸替换
共性
氨基酸
碱性(带正电荷) K=赖氨酸
H=组氨酸
酸性(带负电荷) E=谷氨酸
D=天冬氨酸
极性 Q=谷氨酰胺
N=天冬酰胺
疏水 L=亮氨酸
I=异亮氨酸
V=缬氨酸
M=甲硫氨酸
芳族 F=苯丙氨酸
W=色氨酸
Y=酪氨酸
小的 G=甘氨酸
A=丙氨酸
S=丝氨酸
T=苏氨酸
根据这个原则,包含保守替换的枯草杆菌酶变体预期不会显示出彼此之间具有明显不同的特征。
基于此处公开的和/或示例性的枯草杆菌酶变体,确定可对这些变体进行的合适保守修饰以获得显示出类似改进洗涤性能的其它枯草杆菌酶变体为本领域技术人员的常规工作。
不论是从自然界或人工的多样性创建体中分离酶,还是从亲本枯草杆菌酶设计并产生变体,均优选亲本枯草杆菌酶属于亚组I-S1和I-S2,尤其是亚组I-S2。
对于来自亚组I-S1的变体,亲本枯草杆菌酶优选地选自:BSS168(BSSAS、BSAPRJ、BSAPRN、BMSAMP)、BASBPN、BSSDY、BLSCAR(BLKERA、BLSCA1、BLSCA2、BLSCA3)、BSSPRC和BSSPRD,或它们的保留了亚组I-S1特征的功能性变体。
对于来自亚组I-S2的变体,亲本枯草杆菌酶优选地选自:BSAPRQ、BLS147(BSAPRM、BAH101)、BLSAVI(BSKSMK、BAALKP、BLSUBL)、BYSYAB、BAPB92、TVTHER和BSAPRS,或它们的保留了亚组I-S2特征的功能性变体。尤其是,亲本枯草杆菌酶是BLSAVI(Savinase,NOVOZYMES A/S),并且本发明优选的枯草杆菌酶变体因此为Savinase的变体。
本发明也包括其氨基酸序列中组合有任何其它修饰的任一上述枯草杆菌酶变体。特别是可以想到与本领域公知的能为该酶提供改良性质的其它修饰进行组合。现有技术描述了大量具有不同改良性质的枯草杆菌酶变体,其中的一些在本文的“发明背景”部分中已有提及(见上)。这些参考文献在此处公开作为参考,用来鉴定可有利地与此处描述的枯草杆菌酶变体组合的枯草杆菌酶变体。
此类组合包括位置:222(提高氧化稳定性)、218(提高热稳定性)、在稳定酶的Ca2+-结合位点如位置76处的替换、以及从现有技术显而易见的许多其它位置。
在进一步的实施方案中,此处描述的枯草杆菌酶变体可有利地与任一下列位置处的一个或多个修饰组合:27,36,56,76,87,95,96,97,98,99,100,101,102,103,104,120,123,159,167,170,206,218,222,224,232,235,236,245,248,252和274。
尤其是,如下BLSAVI、BLSUBL、BSKSMK和BAALKP修饰被认为适于组合:
K27R,*36D,S56P,N76D,S87N,G97N,S101G,S103A,V104A,V104I,V104N,V104Y,H120D,N123S,G159D,Y167,R170,Q206E,N218S,M222S,M222A,T224S,A232V,K235L,Q236H,Q245R,N248D,N252K和T274A。
包含以下任一种修饰:S101G+V104N,S87N+S101G+V104N,K27R+V104Y+N123S+T274A,N76D+S103A+V104I或N76D+V104A;或包含修饰K27R,N76D,S101G,S103A,V104N,V104Y,V104I,V104A,N123S,G159D,A232V,Q236H,Q245R,N248D,N252K,T274A的其它组合,并组合有任意一种或多种上述修饰的其它变体也显示出改进的特性。
特别令人感兴趣的变体为这样的变体,其中除了根据本发明的修饰外,还包含下列替换:S101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K。
而且,本发明主要方面的枯草杆菌酶变体优选与一个或多个在位置129、131和194之任一处的修饰组合,所述修饰优选地为129K、131H和194P修饰,且最优选地为P129K、P131H和A194P修饰。在产生枯草杆菌酶变体时,预期所有这些修饰都可以提供更高的枯草杆菌酶变体表达水平。
此外,如下具体的本发明变体被认为是有意义的:
BPN′(BASBPN):
I11V,L
L90I,V
M199*(缺失)Savinase(BLSAVI):V11I;V11I+I44V;V11I+L96LA;S9R+V11I+A15T+T22A;V11I+L96LA+A108C+A138C;V11I+N76D+A194P+A230V;V11I+V84I;V11I+V84I+K251R;V11I+V30I;V11I+V139L;V11I+V30I;V30I;V30I+V84I;V30I+V139L;S9R+A15T+T22A+V30I;V30I+V244R;V30I+K251R;V30I+V139L+Y167A+R170S+A194P+N218S;D32A;V68A;V68A+S106A;V68A,+V139I;V68A,+A158V;V68A+V203A;V68A+V139L;A48T+V68A+P131M;V51A+V68A+S106T+A168G;V51A+V68A+S106T+A168G;N76D+M175ML+A194P+A230V;N76D+M175MI+A194P+A230V;
所选择的本发明变体的洗涤性能可用本文实施例3中所公开的“模型洗涤剂洗涤性能试验”进行测试。可以用“模型洗涤剂洗涤性能试验”评价变体在掺入标准的洗涤剂组合物后与参照体系,即亲本枯草杆菌酶进行比较(将亲本枯草杆菌酶掺入同样的标准洗涤剂体系并在相同的条件下测试),除去标准纺织物上的蛋白质性污渍的能力。利用这种试验,可以初步研究所选择的变体的洗涤性能,其原理是:如果所选择的变体在试验中没有表现出相对于亲本枯草杆菌酶的明显改进,则一般不需要进行进一步的试验。
因此,对于此处描述的目的而言,尤其令人感兴趣的变体为这样的变体:当它在标准洗涤剂组合物中如本文“模型洗涤剂洗涤性能试验”中所述进行试验时,与在相同条件下试验的亲本枯草杆菌酶相比表现出改进的洗涤性能,其中所述标准洗涤剂组合物包含:
6.2% LAS(Nansa 80S)
2% C16-C18脂肪酸的钠盐
4% 非离子表面活性剂(Plurafax LF404)
22% 沸石P
10.5% Na2CO3
4% Na2Si2O5
2% 羧甲基纤维素(CMC)
6.8% 丙烯酸盐液体CP5 40%
20% 硼酸钠(经验式NaBO2.H2O2)
0.2% EDTA
21% Na2SO4
水(平衡量)
可以采用在本文的实施例3中定义的所谓“性能因子”对洗涤性能的改进进行定量。
在本发明的一个非常令人感兴趣的实施方案中,本发明的变体在“洗涤性能试验”中测试时,其性能因子至少为1,如至少为1.5,例如至少为2,优选至少为2.5,如至少为3,例如至少为3.5,特别是至少为4,如至少为4.5,例如至少为5。
明显地,优选本发明的变体至少在所述的最低水平上,更优选在所述的中等水平上,最优选在所述的最高水平上符合上述标准。
产生枯草杆菌酶变体
用于克隆枯草杆菌酶的许多方法和用于将替换、缺失或插入导入基因(如枯草杆菌酶基因)的许多方法在本领域是熟知的。
通常,可以采用用于克隆基因和向该基因中引入突变(随机和/或定点)的标准方法以获得本发明的枯草杆菌酶变体。对于适宜的技术的进一步描述,参照本文的实施例(见下文)和(Sambrook等人,(1989),分子克隆,实验室手册,冷泉港实验室,冷泉港,纽约;Ausubel,F.M.等人(编辑)“分子生物学最新方法”(“Current protocols in Molecular Biology”),John Wiley和Sons,1995;Harwood,C.R.和Cutting,S.M.(编辑)“用于芽孢杆菌的分子生物学方法”(“Molecular Biological Methods forBacillus”),John Wiley和Sons,1990);和WO 96/34946。
而且,枯草杆菌酶变体可以通过用于人工产生多样性的标准技术构建,如通过不同枯草杆菌酶基因的DNA改组构建(参见WO 95/22625;Stemmer WPC,自然370:389-91(1994))。例如,将编码Savinase的基因与本质上经过鉴定的一个或多个部分枯草杆菌酶序列进行DNA改组,在随后对具有改进洗涤性能的变体进行筛选后,即可提供适用于此处所述目的的枯草杆菌酶变体。
表达载体
包含编码本发明酶的DNA构建体的重组表达载体可以是任何可方便地进行重组DNA操作的载体。载体的选择一般取决于它将被导入的宿主细胞。因此,载体可以是自主复制载体,即作为染色体外实体而存在的载体,这种载体的复制独立于染色体的复制,如质粒。
另外,所述载体可以在导入宿主细胞后部分或全部整合入宿主细胞基因组中,并与其整合的染色体一起复制。
所述载体优选为这样的表达载体,其中编码本发明酶的DNA序列可操作地与此DNA转录所需的其它片段相连接。一般来说,表达载体来自质粒或病毒DNA,或可以含有两者的元件。术语“可操作地连接”指将诸片段以使其能按它们的预期目的协同发挥功能的方式排列,如转录在启动子中引发并前行通过编码此酶的DNA序列。
所述的启动子可以是在所选择的宿主细胞内表现出转录活性的任何DNA序列,并可以得自编码与宿主细胞同源或异源的蛋白质的基因。
用于细菌宿主细胞的适宜启动子的实例包括以下基因的启动子:嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)生麦芽糖淀粉酶基因、地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶基因、枯草芽孢杆菌(Bacillussubtilis)碱性蛋白酶基因或短小芽孢杆菌(Bacillus pumilus)木糖苷酶基因,或为λ噬菌体的PR或PL启动子或大肠杆菌的
lac、
trp或
tac启动子。
如果需要,编码本发明酶的DNA序列还可以可操作地连接到适宜的终止子上。
本发明的重组载体还可包含能够使所述载体在所讨论的宿主细胞中复制的DNA序列。
所述载体还可以包含可选择的标记,例如其产物弥补宿主细胞缺陷的基因,或编码如对抗生素像卡那霉素、氯霉素、红霉素、四环素、壮观霉素等的抗性,或对重金属或除草剂的抗性的基因。
为了指导本发明的酶进入宿主细胞的分泌途径,可以在重组载体中提供分泌信号序列(也称为前导序列、前原序列或前序列)。可以将分泌信号序列以正确的读框连接编码酶的DNA序列。分泌信号序列一般位于编码此酶的DNA序列的5’端。所述分泌信号序列可以通常与该酶相关或可以来自编码另一种分泌蛋白质的基因。
用于分别连接编码本发明酶的DNA序列、启动子和任选地终止子和/或分泌信号序列,或通过适宜的PCR扩增方案组装这些序列和将它们插入含有复制或整合所需信息的适宜载体中的方法对本领域技术人员来说是已知的(参见例如,Sambrook等人,前引书)。
宿主细胞
导入宿主细胞中的编码本发明酶的DNA序列可以与此宿主同源或异源。如果与所述宿主细胞同源,即由该宿主细胞天然产生,则该DNA序列一般可操作地与非其天然环境中的另一启动子序列相连接,或者适当时与另一分泌信号序列和/或终止子序列相连接。术语″同源″旨在包括编码天然存在于所讨论的宿主生物体中的酶的DNA序列。术语“异源的”旨在包括所述宿主细胞本身不表达的DNA序列。因此该DNA序列可以来源于另一生物体或者是合成的序列。
可导入本发明的DNA构建体或重组载体的宿主细胞可以是任何能够产生本发明的酶的细胞,包括细菌、酵母、真菌和高等真核细胞,包括植物。
通过培养能够产生本发明的酶的细菌宿主细胞的实例是革兰氏阳性细菌,如芽孢杆菌菌株,如枯草芽孢杆菌(B.subtilis)、地衣芽孢杆菌(B.licheniformis)、迟缓芽孢杆菌(B.lentus)、短芽孢杆菌(B.brevis)、嗜热脂肪芽孢杆菌(B.stearothermophilus)、嗜碱芽孢杆菌(B.alkalophilus)、解淀粉芽孢杆菌(B.amyloliquefaciens)、凝结芽孢杆菌(B.coagulans)、环状芽孢杆菌(B.circulans)、灿烂芽孢杆菌(B.lautus)、巨大芽孢杆菌(B.megaterium)或苏云金芽孢杆菌(B.thuringiensis)菌株,或链霉菌(streptomyces)菌株,如变铅青链霉菌(S.lividans)或鼠灰链霉菌(S.murinus),或革兰氏阴性细菌如大肠杆菌(Echerichiacoli)。
细菌的转化可以通过本身已知的方式用原生质体转化、电穿孔、接合或使用感受态细胞进行(参见Sambrook等人,见上)。
当在细菌如大肠杆菌中表达酶时,该酶一般可以以不溶性颗粒形式驻留于细胞质中(称作包涵体),或可以由细菌分泌序列引导到周质间隙。在前一种情况下,将细胞裂解,回收所述颗粒,变性,其后通过稀释变性试剂使酶重新折叠。后一种情况下,该酶可以通过以下操作从周质间隙中回收:如利用超声或渗透压休克破坏细胞,使所述的周质间隙的内容物释放,再回收该酶。
当在革兰氏阳性细菌如芽孢杆菌属或链霉菌属菌株中表达该酶时,该酶可以驻留于细胞质中,或由细菌分泌序列引导到胞外培养基中。在后一种情况下,该酶可按下述方法从培养基中回收。
产生枯草杆菌酶变体的方法
本发明提供了产生本发明的分离酶的方法,其中在允许该酶产生的条件下培养已用编码该酶的DNA序列转化的合适宿主细胞,以及从培养物中回收所得的酶。
当将含有编码该酶的DNA序列的表达载体转化入异源宿主细胞中时,即可使本发明的酶异源重组产生。
由此可以产生以不含同源杂质为特征的高度纯化的枯草杆菌酶组合物。
本文中,同源杂质是指来源于最初获得本发明的酶的同源细胞的任何杂质(例如除本发明的酶之外的其他多肽)。
用于培养转化的宿主细胞的培养基可以是任何适合于所讨论的宿主细胞生长的常规培养基。所表达的枯草杆菌酶可方便地分泌到培养基中,然后可以通过已知的方法回收,所述方法包括通过离心或过滤从培养基中分离细胞,再利用盐如硫酸铵沉淀培养基中的蛋白质成分,接下来进行层析,如离子交换层析、亲和层析等。
清洁和洗涤剂组合物
一般来说,清洁和洗涤剂组合物已在本领域中有充分描述,对合适的清洁和洗涤剂组合物的进一步描述可以参见WO 96/34946;WO97/07202;WO 95/30011。
洗涤剂组合物
枯草杆菌酶变体可以添加到洗涤剂组合物中而成为其中的组分。
例如,本发明的洗涤剂组合物可以制成手洗或机洗洗衣洗涤剂组合物,包括适用于预处理污染织物的洗衣添加剂组合物和漂洗时添加的织物柔软剂组合物,或制成用于普通家庭硬表面清洁操作的洗涤剂组合物,或配制以用于手洗或机洗洗盘操作。
在一特定的方面,本发明提供包含本发明枯草杆菌酶的洗涤添加剂。所述的洗涤添加剂和洗涤剂组合物可以包含一种或多种其它的酶如另一种蛋白酶、脂肪酶、角质酶、淀粉酶、糖酶、纤维素酶、果胶酶、甘露聚糖酶、阿拉伯糖酶、半乳聚糖酶、木聚糖酶、氧化酶、例如漆酶,和/或过氧化物酶。
一般来说,所选择的酶的性质应当与所选择的洗涤剂相适应(即最适pH,与其它酶和非酶组分具相容性等),且所述的酶应以有效量存在。
蛋白酶:合适的蛋白酶包括来自动物、植物或微生物的蛋白酶。来源于微生物的蛋白酶是优选的。也包括经化学修饰或蛋白质工程化的突变体。所述的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。碱性蛋白酶的实例为枯草杆菌蛋白酶,特别是来自芽孢杆菌的枯草杆菌蛋白酶,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(WO 89/06279中描述)。胰蛋白酶样蛋白酶的实例为胰蛋白酶(例如来自猪或牛的)和如WO 89/06270和WO 94/25583中所公开的镰孢霉属(Fusarium)的蛋白酶。
有用的蛋白酶的实例是如WO 92/19729,WO 98/20115,WO98/20116和WO 98/34946所描述的变体,特别是在一个或多个下述位置发生替换的变体:27,36,57,76,87,97,101,104,120,123,167,170,194,206,218,222,224,235和274。
优选的可商购的蛋白酶包括AlcalaseTM,SavinaseTM,PrimaseTM,DuralaseTM,EsperaseTM和KannaseTM(Novozymes A/S),MaxataseTM,MaxacalTM,MaxapemTM,ProperaseTM,PurafectTM,Purafect OxPTM,FN2TM和FN3TM(Genencor International Inc.)。
脂肪酶:合适的脂肪酶包括那些来源于细菌或真菌的脂肪酶。也包括经化学修饰或蛋白质工程化的突变体。有用的脂肪酶的实例包括来自腐质霉属(Humicola)(Thermomyces与之同物异名),如EP 258 068和EP 305 216中所述的来自H.lanuginosa(T.lanuginosus)的脂肪酶或如WO 96/13580中所述的来自H.insolens的脂肪酶;假单胞菌(Pseudomonas)脂肪酶,例如来自产碱假单胞菌(P.alcaligenes)或类产碱假单胞菌(P.pseudoalcaligenes)(EP 218 272),洋葱假单胞菌(P.cepacia)(EP 331 376),施氏假单胞菌(P.stutzeri)(GB 1,372,034),荧光假单胞菌(P.fluorescens),假单胞菌菌株SD 705(WO 95/06720和WO 96/27002),P.wisconsinensis(WO 96/12012)的脂肪酶;芽孢杆菌脂肪酶,例如来自枯草芽孢杆菌(Dartois等人(1993),生物化学与生物物理学学报(Biochemica et Biophysica Acta),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。
其它的实例为如WO 92/05249,WO 94/01541,EP 407 225,EP 260105,WO 95/35381,WO 96/00292,WO 95/30744,WO 94/25578,WO95/14783,WO 95/22615,WO 97/04079和WO 97/07202中所述的脂肪酶变体。
优选的可商购的脂肪酶包括LipolaseTM和LipolaseUltraTM(Novozymes A/S)。
淀粉酶:合适的淀粉酶(α和/或β)包括那些来源于细菌或真菌的淀粉酶。也包括经化学修饰或蛋白质工程化的突变体。淀粉酶包括例如来源于芽孢杆菌的α-淀粉酶,例如来自地衣芽孢杆菌的特定菌株,详细内容参见GB 1,296,839。
有用的淀粉酶的实例是如WO 94/02597,WO 94/18314,WO96/23873和WO 97/43424中所描述的变体,尤其是那些在一个或多个下列位置发生替换的变体:15,23,105,106,124,128,133,154,156,181,188,190,197,202,208,209,243,264,304,305,391,408和444。
可商购的淀粉酶是DuramylTM,TermamylTM,FungamylTM和BANTM(Novozymes A/S),RapidaseTM和PurastarTM(来自GenencorInternational Inc.)。
纤维素酶:合适的纤维素酶包括来源于细菌或真菌的纤维素酶。也包括其经化学修饰或蛋白质工程化的突变体。合适的纤维素酶包括来自芽孢杆菌属、假单胞菌属、腐质霉属(Humicola)、镰孢霉属(Fusarium)、草根霉属(Thielavia)、枝顶孢霉属(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WO89/09259中公开的由Humicola insolens、嗜热毁丝霉(Myceliophthorathermophila)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保护优势的碱性或中性纤维素酶。这样的纤维素酶的实例为如EP 0 495 257,EP 0 531 372,WO 96/11262,WO 96/29397,WO 98/08940中所公开的纤维素酶。其它的实例为如那些在WO 94/07998,EP 0 531 315,US 5,457,046,US 5,686,593,US5,763,254,WO 95/24471,WO 98/12307和PCT/DK98/00299中所公开的纤维素酶变体。
可商购的纤维素酶包括CelluzymeTM和CarezymeTM(NovozymesA/S),ClazinaseTM和Puradax HATM(Genencor International Inc.)和KAC-500(B)TM(Kao Corporation)。
过氧化物酶/氧化酶:合适的过氧化物酶/氧化酶包括那些来源于植物、细菌或真菌的过氧化物酶/氧化酶。也包括化学修饰或蛋白质工程化的突变体。有用的过氧化物酶的实例包括来自鬼伞属(Coprinus)(例如来自灰盖鬼伞(C.cinereus))的过氧化物酶和其变体,如WO93/24618,WO 95/10602和WO 98/15257所述的那些。
可商购的过氧化物酶包括GuardzymeTM(Novozymes A/S)。
这些洗涤剂酶可以通过添加含一种或多种酶的独立添加剂,或通过添加含所有这些酶的组合添加剂而被包含于洗涤剂组合物中。本发明的洗涤剂添加剂,也就是独立添加剂或组合添加剂可以制成例如颗粒状、液体、浆状等等。优选的洗涤剂添加剂制剂为颗粒(特别是无粉尘颗粒)、液体(特别是被稳定化的液体)或浆。
无粉尘颗粒可依照例如US 4,106,991和4,661,452所述进行生产并可任选地用本领域已知的方法加包衣。蜡质包衣材料的实例是平均分子量为1000到20000的聚(环氧乙烷)产物(聚乙二醇,PEG);含有16到50个环氧乙烷单位的乙氧基化的壬基酚;乙氧基化脂肪醇,其中,醇含12到20个碳原子且其中存在15到80个环氧乙烷单位;脂肪醇;脂肪酸;和脂肪酸的单-、二-和三酸甘油酯。GB 1483591中给出了适合于通过流化床技术应用的成膜包衣材料的实例。液体酶制剂可以例如依照现成的方法通过添加多元醇如丙二醇、糖或糖醇、乳酸或硼酸得以稳定。受保护的酶可依照EP 238,216中所公开的方法进行制备。
本发明的洗涤剂组合物可以是任何方便的形式,例如棒状、片状、粉末、颗粒、糊或液体。液体洗涤剂可以是含水的,一般含高达70%的水和0-30%的有机溶剂,或是不含水的。
所述的洗涤剂组合物一般包含一种或多种表面活性剂,它们可以是非离子(包括半极性的)和/或阴离子和/或阳离子和/或两性离子表面活性剂。所述的表面活性剂一般占到0.1%到60%的重量。当包含在所述洗涤剂中时,洗涤剂通常包含约1%到约40%的阴离子表面活性剂,如直链烷基苯磺酸盐、α-烯属磺酸盐、烷基硫酸盐(脂肪醇硫酸盐)、醇乙氧化硫酸盐、仲链烷磺酸盐、α-磺基脂肪酸甲酯、烷基-或链烯基琥珀酸或皂。
当包含在所述洗涤剂中时,通常含有约0.2%到约40%的非离子表面活性剂,例如醇乙氧基化物、壬基酚乙氧基化物、烷基多苷、烷基二甲基胺氧化物、乙氧基化脂肪酸单乙醇酰胺、脂肪酸单乙醇酰胺、多羟基烷基脂肪酰胺或葡糖胺的N-酰基N-烷基衍生物(“葡糖酰胺”)。
所述的洗涤剂可以包含0-65%的洗涤剂助洗剂或络合剂例如沸石、二磷酸盐、三磷酸盐、膦酸酯、碳酸盐、柠檬酸盐、次氮基三乙酸、乙二胺四乙酸、二亚乙基三胺五乙酸、烷基-或链烯基琥珀酸、可溶性硅酸盐或层叠式硅酸盐(例如购自Hoechst的SKS-6)。
所述的洗涤剂可以包含一种或多种聚合物。实例为羧甲基纤维素、聚(乙烯吡咯烷酮)、聚(乙二醇)、聚(乙烯醇)、聚(乙烯基吡啶-N-氧化物)、聚(乙烯基咪唑)、聚羧酸酯如聚丙烯酸酯、马来酸/丙烯酸共聚物和甲基丙烯酸月桂醇酯/丙烯酸共聚物。
所述的洗涤剂可以含有漂白体系,所述漂白体系可以包含H2O2来源如过硼酸盐或过碳酸盐,其可以与形成过酸的漂白活化剂如四乙酰基乙二胺或壬酰氧基苯磺酸盐相结合。可选择地,所述的漂白体系可以包含例如酰胺、二酰亚胺或砜类的过氧酸。
本发明的洗涤剂组合物中的酶可以通过诸如以下的常规稳定剂稳定:多元醇如丙二醇或丙三醇、糖或糖醇、乳酸、硼酸或硼酸衍生物如芳香硼酸酯或苯基硼酸衍生物如4-甲酰基苯基硼酸,且所述的组合物可以按例如WO 92/19709和WO 92/19708中所述配制。
所述的洗涤剂还可以包含其它的常规洗涤剂成分,例如织物调理剂包括粘土、发泡剂、泡沫抑制剂、防腐蚀剂、污垢悬浮剂、抗污物再沉淀剂、染料、杀菌剂、光学增白剂、增溶剂、晦暗抑制剂或香料。
目前认为在所述洗涤剂组合物中任何酶,特别是本发明的酶可以以相当于每升洗涤液中具有0.01-100mg酶蛋白质,优选每升洗涤液0.05-5mg酶蛋白质,特别优选每升洗涤液中具有0.1-1mg酶蛋白质的量加入。
此外,本发明的酶可以额外地添加到如WO 97/07202所公开的洗涤剂制剂中,该文献引入本文作为参考。
通过下述的实施例对本发明进行更详细说明,这些实施例不作为对本发明请求保护范围的任何限制。
在洗涤剂组合物中,简写成分的意义如下:
LAS: 直链C12烷基苯磺酸钠
TAS: 牛油烷基硫酸钠
XYAS: C1x-C1y烷基硫酸钠
SS: 式2-丁基辛酸的仲皂化表面活性剂
25EY: 与平均Y摩尔的环氧乙烷缩合的主要为C12-C15
的直链伯醇
45EY: 与平均Y摩尔的环氧乙烷缩合的主要为
C14-C15的直链伯醇
XYEZS: 每摩尔与平均Z摩尔的环氧乙烷缩合的C1x-C1y
烷基硫酸钠
非离子表面活性剂:平均乙氧基化程度为3.8而平均丙氧基化程度
为4.5的C13-C15混合的乙氧基化/丙氧基化脂
肪醇,由BASF GmbH售出,商品名为Plurafax
LF404
CFAA: C12-C14烷基N-甲基葡糖酰胺
TFAA: C16-C18烷基N-甲基葡糖酰胺
硅酸盐: 无定型硅酸钠(SiO2∶Na2O比率=2.0)
NaSKS-6: 分子式为δ-Na2Si2O5的晶状多层硅酸盐
碳酸盐: 无水碳酸钠
磷酸盐: 三聚磷酸钠
MA/AA: 1∶4马来酸/丙烯酸共聚物,平均分子量约为
80,000
聚丙烯酸: 平均分子量为8,000的聚丙烯酸均聚物,由
BASF GmbH销售,商品名为PA30
沸石A: 基本颗粒大小在1-10微米范围内的分子式为
Na12(AlO2SiO2)12·27H2O的水合硅酸铝钠
柠檬酸盐: 二水合柠檬酸三钠
Citric: 柠檬酸
过硼酸盐: 无水过硼酸钠一水合物漂白剂,经验式为
NaBO2·H2O2
PB4: 无水过硼酸钠四水合物
过碳酸盐: 经验式为2Na2CO3·3H2O2的无水过碳酸钠漂白剂
TAED: 四乙酰基乙二胺
CMC: 羧甲基纤维素钠
DETPMP 二亚乙基三胺五(亚甲基膦酸),由Monsanto出
售,商品名为Dequest 2060
PVP: 聚乙烯吡咯烷酮聚合物
EDDS: 钠盐形式的乙二胺-N,N′-二琥珀酸[S,S]异构
体
抑泡剂: 25%固体石蜡,熔点温度50℃,17%疏水二
氧化硅,58%石蜡油
粒状抑泡剂:12%硅氧烷/二氧化硅,18%十八烷醇,70%
粒状淀粉
硫酸盐: 无水硫酸钠
HMWPEO: 高分子量聚环氧乙烷
TAE 25: 乙氧基(25)化牛油醇
洗涤剂实施例I
本发明的粒状织物清洁组合物可如下制备:
直链C12烷基苯磺酸钠 6.5
硫酸钠 15.0
沸石A 26.0
次氮基三乙酸钠 5.0
酶 0.1
PVP 0.5
TAED 3.0
硼酸 4.0
过硼酸盐 18.0
酚磺酸盐 0.1
次要组分 补充至100%
洗涤剂实施例II
本发明的致密颗粒织物清洁组合物(密度800g/l)可依照下述制备:
45AS 8.0
25E3S 2.0
25E5 3.0
25E3 3.0
TFAA 2.5
沸石A 17.0
NaSKS-6 12.0
柠檬酸 3.0
碳酸盐 7.0
MA/AA 5.0
CMC 0.4
酶 0.1
TAED 6.0
过碳酸盐 22.0
EDDS 0.3
粒状抑泡剂 3.5
水/次要组分 补充至100%
洗涤剂实施例III
本发明的对洗涤有色织物特别有用的粒状织物清洁组合物可依照下述制备:
LAS 10.7 -
TAS 2.4 -
TFAA - 4.0
45AS 3.1 10.0
45E7 4.0 -
25E3S - 3.0
68E11 1.8 -
25E5 - 8.0
柠檬酸盐 15.0 7.0
碳酸盐 - 10.0
柠檬酸 2.5 3.0
沸石A 32.1 25.0
Na-SKS-6 - 9.0
MA/AA 5.0 5.0
DETPMP 0.2 0.8
酶 0.10 0.05
硅酸盐 2.5 -
硫酸盐 5.2 3.0
PVP 0.5 -
聚(4-乙烯基吡啶)-N-氧化物/ - 0.2
乙烯基咪唑和乙烯基吡咯烷
酮的共聚物
过硼酸盐 1.0 -
酚磺酸盐 0.2 -
水/次要组分 补充至100%
洗涤剂实施例IV
提供“洗涤过程中的柔顺处理”能力的本发明粒状织物清洁组合物可依照下述制备:
45AS - 10.0
LAS 7.6 -
68AS 1.3 -
45E7 4.0 -
25E3 - 5.0
椰油-烷基-二甲基羟基乙基氯化铵 1.4 1.0
柠檬酸盐 5.0 3.0
Na-SKS-6 - 11.0
沸石A 15.0 15.0
MA/AA 4.0 4.0
DETPMP 0.4 0.4
过硼酸盐 15.0 -
过碳酸盐 - 15.0
TAED 5.0 5.0
绿土粘土(smectite clay) 10.0 10.0
HMWPEO - 0.1
酶 0.10 0.05
硅酸盐 3.0 5.0
碳酸盐 10.0 10.0
粒状抑泡剂 1.0 4.0
CMC 0.2 0.1
水/次要组分 补充至100%
洗涤剂实施例V
本发明的强效型液体织物清洁组合物可依照下述制备:
酸形式的LAS - 25.0
柠檬酸 5.0 2.0
酸形式的25AS 8.0 -
酸形式的25AE2S 3.0 -
25AE7 8.0 -
CFAA 5 -
DETPMP 1.0 1.0
脂肪酸 8 -
油酸 - 1.0
乙醇 4.0 6.0
丙二醇 2.0 6.0
酶 0.10 0.05
椰油-烷基二甲基羟基乙基氯化铵 - 3.0
绿土粘土 - 5.0
PVP 2.0 -
水/次要组分 补充至100%
粉末自动洗盘组合物I
非离子表面活性剂 | 0.4-2.5% |
偏硅酸钠 | 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% |
四乙酰基乙二胺(TAED) | 1-4% |
硫酸钠 | 5-33% |
酶 | 0.0001-0.1% |
粉末自动洗盘组合物II
非离子表面活性剂(如醇乙氧基化物) | 1-2% |
二硅酸钠 | 2-30% |
碳酸钠 | 10-50% |
磷酸钠 | 0-5% |
柠檬酸三钠二水合物 | 9-30% |
乙酸次氮基三钠(NTA) | 0-20% |
过硼酸钠一水合物 | 5-10% |
四乙酰基乙二胺(TAED) | 1-2% |
聚丙烯酸酯聚合物(例如马来酸/丙烯酸共聚物) | 6-25% |
酶 | 0.0001-0.1% |
香料 | 0.1-0.5% |
水 | 5-10 |
粉末自动洗盘组合物III
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% |
过硼酸钠一水合物 | 0-15% |
四乙酰基乙二胺(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% |
粉末自动洗盘组合物IV
非离子表面活性剂 | 1-2% |
沸石MAP | 15-42% |
二硅酸钠 | 30-34% |
柠檬酸钠 | 0-12% |
碳酸钠 | 0-20% |
过硼酸钠一水合物 | 7-15% |
四乙酰基乙二胺(TAED) | 0-3% |
多聚物 | 0-4% |
马来酸/丙烯酸共聚物 | 0-5% |
有机膦酸酯 | 0-4% |
粘土 | 1-2% |
酶 | 0.0001-0.1% |
硫酸钠 | 平衡 |
粉末自动洗盘组合物V
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% |
单过硫酸盐(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五钠盐 | 0-2.5% |
酶 | 0.0001-0.1% |
硫酸钠,水 | 平衡 |
粉末和液体的具清洁表面活性剂系统的洗盘组合物VI
非离子表面活性剂 | 0-1.5% |
十八烷基二甲基胺N-氧化物二水合物 | 0-5% |
十八烷基二甲基胺N-氧化物二水合物和十六烷基二甲基胺N-氧化物二水合物的80∶20wt.C18/C16混合物 | 0-4% |
无水十八烷基双(羟乙基)胺N-氧化物和无水十六烷基双(羟乙基)胺N-氧化物的70∶30wt.C18/C16混合物 | 0-5% |
平均乙氧基化程度为3的C13-C15烷基乙氧基化硫酸盐 | 0-10% |
平均乙氧基化程度为3的C12-C15烷基乙氧基化硫酸盐 | 0-5% |
平均乙氧基化程度为12的C13-C15乙氧基化醇 | 0-5% |
平均乙氧基化程度为9的C12-C15乙氧基化醇的混合物 | 0-6.5% |
平均乙氧基化程度为30的C13-C15乙氧基化醇的混合物 | 0-4% |
二硅酸钠 | 0-33% |
三聚磷酸钠 | 0-46% |
柠檬酸钠 | 0-28% |
柠檬酸 | 0-29% |
碳酸钠 | 0-20% |
过硼酸钠一水合物 | 0-11.5% |
四乙酰基乙二胺(TAED) | 0-4% |
马来酸/丙烯酸共聚物 | 0-7.5% |
硫酸钠 | 0-12.5% |
酶 | 0.0001-0.1% |
非水性液态自动洗盘组合物VII
液体非离子表面活性剂(例如醇乙氧基化物) | 2.0-10.0% |
碱金属硅酸盐 | 3.0-15.0% |
碱金属磷酸盐 | 20.0-40.0% |
选自高级二元醇、聚二醇、多氧化物、二醇醚的液体载体 | 25.0-45.0% |
稳定剂(例如磷酸和C16-C18链烷醇的部分酯) | 0.5-7.0% |
泡沫抑制剂(例如硅氧烷) | 0-1.5% |
酶 | 0.0001-0.1% |
非水性液态洗盘组合物VIII
液体非离子表面活性剂(例如醇乙氧基化物) | 2.0-10.0% |
硅酸钠 | 3.0-15.0% |
碱金属碳酸盐 | 7.0-20.0% |
柠檬酸钠 | 0.0-1.5% |
稳定系统(例如细碎硅氧烷和低分子量二烷基聚二醇醚的混合物) | 0.5-7.0% |
低分子量聚丙烯酸聚合物 | 5.0-15.0% |
粘土凝胶增稠剂(例如斑脱土) | 0.0-10.0% |
羟丙基纤维素聚合物 | 0.0-0.6% |
酶 | 0.0001-0.1% |
选自高级二元醇、聚二醇、多氧化物和二醇醚的液体载体 | 平衡量 |
触变性液体自动洗盘组合物IX
C12-C14脂肪酸 | 0-0.5% |
嵌段共聚物表面活性剂 | 1.5-15.0% |
柠檬酸钠 | 0-12% |
三聚磷酸钠 | 0-15% |
碳酸钠 | 0-8% |
三硬脂酸铝 | 0-0.1% |
枯烯磺酸钠 | 0-1.7% |
聚丙烯酸酯增稠剂 | 1.32-2.5% |
聚丙烯酸钠 | 2.4-6.0% |
硼酸 | 0-4.0% |
甲酸钠 | 0-0.45% |
甲酸钙 | 0-0.2% |
正癸基二苯基氧化物二磺酸钠 | 0-4.0% |
一乙醇胺(MEA) | 0-1.86% |
氢氧化钠(50%) | 1.9-9.3% |
1,2-丙二醇 | 0-9.4% |
酶 | 0.0001-0.1% |
抑泡剂、染料、香料、水 | 平衡量 |
液体自动洗盘组合物X
醇乙氧基化物 | 0-20% |
脂肪酸酯磺酸盐 | 0-30% |
十二烷基硫酸钠 | 0-20% |
烷基多苷 | 0-21% |
油酸 | 0-10% |
二硅酸钠一水合物 | 18-33% |
柠檬酸钠二水合物 | 18-33% |
硬脂酸钠 | 0-2.5% |
过硼酸钠一水合物 | 0-13% |
四乙酰基乙二胺(TAED) | 0-8% |
马来酸/丙烯酸共聚物 | 4-8% |
酶 | 0.0001-0.1% |
含被保护的漂白颗粒的液体自动洗盘组合物XI
硅酸钠 | 5-10% |
焦磷酸四钾 | 15-25% |
三磷酸钠 | 0-2% |
碳酸钾 | 4-8% |
被保护的漂白颗粒,如氯 | 5-10% |
聚合增稠剂 | 0.7-1.5% |
氢氧化钾 | 0-2% |
酶 | 0.0001-0.1% |
水 | 平衡 |
XII:如I、II、III、IV、VI和X所述的自动洗盘组合物,其中,由过碳酸盐替代过硼酸盐。
XIII:如I-VI所述的自动洗盘组合物,其还含有锰催化剂。所述的锰催化剂可以是例如,在“用于低温漂白的高效锰催化剂”,自然,(1994),369,637-639中所述的化合物中的一种。
材料和方法
织物:
标准织物片获得自德国的WFK Testgewebe GmbH(Christenfeld 10,D-41379 Brüggen-Bracht)。
菌株:
枯草芽孢杆菌DN1885(Diderichsen等人,1990)。
迟缓芽孢杆菌309和147为迟缓芽孢杆菌的特定菌株,保藏在NCIB且保藏号为NCIB 10309和10147,在美国专利号3,723,250(此处引用作为参考)中进行了描述。
大肠杆菌MC 1000(M.J.Casadaban和S.N.Cohen(1980);分子生物学杂志(J.Mol.Biol.)138 179-207)通过常规方法成为r-,m+,也在美国专利申请系列号039,298中进行了描述。
质粒:
pJS3:包含编码枯草杆菌酶309的合成基因的大肠杆菌-枯草芽孢杆菌穿梭载体(Jacob Schiφdt等人在《蛋白质和肽通讯》(Protein and Peptideletters)3:39-44(1996)中描述)。
pSX222:枯草芽孢杆菌表达载体(描述见WO 96/34946)。
常规的分子生物学方法:
除非另外指出,DNA操作和转化使用分子生物学标准方法进行(Sambrook等人,(1989),分子克隆,实验室手册,冷泉港实验室,冷泉港,纽约;Ausubel,F.M.等人(编辑)“分子生物学最新方法”(“Currentprotocols in Molecular Biology”),John Wiley和Sons,1995;Harwood,C.R.和Cutting,S.M.(编辑)“用于芽孢杆菌的分子生物学方法”(“Molecular Biological Methods for Bacillus”),John Wiley和Sons,1990)。
DNA操作中使用的酶按照供应商的说明书进行操作。
用于DNA操作的酶:
除非另外指出,所有用于DNA操作的酶如限制性内切酶、连接酶等均来自New England Biolabs,Inc。
蛋白水解活性
在本发明的上下文中蛋白水解活性是用Kilo NOVO蛋白酶单位(KNPU)表示的。所述的活性相对于酶标准品(SAVINASE)而确定,所述的测定是基于在标准条件下蛋白水解酶对二甲基酪蛋白(DMC)溶液的消化进行的,所述的标准条件为50℃,pH 8.3,反应时间9分钟,测定时间3分钟。可向Novozymes A/S(Denmark)索取AF 220/1文件夹(folder),该文件夹包括在此作为参考。
GU是甘氨酸单位,定义为蛋白水解酶活性,即在标准条件下,N-乙酰基酪蛋白作为底物在40℃温育15分钟,产生相当于1毫摩尔甘氨酸的NH2-基团量。
也可以用PNA试验根据与可溶性底物琥珀酰丙氨酸-丙氨酸-脯氨酸-苯基-丙氨酸-对硝基苯酚的反应测定酶活性,参见美国油脂化学家学会会志(Journal of American Oil Chemists Society),Rothgeb,T.M.,Goodlander,B.D.,Garrison,P.H.和Smith,L.A.,(1988)中的描述。
发酵:
产生枯草杆菌酶的发酵是通过将含有100ml BPX培养基的500ml带挡板的三角瓶在回转式摇床上(300转/分钟)于30℃培养5天而进行的。
因此,为了制备如2升的肉汤,同时发酵了20个三角瓶。
培养基:
BPX培养基组成(每升)
马铃薯淀粉 100g
大麦粉 50g
大豆粉 20g
Na2HPO4×12H2O 9g
泊洛尼克(Pluronic) 0.1g
酪蛋白酸钠 10g
培养基中的淀粉用α-淀粉酶液化,并将培养基于120℃加热灭菌45分钟。灭菌后通过加入NaHCO3至0.1M,将培养基的pH调节至9。
实施例1
酶变体的构建和表达:
定点诱变:
包含特定插入/缺失/替换的本发明枯草杆菌酶309(savinase)定点变体通过DNA片段的常规克隆(Sambrook等人,分子克隆:实验室手册,第二版,冷泉港,1989)制备,其中所述DNA片段通过用包含所需突变的寡聚物进行PCR而产生(见下面)。
模板质粒DNA可以为pJS3(见下面),或为其包含枯草杆菌酶309的变体的类似物。
通过寡聚物定向诱变导入突变来构建变体。
将枯草杆菌酶309变体转化入大肠杆菌。从这些转化体的过夜培养物中纯化的DNA通过限制性内切酶消化、DNA片段的纯化、连接、枯草芽孢杆菌的转化,从而转化入枯草芽孢杆菌。枯草芽孢杆菌的转化如Dubnau等人,1971,分子生物学杂志,56,209-221页中所述进行。
定点诱变以在特定区域导入突变:
用于进行定点诱变的总体策略是:
合成诱变引物(寡核苷酸),该引物对应于被限定插入/缺失/替换的DNA碱基对分隔开的突变位点两翼的DNA序列。
接下来,所得的诱变引物与改变了的质粒pJS3(见上面)用于PCR反应。纯化得到的PCR片段,并在第二轮PCR反应中延伸,纯化所得的PCR产物并在第三轮PCR反应中延伸,然后用核酸内切酶消化,并克隆入大肠杆菌-枯草芽孢杆菌穿梭载体(见下面)。PCR反应在通常条件下进行。
利用熟知技术将质粒DNA转化至大肠杆菌中,并测序一个大肠杆菌菌落以验证所设计的突变。
按上述在亲本枯草杆菌蛋白酶309中构建下列变体,亲本枯草杆菌蛋白酶309的序列显示在图1中:
V11{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y},缺失,插入;
V30{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y},缺失,插入;
L31{R,N,D,Q,E,H,K,M,P,W,Y},缺失,插入;
D32{A,R,C,E,G,H,I,L,K,M,F,P,T,W,Y,V},插入;
G34{A,R,N,D,C,Q,E,H,I,L,K,M,F,S,T,W,Y,V},缺失;
G65{A,R,C,H,I,L,K,M,F,T,W,Y,V},缺失,插入;
T66{A,R,C,H,I,L,K,M,F,W,Y,V},缺失,插入;
H67{A,R,N,Q,G,I,K,M,F,P,S,T,W,Y,V};
V68{R,N,D,Q,E,G,H,I,L,K,F,P,S,T,W,Y},缺失,插入;
A69{R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;G83插入;
A85{R,N,D,Q,E,G,H,I,L,M,F,S,T,W,Y,V};
L90{A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V},缺失,插入;
V95{R,K,F,W,Y},缺失;
G110插入;
V121插入;
A122插入;
V150{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y},缺失,插入;
A152{R,N,D,Q,E,H,K,F,P,W,Y,V};
S153{R,N,D,Q,E,G,H,I,L,K,M,F,T,W,Y,V};
G154{A,R,H,I,L,M,F,W,Y,V};
*164{A,R,H,T,W};
*165{R,L,F,W,Y};
*166{W,Y},插入;
M175{R,N,D,Q,E,G,H,K,F,P,T,W,Y,V};
V177{R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y},缺失,插入;
G178{A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
T180{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V},缺失,插入;
V199{R,L,K,F,W,Y},缺失,插入;
A200{R,I,L,K,M,F,W,Y,V},缺失,插入;
P201{A,R,H,I,L,K,M,F,T,W,Y,V},缺失,插入;
G202{A,R,C,H,I,L,K,M,F,T,W,Y,V};
S207{A,R,N,C,Q,G,H,I,L,K,M,F,P,T,W,Y,V};
T220插入;
A223插入;
P225插入;
H226插入;
V227插入;
A228插入;
G229插入;
A230插入;
A231A,插入;
T253{A,R,N,D,Q,E,G,H,I,L,M,F,S,W,Y,V},缺失,插入;
G264{A,R,N,Q,H,I,L,M,F,S,T,W,Y,V},缺失,插入;
G266{A,R,N,D,C,Q,E,H,I,L,K,M,F,S,T,W,Y,V},缺失,插入。
为了纯化本发明的枯草杆菌酶变体,将包含本发明变体的枯草芽孢杆菌pJS3表达质粒转化入感受态枯草芽孢杆菌菌株,并如上所述进行发酵。
实施例2
酶变体的纯化:
该过程涉及到对用于在芽孢杆菌宿主细胞中产生本发明枯草杆菌酶的2升规模发酵物进行纯化。
用1升的烧杯于5000转/分钟离心约1.6升发酵肉汤35分钟。用10%醋酸将上清调节至pH 6.5,并通过Seitz Supra S100过滤板过滤。
用配备有Amicon S1Y10 UF柱体的Amicon CH2A UF单元浓缩滤液至约400ml。离心并过滤该UF浓缩物,然后室温下吸附于pH 7的杆菌肽亲和柱上。使用pH调节至7的25%异丙醇和1M氯化钠(在具有0.01M二甲基戊二酸、0.1M硼酸和0.002M氯化钙的缓冲液中)将蛋白酶从该杆菌肽柱上室温洗脱下来。
合并来自杆菌肽纯化步骤的具蛋白酶活性的级分,并加至用pH调节至6.5的含0.01M二甲基戊二酸、0.2M硼酸和0.002M氯化钙的缓冲液平衡过的750ml Sephadex G25柱(直径为5cm)上。
合并来自Sephadex G25柱的具蛋白水解活性的级分,并加至用pH调节至6.5的含0.01M二甲基戊二酸、0.2M硼酸和0.002M氯化钙的缓冲液平衡过的150ml CM Sepharose CL 6B阳离子交换柱(直径为5cm)上。
使用在2升同一缓冲液中的0-0.1M氯化钠线性梯度洗脱蛋白酶(对于枯草杆菌蛋白酶147,用0-0.2M氯化钠)。
在最后的纯化步骤中,合并来自CM Sepharose柱的含蛋白酶的级分,并在配备有GR81PP膜(来自Danish Sugar Factories Inc.)的Amicon超滤室中浓缩。
根据上述方法制备和纯化了如下的本发明变体:
BPN′(BASBPN):
I11V,L
L90I,V
M199*(缺失)Savinase(BLSAVI):V11I;V11I+I44V;V11I+L96LA;S9R+V11I+A15T+T22A;V11I+L96LA+A108C+A138C;V11I+N76D+A194P+A230V;V11I+V84I;V11I+V84I+K251R;V11I+V30I;V11I+V139L;V11I+V30I;V30I;V30I+V84I;V30I+V139L;S9R+A15T+T22A+V30I;V30I+V244R;V30I+K251R;V30I+V139L+Y167A+R170S+A194P+N218S;D32A;V;68A;V68A+S106A;V68A,+V139I;V68A,+A158V;V68A+V203A;V68A+V139L;A48T+V68A+P131M;V51A+V68A+S106T+A168G;V51A+V68A+S106T+A168G;N76D+M175ML+A194P+A230V;N76D+M175MI+A194P+A230V;
所有这些变体均表现出以上所述蛋白水解活性。
实施例3:
“模型洗涤剂洗涤性能试验”
为评估所选择的枯草杆菌酶变体在标准洗涤剂组合物中的洗涤性能,可以用下述的实验条件进行标准洗涤实验:
洗涤剂: 模型(model)洗涤剂
洗涤剂剂量 4.0g/l
pH 10.1
洗涤时间 20分钟
温度: 30℃
水硬度: 15°dH
酶浓度: 10nm(在洗涤剂溶液中)
测试系统: 10ml烧杯和搅拌棒
织物/体积: 5件织物(2.5cm)/50ml洗涤剂溶液
测试材料: 适宜的织物样本,如WFK10N(卵污
渍)
所述的模型洗涤剂的组成如下:
6.2% LAS(Nansa 80S)
2% C16-C18脂肪酸钠盐
4% 非离子表面活性剂(Plurafax LF404)
22% 沸P
10.5% Na2CO3
4% Na2Si2O5
2% 羧甲基纤维素(CMC)
6.8% 丙烯酸液体CP5 40%
20% 过硼酸钠(经验式NaBO2·H2O2)
0.2% EDTA
21% Na2SO4
水(平衡量)
通过添加HCl或NaOH将洗涤剂溶液的pH调节到10.1。通过向测试系统中添加CaCl2和MgCl2(Ca2+∶Mg2+=4∶1)将水硬度调节到15°dH。洗涤后织物用自来水冲洗并于空气中干燥。
对试验材料的反射能力(R变体)的测定是用Macbeth ColorEye 7000光度计(Macbeth,Division of Kollmorgen Instruments Corporation,德国)在460nm下进行的。测定按照制造商的方法进行。
为确定空白值,进行了不添加酶的类似洗涤实验。接下来如上述进行反射能力(R空白)的测定。
然后如上所述进行参考实验,其中测定亲本酶的洗涤性能。接下来,如上所述测定了反射能力(R亲本)。
洗涤性能用下述公式定义的性能因子(P)进行评估:
P=(R变体-R空白)-(R亲本-R空白)
=R变体-R亲本
序列表
<110>诺和酶股份有限公司
<120>枯草杆菌酶变体
<130>10183.204-WO
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<141>
<160>4
<170>PatentIn版本2.1
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<213>人工序列
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<223>人工序列的描述:专门用语的例子
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<213>解淀粉芽孢杆菌(B.amyloliquefaciens)(枯草杆菌蛋白酶BPN’)
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His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp
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Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala
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Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His
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Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly
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Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala
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<213>迟缓芽孢杆菌(Bacillus lentus)(枯草杆菌蛋白酶309)
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His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
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Claims (17)
1、包含至少一个或多个如下改变的枯草杆菌酶变体:
X11{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失,插入;
X30{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X31{R,N,D,Q,E,H,K,M,P,W,Y},缺失,插入;
X32{A,R,D,C,E,G,H,I,L,K,M,F,P,T,W,Y,V},插入;
X34{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失;
X65{A,R,C,G,H,I,L,K,M,F,T,W,Y,V},缺失,插入;
X66{A,R,C,H,I,L,K,M,F,T,W,Y,V},缺失,插入;
X67{A,R,N,Q,G,H,I,K,M,F,P,S,T,W,Y,V};
X68{R,N,D,Q,E,G,H,I,L,K,F,P,S,T,W,Y,V},缺失,插入;
X69{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X70G;
X71T;
X77N;
X83G,插入;
X84V;
X85{A,R,N,D,Q,E,G,H,I,L,M,F,S,T,W,Y,V};
X90{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X95{R,K,F,W,Y,V},缺失;
X107I
X110G,插入;
X121V,插入;
X122插入;
X123N;
X125S;
X150{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X152{A,R,N,D,Q,E,H,K,F,P,W,Y,V};
X153{R,N,D,Q,E,G,H,I,L,K,M,F,T,W,Y,V};
X154{A,R,G,H,I,L,M,F,W,Y,V};
X164{A,R,H,T,W};
X165{R,L,F,W,Y};
X166{W,Y},插入;
X175{R,N,D,Q,E,G,H,K,M,F,P,T,W,Y,V};
X177{R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X178{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X180{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V},缺失,插入;
X199{R,L,K,F,W,Y,V},缺失,插入;
X200{A,R,I,L,K,M,F,W,Y,V},缺失,插入;
X201{A,R,H,I,L,K,M,F,P,T,W,Y,V},缺失,插入;
X202{A,R,C,G,H,I,L,K,M,F,T,W,Y,V};
X207{A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V};
X220T,插入;
X223A,插入;
X225P,插入;
X226H,插入;
X227V,插入;
X228A,插入;
X229G,插入;
X230插入;
X231A,插入;
X253{A,R,N,D,Q,E,G,H,I,L,M,F,S,T,W,Y,V},缺失,插入;
X264{A,R,N,Q,G,H,I,L,M,F,S,T,W,Y,V},缺失,插入;
X266{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V},缺失,插入;
其中,
(a)所述改变各自独立地为
(i)以不同的氨基酸替换占据所述位置的氨基酸,
(ii)缺失占据所述位置的氨基酸,或
(iii)在占据所述位置的氨基酸的下游插入至少一个额外氨基酸,
(b)所述变体具有蛋白酶活性,且
(c)每个位置都对应于图1所示枯草杆菌蛋白酶BPN,氨基酸序列的位置。
2、根据权利要求1的变体,其中亲本枯草杆菌酶属于I-S1亚组。
3、根据权利要求2的变体,其中亲本枯草杆菌酶选自:BASBPN、BSS168、BSSDY和BLSCAR,或它们的保留了I-S1亚组特征的功能性变体。
4、根据权利要求1的变体,其中亲本枯草杆菌酶属于I-S2亚组。
5、根据权利要求4的变体,其中亲本枯草杆菌酶选自:BLS147、BLSAVI(SaVinase)、BAPB92、TVTHER和BYSYAB,或它们的保留了I-S2亚组特征的功能性变体,优选BLSAVI。
6、根据权利要求4或5之任一项的变体,其中所述变体为:
V11I;V11I+I44V;V11I+L96LA;S9R+V11I+A15T+T22A;V11I+L96LA+A108C+A138C;V11I+N76D+A194P+A230V;V11I+V84I;V11I+V84I+K251R;V11I+V30I;V11I+V139L;V11I+V30I;V30I;V30I+V84I;V30I+V139L;S9R+A15T+T22A+V30I;V30I+V244R;V30I+K251R;V30I+V139L+Y167A+R170S+A194P+N218S;D32A;V68A;V68A+S106A;V68A,+V139I;V68A,+A158V;V68A+V203A;V68A+V139L;A48T+V68A+P131M;V51A+V68A+S106T+A168G;V51A+V68A+S106T+A168G;N76D+M175ML+A194P+A230V;N76D+M175MI+A194P+A230V.
7、权利要求4-6之任一项的变体,其中所述变体还包含至少一个在如下位置处的修饰:27、36、56、76、87、95、96、97、98、99、100、101、102、103、104、120、123、159、167、170、206、218、222、224、232、235、236、245、248、252或274(BASBPN编号)。
8、权利要求7的变体,其中,所述变体还包含如下修饰:S101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K。
9、编码如权利要求1-8之任一项所定义的枯草杆菌酶变体的分离的DNA序列。
10、包含权利要求9的分离的DNA序列的表达载体。
11、用权利要求10的表达载体转化的微生物宿主细胞。
12、根据权利要求11的微生物宿主细胞,其为细菌,优选地为芽孢杆菌,尤其为迟缓芽孢杆菌。
13、根据权利要求11的微生物宿主细胞,其为真菌或酵母,优选地为丝状真菌,尤其为曲霉。
14、产生如权利要求1-8之任一项所定义的枯草杆菌酶变体的方法,其中将权利要求11-13之任一项的宿主细胞在利于该变体表达和分泌的条件下培养,并回收该变体。
15、清洁或洗涤剂组合物,优选地洗衣或洗盘组合物,其包含如权利要求1-8之任一项所定义的变体。
16、根据权利要求15的组合物,其还包含纤维素酶、脂肪酶、角质酶、氧化还原酶、其它蛋白酶、淀粉酶或它们的混合物。
17、如权利要求1-8之任一项定义的变体在洗衣和/或洗盘洗涤剂中的用途。
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-
2001
- 2001-07-12 DK DK200101090A patent/DK200101090A/da not_active Application Discontinuation
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2002
- 2002-07-11 WO PCT/DK2002/000485 patent/WO2003006602A2/en active IP Right Grant
- 2002-07-11 DE DE60229136T patent/DE60229136D1/de not_active Expired - Lifetime
- 2002-07-11 AT AT02745188T patent/ATE409744T1/de not_active IP Right Cessation
- 2002-07-11 US US10/481,723 patent/US20040197894A1/en not_active Abandoned
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- 2002-07-11 CN CNA028137477A patent/CN1537163A/zh active Pending
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- 2008-02-22 US US12/035,766 patent/US20080167213A1/en not_active Abandoned
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- 2013-09-24 US US14/035,241 patent/US20140018283A1/en not_active Abandoned
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- 2014-09-30 US US14/502,234 patent/US9528100B2/en not_active Expired - Fee Related
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108012544A (zh) * | 2015-06-18 | 2018-05-08 | 诺维信公司 | 枯草杆菌酶变体以及编码它们的多核苷酸 |
WO2017107980A1 (en) * | 2015-12-23 | 2017-06-29 | Novozymes A/S | Methods for enhancing the dewaterability of sludge with enzyme treatment |
CN108713002A (zh) * | 2015-12-23 | 2018-10-26 | 诺维信公司 | 用于用酶处理增强污泥的脱水性能的方法 |
WO2022134236A1 (zh) * | 2020-12-21 | 2022-06-30 | 天津科技大学 | 一种碱性蛋白酶突变体及其基因、工程菌、制备方法和应用 |
US11655464B2 (en) | 2020-12-21 | 2023-05-23 | Tianjin University Of Science And Technology | Alkaline protease mutant, and gene, engineered strain, preparation method and application thereof |
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US20140018283A1 (en) | 2014-01-16 |
EP1409659A2 (en) | 2004-04-21 |
US20170096654A1 (en) | 2017-04-06 |
AU2002316816A1 (en) | 2003-01-29 |
US20110152155A1 (en) | 2011-06-23 |
WO2003006602A3 (en) | 2003-11-06 |
DK200101090A (da) | 2001-08-16 |
US20080167213A1 (en) | 2008-07-10 |
WO2003006602A2 (en) | 2003-01-23 |
US20130225466A1 (en) | 2013-08-29 |
JP2004537994A (ja) | 2004-12-24 |
NZ530237A (en) | 2006-05-26 |
ZA200309666B (en) | 2004-08-03 |
US20040197894A1 (en) | 2004-10-07 |
DE60229136D1 (de) | 2008-11-13 |
EP1409659B1 (en) | 2008-10-01 |
US8569035B2 (en) | 2013-10-29 |
NZ541095A (en) | 2008-02-29 |
JP4383858B2 (ja) | 2009-12-16 |
US10351837B2 (en) | 2019-07-16 |
US9528100B2 (en) | 2016-12-27 |
US20150024990A1 (en) | 2015-01-22 |
ATE409744T1 (de) | 2008-10-15 |
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