CN101857858A - 具有改变特性的α-淀粉酶突变体 - Google Patents
具有改变特性的α-淀粉酶突变体 Download PDFInfo
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Abstract
本发明涉及具有改变特性的α-淀粉酶突变体，具体地涉及亲本Termamyl样α-淀粉酶的变体(突变体)，其中相对于亲本α-淀粉酶该变体具有α-淀粉酶的活性并且显示改变的稳定性，尤其是在高温和/或低pH条件，和/或低Ca2+浓度下。
Description
本发明申请是基于申请日为2001年7月12日，申请号为01815680.0(国际申请号为PCT/DK01/00488)，发明名称为“具有改变特性的α-淀粉酶突变体”的专利申请的分案申请。
发明领域
本发明涉及亲本Termamyl样(Termamyl-like)α-淀粉酶的变体(突变体)，变体具有α-淀粉酶的活性，并且相对于所述亲本α-淀粉酶其显示出下述性质中至少一种性质的改变：例如，高温和/或低pH条件，尤其是低钙浓度下的稳定性。本发明的变体适合于淀粉转化，制备乙醇，洗衣冲洗剂，餐具冲洗剂，硬表面清洗剂，纺织品退浆，和/或制备增甜剂(sweetner)。
背景技术
α(-淀粉酶(α-1，4-葡聚糖-4-葡聚糖水解酶，EC3.2.1.1)构成这样一组酶，它们催化淀粉和其他线性及分枝1，4-葡萄糖苷寡-糖和多糖的水解。
发明简述
本发明的目的是提供亲本Termamyl样α-淀粉酶的变体，与相应的亲本α-淀粉酶即未成熟的α-淀粉酶相比，具有α-淀粉酶的活性，并且相对于所述亲本α-淀粉酶其显示出下述性质中至少一种性质的改变：稳定性，例如在高温和/或低pH条件，尤其是低钙浓度下。
本发明具体涉及如下方面：
1)一种亲本Termamyl样α-淀粉酶的变体，所述变体包含一个或多个选自下述位点的改变：
49，60，104，132，161，170，176，179，180，181，183，200，203，204，207，212，237，239，250，280，298，318，374，385，393，402，406，427，430，440，444，447，482，
其中
(a)所述改变分别是
(i)占据该位点的氨基酸下游的氨基酸插入，
(ii)占据该位点的氨基酸的缺失，或
(iii)以不同的氨基酸取代占据该位点的氨基酸，
(b)变体具有α-淀粉酶活性，和(c)每个位点相应于具有SEQ ID NO：8所示的氨基酸序列的亲本Termamyl样α-淀粉酶的氨基酸序列的位点。
2)项1所述的变体，其中该变体具有一个或多个下述的突变：
T49I；D60N；N104D；E132A，V，P；D161N；K170Q；K176R；G179N；K180T；A181N；D183N；D200N；X203Y；D204S；D207V，E，L，G；X212I；K237P；S239W；E250G，F；N280S；X298Q；L318M；Q374R；E385V；Q393R；Y402F；H406L，W；L427I D430N；V440A；N444R，K；E447Q，K；Q482K，使用SEQ ID NO：8的编号。
3)项1或2所述的变体，其中该变体具有下述的突变：
K170Q+D207V+N280S；E132A+D207V；D207E+E250G+H406L+L427I；D207V+L318M；D60N+D207V+L318M；T49I+E132V+V440A；T49I+K176R+D207V+Y402F；Q374R+E385V+Q393R；N190F+A209V+Q264S；G48A+T49I+G107A+I201F；T49I+G107A+I201F；G48A+T49I+I201F；G48A+T49I+G107A；T49I+I201F；T49I+G107A；G48A+T49I；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K；
D161N+A181N+D 183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K；
D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D43ON；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H 406W+D430N；
H406W+D43ON；N444K+E447Q+Q482K；E447Q+Q482K；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W；
H406W+D430N；N444K+E447K+Q482K；E447K+Q482K；
N104D+D161N+A181N+D183N+D200N+D204S+K237P+S239W；
N104D+D161N+A181N+D183N+D200N+D204S+K237P；
N104D+D161N+A181N+D183N+D200N+D204S；
D161N+A181N+D183N+D200N+D204S+K237P+S239W；
D161N+A181N+D183N+D200N+D204S+K237P；
D161N+A181N+D183N+D200N+D204S；K237P+S239W，使用SEQ ID NO：8的编号。
4)项1-3中任一项所述的变体，其中亲本Termamyl样α-淀粉酶衍生于地衣芽孢杆菌(SEQ ID NO：8)，解淀粉芽孢杆菌(SEQ ID NO：10)，嗜热脂肪芽孢杆菌(SEQ ID NO：6)的菌株。
5)项1-4中任一项所述的变体，其中亲本Termamyl样α-淀粉酶是：LE174；LE174+G48A+T49I+G107A+I201F；LE174+M197L；或LE174+G48A+T49I+G107A+M197L+I201F。
6)项1中所述的变体，其中该变体在下述一或多个位点发生突变：T51I；D62N；N106D；D134A，V，P；D163N；X172Q；K179R；G184N；K185T；A186N；D188N；D205N；M208Y；D209S；X212V，E，L，G；L217I，K242P，S244W，N255G，F，N285S，S303Q，X323M；D387V，N395R；Y404F；H408L，W；X429I；D432N；V442A；X446R，K；X449Q，K；X484K，使用SEQ ID NO：4(SP722)的编号。
7)项1或6中所述的变体，该变体具有下述的突变：E212V+N285S；D134A+E212V；N255G+H408L+X429I；E212V+X323M；D62N+E212V+X323M；T51I+D 134V+V442A；T51I+K179R+E212V+Y404F；D387V+N395R；N195F+X212V+K269S，使用SEQ ID NO：4(SP722)的编号。
8)项1-7中任一项所述的变体，其中亲本Termamyl样α-淀粉酶选自：SP690(SEQ ID NO：2)；SP722(SEQ ID NO：4；AA560(SEQ ID NO：12)；#707α-淀粉酶(SEQ ID NO：13)；KSM-AP1378。
9)项1-8中任一项所述的变体，其中亲本Termamyl样α-淀粉酶是下述任何一种：SP722+D183*+G184*；SP722+D183*+G184*+N195F；SP722+D183*+G184*+M202L；SP722+D183*+G184*+N195F+M202L；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。
10)项1-9中任一项所述的变体，其中亲本Termamyl样α-淀粉酶具有与SEQ ID NO：8有至少60％相同的氨基酸序列，优选70％，更优选至少80％，甚至更优选至少约90％，甚至更优选至少95％，甚至更优选至少97％，甚至更优选至少99％。
11)项1-10中任一项所述的变体，其中亲本Termamyl样α-淀粉酶由核酸序列编码，该核酸序列与SEQ ID NO：7的核酸序列可在低严谨度条件下，优选中度严谨度条件下，更优选高严谨度条件下杂交。
12)项1-11中任一项所述的变体，其中变体具有改变的稳定性，尤其在70-120℃高温下和/或pH 4-6的低pH下。
13)一种DNA构建体，包含编码项1-12中任一项所述的α-淀粉酶变体的DNA序列。
14)一种重组表达载体，其携带项13的DNA构建体。
15)一种由项13的DNA构建体或项14的载体转化的细胞。
16)项15的细胞，其是微生物，优选是细菌或真菌。
17)项16的细胞，该细胞是革兰氏阳性细菌，比如枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌、短芽孢杆菌、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌、解淀粉芽孢杆菌、凝结芽孢杆菌、环状芽孢杆菌、灿烂芽孢杆菌、或苏云金芽孢杆菌。
18)一种组合物，其包含项1-12中任一项所述的α-淀粉酶变体。
19)项18的组合物，进一步包括嗜热脂肪芽孢杆菌(BSG)α-淀粉酶，尤其是SP961，尤其比例为1∶10-10∶1，优选1∶2。
20)项18或19的组合物，其中组合物进一步包括葡糖淀粉酶，支链淀粉酶和/或植酸酶。
21)一种洗涤剂组合物，包含项1-12中任一项所述的α-淀粉酶变体。
22)项21的洗涤剂组合物，还包含另一种酶，例如蛋白酶、脂酶、过氧化物酶，另一种淀粉分解酶、葡糖淀粉酶、麦芽糖淀粉酶、CGTase、甘露聚糖酶、角质酶、漆酶、和/或纤维素酶。
23)项1-12中任一项所述α-淀粉酶变体或项18-20中任一项所述的组合物在淀粉液化中的用途。
24)项1-12中任一项所述α-淀粉酶变体或项18-20中任一项所述组合物在乙醇生产中的用途。
25)项1-12中任一项所述α-淀粉酶变体或项18-20中任一项所述组合物在冲洗和/或餐具清洗中的用途。
26)项1-12中任一项所述α-淀粉酶变体或项18-20中任一项所述组合物在纺织品退浆中的用途。
命名法则
在本说明书和权利要求书中，使用了氨基酸残基的常规单字母和三字母编码。为了便于参考，采用以下命名法则来描述本发明的α-淀粉酶变体：
原氨基酸：位点：取代氨基酸
根据这一命名原则，例如在第30位丙氨酸到天冬酰胺的取代应表示为：
Ala30Asn 或A30N
在同一位点的丙氨酸缺失表示为：
Ala30*或A30*
添加的氨基酸残基，比如赖氨酸插入表示为：
Ala30AlaLys或A30AK
连续的氨基酸残基片段，比如30-33位氨基酸残基缺失表示为(30-33)*或Δ(A30-N33)。
当特定α-淀粉酶与其他α-淀粉酶相比含有一个“缺失”，并且在该位点做了一个插入，这种情况表示为(对于36位插入了一个天冬氨酸)：
*36Asp或*36D
多重突变用加号隔开，即：
Ala30Asp+Glu34Ser或A30N+E34S
代表在30和34位分别将丙氨酸和谷氨酸取代为天冬氨酸和丝氨酸。
当在一个给定位点可以插入1或多个可选择的氨基酸残基时，将其表示为：
A30N，E或
A30N或A30E
另外，当本文中确定了一个适合进行修饰的位点而没有建议具体的修饰方式，应当理解为可以用任何氨基酸残基来取代该位点的氨基酸残基。因此，例如，如果提及30位的丙氨酸修饰，但没有具体化，应当理解可以将该丙氨酸缺失或用任何其他氨基酸来取代它，即下列之一的氨基酸：R、N、D、A、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y、V。
而且，“A30X”表示下述的任何一个取代：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。
若所用编号的亲本酶已经含有在该位置取代的所研究氨基酸残基，则用下面的命名：
这种情况中″X30N″或″X30N，V″其中例如一或N或V表示野生型。
因此，其它相应的亲本酶在位点30被取代为″Asn″或″Val″。
附图简述
图1是5个亲本Termamyl样α-淀粉酶的氨基酸序列对比。最左端的数字代表下列各氨基酸序列：
1：SEQ ID NO：4(SP722)
2：SEQ ID NO：2(SP690)
3：SEQ ID NO：10(BAN)
4：SEQ ID NO：8(BLA)
5：SEQ ID NO：6(BSG)
发明详述
本发明的目的是提供Termamyl样α-淀粉酶，该变体具有α-淀粉酶的活性，并且其在高温和/或低pH条件，尤其是低钙浓度下显示改变的稳定性。
Termamyl样α-淀粉酶
许多由芽孢杆菌类制备的α-淀粉酶在氨基酸水平上具有高度同源性(同一性)。下表显示了许多已知的芽孢杆菌属α-淀粉酶的同一性：
表1
百分比
同一性
707 AP137 BAN BSG SP690 SP722 AA560 Termamy
8 1
707 100.0 86.4 66.9 66.5 87.6 86.2 95.5 68.1
AP1378 86.4 100.0 67.1 68.1 95.1 86.6 86.0 69.4
BAN 66.9 67.1 100.0 65.6 67.1 68.8 66.9 80.7
BSG 66.5 68.1 65.6 100.0 67.9 67.1 66.3 65.4
SP690 87.6 95.1 67.1 67.9 100.0 87.2 87.0 69.2
SP722 86.2 86.6 68.8 67.1 87.2 100.0 86.8 70.8
AA560 95.5 86.0 66.9 66.3 87.0 86.8 100.0 68.3
Terma- 68.1 69.4 80.7 65.4 69.2 70.8 68.3 100.0
myl
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(商品名为TermamylTM)与包含SEQ ID NO：10所示氨基酸序列的解淀粉芽孢杆菌α-淀粉酶大约81％同源，与包含SEQ ID NO：6所示氨基酸序列的嗜热脂肪芽孢杆菌α-淀粉酶(BSG)大约65％同源。另外同源的α-淀粉酶包括公开于WO 95/26397的SP690和SP722，并进一步分别在SEQ ID NO：2和SEQ ID NO：4中描述。本文中其它的淀粉酶有衍生于芽孢杆菌属的AA560α-淀粉酶并表示为SEQ ID NO：12，以及在SEQ ID NO：13中显示并由Tsukamoto等(生物化学和生物物理研究通讯，151(1988)，25-31页)描述过的衍生于芽孢杆菌的#707α-淀粉酶。
KSM AP1378α-淀粉酶公开于WO 97/00324(自KAO Corporation)。
其他同源α-淀粉酶包括EP0252666中描述的地衣芽孢杆菌菌株(ATCC27811)产生的α-淀粉酶，以及WO91/00353和WO94/18314中鉴定到的α-淀粉酶。在产品OptithermTM和TakathermTM(Solvay有售)、MaxamylTM(Gist-brocades/Genencor有售)、Spezym AATM和Spezym Delta AATM(Genencor有售)以及KeistaseTM(Daiwa有售)，Dex lo，GC 521(Genencor有售)和Ultraphlow(得自Enzyme Biosystems)中含有其他一些商品Termamyl样α-淀粉酶。
由于发现这些α-淀粉酶之间有极大的同源性，可以认为它们属于同类α-淀粉酶，即“Termamyl样α-淀粉酶”类。
因此，在本文中，术语“Termamyl样α-淀粉酶”意指一种α-淀粉酶，具体是芽孢杆菌属α-淀粉酶，它在氨基酸水平上与TermamylTM(即具有文中SEQ ID NO：8所示氨基酸序列的地衣芽孢杆菌α-淀粉酶)有极大的同源性。
换句话说，以下所有具有SEQ ID NO：2、4、6、8、10、12或13所示氨基酸序列的α-淀粉酶都被认为是“Termamyl样α-淀粉酶”。其他的Termamyl样α-淀粉酶是这样一些α-淀粉酶：i)与SEQ ID NO：2、4、6、8、10、12和13所示氨基酸序列中的至少一个有至少60％、比如至少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(同一性)，和/或由这样一种DNA序列编码，这些序列能与编码以上的α-淀粉酶的DNA序列进行杂交，其很明显于SEQ ID NO：1、3、5、7、9和本说明书(分别编码此处SEQ ID NO：2、4、6、8、10、12和13所示的氨基酸序列)。
同源性
同源性可以测定为两序列间表示第一序列衍生自第二序列的同一性的程度。同源性可以通过本领域已知的计算机程序诸如GCG程序包的GAP程序(上述)。因此，Gap GCGv8可用用于同一性的默认得分矩阵(scoringmatrix)和下述默认参数：对于核酸序列比较，分别是GAP生成罚分为5.0，GAP延伸罚分为0.3，以及对于蛋白质序列比较分别是GAP生成罚分为3.0，GAP延伸罚分为0.1。GAP用Needleman和Wunsch，(1970)，J.Mol.Biol.48，p.443-453的方法进行序列对比并推断出同一性。
可以利用Termamyl(SEQ ID NO：8)和，例如另一α-淀粉酶之间的结构对比来鉴定其他Termamyl样α-淀粉酶中的等同/相应位点。获得所述结构对比的一个方法是利用GCG程序包中的Pile Up程序，该程序中GAP罚分采用默认值，即GAP生成罚分为3.0，GAP延伸罚分为0.1。其他结构对比方法包括疏水簇分析(Gaboriaud等，(1987)，FEBS快报224，149-155页)和反相成丝技术(reverse threading)(Huber T，Torda，AE，蛋白质科学，7卷，1期：142-149(1998))。
杂交
基于所研究α-淀粉酶的全部或部分核苷酸或氨基酸序列，适当地制备上述Termamyl样α-淀粉酶特征中所用到的寡核苷酸探针。
检测杂交的适宜条件包括在5×SSC中预浸泡，于40℃在含有20％甲酰胺、5×Denhardt′s溶液、50mM磷酸钠(pH6.8)以及50mg超声变性小牛胸腺DNA的溶液中预杂交1小时，然后于40℃在补充有100mM ATP的同一溶液中杂交18小时，随之将滤膜洗3次，每次在2×SSC、0.2％SDS中于40℃(低严谨性)，优选50℃(中等严谨性)，更优选65℃(高严谨性)，还要优选的是75℃(极高严谨性)洗30分钟。有关杂交方法的其他细节可见Sambrook等，分子克隆：实验手册，第2版，Cold Spring Harbor，1989。
在本文中，“衍生于”不仅是指由所研究的微生物菌株产生或能产生的α-淀粉酶，也指分离自该菌株的DNA序列所编码的、并在用所述DNA序列转化的宿主微生物中产生的α-淀粉酶。最后，该术语意指这样的α-淀粉酶，它由合成的和/或cDNA来源的DNA序列编码并且具备所述α-淀粉酶的鉴定特征。该术语还用来表示亲本α-淀粉酶可以是天然存在的α-淀粉酶的变异体，即由天然存在的α-淀粉酶的1或多个氨基酸残基经过修饰(插入、取代、缺失)所产生的变体。
亲本Termamyl样α-淀粉酶
本发明所有上述的定义的Termamyl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-淀粉酶衍生于地衣芽孢杆菌，例如，上述所指的其中之一诸如具有如SEQ ID NO：8所示氨基酸序列的地衣芽孢杆菌α-淀粉酶。
亲本杂种α-淀粉酶
亲本α-淀粉酶(即主链α-淀粉酶)可以是一种杂种α-淀粉酶，即包含组合在一起的衍生于至少两个α-淀粉酶的部分氨基酸序列。
亲本杂种α-淀粉酶可以是这样的酶，在氨基酸同源性(同一性)和/或DNA杂交(如上所述)的基础上可以确定它属于Termamyl样α-淀粉酶家族。在这种情况中，杂种α-淀粉酶通常包含Termamyl样α-淀粉酶的至少一部分和1或多个其他α-淀粉酶的1个或多个部分，后者选自微生物(细菌或真菌)和/或哺乳动物来源的Termamyl样α-淀粉酶或非Termamyl样α-淀粉酶。
因此，亲本杂种α-淀粉酶可以包括来源于至少两个Termamyl样α-淀粉酶，或者来源于至少一个Termamyl样α-淀粉酶和至少一个非Termamyl样细菌α-淀粉酶，或者来源于至少一个Termamyl样和至少一个真菌α-淀粉酶的部分氨基酸序列的组合。部分氨基酸序列衍生自的Termamyl样α-淀粉酶可以是，例如文中提到的那些特定Termamyl样α-淀粉酶中的任何一个。
例如，亲本α-淀粉酶可以包括衍生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-淀粉酶可以包括地衣芽孢杆菌α-淀粉酶的C-末端部分的至少430个氨基酸残基，还可以，例如，包括a)与具有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-末端氨基酸残基相对应的氨基酸片段和与具有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-末端氨基酸残基相对应的氨基酸片段，或者与Termamyl序列(即SEQ ID NO：8所示的地衣芽孢杆菌α-淀粉酶)相同的杂种Termamyl样α-淀粉酶，除了N-末端(成熟蛋白质)的35个氨基酸残基被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(成熟蛋白质)取代；或者b)与具有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-末端氨基酸残基相对应的氨基酸片段和与具有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-末端氨基酸残基相对应的氨基酸片段。
另一合适的亲本杂种α-淀粉酶是先前在WO96/23874(Novo Nordisk申请)中描述的酶，该酶由BAN(解淀粉芽孢杆菌α-淀粉酶)的N-末端(成熟蛋白质的1-300位氨基酸)和Termamyl的C-末端(成熟蛋白质的301-483位氨基酸)构成。
本发明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α-淀粉酶的杂种。特别是，亲本Termamyl样α-淀粉酶的杂种可以是杂种α-淀粉酶，该α-淀粉酶包含SEQ ID 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所示的解淀粉芽孢杆菌α-淀粉酶的37个N-末端氨基酸残基，其进一步适合含有下面的突变：H156Y+A181T+N190F+A209V+Q264S(使用SEQ ID NO：8中的编号)。后面所提到的杂种用在下述的实施例中，并指定为LE174。
其它需要特定考虑的亲本α-淀粉酶包括具有较少突变的LE174，即恰上述的具有下面突变的杂种：A181T+N190F+A209V+Q264S；N190F+A209V+Q264S；A209V+Q264S；Q264S；H 156Y+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+N19OF+A209V。这些杂交也被认为是本发明的部分。
在优选的实施方案中，亲本Termamyl样α-淀粉酶是LE174，SP722，或AA560包括任何LE174+G48A+T49I+G107A+I201F；LE174+M197L；LE174+G48A+T49I+G107A+M197L+I201F，or SP722+D183*+G184*；SP722+D183*+G184*+N195F；SP722+D183*+G184*+M202L；SP722+D183*+G184*+N195F+M202L；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。
其它要考虑的亲本α-淀粉酶包括LE429，其是具有I201F的附加取代的LE174。基于本发明LE335是这样的α-淀粉酶，其与LE429相比，在T49I+G107A中有附加的取代；LE399是LE335+G48A，即LE174，具有G48A+T49I+G107A+I201F。
改变特性
以下讨论存在于本发明的变体中的突变与可能由该突变导致的希望的性质变化(相对亲本Termamyl样α-淀粉酶的这些性质)之间的关系。
如上所述本发明涉及具有改变特性(上述)的亲本Termamyl样α-淀粉
酶，尤其是在高温和/或在低pH下，特别是低钙浓度下。
本发明中“高温”指的是70-120℃，优选80-100℃，尤其是85-95℃。
本发明中“低pH”指的是pH的范围为4-6，优选4.2-5.5，尤其是4.5-5。
本发明中“高pH”指的是pH的范围为8-11，优选8.5-10.6。
本发明中“低钙浓度”指的是低于60ppm的游离钙的水平，优选40ppm，更优选25ppm，尤其5ppm。
特别值得研究的亲本Termamyl样α-淀粉酶具有经过特别研究的改变特性，是上述所提到的亲本Termamyl样α-淀粉酶和亲本杂种Termamyl样α-淀粉酶。
在优选的实施方案中，本发明的变体尤其在具有高温和/或低pH下。
本发明一方面涉及具有上述的改变特性的变体。
第一方面亲本Termamyl样α-淀粉酶包含在选自下述的一或更多的位点改变(采用SEQ ID NO：8用于氨基酸编号)：49，60，104，132，161，170，176，179，180，181，183，200，203，204，207，212，237，239，250，280，298，318，374，385，393，402，406，427，430，440，444，447，482，其中
(a)改变分别是
(i)在占据该位点的氨基酸下游的氨基酸插入，
(ii)占据该位点的氨基酸的缺失，或
(iii)以不同的氨基酸取代占据该位点的氨基酸，
(b)变体具有α-淀粉酶活性(c)每个位点相应于具有SEQ ID NO：8中所
示的氨基酸序列的亲本Termamyl样α-淀粉酶氨基酸序列的位点。
在
(SEQ ID NO：8)，所述相应的位点是：
T49；D60；N104；E132；D161；K170；K176；G179；K180；A181；D183；D200；Y203；D204；D207；I212；K237；S239；E250；N280；Q298；L318；Q374；E385；Q393；Y402；H406；L427D430；V440；N444；E447；Q482。
SP722(SEQ ID NO：4)中相应的位点是：T51；D62；N106；D134；D163；Q172；K179；G184；K185；A186；D188；D205；M208；D209；X212；L217，K242，S244，N255，N285，S303，M323；D387，N395；Y404；H408；I429；D432；V442；K446；Q449；K484。
通过如上所述的序列对比和在图1所示的排列发现其它亲本α-淀粉酶相应的位点。
在优选的实施方案中，本发明的变体(用SEQ ID NO：8(TermamylTM)编号)具有下述的一或多个取代：
T49I；D60N；N104D；E132A，V，P；D161N；K170Q；K176R；G179N；K180T；A181N；D183N；D200N；X203Y；D204S；D207V，E，L，G；X212I；K237P；S239W；E250G，F；N280S；X298Q；L318M；Q374R；E385V；Q393R；Y402F；H406L，W；L427I D430N；V440A；N444R，K；E447Q，K；Q482K.
在优选的实施方案中，本发明的变体(使用SEQ ID NO：4(SP722)编号)具有下述的一或多个取代：
T51I；D62N；N106D；D134A，V，P；D163N；X172Q；K179R；G184N；K185T；A186N；D188N；D205N；M208Y；D209S；X212V，E，L，G；L217I，K242P，S244W，N255G，F，N285S，S303Q，X323M；D387V，N395R；Y404F；H408L，W；X429I；D432N；V442A；X446R，K；X449Q，K；X484K，使用SEQ ID NO：4(SP722)编号。
优选的是，二重，三重或多重突变-使用SEQ ID NO：8作为编号基础-所述突变选自：T49I+D60N；T49I+D60N+E132A；T49I+D60N+E132V；
T49I+D60N+E132V+K170Q；T49I+D60N+E132A+K170Q；
T49I+D60N+E132V+K170Q+K176R；T49I+D60N+E132A+K170Q+K176R；
T49I+D60N+E132V+K170Q+K176R+D207V；
T49I+D60N+E132A+K170Q+K176R+D207V；
T49I+D60N+E132V+K170Q+K176R+D207E；
T49I+D60N+E132A+K170Q+K176R+D207E；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A；
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；D60N+E132A；
D60N+E132V；D60N+E132V+K170Q；D60N+E132A+K170Q；
D60N+E132V+K170Q+K176R；T49I+D60N+E132A+K170Q+K176R；
D60N+E132V+K170Q+K176R+D207V；
T49I+D60N+E132A+K170Q+K176R+D207V；
D60N+E132V+K170Q+K176R+D207E；
T49I+D60N+E132A+K170Q+K176R+D207E；
D60N+E132V+K170Q+K176R+D207V+E250G；
D60N+E132A+K170Q+K176R+D207V+E250G；
D60N+E132V+K170Q+K176R+D207E+E250G；
D60N+E132A+K170Q+K176R+D207E+E250G；
D60N+E132V+K170Q+K176R+D207V+E250G+N280S；
D60N+E132A+K170Q+K176R+D207V+E250G+N280S；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S；
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M；
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M；
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R；
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F；
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L；
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I；
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
D60N+E 132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A；
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；E132V+K170Q；E132A+K170Q；
E132V+K170Q+K176R；E132A+K170Q+K176R；
E132V+K170Q+K176R+D207V；E132A+K170Q+K176R+D207V；
E132V+K170Q+K176R+D207E；E132A+K170Q+K176R+D207E；
E132V+K170Q+K176R+D207V+E250G；
E132A+K17OQ+K176R+D207V+E25OG；
E132V+K17OQ+K176R+D207E+E25OG；
E132A+K170Q+K176R+D207E+E250G；
E132V+K170Q+K176R+D207E+E250G+N280S；
E132A+K170Q+K176R+D207E+E250G+N280S；
E132V+K170Q+K176R+D207V+E250G+N280S；
E132A+K170Q+K176R+D207V+E250G+N280S；
E132V+K170Q+K176R+D207V+E250G+N280S+L318M；
E132A+K170Q+K176R+D207V+E250G+N280S+L318M；
E132V+K170Q+K176R+D207E+E250G+N280S+L318M；
E132A+K170Q+K176R+D207E+E250G+N280S+L318M；
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R；
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R；
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R；
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R；
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V；
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V；
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R；
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R；
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R；
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R；
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F；
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L；
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I；
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A；
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；K170Q+K176R；
K170Q+K176R+D207V；K170Q+K176R+D207E；
K170Q+K176R+D207V+E250G；K170Q+K176R+D207E+E250G；
K170Q+K176R+D207V+E250G+N280S；
K170Q+K176R+D207E+E250G+N280S；
K170Q+K176R+D207E+E250G+N280S+L318M；
K170Q+K176R+D207V+E250G+N280S+L318M；
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R；
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R；
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V；
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R；
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R；
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+385V+Q393R+Y402F+H406L；
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I；
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A；
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
K176R+D207V；K176R+D207E；K176R+D207V+E25OG；
K176R+D207E+E250G；K176R+D207V+E250G+N280S；
K176R+D207E+E250G+N280S；K176R+D207E+E250G+N280S+L318M；
K176R+D207V+E250G+N280S+L318M；
K176R+D207E+E250G+N280S+L318M+Q374R；
K176R+D207V+E250G+N280S+L318M+Q374R；
K176R+D207E+E250G+N280S+L318M+Q374R+E385V；
K176R+D207V+E250G+N280S+L318M+Q374R+E385V；
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R；
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R；
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A；
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；D207V+E250G；
D207E+E250G；D207V+E250G+N280S；D207E+E250G+N280S+L318M；
D207V+E250G+N280S+L318M；D207E+E250G+N280S+L318M+Q374R；
D207V+E250G+N280S+L318M+Q374R；
D207E+E250G+N280S+L318M+Q374R+E385V；
D207V+E250G+N280S+L318M+Q374R+E385V；
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R；
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R；
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；E250G+N280S；E250G+N280S+L318M；
E250G+N280S+L318M+Q374R；
E250G+N280S+L318M+Q374R+E385V；
E250G+N280S+L318M+Q374R+E385V+Q393R；
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F；
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A；N280S+L318M；N280S+L318M+Q374R；N280S+L318M+Q374R+E385V；
N280S+L318M+Q374R+E385V+Q393R；
N280S+L318M+Q374R+E385V+Q393R+Y402F；
N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L；
N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
L318M+Q374R；L318M+Q374R+E385V；L318M+Q374R+E385V+Q393R；
L318M+Q374R+E385V+Q393R+Y402F；
L318M+Q374R+E385V+Q393R+Y402F+H406L；
L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I；
L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
Q374R+E385V；Q374R+E385V+Q393R；Q374R+E385V+Q393R+Y402F；
Q374R+E385V+Q393R+Y402F+H406L；
Q374R+E385V+Q393R+Y402F+H406L+L427I；
Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A；
E385V+Q393R；E385V+Q393R+Y402F；E385V+Q393R+Y402F+H406L；
E385V+Q393R+Y402F+H406L+L427I；
E385V+Q393R+Y402F+H406L+L427I+V440A；
Q393R+Y402F；Q393R+Y402F+H406L；Q393R+Y402F+H406L+L427I；
Q393R+Y402F+H406L+L427I+V440A；Y402F+H406L；
Y402F+H406L+L427I；Y402F+H406L+L427I+V440A；H406L+L427I；H406L+L427I+V440A；L427I+V440A；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K；
D161N+A181N+D183N+D20ON+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K；
D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D43ON；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N；H406W+D43ON；N444K+E447Q+Q482K；E447Q+Q482K；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K；
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W；
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W；
H406W+D43ON；N444K+E447K+Q482K；E447K+Q482K；
N104D+D161N+A181N+D183N+D200N+D204S+K237P+S239W；
N104D+D161N+A181N+D183N+D200N+D204S+K237P；
N104D+D161N+A181N+D183N+D200N+D204S；
D161N+A181N+D183N+D200N+D204S+K237P+S239W；
D161N+A181N+D183N+D200N+D204S+K237P；
D161N+A181N+D183N+D200N+D204S；K237P+S239W，使用SEQ ID NO：8编号。
优选的实施方案中变体具有下述取代：K170Q+D207V+N280S；E132A+D207V；D207E+E250G+H406L+L427I；D207V+L318M；D60N+D207V+L318M；T49I+E132V+V440A；T49I+K176R+D207V+Y402F；Q374R+E385V+Q393R；N190F+A209V+Q264S；G48A+T49I+G107A+I201F；T49I+G107A+I201F；G48A+T49I+I201F；G48A+T49I+G107A；T49I+I201F；T49I+G107A；G48A+T49I；
D161N+G179N+K18OT+A181N+D183N+D20ON+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K使用SEQ ID NO：8编号。特定的变体包括：LE399；LE174+G48A+T49I+G107A；LE174+G48A+T49I+I201F；LE174+G48A+G 107A+I201F；LE174+T49I+G 107A+I201F；LE 174+G48A+T49I；LE174+G48A；LE174+G107A+I201F；LE174+I201F，是本发明具体考虑的变体。
稳定性
本发明中，重要的突变(包括氨基酸)以达到改变的稳定性，尤其是改进的稳定性(即高或低)，尤其是高温(即70-120℃)和/或极端pH(即低或高pH，即分别为pH4-6或pH8-11)，尤其是在游离的(即未结合的，在溶液中)低于60ppm钙浓度，包括“改变特性”那节所列的任何突变。稳定性可按照“材料和方法”那节所述进行测定。
本发明变体的一般突变
本发明的变体在一实施方案中可包括除了上述那些之外的一或多个突变。因此，将被修饰的α-淀粉酶变体某部分存在的1或多个脯氨酸取代为非脯氨酸残基可能是有益的，所述非脯氨酸可以是任何可能的、天然非脯氨酸残基，优选是Ala、Gly、Ser、Thr、Val或Leu。
类似地，可以优选将亲本α-淀粉酶被修饰的那些氨基酸残基中存在的1或多个Cys残基取代为非半胱氨酸残基，比如Ser、Ala、Thr、Gly、Val或Leu。
另外，可以将本发明的变体进行修饰—作为唯一的修饰或者结合上面概括的任何修饰—以便将与SEQ ID NO：10中的185-209位氨基酸片段所对应的氨基酸片段中存在的1或多个Asp和/或Glu分别取代为Asn或Gln。同样有意义的是在Termamyl样α-淀粉酶中，将与SEQ ID NO：10中的185-209位氨基酸片段所对应的氨基酸片段中存在的1或多个Lys残基取代为Arg。
应当明白，本发明涵盖了含有两个或多个上述修饰的变体。
另外，向此处描述的一或多个位点导入突变可能是有益的(使用SEQ IDNO：8(Termamyl)编号)。
M15，V128，A111，H133，W138，T149，M197，N188，A209，A210，H405，T412，尤其是下述的单一的，两重或三重或多重突变：
M15X，尤其是M15T，L；
V128X，尤其是V128E；
H133X，尤其是H133Y；
N188X，尤其是N188S，T，P；
M197X，尤其是M197T，L；
A209X，尤其是A209V；M197T/W138F；M197T/W138Y；M15T/H133Y/N188S；M15/V128E/H133Y/N188S；E119C/S130C；D124C/R127C；H133Y/T149I；G475R，H133Y/S187D；H133Y/A209V.
制备本发明α-淀粉酶变体的方法
几种将突变导入基因的方法是本领域已知的。简要讨论α-淀粉酶-编码DNA序列之后，将讨论在α-淀粉酶-编码序列中的特定位点产生突变的方法。
克隆编码α-淀粉酶的DNA序列
可以利用多种本领域的公知方法，从任何能产生目的α-淀粉酶的细胞或微生物中分离编码亲本α-淀粉酶的DNA序列。首先，应当利用得自能产生目的α-淀粉酶的有机体的染色体DNA或信使RNA来构建基因组DNA和/或cDNA文库。然后，如果α-淀粉酶的氨基酸序列是已知的，可以合成并合成并用同源的、标记的寡核苷酸来由从目的有机体制备的基因组文库鉴定α-淀粉酶-编码克隆。另外，可以使用标记的寡核苷酸探针(该探针含有与已知α-淀粉酶基因同源的序列)作为探针，采用低严谨性杂交和洗涤条件来鉴定α-淀粉酶-编码克隆。
另一种鉴定α-淀粉酶-编码克隆的方法包括将基因组DNA片段插入表达载体(比如质粒)，用所得基因组DNA文库转化α-淀粉酶阴性的细菌，以及随后将转化细菌铺到含有α-淀粉酶底物的琼脂上，从而能鉴定表达α-淀粉酶的克隆。
或者，可以通过已确立的标准方法来合成制备编码酶的DNA序列，例如S.L.Beaucage和M.H.Caruthers，Terahedron Letters，22，1981，PP.1859-1869描述的磷酸脒方法或Matthes等The EMBO J.3，1984，pp.801-805描述的方法。在磷酸脒方法中，在例如自动DNA合成仪中合成寡核苷酸，将其纯化、退火、连接和克隆到合适的载体中。
最后，DNA序列可以是基因组和合成来源混合的、合成和cDNA来源混合的，或者基因组和cDNA来源混合的，其是依照标准技术，将合成的、基因组或cDNA来源的片段连接在一起而制备的(在合适时，对应完整DNA序列各部分的片段)。还可以如US4683202或R.K.Saiki等Science 239，1988，pp.487-491描述的，使用特异引物通过聚合酶链反应(PCR)来制备DNA序列。
定点突变
一旦分离出α-淀粉酶编码的DNA序列，利用合成的寡核苷酸引入突变，鉴定用于突变的所希望的位点。这些寡核苷酸包含侧接目的突变位点的核苷酸序列。将突变的核苷酸在寡核苷酸合成期间插入。在特定方法中，DNA单链缺口，连接(bridging)α-淀粉酶编码序列，在携带有α-淀粉酶基因的载体中产生。然后含有目的突变的合成核苷酸退火至单链DNA的同源部分。所保留的缺口利用DNA聚合酶I(Klenow片段)填充，利用T4连接酶连接构建体。该方法的特异性实施例在Morinaga等(1984)中描述。US4760025公开了通过实施较小改变盒寡编码多重突变核苷酸。然而，由于多数各种长度的寡核苷酸可被引入甚至更多的突变可通过Morinaga等方法在任何时候引入。
将突变引入编码α-淀粉酶的DNA序列的另一种方法在Nelson和Long(1989)中描述。其涉及PCR片段的3-步产生(3-step generation)，该PCR片段含有导入的目的突变，利用化学合成的DNA链作为PCR反应中的之一引物。PCR生成的片段，通过限制性内切酶的切割分离出携带突变的DNA片段并将其重新插入到表达质粒。
本发明提供变体的可选的方法，例如，如WO 95/22625(自AffymaxTechnologies N.V.)或WO96/00343(Novo Nordisk A/S)或其它相应的技术导致包含该要研究的突变例如取代和/或缺失的杂交酶，包括基因改组。亲本α-淀粉酶变体的实施例可以合适地用于提供具有所述突变的杂交，根据本发明包括的在EP1022334中公开的KSM-K36和KSM-K38α-淀粉酶(在此引入作为参考)。
表达α-淀粉酶变体
根据本发明，可以利用表达载体将通过上述方法，或者通过本领域已知的任何替代方法制备的编码变体的DNA序列表达为酶的形式，载体通常包括调控序列，该序列编码启动子、操纵子、核糖体结合位点、翻译起始信号以及任选地，阻遏子基因或各种激活子基因。
携带编码本发明所述α-淀粉酶变体之DNA序列的重组表达载体可以是任何能方便地进行重组DNA操作的载体，且载体的选择通常取决于它将要导入的宿主细胞。因此，载体可以是自主复制的载体，即以染色体外个体存在的载体，其复制独立于染色体的复制，例如质粒、噬菌体或染色体外元件、微小染色体或人工染色体。可选择地，载体可以是这样的，当被导入宿主细胞时，它会整合到宿主基因组中，并与所整合到其中的染色体一起复制。
在所述载体中，DNA序列应当可操纵地连接到合适的启动子序列。该启动子可以是在所选宿主细胞中显示转录活性的任何DNA序列，并可以来源于编码宿主细胞的同源或异源蛋白质的基因。适于引导编码本发明所述α-淀粉酶变体的DNA序列进行转录(特别是在细菌宿主中)的启动子的例子是大肠杆菌lac操纵子的启动子、天蓝色链霉菌琼脂糖酶基因dagA启动子、地衣芽孢杆菌α-淀粉酶(amyL)的启动子、嗜热脂肪芽孢杆菌麦芽糖淀粉酶(maltogenic amylase)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。在真菌宿主中进行转录时，有用的启动子的例子是来源于这样一些基因的启动子，所述基因编码米曲霉TAKA淀粉酶、米赫氏根霉天冬氨酸蛋白酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定α-淀粉酶、黑曲霉葡糖淀粉酶、米赫氏根霉脂酶、米曲霉碱性蛋白酶、米曲霉丙糖磷酸异构酶或构巢曲霉乙酰胺酶。
本发明的表达载体还可以包含合适的转录终止子以及，在真核细胞中时的多腺苷酸化序列，它们与编码发明所述α-淀粉酶变体的DNA序列可操纵地连接在一起。终止和多腺苷酸化序列可以适当地来自与启动子相同的来源。
载体还可以包含能使载体在目的宿主细胞中进行复制的DNA序列。这类序列的例子是质粒pUC19、pACYC177、pUB110、pE194、pAMB1和pIJ702的复制原点。
载体还可以包含选择标记，例如一个其产物能补偿宿主细胞缺陷的基因，比如来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因；或者赋予抗生素抗性(比如氨苄青霉素、卡那霉素、氯霉素或四环素抗性)的基因。另外，载体可以包含曲霉选择标记，比如amdS、argB、niaD和sC，产生潮霉素抗性的标记，或者可以通过共转化(例如WO91/17243中描述的)来实现选择。
胞内表达在某些方面可能是有益的，例如用某种细菌作为宿主细胞时，但通常优选表达是胞外的。总之，此处提及的芽孢杆菌α-淀粉酶包含一个允许被表达的蛋白酶分泌到培养基中的前导区。如果需要，可以方便地通过取代编码该前区的DNA序列来将该前区替换为不同的前导区或信号序列。
用于分别将编码α-淀粉酶变体、启动子、终止子和其他元件的本发明所述DNA构建体连接在一起，并将其插入合适的包含复制所用的必要信息之载体的步骤是本领域技术人员所熟知的(参考，例如，Sambrook等，分子克隆：实验指南，第2版，Cold Spring Harbor，1989)。
本发明的细胞，它包含如上所述的本发明DNA构建体或表达载体，可以有效地作为重组制备本发明α-淀粉酶变体的宿主细胞。可以用本发明编码变体的DNA构建体，方便地通过将该构建体(以1或多拷贝)整合到宿主染色体中来转化所述细胞。通常认为整合是有益的，因为这样DNA序列更可能稳定地保持在细胞中。可以依照常规方法，例如通过同源或异源重组将DNA构建体整合到宿主染色体中。可选择地，可以根据宿主细胞的不同类型用上面描述过的表达载体来转化细胞。
本发明的细胞可以是更高等的生物，比如哺乳动物或昆虫的细胞，但优选是微生物细胞，比如细菌或真菌(包括酵母)细胞。
合适的细菌的例子是革兰氏阳性细菌，比如枯草芽孢杆菌、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(Bacillcus alkalophilus)、解淀粉芽孢杆菌、凝结芽孢杆菌、环状芽孢杆菌、灿烂芽孢杆菌、巨大芽孢杆菌、苏云金芽孢杆菌，或者浅青紫链霉菌或鼠灰链霉菌，或者是革兰氏阴性细菌，比如大肠杆菌。可以通过例如原生质体转化或利用竞争细胞以已知方式来实现细菌的转化。
可以有利地从酵母属或裂殖酵母属中选择酵母微生物，例如酿酒酵母。丝状真菌最好是属于曲霉属的种，例如米曲霉或黑曲霉。可以通过一个包括原生质体形成和转化，以及随后以已知方式再生细胞壁的方法来转化真菌细胞。在EP238023中描述了转化曲霉宿主细胞的适用步骤。
再一方面，本发明涉及制备发明所述α-淀粉酶变体的方法，该方法包括在有助于变体生产的条件下培养以上描述的宿主细胞，以及从细胞和/或培养基中回收变体。
用于培养细胞的培养基可以是任何适合目的宿主细胞生长以及本发明所述α-淀粉酶变体进行表达的常规培养基。可以从供应商那里获得合适的培养基或者可以根据公开的配方(例如，美国典型培养物保藏中心的目录中所描述的)来制备。
可以通过公知方法从培养基中方便地回收宿主细胞所分泌的α-淀粉酶变体，这些方法包括通过离心或过滤从培养基中分离细胞，以及借助盐(比如硫酸铵)来沉淀培养基中的蛋白类成分，然后利用层析操作，比如离子交换层析、亲合层析等。
工业应用
本发明的α-淀粉酶变体具备适合多种工业应用的有价值的特性。具体来说，本发明的酶变体可以作为洗涤、餐具清洗和硬表面清洁的洗涤剂组合物的成分。
本发明具有改变特性的变体可用于淀粉加工，尤其是淀粉转化，特别是淀粉液化作用(参见，例如US3912590，EP专利252730和63909，WO99/19467，WO96/28567此处所有的文献引入作为参考)。还要考虑的是用于淀粉转化的组合物，除了本发明的变体外还包括AMG，支链淀粉酶和其它α-淀粉酶。
而且，本发明的变体还在由淀粉生产增甜剂和乙醇(参见例如在此引入参考的美国专利5231017)，例如燃料，饮料和工业乙醇，来自淀粉或全部谷物。
本发明的变体还在纺织品退浆时有用(参见例如在此引入参考的，WO95/21247，美国专利4643736，EP119920)。
洗涤剂组合物
如上所述，可以适当地将本发明的变体加入洗涤剂组合物中。涉及洗涤剂组合物相关成分(如洗衣洗涤剂或餐具洗涤剂)的进一步的细节、在这类洗涤剂组合物中配制变体的合适方法以及洗涤剂组合物的有关类型，可以参考例如，WO96/23874和WO97/07202。
包含本发明变体的洗涤剂组合物可以另外含有一或多种其他酶，比如蛋白酶、脂酶、过氧化物酶，另一种淀粉分解酶、葡糖淀粉酶、麦芽糖淀粉酶、CGTase和/或纤维素酶、甘露聚糖酶(如自Novozymes的MannawayTM，Denmark))，果胶酶，pectine裂解酶，角质酶、漆酶，和/或另一种α淀粉酶。
可以将本发明的α-淀粉酶变体以常规使用的浓度加入洗涤剂中。目前考虑可以将本发明的变体以相当于每升使用常规剂量水平洗涤剂的洗涤/餐具清洗液中含0.00001-1mg α-淀粉酶的用量(以纯品计算，活性酶蛋白)加入。
组合物
本发明还涉及包含本发明变体的组合物，在优选的实施方案中还有嗜热脂肪芽孢杆菌α-淀粉酶(BSG)，特别是其变体。
在另一实施方案中，组合物除了本发明葡糖淀粉酶变体，包括尤其是衍生于黑曲霉的葡糖淀粉酶(例如公开于Boel et al.(1984)，G1或G2黑曲霉AMG“衍生于黑曲霉的葡糖淀粉酶G1和G2合成自两种不同但很相关的mRNA”EMBO J.3(5)，P.1097-1102，或其变体，尤其公开于WO 00/04136或WO 01/04273或公开于WO 99/28448Talaromyces emersonii AMG。
特定组合是LE399和公开于WO 00/04136或WO 01/04273的变体，尤其是具有下述一或更多个取代的变体：
N9A，S56A，V59A，S119P，A246T，N313G，E342T，A393R，S394R，Y402F，E408R，尤其是具有所有突变的变体。
在实施方案中，本发明的组合物还包括支链淀粉酶，尤其是芽孢杆菌属的支链淀粉酶。
材料和方法
酶
SEQ ID NO：8所示的地衣芽孢杆菌α-淀粉酶，也可从Novozyme获得。
AA560：SEQ ID NO：12；公开于WO 00/60060；于1999年1月25日在DSMZ保藏并且保藏号为DSMZ.12649。本发明人将AA560保藏于国际承认用于专利程序的微生物保藏布达佩斯条约下的德意志微生物和细胞培养物保藏中心(Deutshe Sammmlung von Microorganismen und ZellkulturenGmbH(DSMZ))，Mascheroder Weg lb，D-38124Braunschweig DE。
LB培养基(1升H20中：10g bacto-胰蛋白胨e，5g bactoyeast提取物，10g NaCl，pH调为7.0w.NaOH，高压灭菌)。
TY琼脂板(1升H20：16g bacto-胰蛋白胨，10g bacto-yeast提取物，5gNaCl，pH调为7.0w.NaOH，和高压灭菌前添加15g bacto-agar)。
10％Lugol溶液(碘/碘化钾溶液；通过储藏的H20稀释(dil.)10倍制备：Sigma Cat.no.L6146)。
枯草芽孢杆菌SHA273：见WO 95/10603
质粒
pDN1528包含编码Termamyl的完整基因，amyL，其自身启动子指导其表达。而且，该质粒还包含来自质粒pUB110的复制原点，ori，以及来自质粒pUC194的cat基因，赋予对氯霉素的抗性。pDN1528在WO 96/23874的图9中显示。
方法
低pH滤膜检测
将芽孢杆菌文库铺在含有10μg/ml氯霉素的TY琼脂板上的醋酸纤维素(OE67，Schleicher&Schuell，Dassel，Germany)和硝酸纤维素滤膜(Protran-Ba85，Schleicher&Schuell，Dassel，Germany)夹心上，于37℃保持至少21小时。将醋酸纤维素层放在TY琼脂板上。
铺板之后，但要在保温之前用针将每个滤膜夹心特异地标记，以便能够确定阳性变体在滤膜上的位置，并将结合了变体的硝酸纤维素滤膜转移到盛有柠檬酸盐缓冲液(pH4.5)的容器中，于80℃温育20分钟(筛选野生型主链变体时)或85℃60分钟(筛选LE399主链变体时)。将带有菌落的醋酸纤维素滤膜于室温保存在TY平板上待用。温育后，在含有1％琼脂糖、0.2％淀粉的柠檬酸盐缓冲液(pH6.0)的平板上检测残存的活性。用与滤膜夹心相同的方式标记带有硝酸纤维素滤膜的检测平板，并于50℃培养2小时。移去滤膜后，用10％Lugol溶液将检测平板染色。降解淀粉的变体检测为深蓝色背景上的白色斑点，然后在保存平板上鉴定。在与第一次筛选相同的条件下将阳性变体重筛两次。
次级筛选
将在筛选后的阳性转化体自储藏板中挑起并在次级板试验中检测。阳性转化体在5ml LB+氯霉素于37℃生长22小时。芽胞杆菌属每个阳性转化体和作为对照的表达相应主链的克隆在柠檬酸盐缓冲液，pH 4.5中90℃培养，样品于0，10，20，30，40，60和80分钟时取出。3微升样品在试验板上点样。试验板用10％Lugol溶液染色。观察到改进的变体具有比主链高的残留活性(试验板上检测为晕圈)。通过核苷酸测序测定改进的变体。
未纯化变体的稳定试验
要分析的表达变体的芽孢杆菌属培养物在10ml LB+氯霉素中37℃生长21小时。800微升培养物与200微升柠檬酸盐缓冲液pH 4.5混合。相应于大量样品时间点的多个70微升等分试样在PCR管中制备，并于PCR仪器70℃培养((wt主链(backbone)变体)或90℃(对于LE399的变体)各时间点(通常5，10，15，20，25和30分钟)培养。0分钟样品未在高温培养。样品的活性通过转移20微升到200微升α-淀粉酶PNP-G7底物MPR3的(BoehringerMannheim Cat.no.1660730)，如下“α-淀粉酶活性的试验”所述。结果显示为百分比活性(相对与0时间点)对时间作图，或表述为培养一段时间之后百分比剩余活性。
α-淀粉酶变体的发酵和纯化
携带相关表达质粒的枯草芽孢杆菌菌株在自-80℃储藏的有10微克/ml卡那霉素的LB-琼脂板上接种，将菌斑转移到100ml PS-1添有chloamphinicol 10micro g/ml培养基的500ml摇瓶中。
PS-1培养基组合物：
Pearl sugar 100g/l
Soy Bean Meal 40g/l
Na2HPO4，12H2O 10g/l
PluronicTM PE 6100 0.1g/l
CaCO3 5g/l
培养物在37℃于270rpm振荡5天。
通过4500rpm离心20-25分钟从发酵液中除去细胞或细胞碎片。然后将上清过滤，以获得彻底清晰的溶液。浓缩滤液并在UF-过滤器(10000cut off膜)冲洗。缓冲液变为20mM醋酸盐pH 5.5。将UF-滤过液应用到S-sepharose F.F.上并且通过用0.2M NaCl的相同缓冲液进行分步洗脱。10mMTris，pH 9.0透析洗脱液并应用到Q-sepharose F.F.，用线性梯度0-0.3M NaCl6柱体积洗脱。含有活性的(由Phadebas试验测定)级分被汇集，调pH到pH 7.5通过5分钟内0.5％W/vol.活性炭处理除去剩余的颜色。
纯化变体的稳定性测定
全部纯化变体稳定性实验使用相同的设置进行。方法是：
在相应条件(1-4)下温育酶。于不同时刻取样品，例如0、5、10、15和30分钟后，将样品在检测缓冲液(0.1M 50mM Britton缓冲液pH7.3)中稀释25倍(所有取样稀释度相同)，并采用Phadebas检测法(Pharmacia)在标准条件(pH7.3，37℃)下测量活性。
以温育前(0分钟)测量的活性作为对照(100％)。将下降的百分比作为温育时间的函数来计算活性。表中显示在温育例如30分钟后的残存活性。
比活性测定
采用Phadebas检测法(Pharmacia)将比活性确定为活性/mg酶。测定依照产品说明书进行(还可见以下“α-淀粉酶活性检测”)。
α-淀粉酶活性检测法
1.Phadebas检测法
通过一种采用
片剂作为底物的方法来测定α-淀粉酶的活性。Phadebas片剂(
Amylase Test，由Pharmacia Diagnostic提供)含有一种交联的不溶性兰色淀粉聚合物，该聚合物与牛血清白蛋白和缓冲物质混合在一起并做成片剂。
进行每次测量时，将一个药片悬浮于含有5ml 50mM Britton-Robinson缓冲液(50mM醋酸、50mM磷酸、50mM硼酸、0.1mM CaCl2、用NaOH调至所需pH)的试管中。于所需温度在水浴中进行实验。将待测α-淀粉酶在xml 50mM Britton-Robinson缓冲液中进行稀释。将1ml该α-淀粉酶溶液加入5ml 50mM Britton-Robinson缓冲液中。α-淀粉酶水解淀粉产生水溶性兰色碎片。在620nm比色测定到的所得兰色溶液的吸光度是α-淀粉酶活性的函数。
重要的是温育10或15分钟后测量的620nm吸光度应在0.2到2.0吸收单位内。在这样的吸光度范围内，活性和吸光度是线性关系(Lambert-Beer定律)。因此，必须调节酶的稀释度以便符合该法则。在特别设置的条件(温度、pH 、反应时间、缓冲液条件)下，1mg给定α-淀粉酶将水解一定量的底物并产生兰色。在620nm测量色度。在给定条件下，所测得的吸光度与受测α-淀粉酶的特异活性直接成比例(活性/毫克纯α-淀粉酶蛋白质)。
2.替代方法
通过一种采用PNP-G7作为底物的方法来确定α-淀粉酶的活性。PNP-G7(邻-硝基苯-α，D-麦芽庚糖苷的缩写)是一种被封闭的寡糖，可以被内-淀粉酶切割。切割之后，试剂盒中包含的α-葡糖苷酶消化底物释放出游离的PNP分子，它呈现黄色，因此可以在λ＝405nm(400-420nm)的可见光处比色测量。包含PNP-G7底物和α-葡糖苷酶的试剂盒由Boehringer-Mannheim cat.No.1054635)制造。
按照生产商所推荐将10ml底物/冲液加入50ml酶/缓冲液来制备试剂缓冲液，该试验是通过下述进行的：将20μl酶溶液转移到一个96孔微量滴定板上并于25℃温育来进行检测。加入200μl试剂溶液(25℃)。将溶液混匀，预保温1分钟，并在ELISA reader上4分钟内每30秒测量一次OD405nm处的吸光度。
在给定条件设置下，随时间变化的吸光度曲线的斜率与受测α-淀粉酶活性(活性/毫克酶)成正比。
实施例
实施例1
与亲本酶相比，在高温和低钙离子浓度，低pH下具有在改进稳定性的地衣芽孢杆菌α-淀粉酶变体通过易错RCR诱变构建。
易错PCR诱变和文库构建
为改进在高温和低钙离子浓度，低pH下地衣芽孢杆菌α-淀粉酶变体的稳定性，实施易错PCR诱变。利用编码野生型地衣芽孢杆菌α-淀粉酶基因的质粒PDN1528作为模板，以用下面引物在PCR条件下扩增该基因，22149：5′-CGA TTG CTG ACG CTG TTA TTT GCG3′(SEQID NO：14)和24814：5′-GAT CAC CCG CGA TAC CGT C-3′(SEQ ID NO：15)。其中升高的错率导致引入随机点突变。所用的PCR条件是：10mM Tris-HCl，pH 8.3，50mM KCl，4mM MgCl2，0.3mM MnCl2，0.1mM dGTP/dATP，0.5mMdTTP/dCTP，和每100微升(micro 1)反应液2.5单位的Taq聚合酶。
所得的PCR片段在胶上纯化，并在基于PCR多聚化步骤中使用，通过下述引物的pDN1528PCR扩增产生的胶纯化载体片段，#24：5′-GAA TGTATG TCG GCC GGC AAA ACG CCG GTG A-3′(SEQ ID NO：16)和#27：5-GCC GCC GCT GCT GCA GAA TGA GGC AGC AAG-3′(SEQ ID NO：17)，由此形成重叠以插入片段。多聚化反应随后被引入枯草芽孢杆菌(Shafikhaniet al.，Biotechniques，23(1997)，304～310)。
筛选
上述易错文库在低pH滤膜试验中筛选(见″材料与方法″)。对重筛选阳性的克隆检测进行“材料与方法”的液体试验中其稳定性次级筛选。
结果
在pH 4.5，5ppm钙下于90℃温育升高的稳定性
| 名称 | wt | LE488 | LE489 | 7.19.1 | 8.9.1 |
| 突变 | - | D207V | K170QD207VN280S | E132AD207V | D207EE250GH406LL427I |
| 稳定性 | - | + | + | + | + |
1)“+”表示相对于野生型(wt)稳定性显著升高
在pH 4.5，5ppm钙下于90℃温育升高的稳定性
| 名称 | wt | LE491 | LE492 | LE493 | LE494 | 19.3.1 |
| 突变 | - | D60ND207VL318M | T49IE132VV440A | T49IK176RD207VY402F | Q374RE385VQ393R | N190FA209VQ264S |
| 稳定性 | - | + | + | + | + | + |
1)“+”表示相对于野生型稳定性显著升高
在pH4.5，5ppm钙下于90℃温育升高的稳定性
| 名称 | wt | E132-1 | D207-7 | D207-6 | E250-8 |
| 突变 | - | E132P | D207L | D207G | E250F |
| 稳定性 | - | + | + | + | + |
1)“+”表示相对于野生型稳定性显著升高
实施例2
通过定点诱变，与亲本酶从实施例1到新(非野生型)主链的进行选择以改进在低pH和低钙离子浓度稳定性。
定点诱变
将自LE493(K176R+D207V+Y402F)突变转移到LE399生产的LE495。这是通过重叠PCR方法(Kirchhoff和Desrosiers，PCR Methods andApplications，2(1993)，301-304)。通过下述引物及LE399模板产生2个重叠PCR片段，片段A：#312Mut1765′-CCC GAAAGC TGAACC GCA TCT ATAGGT TTC AAG GGA AGA CTT GGG ATT-3′(SEQ ID NO：18)(粗体表示突变密码子)和#290D207重叠5′-AGG ATG GTC ATA ATC AAA GTC GG-3′(SEQ ID NO：19)；片段B：#313Mut2075′-CCG ACT TTG ATT ATG ACCATC CTG TTG TCG TAG CAG AGA TTA AGA GAT GGG G-3′(SEQ ID NO：20)和#314Mut4025′-CGA CAA TGT CAT GGT GGT CGA AAA AAT CATGCT GTG CTC CGT ACG-3′(SEQ ID NO：21)。片段A和B等克分子的比例混合并接着用下面外引物扩增全长片段，#312Mut176和#314Mut402。该片段用下述引物产生的载体PCR片段用于多聚化反应。#296Y402multi5′-TTT CGA CCA CCA TGA CAT TGT CG-3′(SEQ ID NO：22)和#305399Multi1765′-TAT AGA TGC GGT TCA GCT TTC GGG-3′(SEQ ID NO：23)，用上述的模板LE399。接着用多聚化反应转化入枯草芽孢杆菌。对于上述试验中的稳定性筛选克隆。一些克隆中具有改进稳定性自LE493的突变存在通过测序证实。
以相似的方式通过采用利用引物#312Mut176和#314Mut402扩增LE399编码模板获得LE 497，并在采用载体片段多聚化反应中使用所得PCR片段，而载体片段是通过LE399PCR扩增，采用引物#296Y402multi和#305399Multi176获得。
结果
LE399变体在pH4.5，5ppm钙于90℃培养升高的稳定性
| 名称 | LE399 | LE495 | LE497 |
| 突变 | -(主链) | K176RD207VY402F | K176RY402F |
| 稳定性 | - | + | + |
1)“+”表示相对于主链稳定性显著升高
序列表
<110>诺维信公司(Novozymes A/S)
<120>具有改变特性的α-淀粉酶突变体
<130>
<160>28
<170>PatentIn Ver.2.1
<210>1
<211>1455
<212>DNA
<213>芽孢杆菌属菌种(Bacillus sp.)
<220>
<221>CDS
<222>(1)..(1455)
<223>SP690
<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
35 40 45
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
50 55 60
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
65 70 75 80
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
85 90 95
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
100 105 110
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
115 120 125
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
130 135 140
ttt cct gga aga gga aat aac cat tcc agc ttt aag tgg cgc tgg tat 480
Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
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
165 170 175
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
180 185 190
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
195 200 205
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
210 215 220
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
225 230 235 240
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
245 250 255
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
260 265 270
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
275 280 285
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
290 295 300
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
305 310 315 320
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
325 330 335
ggg gaa gca ttg gaa tcc ttt gtt caa caa tgg ttt aaa cca ctt gca 1056
Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala
340 345 350
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
355 360 365
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
370 375 380
aaa ata gac cct ctt ctg cag gca cgt caa act ttt gcc tat ggt acg 1200
Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr
385 390 395 400
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
405 410 415
gga aat agc tcc cat cca aat tca ggc ctt gcc acc att atg tca gat 1296
Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
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
450 455 460
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
465 470 475 480
gtt tgg gtg aag caa 1455
Val Trp Val Lys Gln
485
<210>2
<211>485
<212>PRT
<213>芽孢杆菌属菌种(Bacillus sp.)
<400>2
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala
20 25 30
Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
115 120 125
Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys
165 170 175
Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255
Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300
Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys
305 310 315 320
His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380
Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr
385 390 395 400
Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly
435 440 445
Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Val Trp Val Lys Gln
485
<210>3
<211>1455
<212>DNA
<213>芽孢杆菌属菌种(Bacillus sp.)
<220>
<221>CDS
<222>(1)..(1455)
<223>SP722
<400>3
cat cat aat ggg aca aat ggg acg atg atg caa tac ttt gaa tgg cac 48
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
1 5 10 15
ttg cct aat gat ggg aat cac tgg aat aga tta aga gat gat gct agt 96
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser
20 25 30
aat cta aga aat aga ggt ata acc gct att tgg att ccg cct gcc tgg 144
Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp
35 40 45
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
50 55 60
gat tta ggg gaa ttt aat caa aag ggg acg gtt cgt act aag tat ggg 240
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
aca cgt agt caa ttg gag tct gcc atc cat gct tta aag aat aat ggc 288
Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly
85 90 95
gtt caa gtt tat ggg gat gta gtg atg aac cat aaa gga gga gct gat 336
Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
gct aca gaa aac gtt ctt gct gtc gag gtg aat cca aat aac cgg aat 384
Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
caa gaa ata tct ggg gac tac aca att gag gct tgg act aag ttt gat 432
Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
ttt cca ggg agg ggt aat aca tac tca gac ttt aaa tgg cgt tgg tat 480
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
tcg gaa aat gga aat tat gat tat tta atg tat gca gat gta gat atg 624
Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
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
210 215 220
aca aat aca tta aat ctt gat gga ttt agg atc gat gcg gtg aag cat 720
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
att aaa tat agc ttt aca cgt gat tgg ttg acc cat gta aga aac gca 768
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala
245 250 255
acg gga aaa gaa atg ttt gct gtt gct gaa ttt tgg aaa aat gat tta 816
Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
ggt gcc ttg gag aac tat tta aat aaa aca aac tgg aat cat tct gtc 864
Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285
ttt gat gtc ccc ctt cat tat aat ctt tat aac gcg tca aat agt gga 912
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300
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
305 310 315 320
cat cca atg cat gcc gta act ttt gtg gat aat cac gat tct caa cct 1008
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
ggg gaa tca tta gaa tca ttt gta caa gaa tgg ttt aag cca ctt gct 1056
Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala
340 345 350
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
355 360 365
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
370 375 380
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
405 410 415
gga aat acc acg cat ccc aat tca gga ctt gcg act atc atg tcg gat 1296
Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
ggg cca ggg gga gag aaa tgg atg tac gta ggg caa aat aaa gca ggt 1344
Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly
435 440 445
caa gtt tgg cat gac ata act gga aat aaa cca gga aca gtt acg atc 1392
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
465 470 475 480
att tgg gtg aaa cga 1455
Ile Trp Val Lys Arg
485
<210>4
<211>485
<212>PRT
<213>芽孢杆菌属菌种(Bacillus sp.)
<400>4
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser
20 25 30
Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly
85 90 95
Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr
210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala
245 250 255
Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly
290 295 300
Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys
305 310 315 320
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala
370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr
385 390 395 400
Gln His Asp Tyr Phe Asp His His Asn Ile lle Gly Trp Thr Arg Glu
405 410 415
Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly
435 440 445
Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile
450 455 460
Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser
465 470 475 480
Ile Trp Val Lys Arg
485
<210>5
<211>1548
<212>DNA
<213>嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)
<220>
<221>CDS
<222>(1)..(1548)
<223>BSG
<400>5
gcc gca ccg ttt aac ggc acc atg atg cag tat ttt gaa tgg tac ttg 48
Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu
1 5 10 15
ccg gat gat ggc acg tta tgg acc aaa gtg gcc aat gaa gcc aac aac 96
Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn
20 25 30
tta tcc agc ctt ggc atc acc gct ctt tgg ctg ccg ccc gct tac aaa 144
Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
35 40 45
gga aca agc cgc agc gac gta ggg tac gga gta tac gac ttg tat gac 192
Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60
ctc ggc gaa ttc aat caa aaa ggg acc gtc cgc aca aaa tac gga aca 240
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>嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)
<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)
<223>Termamyl
<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(Bacillus licheniformis)
<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>解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<220>
<221>CDS
<222>(343)..(1794)
<223>BAN
<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 gt ctgg 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
5 560 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>解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<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>芽孢杆菌属菌种(Bacillus sp.)
<220>
<221>CDS
<222>(1)..(1458)
<223>AA560
<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>芽孢杆菌属菌种(Bacillus sp.)
<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>485
<212>PRT
<213>芽孢杆菌属菌种707(Bacillus sp.707)
<400>13
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser
20 25 30
Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly
85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125
Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly His Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205
Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220
Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His
225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala
245 250 255
Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270
Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val
275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly
290 295 300
Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg
305 310 315 320
His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335
Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala
340 345 350
Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser
370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys
385 390 395 400
Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430
Gly Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly
435 440 445
Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
450 455 460
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>14
<211>27
<212>DNA
<213>人工序列
<220>
<223>引物22149
<400>14
CGATTGCTGA CGCTGTTATT TGCG 24
<210>15
<211>29
<212>DNA
<213>人工序列
<220>
<223>引物24814
<400>15
GATCACCCGC GATACCGTC 29
<210>16
<211>31
<212>DNA
<213>人工序列
<220>
<223>引物#24
<400>16
GAATGTATGT CGGCCGGCAA AACGCCGGTG A 31
<210>17
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物#27
<400>17
GCCGCCGCTG CTGCAGAATG AGGCAGCAAG 30
<210>18
<211>48
<212>DNA
<213>人工序列
<220>
<223>引物#312
<400>18
CCCGAAAGCT GAACCGCATC TATAGGTTTC AAGGGAAGAC TTGGGATT 48
<210>19
<211>23
<212>DNA
<213>人工序列
<220>
<223>引物#290
<400>19
AGGATGGTCA TAATCAAAGT CGG 23
<210>20
<211>52
<212>DNA
<213>人工序列
<220>
<223>引物#313
<400>20
CCGACTTTGA TTATGACCAT CCTGTTGTCG TAGCAGAGAT TAAGAGATGG GG 52
<210>21
<211>45
<212>DNA
<213>人工序列
<220>
<223>引物#314
<400>21
CGACAATGTC ATGGTGGTCG AAAAAATCAT GCTGTGCTCC GTACG 45
<210>22
<211>23
<212>DNA
<213>人工序列
<220>
<223>引物#296
<400>22
TTTCGACCAC CATGACATTG TCG 23
<210>23
<211>24
<212>DNA
<213>人工序列
<220>
<223>引物#305
<400>23
TATAGATGCG GTTCAGCTTT CGGG 24
<210>24
<211>1650
<212>DNA
<213>芽孢杆菌属菌种(Bacillus sp.)
<220>
<221>CDS
<222>(65)..(1567)
<220>
<221>成熟肽
<222>(128)..()
<220>
<221>信号肽
<222>(65)..(128)
<400>24
cttgaatca ttatttaaagc tggttatgat atatgtaagc gttatcatta aaaggaggta 60
tttg atg aaa aga tgg gta gta gca atg ctg gca gtg tta ttt tta ttt 109
Met Lys Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe
-20 -15 -10
cct tcg gta gta gtt gca gat ggc ttg aat gga acg atg atg cag tat 157
Pro Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr
-5 -1 1 5 10
tat gag tgg cat cta gag aat gat ggg caa cac tgg aat cgg ttg cat 205
Tyr Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His
15 20 25
gat gat gcc gaa gct tta agt aat gcg ggt att aca gct att tgg ata 253
Asp Asp Ala Glu Ala Leu Ser Asn Ala Gly Ile Thr Ala Ile Trp Ile
30 35 40
ccc cca gcc tac aaa gga aat agt cag gct gat gtt ggg tat ggt gca 301
Pro Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala
45 50 55
tac gac ctt tat gat tta ggg gag ttt aat caa aaa ggt acc gtt cga 349
Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg
60 65 70
acg aaa tac ggg aca aag gct cag ctt gag cga gct ata ggg tcc cta 397
Thr Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu
75 80 85 90
aag tcg aat gat atc aat gtt tat ggg gat gtc gta atg aat cat aaa 445
Lys Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys
95 100 105
tta gga gct gat ttc acg gag gca gtg caa gct gtt caa gta aat cct 493
Leu Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro
110 115 120
tcg aac cgt tgg cag gat att tca ggt gtc tac acg att gat gca tgg 541
Ser Asn Arg Trp Gln Asp Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp
125 130 135
acg gga ttt gac ttt cca ggg cgc aac aat gcc tat tcc gat ttt aaa 589
Thr Gly Phe Asp Phe Pro Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys
140 145 150
tgg aga tgg ttc cat ttt aat ggc gtt gac tgg gat caa cgc tat caa 637
Trp Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln
155 160 165 170
gaa aac cat ctt ttt cgc ttt gca aat acg aac tgg aac tgg cga gtg 685
Glu Asn His Leu Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val
175 180 185
gat gaa gag aat ggt aat tat gac tat tta tta gga tcg aac att gac 733
Asp Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp
190 195 200
ttt agc cac cca gag gtt caa gag gaa tta aag gat tgg ggg agc tgg 781
Phe Ser His Pro Glu Val Gln Glu Glu Leu Lys Asp Trp Gly Ser Trp
205 210 215
ttt acg gat gag cta gat tta gat ggg tat cga ttg gat gct att aag 829
Phe Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys
220 225 230
cat att cca ttc tgg tat acg tca gat tgg gtt agg cat cag cga agt 877
His Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Ser
235 240 245 250
gaa gca gac caa gat tta ttt gtc gta ggg gag tat tgg aag gat gac 925
Glu Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp
255 260 265
gta ggt gct ctc gaa ttt tat tta gat gaa atg aat tgg gag atg tct 973
Val Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser
270 275 280
cta ttc gat gtt ccg ctc aat tat aat ttt tac cgg gct tca aag caa 1021
Leu Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Lys Gln
285 290 295
ggc gga agc tat gat atg cgt aat att tta cga gga tct tta gta gaa 1069
Gly Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu
300 305 310
gca cat ccg att cat gca gtt acg ttt gtt gat aat cat gat act cag 1117
Ala His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln
315 320 325 330
cca gga gag tca tta gaa tca tgg gtc gct gat tgg ttt aag cca ctt 1165
Pro Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu
335 340 345
gct tat gcg aca atc ttg acg cgt gaa ggt ggt tat cca aat gta ttt 1213
Ala Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe
350 355 360
tac ggt gac tac tat ggg att cct aac gat aac att tca gct aag aag 1261
Tyr Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys
365 370 375
gat atg att gat gag ttg ctt gat gca cgt caa aat tac gca tat ggc 1309
Asp Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly
380 385 390
aca caa cat gac tat ttt gat cat tgg gat atc gtt gga tgg aca aga 1357
Thr Gln His Asp Tyr Phe Asp His Trp Asp Ile Val Gly Trp Thr Arg
395 400 405 410
gaa ggt aca tcc tca cgt cct aat tcg ggt ctt gct act att atg tcc 1405
Glu Gly Thr Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser
415 420 425
aat ggt cct gga gga tca aaa tgg atg tac gta gga cag caa cat gca 1453
Asn Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Gln Gln His Ala
430 435 440
gga caa acg tgg aca gat tta act ggc aat cac gcg gcg tcg gtt acg 1501
Gly Gln Thr Trp Thr Asp Leu Thr Gly Asn His Ala Ala Ser Val Thr
445 450 455
att aat ggt gat ggc tgg ggc gaa ttc ttt aca aat gga gga tct gta 1549
Ile Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val
460 465 470
tcc gtg tat gtg aac caa taataaaaag ccttgagaag ggattcctcc 1597
Ser Val Tyr Val Asn Gln
475 480
ct aact caag gct ttctt ta tgtcgtttag ctcaacgctt ctacgaagct tta 1650
<210>25
<211>501
<212>PRT
<213>芽孢杆菌属菌种(Bacillus sp.)
<400>25
Met Lys Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro
-20 -15 -10
Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr
-5 -1 1 5 10
Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp
15 20 25
Asp Ala Glu Ala Leu Ser Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro
30 35 40
Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr
45 50 55
Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
60 65 70 75
Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys
80 85 90
Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Leu
95 100 105
Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Ser
110 115 120
Asn Arg Trp Gln Asp Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp Thr
125 130 135
Gly Phe Asp Phe Pro Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp
140 145 150 155
Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu
160 165 170
Asn His Leu Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp
175 180 185
Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe
190 195 200
Ser His Pro Glu Val Gln Glu Glu Leu Lys Asp Trp Gly Ser Trp Phe
205 210 215
Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His
220 225 230 235
Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Ser Glu
240 245 250
Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val
255 260 265
Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu
270 275 280
Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Lys Gln Gly
285 290 295
Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala
300 305 310 315
His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro
320 325 330
Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala
335 340 345
Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr
350 355 360
Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp
365 370 375
Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr
380 385 390 395
Gln His Asp Tyr Phe Asp His Trp Asp Ile Val Gly Trp Thr Arg Glu
400 405 410
Gly Thr Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn
415 420 425
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Gln Gln His Ala Gly
430 435 440
Gln Thr Trp Thr Asp Leu Thr Gly Asn His Ala Ala Ser Val Thr Ile
445 450 455
Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser
460 465 470 475
Val Tyr Val Asn Gln
480
<210>26
<211>1745
<212>DNA
<213>芽孢杆菌属菌种(Bacillus sp.)
<220>
<221>CDS
<222>(190)..(1692)
<220>
<221>成熟肽
<222>(253)..()
<220>
<221>信号肽
<222>(190)..(253)
<400>26
aactaagtaa catcgattca ggataaaagt atgcgaaacg atgcgcaaaa ctgcgcaact 60
actagcactc ttcagggact aaaccacctt ttttccaaaa atgacatcat ataaacaaat 120
ttgtctacca atcactattt aaagctgttt atgatatatg taagcgttat cattaaaagg 180
aggtatttg atg aga aga tgg gta gta gca atg ttg gca gtg tta ttt tta 231
Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu
-20 -15 -10
ttt cct tcg gta gta gtt gca gat gga ttg aac ggt acg atg atg cag 279
Phe Pro Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln
-5 -1 1 5
tat tat gag tgg cat ttg gaa aac gac ggg cag cat tgg aat cgg ttg 327
Tyr Tyr Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu
10 15 20 25
cac gat gat gcc gca gct ttg agt gat gct ggt att aca gct att tgg 375
His Asp Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp
30 35 40
att ccg cca gcc tac aaa ggt aat agt cag gcg gat gtt ggg tac gtt 423
Ile Pro Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly
45 50 55
gca tac gat ctt tat gat tta gga gag ttc aat caa aag ggt act gtt 471
Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val
60 65 70
cga acg aaa tac gga act aag gca cag ctt gaa cga gct att ggg tcc 519
Arg Thr Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser
75 80 85
ctt aaa tct aat gat atc aat gta tac gga gat gtc gtg atg aat cat 567
Leu Lys Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His
90 95 100 105
aaa atg gga gct gat ttt acg gag gca gtg caa gct gtt caa gta aat 615
Lys Met Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn
110 115 120
cca acg aat cgt tgg cag gat att tca ggt gcc tac acg att gat gcg 663
Pro Thr Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala
125 130 135
tgg acg ggt ttc gac ttt tca ggg cgt aac aac gcc tat tca gat ttt 711
Trp Thr Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe
140 145 150
aag tgg aga tgg ttc cat ttt aat ggt gtt gac tgg gat eag cgc tat 759
Lys Trp Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr
155 160 165
caa gaa aat cat att ttc cgc ttt gca aat acg aac tgg aac tgg cga 807
Gln Glu Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg
170 175 180 185
gtg gat gaa gag aac ggt aat tat gat tac ctg tta gga tcg aat atc 855
Val Asp Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile
190 195 200
gac ttt agt cat cca gaa gta caa gat gag ttg aag gat tgg ggt agc 903
Asp Phe Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser
205 210 215
tgg ttt acc gat gag tta gat ttg gat ggt tat cgt tta gat gct att 951
Trp Phe Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile
220 225 230
aaa cat att cca ttc tgg tat aca tct gat tgg gtt cgg cat cag cgc 999
Lys His Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg
235 240 245
aac gaa gca gat caa gat tta ttt gtc gta ggg gaa tat tgg aag gat 1047
Asn Glu Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp
250 255 260 265
gac gta ggt gct ctc gaa ttt tat tta gat gaa atg aat tgg gag atg 1095
Asp Val Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met
270 275 280
tct cta ttc gat gtt cca ctt aat tat aat ttt tac cgg gct tca caa 1143
Ser Leu Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln
285 290 295
caa ggt gga agc tat gat atg cgt aat att tta cga gga tct tta gta 1191
Gln Gly Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val
300 305 310
gaa gcg cat ccg atg cat gca gtt acg ttt gtt gat aat cat gat act 1239
Glu Ala His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr
315 320 325
cag cca ggg gag tca tta gag tca tgg gtt gct gat tgg ttt aag cca 1287
Gln Pro Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro
330 335 340 345
ctt gct tat gcg aca att ttg acg cgt gaa ggt ggt tat cca aat gta 1335
Leu Ala Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val
350 355 360
ttt tac ggt gat tac tat ggg att cct aac gat aac att tca gct aaa 1383
Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys
365 370 375
aaa gat atg att gat gag ctg ctt gat gca cgt caa aat tac gca tat 1431
Lys Asp Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr
380 385 390
ggc acg cag cat gac tat ttt gat cat tgg gat gtt gta gga tgg act 1479
Gly Thr Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr
395 400 405
aag gaa gga tct tcc tcc aga cct aat tca ggc ctt gcg act att atg 1527
Arg Glu Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met
410 415 420 425
tcg aat gga cct ggt ggt tcc aag tgg atg tat gta gga cgt cag aat 1575
Ser Asn Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn
430 435 440
gca gga caa aca tgg aca gat tta act ggt aat aac gga gcg tcc gtt 1623
Ala Gly Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val
445 450 455
aca att aat ggc gat gga tgg ggc gaa ttc ttt acg aat gga gga tct 1671
Thr Ile Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser
460 465 470
gta tcc gtg tac gtg aac caa taacaaaaag ccttgagaag ggattcctcc 1722
Val Ser Val Tyr Val Asn Gln
475 480
ctaactcaag gctttcttta tgt 1745
<210>27
<211>501
<212>PRT
<213>芽孢杆菌属菌种(Bacillus sp.)
<400>27
Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro
-20 -15 -10
Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr
-5 -1 1 5 10
Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp
15 20 25
Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro
30 35 40
Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr
45 50 55
Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
60 65 70 75
Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys
80 85 90
Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Met
95 100 105
Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr
110 115 120
Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr
125 130 135
Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp
140 145 150 155
Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu
160 165 170
Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp
175 180 185
Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe
190 195 200
Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe
205 210 215
Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His
220 225 230 235
Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu
240 245 250
Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val
255 260 265
Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu
270 275 280
Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly
285 290 295
Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala
300 305 310 315
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro
320 325 330
Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala
335 340 345
Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr
350 355 360
Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp
365 370 375
Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr
380 385 390 395
Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr Arg Glu
400 405 410
Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn
415 420 425
Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly
430 435 440
Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile
445 450 455
Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser
460 465 470 475
Val Tyr Val Asn Gln
480
<210>28
<211>1920
<212>DNA
<213>地衣芽孢杆菌(Bacillus licheniformis)
<220>
<221>CDS
<222>(421)..(1872)
<400>28
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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
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Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
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130 135 140
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Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
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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
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Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
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Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
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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 Gln
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
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Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
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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
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Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
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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
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gtt caa aga tag aagagcagag aggacggatt tcctgaagga aatccgtttt 1912
Val Gln Arg
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Claims (28)
1.一种亲本Termamyl样α-淀粉酶的变体，所述变体包含位点207的改变：
其中
(a)所述改变分别是
(i)在紧邻该位点的氨基酸下游的氨基酸插入，
(ii)占据该位点的氨基酸的缺失，或
(iii)以不同的氨基酸取代占据该位点的氨基酸，
(b)变体具有α-淀粉酶活性，和
(c)每个位点相应于具有SEQ ID NO：8所示的氨基酸序列的亲本Termamyl样α-淀粉酶的氨基酸序列的位点。
2.权利要求1所述的变体，其中该变体具有下述的突变：
D207V，L，G；使用SEQ ID NO：8的编号。
3.权利要求1或2所述的变体，其中该变体具有下述的突变：K170Q+D207V+N280S；E132A+D207V；D207V+L318M；D60N+D207V+L318M；T49I+K176R+D207V+Y402F；使用SEQ ID NO：8的编号。
4.权利要求1-3中任一项所述的变体，其中亲本Termamyl样α-淀粉酶具有SEQ ID NO：8所示氨基酸序列且衍生于地衣芽孢杆菌的菌株，亲本Tcrmamyl样α-淀粉酶具有SEQ ID NO：10所示氨基酸序列且衍生于解淀粉芽孢杆菌的菌株，亲本Termamyl样α-淀粉酶具有SEQ ID NO：6所示氨基酸序列且衍生于嗜热脂肪芽孢杆菌的菌株。
5.权利要求1-4中任一项所述的变体，其中亲本Termamyl样α-淀粉酶是：LE174；LE174+G48A+T49I+G107A+I201F；LE174+M197L；或LE174+G48A+T49I+G107A+M197L+I201F。
6.权利要求1中所述的变体，其中该变体在下述位点发生突变：X212V，L，G；使用SEQ ID NO：4的编号。
7.权利要求1或6中所述的变体，该变体具有下述的突变：E212V+N285S；D134A+E212V；E212V+X323M；D62N+E212V+X323M；T51I+K179R+E212V+Y404F；或N195F+X212V+K269S，使用SEQ ID NO：4的编号。
8.权利要求1-7中任一项所述的变体，其中亲本Termamyl样α-淀粉酶选自：SP690(SEQ ID NO：2)；SP722(SEQ ID NO：4)；AA560(SEQ ID NO：12)；#707α-淀粉酶(SEQ ID NO：13)；和KSM-AP1378。
9.权利要求1-8中任一项所述的变体，其中亲本Termamyl样α-淀粉酶是下述任何一种：SP722+D183*+G184*；SP722+D183*+G184*+N195F；SP722+D183*+G184*+M202L；SP722+D183*+G184*+N195F+M202L；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；和AA560+D183*+G184*+N195F+M202L。
10.权利要求1-9中任一项所述的变体，其中亲本Termamyl样α-淀粉酶由核酸序列编码，该核酸序列与SEQ ID NO：7的核酸序列可在低严谨度条件下、中度严谨度条件下或高严谨度条件下杂交。
11.权利要求1-10中任一项所述的变体，其中变体在70-120℃高温和/或pH 4-6的低pH条件下具有改变的稳定性。
12.一种DNA构建体，包含编码权利要求1-11中任一项所述的α-淀粉酶变体的DNA序列。
13.一种重组表达载体，其携带权利要求12的DNA构建体。
14.一种由权利要求12的DNA构建体或权利要求13的载体转化的细胞。
15.权利要求14的细胞，其是微生物。
16.权利要求15的细胞，其是细菌或真菌。
17.权利要求16的细胞，其是革兰氏阳性细菌。
18.权利要求17的细胞，所述革兰氏阳性细菌是枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌、短芽孢杆菌、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌、解淀粉芽孢杆菌、凝结芽孢杆菌、环状芽孢杆菌、灿烂芽孢杆菌、或苏云金芽孢杆菌。
19.一种组合物，其包含权利要求1-11中任一项所述的α-淀粉酶变体。
20.权利要求19的组合物，其中所述α-淀粉酶是SP961。
21.权利要求19-20中任一项所述的组合物，其中组合物进一步包括葡糖淀粉酶、支链淀粉酶和/或植酸酶。
22.一种洗涤剂组合物，包含权利要求1-11中任一项所述的α-淀粉酶变体。
23.权利要求22的洗涤剂组合物，还包含另一种酶。
24.权利要求23的洗涤剂组合物，其中所述另一种酶是脂酶、过氧化物酶、另一种淀粉分解酶、葡糖淀粉酶、麦芽糖淀粉酶、环糊精糖基转移酶、甘露聚糖酶、角质酶、漆酶、和/或纤维素酶。
25.权利要求1-11中任一项所述α-淀粉酶变体或权利要求19-24中任一项所述的组合物在淀粉液化中的用途。
26.权利要求1-11中任一项所述α-淀粉酶变体或权利要求19-24中任一项所述组合物在乙醇生产中的用途。
27.权利要求1-11中任一项所述α-淀粉酶变体或权利要求19-24中任一项所述组合物在清洗和/或餐具清洗中的用途。
28.权利要求1-11中任一项所述α-淀粉酶变体或权利要求19-24中任一项所述组合物在纺织品退浆中的用途。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103649307A (zh) * | 2011-06-30 | 2014-03-19 | 诺维信公司 | α-淀粉酶变体 |
| CN103789285A (zh) * | 2014-02-19 | 2014-05-14 | 江南大学 | 一种热稳定性提高的淀粉酶突变体及其构建方法和应用 |
| CN103805579A (zh) * | 2014-02-19 | 2014-05-21 | 江南大学 | 一种热稳定的淀粉酶突变体及其制备方法和应用 |
| CN103834606A (zh) * | 2014-01-16 | 2014-06-04 | 北京中科星冠生物技术有限责任公司 | 一种表达耐酸高温α-淀粉酶基因突变体的工程菌株 |
Families Citing this family (444)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1975229A3 (en) * | 2000-10-13 | 2009-03-18 | Novozymes A/S | Alpha-amylase variant with altered properties |
| AU2002210380A1 (en) * | 2000-10-13 | 2002-04-22 | Novozymes A/S | Alpha-amylase variant with altered properties |
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| CN100415879C (zh) * | 2005-06-22 | 2008-09-03 | 天津科技大学 | 耐酸耐高温的α-淀粉酶及其制备方法 |
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| WO2009061379A2 (en) * | 2007-11-05 | 2009-05-14 | Danisco Us Inc., Genencor Division | Alpha-amylase variants with altered properties |
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| EP2149786A1 (en) | 2008-08-01 | 2010-02-03 | Unilever PLC | Improvements relating to detergent analysis |
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| US20120252086A1 (en) | 2009-12-22 | 2012-10-04 | Novozymes A/S | Compositions Comprising Boosting Polypeptide And Starch Degrading Enzyme And Uses Thereof |
| CN102918149A (zh) | 2010-01-04 | 2013-02-06 | 诺维信公司 | α-淀粉酶 |
| US20130040354A1 (en) | 2010-01-29 | 2013-02-14 | Novozymes A/S | Biogas Production Process With Enzymatic Pre-Treatment |
| WO2011100161A1 (en) | 2010-02-09 | 2011-08-18 | Novozymes North America, Inc. | Addition of alpha - glucosidase and cobalt for producing fermentation products from starch |
| US9896673B2 (en) * | 2010-02-10 | 2018-02-20 | Novozymes A/S | Compositions of high stability alpha amylase variants |
| EP2357220A1 (en) * | 2010-02-10 | 2011-08-17 | The Procter & Gamble Company | Cleaning composition comprising amylase variants with high stability in the presence of a chelating agent |
| ES2636216T3 (es) | 2010-03-30 | 2017-10-05 | Novozymes North America, Inc. | Procesos de producción de un producto de fermentación |
| MX338068B (es) | 2010-04-14 | 2016-04-01 | Novozymes As | Polipeptidos que tienen actividad de glucoamilasa y polinucleotidos que codifican los mismos. |
| CN103140586A (zh) | 2010-08-02 | 2013-06-05 | 诺维信北美公司 | 产生发酵产物的方法 |
| GB201015672D0 (en) | 2010-09-20 | 2010-10-27 | Unilever Plc | Improvements relating to fabric treatment compositions comprising targeted benefit agents |
| DK2637515T3 (da) | 2010-11-08 | 2017-11-27 | Novozymes As | Polypeptider med glucoamylaseaktivitet og polynukleotider, som koder for dem |
| US9732332B2 (en) | 2010-11-08 | 2017-08-15 | Novozymes A/S | Polypeptides having glucoamylase activity and polynucleotides encoding same |
| EP2640840B1 (en) | 2010-11-19 | 2018-05-30 | Novozymes North America, Inc. | Process for producing a fermentation product employing an amino acid oxidase, an arginase and/or an asparaginase |
| EP2661499A1 (en) | 2011-01-04 | 2013-11-13 | Novozymes A/S | Process for producing biogas from pectin and lignocellulose containing material |
| ES2531247T3 (es) | 2011-02-07 | 2015-03-12 | Novozymes North America Inc | Proceso para licuefacción de almidón en presencia de piridoxamina |
| US9228284B2 (en) | 2011-02-15 | 2016-01-05 | Novozymes North America, Inc. | Mitigation of odor in cleaning machines and cleaning processes |
| US20140106427A1 (en) | 2011-06-28 | 2014-04-17 | Novozymes A/S | Biogas From Enzyme-Treated Bagasse |
| BR112013033524A2 (pt) * | 2011-06-30 | 2017-02-07 | Novozymes As | método para rastrear alfa-amilases, método para selecionar variantes de uma alfa-amilase genitora, polipeptídeo e alfa-amilase variantes, variante, composição detergente, e, utilização de uma alfa-amilase variante |
| EP2540824A1 (en) * | 2011-06-30 | 2013-01-02 | The Procter & Gamble Company | Cleaning compositions comprising amylase variants reference to a sequence listing |
| WO2013006756A2 (en) | 2011-07-06 | 2013-01-10 | Novozymes A/S | Alpha amylase variants and polynucleotides encoding same |
| DK2734633T3 (da) | 2011-07-22 | 2019-06-11 | Novozymes North America Inc | Fremgangsmåder til forbehandling af celluloseholdigt materiale og forbedring af hydrolyse deraf |
| CA2846690A1 (en) | 2011-08-26 | 2013-03-07 | Novozymes A/S | Polypeptides having glucoamylase activity and polynucleotides encoding same |
| EP2748315B1 (en) | 2011-09-06 | 2017-11-15 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
| EP2766476B1 (en) | 2011-10-11 | 2017-05-31 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
| CN113930458A (zh) | 2011-10-11 | 2022-01-14 | 诺维信北美公司 | 用于产生发酵产物的方法 |
| CA2852601C (en) | 2011-10-17 | 2023-05-23 | Novozymes A/S | Alpha-amylase variants and polynucleotides encoding same |
| CA2852603A1 (en) | 2011-10-17 | 2013-04-25 | Novozymes A/S | Alpha-amylase variants and polynucleotides encoding same |
| US20140287477A1 (en) | 2011-10-28 | 2014-09-25 | Danisco Us Inc. | Variant Maltohexaose-Forming Alpha-Amylase Variants |
| ES2644727T3 (es) | 2011-12-02 | 2017-11-30 | Novozymes A/S | Proceso de licuefacción con alfa-amilasas y proteasas seleccionadas |
| WO2013083801A2 (en) | 2011-12-09 | 2013-06-13 | Novozymes A/S | Biogas from substrates comprising animal manure and enzymes |
| WO2013096652A1 (en) | 2011-12-21 | 2013-06-27 | Novozymes, Inc. | Methods for determining the degradation of a biomass material |
| CN104350149A (zh) | 2012-01-26 | 2015-02-11 | 诺维信公司 | 具有蛋白酶活性的多肽在动物饲料和洗涤剂中的用途 |
| EP2859110B1 (en) | 2012-03-30 | 2022-10-26 | Novozymes North America, Inc. | Processes of producing fermentation products |
| AR090971A1 (es) | 2012-05-07 | 2014-12-17 | Novozymes As | Polipeptidos que tienen actividad de degradacion de xantano y polinucleotidos que los codifican |
| CN104302753A (zh) | 2012-05-16 | 2015-01-21 | 诺维信公司 | 包括脂肪酶的组合物及其使用方法 |
| AU2013279440B2 (en) | 2012-06-20 | 2016-10-06 | Novozymes A/S | Use of polypeptides having protease activity in animal feed and detergents |
| US9828595B2 (en) | 2012-08-17 | 2017-11-28 | Novozymes A/S | Thermostable asparaginase variants and polynucleotides encoding same |
| US9315791B2 (en) | 2012-08-22 | 2016-04-19 | Novozymes A/S | Metalloproteases from alicyclobacillus |
| CN104619838A (zh) | 2012-08-22 | 2015-05-13 | 诺维信公司 | 来自微小杆菌属的金属蛋白酶 |
| TR201910918T4 (tr) | 2012-12-07 | 2019-08-21 | Novozymes As | Bakterilerin yapışmasının önlenmesi. |
| US9551042B2 (en) | 2012-12-21 | 2017-01-24 | Novozymes A/S | Polypeptides having protease activity and polynucleotides encoding same |
| WO2014106593A1 (en) | 2013-01-03 | 2014-07-10 | Novozymes A/S | Alpha-amylase variants and polynucleotides encoding same |
| WO2014173980A2 (en) | 2013-04-23 | 2014-10-30 | Novozymes A/S | Liquid automatic dish washing detergent compositions |
| ES2835313T3 (es) | 2013-04-30 | 2021-06-22 | Novozymes As | Variantes de glucoamilasa y polinucleótidos que las codifican |
| WO2014177541A2 (en) | 2013-04-30 | 2014-11-06 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
| EP2992076B1 (en) | 2013-05-03 | 2018-10-24 | Novozymes A/S | Microencapsulation of detergent enzymes |
| MY192746A (en) | 2013-05-14 | 2022-09-06 | Novozymes As | Detergent compositions |
| CN105209613A (zh) | 2013-05-17 | 2015-12-30 | 诺维信公司 | 具有α淀粉酶活性的多肽 |
| EP3004314B1 (en) | 2013-05-29 | 2018-06-20 | Danisco US Inc. | Novel metalloproteases |
| JP2016526880A (ja) | 2013-05-29 | 2016-09-08 | ダニスコ・ユーエス・インク | 新規メタロプロテアーゼ |
| JP6367930B2 (ja) | 2013-05-29 | 2018-08-01 | ダニスコ・ユーエス・インク | 新規メタロプロテアーゼ |
| WO2014194054A1 (en) | 2013-05-29 | 2014-12-04 | Danisco Us Inc. | Novel metalloproteases |
| EP3004315A2 (en) | 2013-06-06 | 2016-04-13 | Novozymes A/S | Alpha-amylase variants and polynucleotides encoding same |
| PE20160799A1 (es) | 2013-06-12 | 2016-09-03 | Earth Alive Clean Tech Inc | Supresor de polvo |
| US10617135B2 (en) | 2013-06-26 | 2020-04-14 | Novozymes A/S | Process for manufacturing a feed composition |
| EP3013955A1 (en) | 2013-06-27 | 2016-05-04 | Novozymes A/S | Subtilase variants and polynucleotides encoding same |
| CN105874067A (zh) | 2013-06-27 | 2016-08-17 | 诺维信公司 | 枯草杆菌酶变体以及编码它们的多核苷酸 |
| US20160152925A1 (en) | 2013-07-04 | 2016-06-02 | Novozymes A/S | Polypeptides Having Anti-Redeposition Effect and Polynucleotides Encoding Same |
| WO2015007639A1 (en) | 2013-07-17 | 2015-01-22 | Novozymes A/S | Pullulanase chimeras and polynucleotides encoding same |
| WO2015014803A1 (en) | 2013-07-29 | 2015-02-05 | Novozymes A/S | Protease variants and polynucleotides encoding same |
| WO2015014790A2 (en) | 2013-07-29 | 2015-02-05 | Novozymes A/S | Protease variants and polynucleotides encoding same |
| EP3063282B1 (en) | 2013-09-11 | 2020-03-25 | Novozymes A/S | Processes for producing fermentation products |
| WO2015049370A1 (en) | 2013-10-03 | 2015-04-09 | Novozymes A/S | Detergent composition and use of detergent composition |
| DK3080262T3 (da) | 2013-12-13 | 2019-05-06 | Danisco Us Inc | Serinproteaser af bacillus-arter |
| EP3910057A1 (en) | 2013-12-13 | 2021-11-17 | Danisco US Inc. | Serine proteases of the bacillus gibsonii-clade |
| MX2016007920A (es) | 2013-12-18 | 2016-09-13 | Du Pont | Eteres de poli-alfa-1,3-glucano cationicos. |
| US10030239B2 (en) | 2013-12-20 | 2018-07-24 | Novozymes A/S | Polypeptides having protease activity and polynucleotides encoding same |
| HUE056192T2 (hu) | 2014-01-22 | 2022-01-28 | Novozymes As | Pullulanázvariánsok és azokat kódoló polinukleotidok |
| WO2015123323A1 (en) | 2014-02-14 | 2015-08-20 | E. I. Du Pont De Nemours And Company | Poly-alpha-1,3-1,6-glucans for viscosity modification |
| WO2015134737A1 (en) | 2014-03-05 | 2015-09-11 | Novozymes A/S | Compositions and methods for improving properties of cellulosic textile materials with xyloglucan endotransglycosylase |
| WO2015134729A1 (en) | 2014-03-05 | 2015-09-11 | Novozymes A/S | Compositions and methods for improving properties of non-cellulosic textile materials with xyloglucan endotransglycosylase |
| MX2016011467A (es) | 2014-03-11 | 2016-11-16 | Du Pont | Poli alfa-1,3-glucano oxidado como mejorador de detergente. |
| WO2015143144A1 (en) | 2014-03-19 | 2015-09-24 | Novozymes A/S | Method for enhancing activity of an x143 polypeptide |
| CN106170546A (zh) | 2014-03-21 | 2016-11-30 | 丹尼斯科美国公司 | 芽孢杆菌属的丝氨酸蛋白酶 |
| EP3126479A1 (en) | 2014-04-01 | 2017-02-08 | Novozymes A/S | Polypeptides having alpha amylase activity |
| WO2015157656A1 (en) * | 2014-04-10 | 2015-10-15 | Novozymes A/S | Alpha-amylase variants and polynucleotides encoding same |
| DK3129457T3 (en) | 2014-04-11 | 2018-09-17 | Novozymes As | detergent |
| AR100606A1 (es) | 2014-05-27 | 2016-10-19 | Novozymes As | Variantes de lipasas y polinucleótidos que las codifican |
| EP3878957A1 (en) | 2014-05-27 | 2021-09-15 | Novozymes A/S | Methods for producing lipases |
| CN113528489A (zh) * | 2014-06-12 | 2021-10-22 | 诺维信公司 | α-淀粉酶变体 |
| EP3155097A1 (en) | 2014-06-12 | 2017-04-19 | Novozymes A/S | Alpha-amylase variants and polynucleotides encoding same |
| WO2015195777A1 (en) | 2014-06-19 | 2015-12-23 | E. I. Du Pont De Nemours And Company | Compositions containing one or more poly alpha-1,3-glucan ether compounds |
| US9714403B2 (en) | 2014-06-19 | 2017-07-25 | E I Du Pont De Nemours And Company | Compositions containing one or more poly alpha-1,3-glucan ether compounds |
| EP3164476A1 (en) | 2014-07-03 | 2017-05-10 | Novozymes A/S | Improved stabilization of non-protease enzyme |
| WO2016001450A2 (en) | 2014-07-04 | 2016-01-07 | Novozymes A/S | Subtilase variants and polynucleotides encoding same |
| BR112017000102B1 (pt) | 2014-07-04 | 2023-11-07 | Novozymes A/S | Variantes de subtilase de uma subtilase progenitora, composição detergente, uso da mesma e método para a produção de uma variante de subtilase que tem atividade de protease |
| US20170233710A1 (en) | 2014-10-17 | 2017-08-17 | Danisco Us Inc. | Serine proteases of bacillus species |
| EP3209788B1 (en) | 2014-10-23 | 2019-06-05 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
| DK3212781T3 (da) | 2014-10-27 | 2019-12-16 | Danisco Us Inc | Serinproteaser |
| EP3212783B1 (en) | 2014-10-27 | 2024-06-26 | Danisco US Inc. | Serine proteases |
| EP3212782B1 (en) | 2014-10-27 | 2019-04-17 | Danisco US Inc. | Serine proteases |
| CN107148472A (zh) | 2014-10-27 | 2017-09-08 | 丹尼斯科美国公司 | 芽孢杆菌属物种的丝氨酸蛋白酶 |
| EP3212662B1 (en) | 2014-10-27 | 2020-04-08 | Danisco US Inc. | Serine proteases |
| WO2016079110A2 (en) | 2014-11-19 | 2016-05-26 | Novozymes A/S | Use of enzyme for cleaning |
| US10287562B2 (en) | 2014-11-20 | 2019-05-14 | Novoszymes A/S | Alicyclobacillus variants and polynucleotides encoding same |
| CN107075493B (zh) | 2014-12-04 | 2020-09-01 | 诺维信公司 | 枯草杆菌酶变体以及编码它们的多核苷酸 |
| EP4067485A3 (en) | 2014-12-05 | 2023-01-04 | Novozymes A/S | Lipase variants and polynucleotides encoding same |
| US10760036B2 (en) | 2014-12-15 | 2020-09-01 | Henkel Ag & Co. Kgaa | Detergent composition comprising subtilase variants |
| WO2016096996A1 (en) | 2014-12-16 | 2016-06-23 | Novozymes A/S | Polypeptides having n-acetyl glucosamine oxidase activity |
| EP3034589A1 (en) | 2014-12-17 | 2016-06-22 | The Procter and Gamble Company | Detergent composition |
| EP3034597A1 (en) | 2014-12-17 | 2016-06-22 | The Procter and Gamble Company | Detergent composition |
| EP3034592A1 (en) | 2014-12-17 | 2016-06-22 | The Procter and Gamble Company | Method of automatic dishwashing |
| EP3034590A1 (en) | 2014-12-17 | 2016-06-22 | The Procter and Gamble Company | Method of automatic dishwashing |
| EP3034591A1 (en) | 2014-12-17 | 2016-06-22 | The Procter and Gamble Company | Method of automatic dishwashing |
| EP3034596B2 (en) | 2014-12-17 | 2021-11-10 | The Procter & Gamble Company | Detergent composition |
| EP3034588B1 (en) | 2014-12-17 | 2019-04-24 | The Procter and Gamble Company | Detergent composition |
| WO2016097352A1 (en) | 2014-12-19 | 2016-06-23 | Novozymes A/S | Protease variants and polynucleotides encoding same |
| ES2813727T3 (es) | 2014-12-19 | 2021-03-24 | Novozymes As | Variantes de proteasa y polinucleótidos que las codifican |
| CA2969241A1 (en) | 2014-12-23 | 2016-06-30 | E.I. Du Pont De Nemours And Company | Enzymatically produced cellulose |
| EP3280791A1 (en) | 2015-04-10 | 2018-02-14 | Novozymes A/S | Laundry method, use of dnase and detergent composition |
| US20180105772A1 (en) | 2015-04-10 | 2018-04-19 | Novozymes A/S | Detergent composition |
| EP3872174B1 (en) | 2015-05-13 | 2023-03-01 | Danisco US Inc. | Aprl-clade protease variants and uses thereof |
| CN107835853B (zh) | 2015-05-19 | 2021-04-20 | 诺维信公司 | 气味减少 |
| ES2670044T3 (es) | 2015-06-04 | 2018-05-29 | The Procter & Gamble Company | Composición detergente líquida para lavado de vajillas a mano |
| EP3101108B1 (en) | 2015-06-04 | 2018-01-31 | The Procter and Gamble Company | Hand dishwashing liquid detergent composition |
| EP3101107B1 (en) | 2015-06-05 | 2019-04-24 | The Procter and Gamble Company | Compacted liquid laundry detergent composition |
| EP3101100B1 (en) | 2015-06-05 | 2018-02-07 | The Procter and Gamble Company | Compacted liquid laundry detergent composition |
| EP3101102B2 (en) | 2015-06-05 | 2023-12-13 | The Procter & Gamble Company | Compacted liquid laundry detergent composition |
| WO2016201069A1 (en) | 2015-06-09 | 2016-12-15 | Danisco Us Inc | Low-density enzyme-containing particles |
| WO2016201040A1 (en) | 2015-06-09 | 2016-12-15 | Danisco Us Inc. | Water-triggered enzyme suspension |
| DK3307427T3 (da) | 2015-06-09 | 2023-11-06 | Danisco Us Inc | Osmotisk sprængnings-kapsler |
| US10858637B2 (en) | 2015-06-16 | 2020-12-08 | Novozymes A/S | Polypeptides with lipase activity and polynucleotides encoding same |
| JP7015695B2 (ja) | 2015-06-17 | 2022-02-03 | ダニスコ・ユーエス・インク | バチルス・ギブソニイ（Ｂａｃｉｌｌｕｓ ｇｉｂｓｏｎｉｉ）クレードセリンプロテアーゼ |
| US10717576B2 (en) | 2015-06-17 | 2020-07-21 | Novozymes A/S | Container for polypeptide |
| WO2016205127A1 (en) | 2015-06-18 | 2016-12-22 | Novozymes A/S | Polypeptides having trehalase activity and the use thereof in process of producing fermentation products |
| WO2016202839A2 (en) | 2015-06-18 | 2016-12-22 | Novozymes A/S | Subtilase variants and polynucleotides encoding same |
| EP3106508B1 (en) | 2015-06-18 | 2019-11-20 | Henkel AG & Co. KGaA | Detergent composition comprising subtilase variants |
| CN107922896A (zh) | 2015-06-30 | 2018-04-17 | 诺维信公司 | 衣物洗涤剂组合物、用于洗涤的方法和组合物的用途 |
| CA3175255A1 (en) | 2015-07-01 | 2017-01-05 | Novozymes A/S | Methods of reducing odor |
| EP3320089B1 (en) | 2015-07-06 | 2021-06-16 | Novozymes A/S | Lipase variants and polynucleotides encoding same |
| CA2991114A1 (en) | 2015-09-17 | 2017-03-23 | Novozymes A/S | Polypeptides having xanthan degrading activity and polynucleotides encoding same |
| AU2016323412B2 (en) | 2015-09-17 | 2021-04-01 | Henkel Ag & Co. Kgaa | Detergent compositions comprising polypeptides having xanthan degrading activity |
| EP3359658A2 (en) | 2015-10-07 | 2018-08-15 | Novozymes A/S | Polypeptides |
| US20180171318A1 (en) | 2015-10-14 | 2018-06-21 | Novozymes A/S | Polypeptides Having Protease Activity and Polynucleotides Encoding Same |
| WO2017064269A1 (en) | 2015-10-14 | 2017-04-20 | Novozymes A/S | Polypeptide variants |
| US10675589B2 (en) | 2015-10-14 | 2020-06-09 | Novozymes A/S | Cleaning of water filtration membranes |
| US11028346B2 (en) | 2015-10-28 | 2021-06-08 | Novozymes A/S | Detergent composition comprising protease and amylase variants |
| US20180320158A1 (en) | 2015-11-05 | 2018-11-08 | Danisco Us Inc. | Paenibacillus and bacillus spp. mannanases |
| EP4141113A1 (en) | 2015-11-05 | 2023-03-01 | Danisco US Inc | Paenibacillus sp. mannanases |
| EP3374401B1 (en) | 2015-11-13 | 2022-04-06 | Nutrition & Biosciences USA 4, Inc. | Glucan fiber compositions for use in laundry care and fabric care |
| WO2017083228A1 (en) | 2015-11-13 | 2017-05-18 | E. I. Du Pont De Nemours And Company | Glucan fiber compositions for use in laundry care and fabric care |
| EP3374488B1 (en) | 2015-11-13 | 2020-10-14 | DuPont Industrial Biosciences USA, LLC | Glucan fiber compositions for use in laundry care and fabric care |
| CN108473974A (zh) | 2015-11-24 | 2018-08-31 | 诺维信公司 | 具有蛋白酶活性的多肽以及编码其的多核苷酸 |
| CN108431217B (zh) | 2015-12-01 | 2022-06-21 | 诺维信公司 | 用于产生脂肪酶的方法 |
| CA3003536A1 (en) | 2015-12-07 | 2017-06-15 | Novozymes A/S | Polypeptides having beta-glucanase activity, polynucleotides encoding same and uses thereof in cleaning and detergent compositions |
| CN108779448B (zh) | 2015-12-09 | 2023-08-18 | 丹尼斯科美国公司 | α-淀粉酶组合变体 |
| US20180362946A1 (en) | 2015-12-18 | 2018-12-20 | Danisco Us Inc. | Polypeptides with endoglucanase activity and uses thereof |
| EP3394275A1 (en) | 2015-12-22 | 2018-10-31 | Novozymes A/S | Process of extracting oil from thin stillage |
| EP3397061A1 (en) | 2015-12-28 | 2018-11-07 | Novozymes BioAG A/S | Heat priming of bacterial spores |
| US20210171927A1 (en) | 2016-01-29 | 2021-06-10 | Novozymes A/S | Beta-glucanase variants and polynucleotides encoding same |
| BR112018069220A2 (pt) | 2016-03-23 | 2019-01-22 | Novozymes As | uso de polipeptídeo que tem atividade de dnase para tratamento de tecidos |
| CN109312270B (zh) | 2016-04-08 | 2022-01-28 | 诺维信公司 | 洗涤剂组合物及其用途 |
| EP3693449A1 (en) | 2016-04-29 | 2020-08-12 | Novozymes A/S | Detergent compositions and uses thereof |
| CA3022875A1 (en) | 2016-05-03 | 2017-11-09 | Danisco Us Inc | Protease variants and uses thereof |
| EP3845642B1 (en) | 2016-05-05 | 2023-08-09 | Danisco US Inc. | Protease variants and uses thereof |
| CN109563450A (zh) | 2016-05-31 | 2019-04-02 | 诺维信公司 | 稳定的液体过氧化物组合物 |
| JP2019523645A (ja) | 2016-05-31 | 2019-08-29 | ダニスコ・ユーエス・インク | プロテアーゼ変異体およびその使用 |
| WO2017207762A1 (en) | 2016-06-03 | 2017-12-07 | Novozymes A/S | Subtilase variants and polynucleotides encoding same |
| JP7152319B2 (ja) | 2016-06-17 | 2022-10-12 | ダニスコ・ユーエス・インク | プロテアーゼ変異体およびその使用 |
| EP3257931A1 (en) | 2016-06-17 | 2017-12-20 | The Procter and Gamble Company | Detergent composition |
| EP3475404A1 (en) | 2016-06-23 | 2019-05-01 | Novozymes A/S | Use of enzymes, composition and method for removing soil |
| CN117721095A (zh) | 2016-06-30 | 2024-03-19 | 诺维信公司 | 脂肪酶变体及包含表面活性剂和脂肪酶变体的组合物 |
| WO2018002261A1 (en) | 2016-07-01 | 2018-01-04 | Novozymes A/S | Detergent compositions |
| WO2018007573A1 (en) | 2016-07-08 | 2018-01-11 | Novozymes A/S | Detergent compositions with galactanase |
| WO2018011276A1 (en) | 2016-07-13 | 2018-01-18 | The Procter & Gamble Company | Bacillus cibi dnase variants and uses thereof |
| US11326152B2 (en) | 2016-07-18 | 2022-05-10 | Novozymes A/S | Lipase variants, polynucleotides encoding same and the use thereof |
| EP3284805B1 (en) | 2016-08-17 | 2020-02-19 | The Procter & Gamble Company | Cleaning composition comprising enzymes |
| EP3504313A1 (en) | 2016-08-24 | 2019-07-03 | Henkel AG & Co. KGaA | Detergent composition comprising gh9 endoglucanase variants i |
| WO2018037064A1 (en) | 2016-08-24 | 2018-03-01 | Henkel Ag & Co. Kgaa | Detergent compositions comprising xanthan lyase variants i |
| WO2018037061A1 (en) | 2016-08-24 | 2018-03-01 | Novozymes A/S | Xanthan lyase variants and polynucleotides encoding same |
| EP3504330A1 (en) | 2016-08-24 | 2019-07-03 | Novozymes A/S | Gh9 endoglucanase variants and polynucleotides encoding same |
| WO2018060216A1 (en) | 2016-09-29 | 2018-04-05 | Novozymes A/S | Use of enzyme for washing, method for washing and warewashing composition |
| WO2018060475A1 (en) | 2016-09-29 | 2018-04-05 | Novozymes A/S | Spore containing granule |
| EP3532592A1 (en) | 2016-10-25 | 2019-09-04 | Novozymes A/S | Detergent compositions |
| US11753605B2 (en) | 2016-11-01 | 2023-09-12 | Novozymes A/S | Multi-core granules |
| WO2018085524A2 (en) | 2016-11-07 | 2018-05-11 | Danisco Us Inc | Laundry detergent composition |
| WO2018098381A1 (en) | 2016-11-23 | 2018-05-31 | Novozymes A/S | Improved yeast for ethanol production |
| KR20190086540A (ko) | 2016-12-01 | 2019-07-22 | 바스프 에스이 | 조성물 중 효소의 안정화 |
| EP3551740B1 (en) | 2016-12-12 | 2021-08-11 | Novozymes A/S | Use of polypeptides |
| CN110312794B (zh) | 2016-12-21 | 2024-04-12 | 丹尼斯科美国公司 | 吉氏芽孢杆菌进化枝丝氨酸蛋白酶 |
| CN110312795A (zh) | 2016-12-21 | 2019-10-08 | 丹尼斯科美国公司 | 蛋白酶变体及其用途 |
| EP3583210B1 (en) | 2017-03-15 | 2021-07-07 | Danisco US Inc. | Trypsin-like serine proteases and uses thereof |
| US20200040283A1 (en) | 2017-03-31 | 2020-02-06 | Danisco Us Inc | Delayed release enzyme formulations for bleach-containing detergents |
| WO2018184004A1 (en) | 2017-03-31 | 2018-10-04 | Danisco Us Inc | Alpha-amylase combinatorial variants |
| EP3601549A1 (en) | 2017-03-31 | 2020-02-05 | Novozymes A/S | Polypeptides having dnase activity |
| WO2018178061A1 (en) | 2017-03-31 | 2018-10-04 | Novozymes A/S | Polypeptides having rnase activity |
| US11208639B2 (en) | 2017-03-31 | 2021-12-28 | Novozymes A/S | Polypeptides having DNase activity |
| EP3606936B1 (en) | 2017-04-03 | 2021-10-20 | Novozymes A/S | Recovery process |
| WO2018185152A1 (en) | 2017-04-04 | 2018-10-11 | Novozymes A/S | Polypeptide compositions and uses thereof |
| WO2018185181A1 (en) | 2017-04-04 | 2018-10-11 | Novozymes A/S | Glycosyl hydrolases |
| EP3607039A1 (en) | 2017-04-04 | 2020-02-12 | Novozymes A/S | Polypeptides |
| EP3385362A1 (en) | 2017-04-05 | 2018-10-10 | Henkel AG & Co. KGaA | Detergent compositions comprising fungal mannanases |
| DK3385361T3 (da) | 2017-04-05 | 2019-06-03 | Ab Enzymes Gmbh | Detergentsammensætninger omfattende bakterielle mannanaser |
| EP3967756A1 (en) | 2017-04-06 | 2022-03-16 | Novozymes A/S | Detergent compositions and uses thereof |
| US10968416B2 (en) | 2017-04-06 | 2021-04-06 | Novozymes A/S | Cleaning compositions and uses thereof |
| EP3626809A1 (en) | 2017-04-06 | 2020-03-25 | Novozymes A/S | Cleaning compositions and uses thereof |
| BR112019020960A2 (pt) | 2017-04-06 | 2020-05-05 | Novozymes As | composições de limpeza e seus usos |
| EP3607043A1 (en) | 2017-04-06 | 2020-02-12 | Novozymes A/S | Cleaning compositions and uses thereof |
| US20200190437A1 (en) | 2017-04-06 | 2020-06-18 | Novozymes A/S | Cleaning compositions and uses thereof |
| WO2018184818A1 (en) | 2017-04-06 | 2018-10-11 | Novozymes A/S | Cleaning compositions and uses thereof |
| EP3607042A1 (en) | 2017-04-06 | 2020-02-12 | Novozymes A/S | Cleaning compositions and uses thereof |
| WO2018202846A1 (en) | 2017-05-05 | 2018-11-08 | Novozymes A/S | Compositions comprising lipase and sulfite |
| WO2018206535A1 (en) | 2017-05-08 | 2018-11-15 | Novozymes A/S | Carbohydrate-binding domain and polynucleotides encoding the same |
| EP3401385A1 (en) | 2017-05-08 | 2018-11-14 | Henkel AG & Co. KGaA | Detergent composition comprising polypeptide comprising carbohydrate-binding domain |
| AU2018275794A1 (en) | 2017-06-02 | 2019-12-05 | Novozymes A/S | Improved yeast for ethanol production |
| WO2018224544A1 (en) | 2017-06-08 | 2018-12-13 | Novozymes A/S | Compositions comprising polypeptides having cellulase activity and amylase activity, and uses thereof in cleaning and detergent compositions |
| EP4015524A1 (en) | 2017-06-28 | 2022-06-22 | Novozymes A/S | Polypeptides having trehalase activity and polynucleotides encoding same |
| JP2020527339A (ja) | 2017-06-30 | 2020-09-10 | ダニスコ・ユーエス・インク | 低凝集の酵素含有粒子 |
| WO2019002356A1 (en) | 2017-06-30 | 2019-01-03 | Novozymes A/S | ENZYMATIC SUSPENSION COMPOSITION |
| CN111094562A (zh) | 2017-08-08 | 2020-05-01 | 诺维信公司 | 具有海藻糖酶活性的多肽及其在产生发酵产物的方法中的用途 |
| CN111212906B (zh) * | 2017-08-18 | 2024-02-02 | 丹尼斯科美国公司 | α-淀粉酶变体 |
| EP3673056A1 (en) | 2017-08-24 | 2020-07-01 | Henkel AG & Co. KGaA | Detergent compositions comprising gh9 endoglucanase variants ii |
| CA3071078A1 (en) | 2017-08-24 | 2019-02-28 | Novozymes A/S | Xanthan lyase variants and polynucleotides encoding same |
| CN111278971A (zh) | 2017-08-24 | 2020-06-12 | 诺维信公司 | Gh9内切葡聚糖酶变体以及编码它们的多核苷酸 |
| US20210130744A1 (en) | 2017-08-24 | 2021-05-06 | Henkel Ag & Co. Kgaa | Detergent composition comprising xanthan lyase variants ii |
| EP3682014A1 (en) | 2017-09-15 | 2020-07-22 | Novozymes A/S | Enzyme blends and processes for improving the nutritional quality of animal feed |
| US20200277553A1 (en) | 2017-09-20 | 2020-09-03 | Novozymes A/S | Use of Enzymes for Improving Water Absorption And/Or Whiteness |
| EP3684899A1 (en) | 2017-09-22 | 2020-07-29 | Novozymes A/S | Novel polypeptides |
| US11286443B2 (en) | 2017-09-27 | 2022-03-29 | The Procter & Gamble Company | Detergent compositions comprising lipases |
| CA3073362A1 (en) | 2017-09-27 | 2019-04-04 | Novozymes A/S | Lipase variants and microcapsule compositions comprising such lipase variants |
| US11732221B2 (en) | 2017-10-02 | 2023-08-22 | Novozymes A/S | Polypeptides having mannanase activity and polynucleotides encoding same |
| US11746310B2 (en) | 2017-10-02 | 2023-09-05 | Novozymes A/S | Polypeptides having mannanase activity and polynucleotides encoding same |
| WO2019076834A1 (en) | 2017-10-16 | 2019-04-25 | Novozymes A/S | PELLETS WITH LOW DUST CONTENT |
| WO2019076800A1 (en) | 2017-10-16 | 2019-04-25 | Novozymes A/S | CLEANING COMPOSITIONS AND USES THEREOF |
| EP3697881A1 (en) | 2017-10-16 | 2020-08-26 | Novozymes A/S | Low dusting granules |
| EP3701037A1 (en) | 2017-10-23 | 2020-09-02 | Novozymes A/S | Processes for reducing lactic acid in a biofuel fermentation system |
| WO2019081515A1 (en) | 2017-10-24 | 2019-05-02 | Novozymes A/S | COMPOSITIONS COMPRISING POLYPEPTIDES HAVING MANNANASE ACTIVITY |
| US10781408B2 (en) | 2017-10-27 | 2020-09-22 | The Procter & Gamble Company | Detergent compositions comprising polypeptide variants |
| US11441136B2 (en) | 2017-10-27 | 2022-09-13 | Novozymes A/S | DNase variants |
| WO2019086532A1 (en) | 2017-11-01 | 2019-05-09 | Novozymes A/S | Methods for cleaning medical devices |
| DE102017125558A1 (de) | 2017-11-01 | 2019-05-02 | Henkel Ag & Co. Kgaa | Reinigungszusammensetzungen, die dispersine i enthalten |
| EP3704219B1 (en) | 2017-11-01 | 2024-01-10 | Novozymes A/S | Polypeptides and compositions comprising such polypeptides |
| DE102017125559A1 (de) | 2017-11-01 | 2019-05-02 | Henkel Ag & Co. Kgaa | Reinigungszusammensetzungen, die dispersine ii enthalten |
| DE102017125560A1 (de) | 2017-11-01 | 2019-05-02 | Henkel Ag & Co. Kgaa | Reinigungszusammensetzungen, die dispersine iii enthalten |
| CN111479919A (zh) | 2017-11-01 | 2020-07-31 | 诺维信公司 | 多肽以及包含此类多肽的组合物 |
| WO2019105781A1 (en) | 2017-11-29 | 2019-06-06 | Basf Se | Storage-stable enzyme preparations, their production and use |
| CN111373039A (zh) | 2017-11-29 | 2020-07-03 | 丹尼斯科美国公司 | 具有改善的稳定性的枯草杆菌蛋白酶变体 |
| WO2019110462A1 (en) | 2017-12-04 | 2019-06-13 | Novozymes A/S | Lipase variants and polynucleotides encoding same |
| US20210222148A1 (en) | 2017-12-21 | 2021-07-22 | Danisco Us Inc. | Enzyme-containing, hot-melt granules comprising a thermotolerant desiccant |
| CN113286871A (zh) | 2018-01-29 | 2021-08-20 | 诺维信公司 | 用于乙醇生产的氮利用提高的微生物 |
| BR112020016068A2 (pt) | 2018-02-08 | 2020-12-08 | Danisco Us Inc. | Partículas cera termicamente resistente matriz para encapsulamento de enzima |
| EP3749760A1 (en) | 2018-02-08 | 2020-12-16 | Novozymes A/S | Lipase variants and compositions thereof |
| CN111868239A (zh) | 2018-02-08 | 2020-10-30 | 诺维信公司 | 脂肪酶、脂肪酶变体及其组合物 |
| CA3107124A1 (en) | 2018-02-15 | 2019-08-22 | Novozymes A/S | Improved yeast for ethanol production |
| WO2019162000A1 (en) | 2018-02-23 | 2019-08-29 | Henkel Ag & Co. Kgaa | Detergent composition comprising xanthan lyase and endoglucanase variants |
| US20210002588A1 (en) | 2018-03-13 | 2021-01-07 | Novozymes A/S | Microencapsulation Using Amino Sugar Oligomers |
| EP3768835A1 (en) | 2018-03-23 | 2021-01-27 | Novozymes A/S | Subtilase variants and compositions comprising same |
| CN112262207B (zh) | 2018-04-17 | 2024-01-23 | 诺维信公司 | 洗涤剂组合物中包含碳水化合物结合活性的多肽及其在减少纺织品或织物中的褶皱的用途 |
| US11661592B2 (en) | 2018-04-19 | 2023-05-30 | Novozymes A/S | Stabilized endoglucanase variants |
| US11566239B2 (en) | 2018-04-19 | 2023-01-31 | Novozymes A/S | Stabilized cellulase variants |
| CN112368375A (zh) | 2018-04-26 | 2021-02-12 | 巴斯夫欧洲公司 | 脂肪酶 |
| US20210115422A1 (en) | 2018-05-03 | 2021-04-22 | Basf Se | Amylase enzymes |
| BR112020024347A2 (pt) | 2018-05-31 | 2021-02-23 | Novozymes A/S | processos para aumentar o crescimento e a produtividade de levedura |
| WO2019238761A1 (en) | 2018-06-15 | 2019-12-19 | Basf Se | Water soluble multilayer films containing wash active chemicals and enzymes |
| US20210363470A1 (en) | 2018-06-19 | 2021-11-25 | Danisco Us Inc | Subtilisin variants |
| US20210214703A1 (en) | 2018-06-19 | 2021-07-15 | Danisco Us Inc | Subtilisin variants |
| EP3814472A1 (en) | 2018-06-28 | 2021-05-05 | Novozymes A/S | Detergent compositions and uses thereof |
| WO2020002608A1 (en) | 2018-06-29 | 2020-01-02 | Novozymes A/S | Detergent compositions and uses thereof |
| WO2020002255A1 (en) | 2018-06-29 | 2020-01-02 | Novozymes A/S | Subtilase variants and compositions comprising same |
| EP3818139A1 (en) | 2018-07-02 | 2021-05-12 | Novozymes A/S | Cleaning compositions and uses thereof |
| WO2020007875A1 (en) | 2018-07-03 | 2020-01-09 | Novozymes A/S | Cleaning compositions and uses thereof |
| WO2020008024A1 (en) | 2018-07-06 | 2020-01-09 | Novozymes A/S | Cleaning compositions and uses thereof |
| US20210253981A1 (en) | 2018-07-06 | 2021-08-19 | Novozymes A/S | Cleaning compositions and uses thereof |
| CN112601818A (zh) | 2018-07-25 | 2021-04-02 | 诺维信公司 | 用于生产乙醇的表达酶的酵母 |
| US20230174962A1 (en) | 2018-07-31 | 2023-06-08 | Danisco Us Inc | Variant alpha-amylases having amino acid substitutions that lower the pka of the general acid |
| EP3844255A1 (en) | 2018-08-30 | 2021-07-07 | Danisco US Inc. | Enzyme-containing granules |
| US20220033737A1 (en) | 2018-09-27 | 2022-02-03 | Danisco Us Inc | Compositions for medical instrument cleaning |
| WO2020070063A2 (en) | 2018-10-01 | 2020-04-09 | Novozymes A/S | Detergent compositions and uses thereof |
| WO2020070209A1 (en) | 2018-10-02 | 2020-04-09 | Novozymes A/S | Cleaning composition |
| EP3861110A1 (en) | 2018-10-02 | 2021-08-11 | Novozymes A/S | Endonuclease 1 ribonucleases for cleaning |
| EP3861094A1 (en) | 2018-10-02 | 2021-08-11 | Novozymes A/S | Cleaning composition |
| WO2020070014A1 (en) | 2018-10-02 | 2020-04-09 | Novozymes A/S | Cleaning composition comprising anionic surfactant and a polypeptide having rnase activity |
| WO2020070199A1 (en) | 2018-10-03 | 2020-04-09 | Novozymes A/S | Polypeptides having alpha-mannan degrading activity and polynucleotides encoding same |
| WO2020070249A1 (en) | 2018-10-03 | 2020-04-09 | Novozymes A/S | Cleaning compositions |
| EP3677676A1 (en) | 2019-01-03 | 2020-07-08 | Basf Se | Compounds stabilizing amylases in liquids |
| BR112021006317A2 (pt) | 2018-10-05 | 2021-07-06 | Basf Se | preparação de enzima, processo para preparar uma preparação de enzima estável, métodos para reduzir perda da atividade amiolítica, de preparação de uma formulação detergente, para remover manchas sensíveis à amilase e para aumentar a estabilidade no armazenamento de uma formulação detergente líquida, usos de um composto e da preparação de enzima, e, formulação detergente |
| US20210395651A1 (en) | 2018-10-05 | 2021-12-23 | Basf Se | Compounds stabilizing hydrolases in liquids |
| MX2021003930A (es) | 2018-10-05 | 2021-06-04 | Basf Se | Compuestos estabilizadores de hidrolasas en liquidos. |
| US11807889B2 (en) | 2018-10-08 | 2023-11-07 | Novozymes A/S | Yeast expressing a heterologous phospholipase for ethanol production |
| CN112996894A (zh) | 2018-10-11 | 2021-06-18 | 诺维信公司 | 清洁组合物及其用途 |
| CN113166745A (zh) | 2018-10-12 | 2021-07-23 | 丹尼斯科美国公司 | 在螯合剂存在下具有可增强稳定性的突变的α-淀粉酶 |
| EP3647397A1 (en) | 2018-10-31 | 2020-05-06 | Henkel AG & Co. KGaA | Cleaning compositions containing dispersins iv |
| EP3647398B1 (en) | 2018-10-31 | 2024-05-15 | Henkel AG & Co. KGaA | Cleaning compositions containing dispersins v |
| CN109439607B (zh) * | 2018-11-22 | 2020-09-04 | 湖南汇升生物科技有限公司 | 一种麦芽糖淀粉酶生产菌株的应用 |
| WO2020112599A1 (en) | 2018-11-28 | 2020-06-04 | Danisco Us Inc | Subtilisin variants having improved stability |
| US20220056379A1 (en) | 2018-12-03 | 2022-02-24 | Novozymes A/S | Powder Detergent Compositions |
| US20220017844A1 (en) | 2018-12-03 | 2022-01-20 | Novozymes A/S | Low pH Powder Detergent Composition |
| EP3898919A1 (en) | 2018-12-21 | 2021-10-27 | Novozymes A/S | Detergent pouch comprising metalloproteases |
| CN113366103A (zh) | 2018-12-21 | 2021-09-07 | 诺维信公司 | 具有肽聚糖降解活性的多肽以及编码其的多核苷酸 |
| BR112021014873A2 (pt) | 2019-01-31 | 2021-10-05 | Novozymes A/S | Polipeptídeo, combinação de enzimas, polinucleotídeo, construto de ácido nucleico ou vetor de expressão recombinante, célula hospedeira recombinante, método de produção de um polipeptídeo, e, processo de produção de um produto de fermentação |
| EP3927837A1 (en) | 2019-02-20 | 2021-12-29 | Basf Se | Industrial fermentation process for bacillus using defined medium and magnesium feed |
| US20220186234A1 (en) | 2019-02-20 | 2022-06-16 | Basf Se | Industrial Fermentation Process for Bacillus Using Defined Medium and Trace Element Feed |
| EP3702452A1 (en) | 2019-03-01 | 2020-09-02 | Novozymes A/S | Detergent compositions comprising two proteases |
| US20220235341A1 (en) | 2019-03-21 | 2022-07-28 | Novozymes A/S | Alpha-amylase variants and polynucleotides encoding same |
| WO2020193532A1 (en) | 2019-03-25 | 2020-10-01 | Basf Se | Cleaning composition having amylase enzymes |
| EP3947665A2 (en) | 2019-03-25 | 2022-02-09 | Basf Se | Amylase enzymes |
| WO2020193534A2 (en) | 2019-03-25 | 2020-10-01 | Basf Se | Amylase enzymes |
| BR112021017085A2 (pt) | 2019-04-02 | 2022-02-08 | Novozymes As | Processo de produção de um produto de fermentação |
| MX2021011981A (es) | 2019-04-03 | 2021-11-03 | Novozymes As | Polipeptidos que tienen actividad de beta-glucanasa, polinucleotidos que los codifican y usos de los mismos en composiciones de limpieza y de detergente. |
| EP3953462A1 (en) | 2019-04-10 | 2022-02-16 | Novozymes A/S | Polypeptide variants |
| EP3953463A1 (en) | 2019-04-12 | 2022-02-16 | Novozymes A/S | Stabilized glycoside hydrolase variants |
| WO2020229480A1 (en) | 2019-05-14 | 2020-11-19 | Basf Se | Compounds stabilizing hydrolases in liquids |
| WO2020242858A1 (en) | 2019-05-24 | 2020-12-03 | Danisco Us Inc | Subtilisin variants and methods of use |
| CN114174486A (zh) | 2019-06-06 | 2022-03-11 | 丹尼斯科美国公司 | 用于清洁的方法和组合物 |
| MX2021015364A (es) | 2019-06-13 | 2022-01-24 | Basf Se | Metodo de recuperacion de una proteina de un caldo de fermentacion mediante el uso de un cation divalente. |
| EP3994255A1 (en) | 2019-07-02 | 2022-05-11 | Novozymes A/S | Lipase variants and compositions thereof |
| WO2021004830A1 (en) | 2019-07-05 | 2021-01-14 | Basf Se | Industrial fermentation process for microbial cells using a fed-batch pre-culture |
| EP3997202A1 (en) | 2019-07-12 | 2022-05-18 | Novozymes A/S | Enzymatic emulsions for detergents |
| WO2021021458A1 (en) | 2019-07-26 | 2021-02-04 | Novozymes A/S | Microorganisms with improved nitrogen transport for ethanol production |
| CA3144423A1 (en) | 2019-08-05 | 2021-02-11 | Kurt Creamer | Enzyme blends and processes for producing a high protein feed ingredient from a whole stillage byproduct |
| BR112021026477A2 (pt) | 2019-08-06 | 2022-02-08 | Novozymes As | Célula hospedeira recombinante, métodos de produção de um produto de fermentação a partir de um material contendo amido ou contendo celulose, de produção do polipeptídeo maduro, de produção de um derivado de uma célula hospedeira recombinante e de produção de etanol, construto de ácido nucleico ou vetor de expressão, composição, e, uso de uma célula hospedeira recombinante |
| WO2021032881A1 (en) | 2019-08-22 | 2021-02-25 | Basf Se | Amylase variants |
| WO2021037878A1 (en) | 2019-08-27 | 2021-03-04 | Novozymes A/S | Composition comprising a lipase |
| US20220325204A1 (en) | 2019-08-27 | 2022-10-13 | Novozymes A/S | Detergent composition |
| EP4031661A1 (en) | 2019-09-16 | 2022-07-27 | Novozymes A/S | Polypeptides having beta-glucanase activity and polynucleotides encoding same |
| WO2021053127A1 (en) | 2019-09-19 | 2021-03-25 | Novozymes A/S | Detergent composition |
| WO2021064068A1 (en) | 2019-10-03 | 2021-04-08 | Novozymes A/S | Polypeptides comprising at least two carbohydrate binding domains |
| BR112022006082A2 (pt) | 2019-10-18 | 2022-06-21 | Basf Se | Preparação enzimática, formulação de detergente, e, uso de pelo menos um diol |
| WO2021080948A2 (en) | 2019-10-24 | 2021-04-29 | Danisco Us Inc | Variant maltopentaose/maltohexaose-forming alpha-amylases |
| CN113891931A (zh) | 2019-11-29 | 2022-01-04 | 巴斯夫欧洲公司 | 组合物和可以用于该类组合物的聚合物 |
| WO2021115912A1 (en) | 2019-12-09 | 2021-06-17 | Basf Se | Formulations comprising a hydrophobically modified polyethyleneimine and one or more enzymes |
| AU2020402990A1 (en) | 2019-12-10 | 2022-06-09 | Novozymes A/S | Microorganism for improved pentose fermentation |
| WO2021126966A1 (en) | 2019-12-16 | 2021-06-24 | Novozymes A/S | Processes for producing fermentation products |
| KR20220119608A (ko) | 2019-12-20 | 2022-08-30 | 헨켈 아게 운트 코. 카게아아 | 디스페르신 viii을 포함하는 세정 조성물 |
| CN114929848A (zh) | 2019-12-20 | 2022-08-19 | 诺维信公司 | 稳定的液体无硼酶组合物 |
| WO2021123307A2 (en) | 2019-12-20 | 2021-06-24 | Novozymes A/S | Polypeptides having proteolytic activity and use thereof |
| EP4077620A1 (en) | 2019-12-20 | 2022-10-26 | Henkel AG & Co. KGaA | Cleaning compositions comprising dispersins vi |
| KR20220121235A (ko) | 2019-12-20 | 2022-08-31 | 헨켈 아게 운트 코. 카게아아 | 디스페르신 및 카르보히드라제를 포함하는 세정 조성물 |
| BR112022015120A2 (pt) | 2020-01-29 | 2022-12-13 | Unilever Ip Holdings B V | Recipiente plástico transparente e processo de fabricação de recipiente plástico transparente |
| JP2023511739A (ja) | 2020-01-31 | 2023-03-22 | ノボザイムス アクティーゼルスカブ | マンナナーゼバリアント及びそれをコードするポリヌクレオチド |
| EP4097226A1 (en) | 2020-01-31 | 2022-12-07 | Novozymes A/S | Mannanase variants and polynucleotides encoding same |
| EP4103709A2 (en) | 2020-02-10 | 2022-12-21 | Novozymes A/S | Polypeptides having alpha-amylase activity and polynucleotides encoding same |
| EP3892708A1 (en) | 2020-04-06 | 2021-10-13 | Henkel AG & Co. KGaA | Cleaning compositions comprising dispersin variants |
| JP2023520312A (ja) | 2020-04-08 | 2023-05-17 | ノボザイムス アクティーゼルスカブ | 炭水化物結合モジュールバリアント |
| WO2021214059A1 (en) | 2020-04-21 | 2021-10-28 | Novozymes A/S | Cleaning compositions comprising polypeptides having fructan degrading activity |
| EP3907271A1 (en) | 2020-05-07 | 2021-11-10 | Novozymes A/S | Cleaning composition, use and method of cleaning |
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| WO2022008732A1 (en) | 2020-07-10 | 2022-01-13 | Basf Se | Enhancing the activity of antimicrobial preservatives |
| EP4189051B1 (en) | 2020-07-27 | 2024-02-28 | Unilever IP Holdings B.V. | Use of an enzyme and surfactant for inhibiting microorganisms |
| JP2023538740A (ja) | 2020-08-25 | 2023-09-11 | ノボザイムス アクティーゼルスカブ | ファミリー４４キシログルカナーゼの変異体 |
| EP4204553A1 (en) | 2020-08-27 | 2023-07-05 | Danisco US Inc. | Enzymes and enzyme compositions for cleaning |
| JP2023538773A (ja) | 2020-08-28 | 2023-09-11 | ノボザイムス アクティーゼルスカブ | 改善された溶解性を有するプロテアーゼバリアント |
| EP4213641A1 (en) | 2020-09-15 | 2023-07-26 | Novozymes A/S | Animal feed comprising insects or insect meal |
| US20240052270A1 (en) | 2020-09-22 | 2024-02-15 | Basf Se | Liquid composition comprising peptide aldehyde |
| MX2023003275A (es) | 2020-09-22 | 2023-04-12 | Basf Se | Combinacion mejorada de proteasa e inhibidor de proteasa con enzima secundaria. |
| EP4225905A2 (en) | 2020-10-07 | 2023-08-16 | Novozymes A/S | Alpha-amylase variants |
| WO2022084303A2 (en) | 2020-10-20 | 2022-04-28 | Novozymes A/S | Use of polypeptides having dnase activity |
| US20230399588A1 (en) | 2020-10-28 | 2023-12-14 | Novozymes A/S | Use of lipoxygenase |
| EP4237552A2 (en) | 2020-10-29 | 2023-09-06 | Novozymes A/S | Lipase variants and compositions comprising such lipase variants |
| US20230399632A1 (en) | 2020-11-02 | 2023-12-14 | Novozymes A/S | Glucoamylase Variants and Polynucleotides Encoding Same |
| CN116670261A (zh) | 2020-11-13 | 2023-08-29 | 诺维信公司 | 包含脂肪酶的洗涤剂组合物 |
| WO2022106404A1 (en) | 2020-11-18 | 2022-05-27 | Novozymes A/S | Combination of proteases |
| WO2022106400A1 (en) | 2020-11-18 | 2022-05-27 | Novozymes A/S | Combination of immunochemically different proteases |
| EP4015629A1 (en) | 2020-12-18 | 2022-06-22 | Basf Se | Polymer mixtures for increasing stability and performance of hydrolase-containing detergents |
| EP4032966A1 (en) | 2021-01-22 | 2022-07-27 | Novozymes A/S | Liquid enzyme composition with sulfite scavenger |
| WO2022162043A1 (en) | 2021-01-28 | 2022-08-04 | Novozymes A/S | Lipase with low malodor generation |
| CN116997642A (zh) | 2021-01-29 | 2023-11-03 | 丹尼斯科美国公司 | 清洁组合物及其相关的方法 |
| EP4039806A1 (en) | 2021-02-04 | 2022-08-10 | Henkel AG & Co. KGaA | Detergent composition comprising xanthan lyase and endoglucanase variants with im-proved stability |
| WO2022171780A2 (en) | 2021-02-12 | 2022-08-18 | Novozymes A/S | Alpha-amylase variants |
| WO2022171872A1 (en) | 2021-02-12 | 2022-08-18 | Novozymes A/S | Stabilized biological detergents |
| MX2023009756A (es) | 2021-02-22 | 2023-09-04 | Basf Se | Variantes de amilasa. |
| EP4047088A1 (en) | 2021-02-22 | 2022-08-24 | Basf Se | Amylase variants |
| EP4305146A1 (en) | 2021-03-12 | 2024-01-17 | Novozymes A/S | Polypeptide variants |
| EP4060036A1 (en) | 2021-03-15 | 2022-09-21 | Novozymes A/S | Polypeptide variants |
| US20240060061A1 (en) | 2021-03-15 | 2024-02-22 | Novozymes A/S | Dnase variants |
| CN117083370A (zh) | 2021-03-26 | 2023-11-17 | 诺维信公司 | 聚合物含量降低的洗涤剂组合物 |
| WO2022261003A1 (en) | 2021-06-07 | 2022-12-15 | Novozymes A/S | Engineered microorganism for improved ethanol fermentation |
| EP4359518A1 (en) | 2021-06-23 | 2024-05-01 | Novozymes A/S | Alpha-amylase polypeptides |
| WO2023278297A1 (en) | 2021-06-30 | 2023-01-05 | Danisco Us Inc | Variant lipases and uses thereof |
| WO2023034486A2 (en) | 2021-09-03 | 2023-03-09 | Danisco Us Inc. | Laundry compositions for cleaning |
| WO2023039270A2 (en) | 2021-09-13 | 2023-03-16 | Danisco Us Inc. | Bioactive-containing granules |
| WO2023114936A2 (en) | 2021-12-16 | 2023-06-22 | Danisco Us Inc. | Subtilisin variants and methods of use |
| WO2023114932A2 (en) | 2021-12-16 | 2023-06-22 | Danisco Us Inc. | Subtilisin variants and methods of use |
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| WO2023117950A1 (en) | 2021-12-21 | 2023-06-29 | Basf Se | Chemical product with environmental attributes |
| WO2023116569A1 (en) | 2021-12-21 | 2023-06-29 | Novozymes A/S | Composition comprising a lipase and a booster |
| EP4206309A1 (en) | 2021-12-30 | 2023-07-05 | Novozymes A/S | Protein particles with improved whiteness |
| EP4234664A1 (en) | 2022-02-24 | 2023-08-30 | Evonik Operations GmbH | Composition comprising glucolipids and enzymes |
| WO2023168234A1 (en) | 2022-03-01 | 2023-09-07 | Danisco Us Inc. | Enzymes and enzyme compositions for cleaning |
| WO2023165507A1 (en) | 2022-03-02 | 2023-09-07 | Novozymes A/S | Use of xyloglucanase for improvement of sustainability of detergents |
| WO2023165950A1 (en) | 2022-03-04 | 2023-09-07 | Novozymes A/S | Dnase variants and compositions |
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| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
| WO2023233025A1 (en) | 2022-06-03 | 2023-12-07 | Unilever Ip Holdings B.V. | Liquid detergent product |
| WO2023250301A1 (en) | 2022-06-21 | 2023-12-28 | Danisco Us Inc. | Methods and compositions for cleaning comprising a polypeptide having thermolysin activity |
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| WO2023247664A2 (en) | 2022-06-24 | 2023-12-28 | Novozymes A/S | Lipase variants and compositions comprising such lipase variants |
| WO2024012894A1 (en) | 2022-07-15 | 2024-01-18 | Basf Se | Alkanolamine formates for enzyme stabilization in liquid formulations |
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| WO2024033136A1 (en) | 2022-08-11 | 2024-02-15 | Basf Se | Amylase variants |
| WO2024050346A1 (en) | 2022-09-02 | 2024-03-07 | Danisco Us Inc. | Detergent compositions and methods related thereto |
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| WO2024094732A1 (en) | 2022-11-04 | 2024-05-10 | Basf Se | Polypeptides having protease activity for use in detergent compositions |
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| WO2024094735A1 (en) | 2022-11-04 | 2024-05-10 | Basf Se | Polypeptides having protease activity for use in detergent compositions |
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| WO2024121070A1 (en) | 2022-12-05 | 2024-06-13 | Novozymes A/S | Protease variants and polynucleotides encoding same |
| WO2024121058A1 (en) | 2022-12-05 | 2024-06-13 | Novozymes A/S | A composition comprising a lipase and a peptide |
| WO2024126483A1 (en) | 2022-12-14 | 2024-06-20 | Novozymes A/S | Improved lipase (gcl1) variants |
| EP4389864A1 (en) | 2022-12-20 | 2024-06-26 | Basf Se | Cutinases |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1217020A (zh) * | 1996-04-30 | 1999-05-19 | 诺沃挪第克公司 | α－淀粉酶变体 |
Family Cites Families (35)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3912590A (en) | 1973-01-03 | 1975-10-14 | Novo Industri As | Procedure for liquefying starch |
| FR2498783B1 (fr) | 1981-01-23 | 1988-03-04 | Decis Mario | Dispositif de controle automatique de presence |
| JPS57174089A (en) | 1981-04-20 | 1982-10-26 | Novo Industri As | Chain dividing enzyme product |
| FR2543181B1 (fr) | 1983-03-22 | 1985-07-26 | Ugine Kuhlmann | Procede ameliore de desencollage-blanchiment simultane des tissus |
| US4760025A (en) | 1984-05-29 | 1988-07-26 | Genencor, Inc. | Modified enzymes and methods for making same |
| US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
| DK122686D0 (da) | 1986-03-17 | 1986-03-17 | Novo Industri As | Fremstilling af proteiner |
| DK311186D0 (da) | 1986-06-30 | 1986-06-30 | Novo Industri As | Enzymer |
| FI89076C (fi) | 1986-07-09 | 1993-08-10 | Novo Nordisk As | Alfa-amylasblandningar foer att oeverfoera staerkelse i vaetskeform och foerfarande foer oeverfoering av staerkelse i flytande form |
| BR9006818A (pt) | 1989-06-29 | 1991-08-06 | Gist Brocades Nv | Amilases microbianas mutantes com maior estabilidade termica,acida e/ou alcalina |
| WO1991017243A1 (en) | 1990-05-09 | 1991-11-14 | Novo Nordisk A/S | A cellulase preparation comprising an endoglucanase enzyme |
| US5231017A (en) | 1991-05-17 | 1993-07-27 | Solvay Enzymes, Inc. | Process for producing ethanol |
| EP0867504B2 (en) | 1993-02-11 | 2011-05-18 | Genencor International, Inc. | Oxidation-stable alpha-amylase |
| AU7807494A (en) | 1993-10-08 | 1995-05-04 | Novo Nordisk A/S | Amylase variants |
| MY111537A (en) | 1994-02-02 | 2000-07-31 | Novo Nordisk As | A combined desizing and bleaching process. |
| US5605793A (en) | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
| MX196038B (es) | 1994-03-29 | 2000-04-14 | Novo Nordisk As | Amilasa de bacillus alcalino. |
| DE4422198C2 (de) | 1994-06-24 | 1997-08-28 | Audi Ag | Verfahren zum Steuern der elektrischen Beheizung eines Katalysators |
| AR000862A1 (es) * | 1995-02-03 | 1997-08-06 | Novozymes As | Variantes de una ó-amilasa madre, un metodo para producir la misma, una estructura de adn y un vector de expresion, una celula transformada por dichaestructura de adn y vector, un aditivo para detergente, composicion detergente, una composicion para lavado de ropa y una composicion para la eliminacion del |
| DE69637940D1 (de) * | 1995-02-03 | 2009-07-09 | Novozymes As | Eine methode zum entwurf von alpha-amylase mutanten mit vorbestimmten eigenschaften |
| KR19980702782A (ko) | 1995-03-09 | 1998-08-05 | 혼 마가렛 에이. | 녹말 액화 방법 |
| JP3025627B2 (ja) | 1995-06-14 | 2000-03-27 | 花王株式会社 | 液化型アルカリα−アミラーゼ遺伝子 |
| ES2221934T3 (es) | 1995-08-11 | 2005-01-16 | Novozymes A/S | Nuevas enzimas lipoliticas. |
| US6080568A (en) * | 1997-08-19 | 2000-06-27 | Genencor International, Inc. | Mutant α-amylase comprising modification at residues corresponding to A210, H405 and/or T412 in Bacillus licheniformis |
| ES2322825T3 (es) * | 1997-10-13 | 2009-06-29 | Novozymes A/S | Mutantes de alfa-amilasa. |
| WO1999028448A1 (en) | 1997-11-26 | 1999-06-10 | Novo Nordisk A/S | Thermostable glucoamylase |
| WO2000004136A1 (en) | 1998-07-15 | 2000-01-27 | Novozymes A/S | Glucoamylase variants |
| US6197565B1 (en) * | 1998-11-16 | 2001-03-06 | Novo-Nordisk A/S | α-Amylase variants |
| US6403355B1 (en) | 1998-12-21 | 2002-06-11 | Kao Corporation | Amylases |
| JP4077095B2 (ja) * | 1998-12-21 | 2008-04-16 | 花王株式会社 | 新規アミラーゼ |
| EP2290060B1 (en) * | 1999-03-30 | 2016-12-07 | Novozymes A/S | Alpha-amylase variants |
| CA2365446C (en) | 1999-03-31 | 2012-07-10 | Novozymes A/S | Polypeptides having alkaline alpha-amylase activity and nucleic acids encoding same |
| JP4417532B2 (ja) * | 1999-06-10 | 2010-02-17 | 花王株式会社 | 変異α−アミラーゼ |
| EP1200566A2 (en) | 1999-07-09 | 2002-05-02 | Novozymes A/S | Glucoamylase variant |
| AU2002210380A1 (en) | 2000-10-13 | 2002-04-22 | Novozymes A/S | Alpha-amylase variant with altered properties |
-
2001
- 2001-07-12 CA CA2416967A patent/CA2416967C/en not_active Expired - Fee Related
- 2001-07-12 EP EP10181164A patent/EP2308980A3/en not_active Withdrawn
- 2001-07-12 AU AU2001278415A patent/AU2001278415A1/en not_active Abandoned
- 2001-07-12 CA CA2778199A patent/CA2778199A1/en not_active Abandoned
- 2001-07-12 WO PCT/DK2001/000488 patent/WO2002010355A2/en active Application Filing
- 2001-07-12 CN CN201010145645A patent/CN101857858A/zh active Pending
- 2001-07-12 JP JP2002516073A patent/JP4855632B2/ja not_active Expired - Fee Related
- 2001-07-12 EP EP10181953A patent/EP2298903A3/en not_active Withdrawn
- 2001-07-12 EP EP09176569A patent/EP2204446A1/en not_active Withdrawn
- 2001-07-12 EP EP01956424A patent/EP1370648A2/en not_active Withdrawn
- 2001-07-12 CA CA2702204A patent/CA2702204C/en not_active Expired - Fee Related
- 2001-07-12 EP EP09178946A patent/EP2180035A1/en not_active Ceased
- 2001-07-12 EP EP10181142A patent/EP2308979A3/en not_active Withdrawn
- 2001-07-12 CN CNA018156800A patent/CN1529752A/zh active Pending
- 2001-08-01 AR ARP010103669A patent/AR028979A1/es not_active Application Discontinuation
-
2010
- 2010-06-01 AR ARP100101923A patent/AR076940A2/es unknown
-
2011
- 2011-07-25 JP JP2011161957A patent/JP2011224015A/ja active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1217020A (zh) * | 1996-04-30 | 1999-05-19 | 诺沃挪第克公司 | α－淀粉酶变体 |
Non-Patent Citations (1)
| Title |
|---|
| MACHIUS M,ET AL: "Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad", 《STRUCTURE》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103649307A (zh) * | 2011-06-30 | 2014-03-19 | 诺维信公司 | α-淀粉酶变体 |
| CN103649307B (zh) * | 2011-06-30 | 2020-03-27 | 诺维信公司 | α-淀粉酶变体 |
| CN103834606A (zh) * | 2014-01-16 | 2014-06-04 | 北京中科星冠生物技术有限责任公司 | 一种表达耐酸高温α-淀粉酶基因突变体的工程菌株 |
| CN103789285A (zh) * | 2014-02-19 | 2014-05-14 | 江南大学 | 一种热稳定性提高的淀粉酶突变体及其构建方法和应用 |
| CN103805579A (zh) * | 2014-02-19 | 2014-05-21 | 江南大学 | 一种热稳定的淀粉酶突变体及其制备方法和应用 |
| CN103789285B (zh) * | 2014-02-19 | 2016-01-20 | 江南大学 | 一种热稳定性提高的淀粉酶突变体及其构建方法和应用 |
| CN103805579B (zh) * | 2014-02-19 | 2016-05-18 | 江南大学 | 一种热稳定的淀粉酶突变体及其制备方法和应用 |
Also Published As
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|---|---|
| AR076940A2 (es) | 2011-07-20 |
| EP2308979A2 (en) | 2011-04-13 |
| AR028979A1 (es) | 2003-05-28 |
| EP1370648A2 (en) | 2003-12-17 |
| EP2308980A3 (en) | 2011-04-27 |
| CN1529752A (zh) | 2004-09-15 |
| JP4855632B2 (ja) | 2012-01-18 |
| CA2702204C (en) | 2011-09-06 |
| CA2416967C (en) | 2014-02-18 |
| EP2298903A3 (en) | 2011-10-05 |
| CA2702204A1 (en) | 2002-02-07 |
| CA2778199A1 (en) | 2002-02-07 |
| JP2011224015A (ja) | 2011-11-10 |
| WO2002010355A3 (en) | 2003-09-12 |
| AU2001278415A1 (en) | 2002-02-13 |
| JP2004508815A (ja) | 2004-03-25 |
| EP2298903A2 (en) | 2011-03-23 |
| EP2308979A3 (en) | 2011-05-04 |
| EP2308980A2 (en) | 2011-04-13 |
| CA2416967A1 (en) | 2002-02-07 |
| EP2180035A1 (en) | 2010-04-28 |
| EP2204446A1 (en) | 2010-07-07 |
| WO2002010355A2 (en) | 2002-02-07 |
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