CN101218343B - 枯草蛋白酶变体 - Google Patents
枯草蛋白酶变体 Download PDFInfo
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- CN101218343B CN101218343B CN2006800247046A CN200680024704A CN101218343B CN 101218343 B CN101218343 B CN 101218343B CN 2006800247046 A CN2006800247046 A CN 2006800247046A CN 200680024704 A CN200680024704 A CN 200680024704A CN 101218343 B CN101218343 B CN 101218343B
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Abstract
本发明涉及新枯草蛋白酶变体，其相对于亲本枯草蛋白酶在一种或多种性质上显示出改进，所述性质包括：洗涤性能、热稳定性、贮藏稳定性或催化活性。本发明的变体适合用于例如清洁或洗涤剂组合物中，例如洗衣洗涤剂组合物和洗碗组合物，包括自动洗碗组合物。
Description
技术领域
本发明涉及新的枯草蛋白酶(subtilase)变体，其相对于亲本枯草蛋白酶在一种或多种性质上显示出改变，所述性质包括：洗涤性能、热稳定性、贮藏稳定性和催化活性。本发明的变体适用于例如清洁或洗涤剂组合物中，例如衣物洗涤剂组合物和洗碗组合物，包括自动洗碗组合物。本发明还涉及编码所述变体的分离的DNA序列、表达载体、宿主细胞和用于产生和使用本发明的变体的方法。此外，本发明涉及包含本发明的变体的清洁和洗涤剂组合物。
发明背景
在洗涤剂工业中，将酶应用在洗涤配方中已超过30年。用在这些配方中的酶包含蛋白酶、脂肪酶、淀粉酶、纤维素酶，以及其它的酶，或它们的混合物。商业上最重要的酶是蛋白酶。
越来越多的商用蛋白酶是蛋白工程的变体或天然存在的野生型蛋白酶，例如Relase
、Alcalase、Savinase
、Primase、Everlase
、Esperase
、Ovozyme
、Coronase
、Polarzyme
和Kannase
(Novozymes A/S)、MaxataseTM、MaxacalTM、MaxapemTM、ProperaseTM、PurafectTM、PurafectOxPTM、FN2TM、FN3TM、FN4TM和Purafect PrimeTM(Genencor International，Inc.)、BLAP X和BLAP S(Henkel)。此外，许多蛋白酶变体在本领域得到描述。现有技术中蛋白酶变体的完整列表在WO 99/27082中列出。
然而，尽管已经描述了大量有用的蛋白酶变体，对于许多工业用途例如洗衣或硬表面清洁仍然存在对新的改进的蛋白酶或蛋白酶变体的需求。因此，本发明的目的是提供改进的枯草蛋白酶变体用于这些用途。
发明概述
因此，在第一方面本发明涉及枯草蛋白酶变体，其包含一个或多个表1中所列的修饰。
表1.枯草蛋白酶变体中的修饰
| T143K，Y167A，R170S，A194P |
| Y167A，R170S，A194P，K251R |
| Y167A，R170S，A194P，S265K |
| Y167A，R170S，A194P，V244R |
| S141E，Y167A，R170S，A194P |
| Y167A，R170S，M175I |
| Y167A，R170S，A172T |
| Y167A，R170S，A174V，M175F |
| Y167A，R170S，A172V，A174V |
| Y167A，R170S，A172E |
| Y167A，R170S，M175L |
| Y167A，R170S，A174T |
| Y167A，R170S，A174T，M175L |
| G53C，G61E |
| A98S，S99D，G100S |
| S9R，T22A，V68A，S99A，*99aD |
| S9R，P14H，R19L，N62D |
| G61P，*99aS |
| N43S，N62D |
| *96aG，P131S，V203A，A228T |
| N62D，A232C，Q236L，Q245N |
| *96aA，A98T，R247K |
| S99D，S101R，S103A，V104I，G160S，A194P，L217D |
| *61aD |
| N62D，S106A |
| V68A，S106M，N184D |
| S9R，A15T，*97aV，H120N |
| A15M，A16P，*99aD |
| *99aE，G160S，S163T，G195S，G211S，K237R，G258A，T260L |
| G23S，*99aD，A194P，S242T，Q245R |
| G100S，N173D |
| Y167A，R170S，A172E |
| A98T，Q137L，Y167A，R170S，M175L |
| *98aA，S99D |
| S99A，*99aD，V203A |
| N62D，K237R |
| V11M，N76D，L126F，K251R |
| S9F，A15L，A16P，T22I，*98aA，S99D，R170H |
| *96aA，*130aG，P131H |
| E54D，N62D |
| *98aA，*98bS，S99G，S101T |
| S9R，A15T，V68A，I79T，G102S，P131H，Q137H |
| *100aA，*100bG，*100cS，*100dG |
| V68A，L111I |
| *98aA，R170H，Q245R |
| I35V，N62D，N183D，T224S |
| *97aG，P131S，V203A，A228T |
| S9R，R10K，P14Q，T22A，Y167A，R170S |
| S9R，*22aL，S57A，G61E，*98aA，V139L，N173S |
| P14T，N18K，Y167A，R170S |
| S9R，Q12E，P14Q，K27R，Y167A，R170S |
| N62D，R170L |
| N62D，R170S，Q245R |
| Y167A，R170S，A194P，K251R，S265K |
| P14T，N18K，Y167A，R170S，A194P |
| N62D，A151G，K237R |
| N62D，A151G，Q245R |
| N62D，A151G，K237R，Q245R |
| S103A，V104I，G159D，A232V，Q236H，Q245R |
| S9R，A15T，T22A，V139L |
| S9R，A15T，G61E，A85T，E89Q，P239L，Q245C |
| S9R，A15T，V68A，H120N，Q245R |
| N248R |
| S9R，A15T，*22aL，V139L，N204D，Q245L |
| N218S |
| S9R，A15T，V68A，Q245R，N252K |
| S9R，A15T，V68A，Q245R，H120N |
| V68A，S106A，H120N |
| V68A，S106A，N252K |
| A15T，V68A，S99G，Q245R，N261D |
| S9R，V68A，S99G，Q245R，N261D |
| V68A，S99G，Q245R，N261D |
| S9R，A15T，V68A，S99G，N261D |
| S9R，A15T，V68A，Q245R，N261D |
| S9R，A15T，*22aL，V139L，S163G，N204D，Q245L |
| Q245R，N252H |
| S9R，*22aL，G61E，*97aA，M119I，Q137H，N173S |
| V68A，S106A，T213A |
| S9R，A15T，V68A，H120N，P131S，Q137H，Q245M |
| S9R，A15T，V68A，I72F，S99G，Q245R，N261D |
| S9R，A15T，V68A，S99D，Q245R，N261D |
| S9R，A15T，V68A，S99G，A194P，Q245R，N261D |
| S9R，A15T，V68A，N76I，S99G，Q245R，N261D |
| S9R，A15T，V68A，S99G，A228V，Q245R，N261D |
表1中所列的变体显示蛋白酶活性，每个位置均相应于在图1和SEQ IDNO：1中所示的枯草溶菌素(subtilisin)BPN’氨基酸序列中的位置。
在第二方面本发明涉及分离的多核苷酸，其编码本发明的枯草蛋白酶变体。
在第三方面本发明涉及表达载体，其包含本发明的分离的多核苷酸。
在第四方面本发明涉及用本发明的表达载体转化的微生物宿主细胞。
在第五方面本发明涉及用于产生根据本发明的枯草蛋白酶变体的方法，其中在有益于表达和分泌所述变体的条件下培养本发明的宿主，并且回收该变体。
在第六个方面本发明涉及包含本发明的变体的清洁或洗涤剂组合物，优选洗衣或洗碗组合物。
关于比对和编号方式，参考图1进行，图1显示枯草溶菌素BPN’(a)(BASBPN)和枯草溶菌素309(b)(BLSAVI)之间的比对。在本专利申请中将该比对用作参考用于对残基进行编号。
定义
在进一步详细讨论本发明之前，将首先对以下术语和惯例进行定义。对于氨基酸和核酸命名法的详细说明，我们参考WO 00/71691中从第5页开始的部分，在此将其并入作为参考。
变体定名的命名法和惯例
在对根据本发明产生或预期的多种枯草蛋白酶酶变体的描述中，为了便于参考已编写出以下命名法和惯例：首先通过将分离的或亲本的酶与枯草溶菌素BPN’(BASBPN)进行比对来定义参照系(frame of reference)。
能够通过GCG软件包9.1版的GAP程序使用以下参数获得比对以对变体进行编号：缺口产生罚分(gap creation penalty)＝8和缺口延伸罚分(gapextension penalty)＝8，并且将全部其它参数保持在它们的默认值。
另一种方法是使用已知的、公认的枯草蛋白酶之间的比对，例如WO91/00345中所示的比对。在大多数情况下，差异不重要。
因此相对于BASBPN(SEQ ID NO.1)来定义许多缺失和插入。在图1中，枯草溶菌素309(SEQ ID NO.2)与BASBPN比较，在位置36、58、158、162、163和164具有6个缺失。这些缺失在图1中以星号表示(*)。对于通过遗传操作引入多肽中的修饰的命名法的详细说明，我们参考WO 00/71691的第7-12页，在此并入作为参考。
蛋白酶将切割蛋白质底物中酰胺键的酶归类为蛋白酶，或(可互换的)肽酶(参见Walsh，1979，Enzymatic Reaction Mechanisms.W.H.Freeman andCompany，San Francisco，Chapter 3)。
氨基酸位置/残基的编号方式如果不进行其它说明，本文所用的氨基酸编号方式对应于枯草蛋白酶BPN’(BASBPN)序列的编号方式。关于BPN’序列的进一步说明，参见图1、SEQ ID NO：1或Siezen et al.，Protein Engng.4(1991)719-737。
丝氨酸蛋白酶丝氨酸蛋白酶是催化肽键水解的酶，并且其中在活性位点存在必需的丝氨酸残基(White，Handler and Smith，1973″Principles ofBiochemistry，″Fifth Edition，McGraw-Hill Book Company，NY，pp.271-272)。细菌丝氨酸蛋白酶具有20,000至45,000道尔顿的分子量。它们受氟磷酸二异丙酯(diisopropylfluorophosphates)抑制。它们水解简单末端酯(simple terminalester)，并且在活性上与同为丝氨酸蛋白酶的真核胰凝乳蛋白酶相似。范围更窄的术语“碱性蛋白酶”，其包含一个亚组，反映出某些丝氨酸蛋白酶的高最适pH，从pH9.0至11.0(关于综述，参见Priest(1977)Bacteriological Rev.41711-753)。
枯草蛋白酶Siezen et al.，Protein Engng.4(1991)719-737和Siezen et al.Protein Science 6(1997)501-523提出了暂时定名为枯草蛋白酶的丝氨酸蛋白酶的亚组。它们通过对先前称作枯草溶菌素-样(subtilisin-like)蛋白酶的丝氨酸蛋白酶的超过170个氨基酸序列的同源性分析来定义。先前通常将枯草溶菌素(subtilisin)定义为由革兰氏阳性细菌或真菌产生的丝氨酸蛋白酶，而根据Siezen et al.目前是枯草蛋白酶的亚组。已鉴定出多种枯草蛋白酶，并且已经测定许多枯草蛋白酶的氨基酸序列。关于对这些枯草蛋白酶和它们的氨基酸序列更详细的说明，参考Siezen et al.(1997)。
枯草蛋白酶的一个亚组I-S1或“真”枯草溶菌素，包含“经典”枯草溶菌素，例如枯草溶菌素168(BSS168)、枯草溶菌素BPN′、枯草溶菌素Carlsberg(ALCALASE，Novozymes A/S)和枯草溶菌素DY(BSSDY)。
枯草蛋白酶的另一个亚组I-S2或高碱性枯草溶菌素，由Siezen et al.(见上文)鉴定。亚组I-S2蛋白酶被描述成高碱性枯草溶菌素，并且包括酶，例如枯草溶菌素PB92(BAALKP)(MAXACAL
，Genencor International Inc.)、枯草溶菌素309(SAVINASE
，Novozymes A/S)、枯草溶菌素147(BLS147)(ESPERASE
，Novozymes A/S)和碱性弹性蛋白酶YaB(BSEYAB)。
“SAVINASE”Savinase由Novozymes A/S在市场上销售。它是来自迟缓芽孢杆菌(B.Lentus)的枯草溶菌素309，并且与BAALKP仅在一个位置(N87S)上有差异。SAVINASE
具有在图1中定名为b)和SEQ ID NO：2中的氨基酸序列。
亲本枯草蛋白酶术语“亲本枯草蛋白酶”描述根据Siezen等人(1991和1997)定义的枯草蛋白酶。其它细节参见以上对“枯草蛋白酶”的描述。亲本枯草蛋白酶也可以是从天然来源分离的枯草蛋白酶，其中在保持枯草蛋白酶特性的同时进行后续的修饰。此外，亲本枯草蛋白酶可以是通过DNA改组技术制备的枯草蛋白酶，例如由J.E.Ness et al.，Nature Biotechnology，17，893-896(1999)所述。可供选择的是，术语“亲本枯草蛋白酶”也可称为“野生型枯草蛋白酶”。
枯草蛋白酶变体的修饰将本文使用的术语“修饰”定义为包括对枯草蛋白酶的化学修饰以及对编码枯草蛋白酶的DNA的遗传操作。所述修饰可以是在感兴趣的氨基酸中或于感兴趣的氨基酸处的取代、缺失、插入和/或对氨基酸侧链的置换。
枯草蛋白酶变体在本发明的上下文中，术语枯草蛋白酶变体或突变的枯草蛋白酶的意思是，由表达突变基因的生物体产生的枯草蛋白酶，所述突变基因源自具有原始或亲本基因并且产生相应亲本酶的亲本微生物，将所述亲本基因突变以产生突变的基因，当在合适的宿主中表达时，从该突变的基因产生突变的枯草蛋白酶蛋白酶(subtilase protease)。
同源枯草蛋白酶序列在本上下文中用参数“同一性”来描述两个氨基酸序列之间的同源性。为了测定两个枯草蛋白酶之间的同一性程度，能够应用GCG软件包9.1版的GAP程序(见上文)，并使用相同的设定。从所述程序输出的数据是除氨基酸比对之外，对两个序列之间“百分比同一性”的计算。基于这种描述，鉴定合适的同源枯草蛋白酶对于本领域技术人员是常规性的，所述合适的同源枯草蛋白酶能够根据本发明进行修饰。
分离的多核苷酸术语“分离的”，当应用于多核苷酸时，表示已将所述多核苷酸从它的天然遗传环境中分离，因此不含其它外来的或不需要的编码序列，并且以适用于遗传工程蛋白质产生体系的形式存在。这些分离的分子分离自它们的天然环境，并且包括cDNA和基因组克隆。本发明的分离的DNA分子不含与它们通常结合的(ordinarily associated)其它基因，但可包括天然存在的5′和3′非翻译区例如启动子和终止子。结合区域的鉴定对于本领域普通技术人员是显而易见的(参见例如，Dynan and Tijan，Nature 316：774-78，1985)。可将术语“分离的多核苷酸”可选地称为“克隆的多核苷酸”。
分离的蛋白质应用于蛋白质时，术语“分离的”表示已将所述蛋白质从它的天然环境中移出。在优选的形式中，分离的蛋白质基本上不含其它蛋白质，特别不含其它同源蛋白质(即“同源杂质”(见下文))。如通过SDS-PAGE所测定的，分离的蛋白质是多于10％纯，优选多于20％纯，更优选多于30％纯。此外优选将所述蛋白质以高度纯化的形式提供，即，如通过SDS-PAGE所测定的，多于40％纯，多于60％纯，多于80％纯，更优选多于95％纯，并且最优选多于99％纯。可将术语“分离的蛋白质”可选地称为“纯化的蛋白质”。
同源杂质术语“同源杂质”的意思可以是起源于同源细胞的任何杂质(例如除本发明的枯草蛋白酶之外的其它多肽)，其中本发明的枯草蛋白酶原先获得自所述同源细胞中。
获得自术语“获得自”用于本文中与具体的微生物来源有关，意思是多核苷酸和/或枯草蛋白酶由所述来源产生，或由其中已插入来自该来源的基因的细胞产生。
底物所用术语“底物”与蛋白酶的底物有关，应理解为其最宽泛的形式，其包括的化合物含有至少一个对枯草溶菌素蛋白酶的水解易感的肽(酰胺)键。
产物所用术语“产物”与源自蛋白酶酶促反应的产物有关，在本发明的上下文中，应理解为包括涉及枯草蛋白酶蛋白酶的水解反应的产物。产物可以是后续水解反应中的底物。
洗涤性能在本上下文中，将术语“洗涤性能”用作在例如洗涤或硬表面清洁的过程中，酶去除存在于待清洁物体上的蛋白质沾污或有机沾污的能力。同样参见本文实施例3中的洗涤性能测试。
附图简述
图1显示枯草溶菌素BPN’(a)和Savinase
(b)之间使用上述GAP程序的比对。
发明详述
本发明涉及新枯草蛋白酶变体，其相对于亲本枯草蛋白酶在一种或多种性质上显示出改变，所述性质包括：洗涤性能、热稳定性、贮藏稳定性或催化活性。预期作为本发明部分的变体是这样的变体：在与野生型枯草蛋白酶比较时，其中已通过取代、缺失或插入对一个或多个氨基酸残基进行了修饰。本发明的变体包含一种或多种表1所列的修饰。
表1所列的变体显示出蛋白酶活性，并且每种位置对应于图1和SEQ IDNO：1中所示的枯草溶菌素BPN’氨基酸序列的位置。
本发明第一方面的枯草蛋白酶变体可以是从自然界鉴定和分离的亲本或野生型枯草蛋白酶。这种亲本野生型枯草蛋白酶可通过本领域已知的标准技术特异性地筛选。
实现这点的一种优选方式可为通过从来自众多不同微生物的枯草蛋白酶中特异性PCR扩增感兴趣的保守DNA区域，所述不同微生物优选为不同的芽孢杆菌属(Bacillus)菌株。
枯草蛋白酶是一组保守的酶，它们的DNA和氨基酸序列是同源的。因此可构建在感兴趣多核苷酸序列侧翼的相关的特异性引物。
使用这些PCR引物从许多不同的微生物(优选从不同的芽孢杆菌属菌株)扩增DNA，接着对扩增的PCR片段进行DNA测序，可鉴定产生本发明的枯草蛋白酶变体的菌株。鉴定菌株和这种感兴趣的枯草蛋白酶的部分DNA序列之后，完成这种枯草蛋白酶的克隆、表达和纯化对本领域技术人员是常规工作。然而，可以想像本发明的枯草蛋白酶变体主要是亲本枯草蛋白酶的变体。
适合于本文所述用途的枯草蛋白酶变体可以通过本领域已知的标准方法构建，例如通过定点/随机诱变或通过不同枯草蛋白酶序列的DNA改组(DNAshuffling)。进一步的细节参见“材料和方法”部分和本文的实施例1(见下文)。
如本领域技术人员公认的，本文所述的变体可包含一个或多个额外的修饰，具体而言是一个或多个额外的取代或插入。此外，本文所述的变体可以在多于一个位置包含突变。例如，根据本发明的变体可在一个位置、两个位置、三个位置或多于三个位置(例如在四至八个位置)含有突变。对于来自自然界或来自人工创造的多样性的酶，同时也对于从亲本枯草蛋白酶设计和产生的酶，均优选亲本枯草蛋白酶属于亚组I-S1或I-S2，特别是亚组I-S2。
关于来自亚组I-S1的变体，优选从下组选择亲本枯草蛋白酶：BSS168(BSSAS、BSAPRJ、BSAPRN、BMSAMP)、BASBPN、BSSDY、BLSCAR(BLKERA、BLSCA1、BLSCA2、BLSCA3)、BSSPRC和BSSPRD，或它们保持了亚组I-S1特性的功能性变体。
关于来自亚组I-S2的变体，优选从下组选择亲本枯草蛋白酶：BSAPRQ、BLS147(BSAPRM、BAH101)、BLSAVI(BSKSMK、BAALKP、BLSUBL)、BYSYAB、BAPB92、TVTHER和BSAPRS，或它们保持了亚组I-S2特性的功能性变体。具体而言，亲本枯草蛋白酶是BLSAVI(Savinase
，NOVOZYMES A/S)，因此本发明优选的枯草蛋白酶变体是Savinase
的变体。
本发明还包含任何上述枯草蛋白酶变体与任何其它对其氨基酸序列的修饰的组合。特别预期的是与本领域已知的其它修饰的组合以将改进的性质提供给酶。本领域对许多具有不同改进性质的枯草蛋白酶变体进行了描述，并且它们中的许多在本文的“发明背景”部分中提及(见上文)。将那些文件在此公开作为鉴定枯草蛋白酶变体的参考，能够将所述变体有利地与本文描述的枯草蛋白酶变体组合。这些组合包含以下位置：222(改进氧化稳定性)，218(改进热稳定性)，使酶稳定的Ca2+-结合位点中的取代，例如位置76，和从现有技术显而易见的许多其它位置。
在其它实施方式中，可有利地将本文所述枯草蛋白酶变体与在任何以下位置中的一个或多个修饰组合：
27、36、56、76、87、95、96、97、98、99、100、101、102、103、104、120、123、159、167、170、206、218、222、224、232、235、236、245、248、252和274。
具体而言，认为以下BLSAVI、BLSUBL、BSKSMK和BAALKP修饰适用于组合：
K27R、*36D、S56P、N62D、V68A、N76D、S87N、G97N、S99SE、S101G、S101R、S103A、V104A、V104I、V104N、V104Y、S106A、H120D、H120N、N123S、G159D、Y167A、R170S、R170L、A194P、N204D、V205I、Q206E、L217D、N218S、N218D、M222S、M222A、T224S、A232V、K235L、Q236H、Q245R、N248D、N252K和T274A。
此外，包含修饰S101G+V104N、S87N+S101G+V104N、K27R+V104Y+N123S+T274A、N76D+S103A+V104I、S99D+S101R+S103A+V104I+G160S、S3T+V4I+S99D+S101R+S103A+V104I+G160S+V199M+V205I+L217D、S3T+V4I+S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217D、S3T+V4I+S99D+S101R+S103A+V104I+G160S+V205I或N76D+V104A中任何修饰的变体，或包含修饰K27R、*36D、S56P、N62D、V68A、N76D、S87N、G97N、S99SE、S101G、S103A、V104A、V104I、V104N、V104Y、S106A、H120D、H120N、N123S、G159D、Y167A、R170S、R170L、A194P、N204D、V205I、Q206E、L217D、N218S、N218D、M222S、M222A、T224S、A232V、K235L、Q236H、Q245R、N248D、N252K和T274A与上述修饰的任何一种或多种组合的其它组合的变体，显示改进的性质。特别感兴趣的变体是其中除了根据本发明的修饰之外，含有以下取代的变体：
S101G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K。
此外，优选将本发明主要方面的枯草蛋白酶变体与在位置129、131和194中任何位置的一个或多个修饰组合，优选如129K、131H和194P修饰，并且最优选如P129K、P131H和A194P修饰。预期那些修饰中的任何修饰将为枯草蛋白酶变体在其产生中提供较高的表达水平。
所选本发明变体的洗涤性能可在本文实施例3中公开的洗涤性能测试中进行测试。所述洗涤性能测试可用于评估当并入标准或商业洗涤剂组合物中时，变体与参照体系相比从标准织物去除蛋白质沾污的能力，所述参照体系即亲本枯草蛋白酶或显示更好洗涤性能的相似枯草蛋白酶(并入相同的洗涤剂体系，并且在相同的条件下测试)。使用自动机械应力试验(AutomaticMechanical Stress Assay(AMSA))测试本申请的酶变体。使用AMSA测试能够迅速检测大量小体积酶洗涤剂溶液的洗涤性能。使用这种测试，能够在最初对所选变体的洗涤性能进行研究，基本原理是如果所选变体在该测试中与亲本枯草蛋白酶相比不显示显著改进，通常不必要进行进一步的测试实验。
因此，用于本文所述目的特别令人感兴趣的变体是这些变体，所述变体当如洗涤性能测试(实施例3)中描述，在商业洗涤剂组合物例如US型洗涤剂、亚洲型、欧洲型或拉丁美洲型洗涤剂中测试时，与相同条件下测试的亲本枯草蛋白酶相比显示改进的洗涤性能。
在洗涤性能上的改进可通过计算本文实施例3中定义的所谓强度值(intensity value)(Int)来定量。
显然，优选本发明的变体至少以所述的最低水平，更优选以所述的最高水平满足以上标准。
产生枯草蛋白酶变体
用于克隆枯草蛋白酶和用于将取代、缺失或插入引入基因(例如枯草蛋白酶基因)中的多种方法是本领域熟知的。
通常可以使用用来克隆基因和将突变(随机突变和/或定位突变)引入所述基因的标准方法以获得本发明的枯草蛋白酶变体。对于合适技术的进一步描述，参照本文的实施例1(见下文)和(Sambrook et al.(1989)Molecular cloning：A laboratory manual，Cold Spring Harbor lab.，Cold Spring Harbor，NY；Ausubel，F.M.et al.(eds.)″Current protocols in Molecular Biology″.John Wiley and Sons，1995；Harwood，C.R.，and Cutting，S.M.(eds.)″Molecular Biological Methodsfor Bacillus″.John Wiley and Sons，1990)，和WO 96/34946。
此外，枯草蛋白酶变体可通过用于人工产生多样性的标准技术构建，例如通过DNA改组不同的枯草蛋白酶基因(WO 95/22625；Stemmer WPC，Nature370：389-91(1994))。用自然界中鉴定的一个或多个部分枯草蛋白酶序列对例如编码Savinase
的基因进行DNA改组，将在后续筛选改进洗涤性能的变体后，提供适合于本文所述目的的枯草蛋白酶变体。
表达载体
包含编码本发明的酶的DNA构建体的重组表达载体可以是任何载体，可对所述载体方便地实施重组DNA方法。载体的选择将通常依赖于待引入该载体的宿主细胞。因此，载体可以是自主复制载体，即作为染色体外实体存在的载体，其复制独立于染色体复制，例如质粒。
可供选择的是，载体可以是在引入宿主细胞中时，以部分或其整体整合到该宿主细胞基因组中，并且与其整合入的染色体一起复制的载体。
载体优选是其中编码本发明的酶的DNA序列与该DNA转录所需的额外片段可操作地连接的表达载体。通常而言，表达载体源自质粒或病毒DNA，或可以含有这二者的元件。术语“可操作地连接”表示排列所述片段从而使它们协同地为了它们预期的目的发挥功能，例如使转录在启动子中起始并且使转录继续经过编码酶的DNA序列。
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，并且可以源自编码与该宿主细胞同源或异源的蛋白质的基因。
用于细菌宿主中合适的启动子的实例包括以下基因的启动子：嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)产麦芽淀粉酶基因、地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因、解淀粉芽孢杆菌(Bacillusamyloliquefaciens)α-淀粉酶基因、枯草芽孢杆菌(Bacillus subtilis)碱性蛋白酶基因或短小芽孢杆菌(Bacillus pumilus)木糖苷酶基因，或噬菌体Lambda PR或PL启动子或大肠杆菌lac、trp或tac启动子。编码本发明的酶的DNA序列如有必要也可以与合适的终止子可操作地连接。
本发明的重组载体可进一步包含使该载体能够在所述宿主细胞中复制的DNA序列。载体也可包含选择性标记，例如其产物补足宿主细胞中缺陷的基因，或编码对于例如抗生素如卡那霉素、氯霉素、红霉素、四环素、壮观霉素等的抗性或对于重金属或除草剂的抗性的基因。
为了指导本发明的酶进入宿主细胞的分泌途径，可在重组载体中提供分泌信号序列(也称为前导序列，前原序列或前序列)。将分泌信号序列以正确的阅读框连接于编码酶的DNA序列。分泌信号序列通常位于编码酶的DNA序列的5′。分泌信号序列通常可与酶有关，或可以来自编码其它分泌蛋白质的基因。
分别用于连接编码本发明酶的DNA序列、启动子和任选的终止子和/或分泌信号序列的方法，或用于通过合适的PCR扩增方案装配这些序列的方法，和用于将它们插入到含有复制或整合所必需信息的合适载体中的方法，对于本领域技术人员是熟知的(参看，例如，Sambrook et al.，在所引文献中)。
宿主细胞
引入宿主细胞中的编码本发明的酶的DNA序列对于所述宿主可以是同源或异源的。如果对于宿主细胞是同源的，即在自然界中由该宿主细胞产生，它将通常与其天然环境之外的另外的启动子序列可操作地连接，或如果适用，与另外的分泌信号序列和/或终止子序列可操作地连接。术语“同源的”意欲包括编码对于所述宿主生物体是天然的酶的DNA序列。术语“异源的”意欲包括在自然界中不由该宿主细胞表达的DNA序列。因此，DNA序列可以来自其它生物体，或它可以是合成序列。
其中引入了本发明的DNA构建体或重组载体的宿主细胞可以是能够产生本发明的酶的任何细胞，并且所述细胞包括细菌、酵母、真菌和高等真核细胞包括植物。
在培养时能够产生本发明的酶的细菌宿主细胞的实例是革兰氏阳性细菌，例如芽孢杆菌属的菌株，例如枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌、短芽孢杆菌、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌(B.alkalophilus)、解淀粉芽孢杆菌、凝结芽孢杆菌(B.coagulans)、环状芽孢杆菌(B.circulans)、灿烂芽孢杆菌(B.lautus)、巨大芽孢杆菌(B.megaterium)或苏云金芽孢杆菌(B.thuringiensis)的菌株，或链霉菌属(Streptomyces)的菌株，例如浅青紫链霉菌(S.lividans)或鼠灰链霉菌(S.murinus)，或革兰氏阴性细菌例如大肠杆菌。
细菌的转化可以通过原生质体转化、电穿孔、接合(conjugation)来完成，或通过使用感受态细胞以本身已知的方式来完成(参看Sambrook et al.，见上文)。
在细菌例如大肠杆菌中表达酶时，可将酶保留在细胞质中，通常作为不溶性颗粒(称为包含体(inclusion body))；或可通过细菌分泌序列将酶引导至周质空间(periplasmic space)。在前者的情况下，将细胞裂解，回收所述颗粒并且变性，之后通过稀释变性剂将酶重折叠(refold)。在后者的情况下，可通过破坏细胞从周质空间中回收酶，例如通过声处理(sonication)或渗压震扰(osmotic shock)，以释放周质空间的内容物和回收酶。
在革兰氏阳性细菌例如芽孢杆菌属或链霉菌属菌株中表达酶时，可将酶保留在细胞质中，或可通过细菌分泌序列将酶引导至胞外介质(extracellularmedium)。在后者的情况下，可如下所述从所述介质中回收酶。
产生枯草蛋白酶变体的方法
本发明提供产生根据本发明的分离的酶的方法，其中在允许产生该酶的条件下培养合适的宿主细胞，所述细胞已用编码所述酶的DNA序列转化，并且将所得的酶从培养物中回收。
将包含编码酶的DNA序列的表达载体转化到异源宿主细胞中时，能够实现本发明的酶的异源重组产生。因此可制备高度纯化的枯草蛋白酶组合物，所述组合物的特征在于不含同源杂质。
用于培养转化的宿主细胞的培养基可以是适合于培养所述宿主细胞的任何常规培养基。可方便地将表达的枯草蛋白酶分泌至培养基中，并且可通过熟知的方法从培养基中回收表达的枯草蛋白酶，所述方法包括：通过离心或过滤从培养基中分离细胞，借助盐例如硫酸铵沉淀培养基的蛋白质成分，随后是层析方法例如离子交换层析、亲和层析等。
洗涤剂应用
可将本发明的酶添加至洗涤剂组合物并且由此成为该洗涤剂组合物的成分。可将本发明的洗涤剂组合物例如配制成手洗或机洗洗涤剂组合物，包括适合于预处理沾污织物的洗衣添加剂组合物和漂洗添加的织物柔软剂组合物；或可配制成用于一般家庭硬表面清洁操作的洗涤剂组合物；或可配制用于手洗或机洗洗碗操作。
在具体的方面，本发明提供包含本发明的酶的洗涤剂添加剂。洗涤剂添加剂以及洗涤剂组合物可包含一种或多种其它的酶，例如蛋白酶、脂肪酶、角质酶(cutinase)、淀粉酶、糖酶、纤维素酶、果胶酶、甘露聚糖酶(mannanase)、阿拉伯糖酶(arabinase)、半乳聚糖酶、木聚糖酶、氧化酶，例如，漆酶和/或过氧化物酶。
通常所选酶的性质应与选择的洗涤剂相容(即最适pH、与其它酶和非酶成分的相容性等)，并且所述酶应该以有效量存在。
蛋白酶：合适的蛋白酶包括动物、植物或微生物来源的那些蛋白酶。微生物来源是优选的。包括化学修饰的或蛋白质工程的变体。蛋白酶可以是丝氨酸蛋白酶或金属蛋白酶，优选是碱性微生物蛋白酶或胰蛋白酶-样蛋白酶。碱性蛋白酶的实例是枯草溶菌素，特别是源自芽孢杆菌属的那些枯草溶菌素，例如枯草溶菌素Novo、枯草溶菌素Carlsberg、枯草溶菌素309、枯草溶菌素147和枯草溶菌素168(在WO 89/06279中有所描述)。胰蛋白酶-样蛋白酶的实例是胰蛋白酶(例如，猪或牛来源的)和WO 89/06270和WO 94/25583中描述的镰孢属(Fusarium)蛋白酶。
有用的蛋白酶的实例是WO 92/19729、WO 98/20115、WO 98/20116和WO 98/34946中描述的变体，特别是在以下位置中的一个或多个具有取代的变体：27、36、57、68、76、87、97、101、104、106、120、123、167、170、194、206、218、222、224、235、245、252和274。优选的商业上使用的蛋白酶包括Relase
、Alcalase
、Savinase、Primase
、Everlase
、Esperase
、Ovozyme
、Coronase
、Polarzyme
和Kannase
(Novozymes A/S)、MaxataseTM、MaxacalTM、MaxapemTM、ProperaseTM、PurafectTM、PurafectOxPTM、FN2TM、FN3TM、FN4TM和Purafect PrimeTM(Genencor International，Inc.)、BLAP X和BLAP S(Henkel)。
脂肪酶：合适的脂肪酶包括细菌或真菌来源的那些脂肪酶。包括化学修饰的或蛋白质工程的突变体。有用的脂肪酶的实例包括来自腐质霉属(Humicola)(同物异名嗜热霉属(Thermomyces))的脂肪酶，例如EP 258068和EP 305216所述来自疏棉状腐质霉(H.lanuginosa)(细毛嗜热霉(T.lanuginosus))的脂肪酶或WO 96/13580中所述来自特异腐质霉(H.insolens)的脂肪酶；假单胞菌属脂肪酶，例如来自产碱假单胞菌(P.alcaligenes)或类产碱假单胞菌(P.pseudoalcaligenes)(EP 218272)、葱头假单胞菌(P.cepacia)(EP 331376)、施氏假单胞菌(P.stutzeri)(GB 1,372,034)、荧光假单胞菌(P.fluorescens)、假单胞菌属菌种菌株SD 705(WO 95/06720和WO 96/27002)、P.wisconsinensis(WO96/12012)；芽孢杆菌属脂肪酶，例如来自枯草芽孢杆菌(Dartois et al.(1993)，Biochemica et Biophysica Acta，1131，253-360)、嗜热脂肪芽孢杆菌(JP64/744992)或短小芽孢杆菌(WO 91/16422)。其它实例是脂肪酶变体，例如在WO 92/05249、WO 94/01541、EP 407225、EP 260105、WO 95/35381、WO96/00292、WO 95/30744、WO 94/25578、WO 95/14783、WO 95/22615、WO97/04079和WO 97/07202中所述的那些。优选的商业上使用的脂肪酶包括Lipolase
、Lipolase Ultra
和Lipex
(Novozymes A/S)。
淀粉酶：合适的淀粉酶(α和/或β)包括细菌或真菌来源的那些淀粉酶。包括化学修饰的或蛋白质工程的突变体。淀粉酶包括，例如，获得自芽孢杆菌属，例如地衣芽孢杆菌的特殊菌株的α-淀粉酶，在GB 1,296,839中有更详细的描述。有用的淀粉酶的实例是WO 94/02597、WO 94/18314、WO 96/23873和WO 97/43424中描述的变体，特别是在以下位置中的一个或多个具有取代的变体：15、23、105、106、124、128、133、154、156、181、188、190、197、202、208、209、243、264、304、305、391、408和444。商业上使用的淀粉酶是Duramyl
、Termamyl
、Stainzyme
、Fungamyl
和BAN
(Novozymes A/S)、RapidaseTM、PurastarTM和Purastar OxAmTM(来自GenencorInternational Inc.)。
纤维素酶：合适的纤维素酶包括细菌或真菌来源的那些纤维素酶。包括化学修饰的或蛋白质工程的突变体。合适的纤维素酶包括来自芽孢杆菌属、假单胞菌属、腐质霉属、镰孢属、梭孢壳属(Thielavia)、枝顶孢霉属(Acremonium)的纤维素酶，例如从特异腐质霉、嗜热毁丝霉(Myceliophthora thermophila)和尖镰孢(Fusarium oxysporum)产生的真菌纤维素酶，如US 4,435,307、US5,648,263、US 5,691,178、US 5,776,757和WO 89/09259中所公开的。特别适合的纤维素酶是具有颜色保护(colour care)和白度保持益处的碱性或中性纤维素酶。这些纤维素酶的实例是在EP 0495257、EP 0531372、WO 96/11262、WO 96/29397、WO 98/08940中描述的纤维素酶。其它实例是纤维素酶变体，例如在WO 94/07998、EP 0531315、US 5,457,046、US 5,686,593、US5,763,254、WO 95/24471、WO 98/12307和PCT/DK98/00299中描述的那些变体。商业上使用的纤维素酶包括Renozyme
、Celluzyme
和Carezyme
(Novozymes A/S)、ClazinaseTM和Puradax HATM(Genencor Int.Inc.)和KAC-500(B)TM(Kao Corporation)。
过氧化物酶/氧化酶：合适的过氧化物酶/氧化酶包括植物、细菌或真菌来源的那些。包括化学修饰的或蛋白质工程的突变体。有用的过氧化物酶的实例包括来自鬼伞属(Coprinus)例如来自灰盖鬼伞(C.cinereus)的过氧化物酶，和它们的变体，如WO 93/24618、WO 95/10602和WO 98/15257中所述。商业上有用的过氧化物酶包括GuardzymeTM(Novozymes A/S)。
半纤维素酶：适合的半纤维素酶包括细菌或真菌来源的那些半纤维素酶。包括化学修饰的或蛋白质工程的突变体。合适的半纤维素酶包括甘露聚糖酶、地衣酶(lichenase)、木聚糖酶、阿拉伯糖酶、半乳聚糖酶、乙酰木聚糖酯酶、葡糖醛酸糖苷酶(glucorunidase)、阿魏酸酯酶、香豆酸酯酶和阿拉伯呋喃糖苷酶，如WO 95/35362中所述。合适的甘露聚糖酶在WO 99/64619中有所描述。
通过添加含有一种或多种酶的单独的添加剂，或通过添加包含全部这些酶的组合添加剂，可将洗涤剂酶包括在洗涤剂组合物中。本发明的洗涤剂添加剂，即单独添加剂或组合添加剂，能够配制成例如颗粒、液体、浆等。优选的洗涤剂添加剂剂型是颗粒，尤其是无粉尘的颗粒；液体，尤其是稳定化的液体；或浆。
无粉尘的颗粒可以例如按US 4,106,991和4,661,452中公开的产生，并且可任选地通过本领域已知的方法进行包覆。蜡包覆材料的实例是具有1000至20000的平均摩尔重量的聚(环氧乙烷)产品(聚乙二醇，PEG)；具有16至50个环氧乙烷单位的乙氧基化壬基酚；乙氧基化脂肪醇，其中所述醇含有12至20个碳原子且其中存在15至80个环氧乙烷单位；脂肪醇；脂肪酸；和脂肪酸的单和二和三酸甘油酯。适于通过流化床技术施用的成膜包覆材料的实例在GB 1483591中给出。例如，可以通过根据已确定的方法添加多元醇来使液体酶制备物稳定，所述多元醇例如丙二醇、糖或糖醇、乳酸或硼酸。保护的酶可根据EP 238,216中公开的方法来制备。
本发明的洗涤剂组合物可以是任何方便的形式，例如，条、片剂、粉剂、颗粒、糊剂(paste)、凝胶或液体。液体洗涤剂可以是含水的，通常含有多至70％水和0-30％有机溶剂，或是非水的。
洗涤剂组合物包含一种或多种表面活性剂，所述表面活性剂可以是非离子的(包括半-极性的)和/或阴离子的和/或阳离子的和/或两性离子的。表面活性剂通常以按重量计0.1％至60％的水平存在。
当包括于其中时，洗涤剂将通常含有大约1％至大约40％的阴离子表面活性剂，例如线性烷基苯磺酸酯(linear alkylbenzenesulfonate)、α-烯烃磺酸酯、烷基硫酸酯(脂肪醇硫酸酯)、醇乙氧基硫酸酯(alcohol ethoxysulfate)、仲烷基磺酸酯(secondary alkanesulfonate)、α-磺基脂肪酸甲酯、烷基-或烯基琥珀酸或肥皂(soap)。
当包括于其中时，洗涤剂将通常含有大约0.2％至大约40％的非离子表面活性剂，例如醇乙氧基化物、壬基酚乙氧基化物、烷基聚苷(alkylpoly-glycoside)、烷基二甲胺氧化物(alkyldimethylamineoxide)、乙氧基化脂肪酸单乙醇酰胺(ethoxylated fatty acid monoethanolamide)、脂肪酸单乙醇酰胺、多羟基烷基脂肪酸酰胺，或葡糖胺的N-酰基N-烷基衍生物(“葡糖酰胺(glucamides)”)。
洗涤剂可含有0-65％的洗涤剂增效剂(builder)或络合剂例如沸石、二磷酸盐/酯、三磷酸盐/酯、膦酸盐/酯(phosphonate)、碳酸盐/酯、柠檬酸盐/酯、氮川三乙酸(nitrilotriacetic acid)、乙二胺四乙酸、二亚乙基三胺五乙酸、烷基-或烯基琥珀酸、可溶性硅酸盐或层状硅酸盐(例如来自Hoechst的SKS-6)。
洗涤剂可包含一种或多种聚合物。实例是羧甲基纤维素、聚(乙烯吡咯烷酮)、聚(乙二醇)、聚(乙烯醇)、聚(乙烯吡啶-N-氧化物)、聚(乙烯基咪唑)、聚羧酸盐/酯(polycarboxylate)例如聚丙烯酸盐/酯、马来酸/丙烯酸共聚物和甲基丙烯酸月桂酯/丙烯酸共聚物。
洗涤剂可含有漂白体系，所述体系可包含H2O2源例如过硼酸盐或过碳酸盐，可将其与形成过酸的漂白活化剂(例如四乙酰乙二胺或壬酰氧苯磺酸酯(nonanoyloxybenzenesulfonate))组合。可供选择的是，漂白体系可包含过氧酸，例如酰胺、二酰亚胺或砜型的过氧酸。
可以使用常规稳定剂使本发明的洗涤剂组合物中的酶稳定化，所述稳定剂例如，多元醇例如丙二醇或甘油，糖或糖醇，乳酸，硼酸或硼酸衍生物，例如，芳香族硼酸酯，或苯基硼酸(phenyl boronic acid)衍生物例如4-甲酰苯基硼酸，并且所述组合物可按例如WO 92/19709和WO 92/19708中所述配制。
洗涤剂也可含有其它常规洗涤剂成分例如织物调节剂，包括粘土、泡沫促进剂、抑泡剂、抗腐蚀剂、悬污剂、防污物再沉积剂、染料、杀菌剂、光学增亮剂、助水溶物(hydrotrope)、晦暗抑制剂(tarnish inhibitors)或香料。
目前预期在洗涤剂组合物中，可以以相当于0.01-100mg酶蛋白每升洗涤液，优选0.05-5mg酶蛋白每升洗涤液，尤其是0.1-1mg酶蛋白每升洗涤液的量添加任何酶，尤其是本发明的酶。
地方性和区域性条件上的差异，例如水硬度和洗涤温度需要区域性的洗涤剂组合物。洗涤剂实例1和2分别提供了用于代表性的拉丁美洲洗涤剂和代表性的欧洲粉末洗涤剂的组合物的范围。
洗涤剂实例1.代表性的拉丁美洲洗涤剂组合物
| 组 | 亚名(Subname) | 含量 |
| 表面活性剂 | 磺酸盐/酯硫酸盐/酯肥皂非离子表面活性剂阳离子表面活性剂FAGA | 0-30％0-30％0-5％0-5％0-5％0-5％0-5％ |
| 漂白剂 | SPT / SPMNOBS，TAED | 0-20％0-15％0-5％ |
| 增效剂 | 磷酸盐/酯沸石Na2OSiO2Na2CO3 | 0-60％0-30％0-5％0-10％0-20％ |
| 填充剂 | Na2SO4 | 0-40％0-40％ |
| 其它 | 聚合物酶泡沫调节剂水助水溶物其它 | 直至100％ |
洗涤剂实例2.典型欧洲粉末洗涤剂组合物
| 组 | 亚名 | 含量 |
| 表面活性剂 | 磺酸盐/酯硫酸盐/酯肥皂非离子表面活性剂阳离子表面活性剂其它 | 0-30％0-20％0-15％0-10％0-10％0-10％0-10％ |
| 漂白 | SPT/SPMNOBS+TAED | 0-30％0-30％0-10％ |
| 增效剂 | 磷酸盐/酯沸石Na2OSiO2Na2CO3 | 0-60％0-40％0-40％0-20％0-20％ |
| 填充剂 | Na2SO4NaCl | 0-40％0-40％0-40％ |
| 其它 | 聚合物酶泡沫调节剂水助水溶物其它 | 直至100％ |
其它应用
本发明的枯草蛋白酶变体可用在食物的加工中，特别在乳制品(例如乳、奶油(cream)和干酪(cheese))的领域中；而且也可用在肉和蔬菜的加工中。本发明的枯草蛋白酶变体也可以用在牛、禽类和猪的饲料的加工中，尤其是用于宠物食品。此外，本发明的枯草蛋白酶变体可用于皮革的处理。本发明的枯草蛋白酶变体也可在医院、诊所和肉加工厂等用于净化设备、表面和其它材料的处理中，以分解朊病毒(prion)或其它传染物(infectious agent)。
材料和方法
产生蛋白酶变体的方法
本发明提供产生本发明的分离的酶的方法，其中在允许产生该酶的条件下培养已用编码所述酶的DNA序列转化的合适的宿主细胞，并且从培养物中回收所得的酶。
将包含编码酶的DNA序列的表达载体转化到异源宿主细胞中时，可使异源重组产生本发明的酶成为可能。因此可制造高度纯化的RP-II蛋白酶组合物，所述组合物的特征在于不含同源杂质。
用于培养经转化的宿主细胞的培养基可以是适合于培养所述宿主细胞的任何常规培养基。可方便地将表达的枯草蛋白酶变体分泌至培养基中，并且可通过熟知的方法从培养基中回收，所述方法包括：通过离心或过滤从培养基中分离细胞，通过盐例如硫酸铵沉淀培养基的蛋白质成分，随后是层析步骤例如离子交换层析、亲和层析等。
蛋白水解活性
能够使用PNA试验来测量酶活性，所述试验使用琥珀酰-丙氨酸-丙氨酸-脯氨酸-谷氨酸-对硝基苯胺作为底物。PNA试验的原理在Joumal of AmericanOil Chemists Society，Rothgeb，T.M.，Goodlander，B.D.，Garrison，P.H.，and Smith，L.A.，(1988)中描述。
织物
标准织物件从EMPA St.Gallen，Lerchfeldstrasse 5，CH-9014 St.Gallen，Switzerland或CFT，Center For Testmaterials，Vlaardingen，Netherlands获得。特别重要的是EMPA 116(用血液、乳和墨水沾污的棉织物)、EMPA 117(用血液、乳和墨水沾污的聚酯/棉织物)、C-03(用巧克力奶和烟灰沾污的棉织物)、C-05(用血液、乳和墨水沾污的棉织物)和C-10(用乳、油和颜料沾污的棉织物)。
洗涤条件
| 地域 | 拉丁美洲 | 北美洲 | 欧洲 | 亚洲，除日本 | 日本 |
| 温度 | 20-25℃ | 20-32℃ | 30-60℃ | 15-30℃ | 15-20℃ |
| 洗涤时间 | 14-16分钟 | 12分钟 | 20-40分钟 | 14-20分钟 | 15分钟 |
| 水硬度* | 6-12°dH | 6°dH | 15°dH | 14°dH | 3°dH |
| 洗涤剂剂量 | 1.5-4g/l | 1.0-1.5g/l | 4-10g/l | 1.5-2.5g/l | 0.5-0.7g/l |
| 洗涤pH | 依照现状 | 依照现状 | 依照现状 | 依照现状 | 依照现状 |
*°dH：通过将CaCl2*2H2O，MgCl2*6H2O和NaHCO3添加至Milli-Q水来调节。
洗涤剂
可在WO 97/07202中公开的洗涤剂配制物中或上述洗涤剂实例中测试本发明的酶。此外，能够在购自wfk testgewebe GmbH(Germany)或类似供应商的洗涤剂配制物中，或在商业洗涤剂中进行测试。
来自wfk testgewebe的测试洗涤剂列表：
●IEC 60456 Type A*Base Detergent
●IEC 60456 Type B Base Detergent
●IEC 60456 Type C Detergent
●ECE Reference Detergent with Phosphate(1977)
●ECE Reference Detergent without Phosphate(1998)
●AHAM Standard Detergent
●EU ECOLABEL(洗涤剂)Light Duty Detergent
●EU ECOLABEL(洗涤剂)PVP
然而，也可在洗涤试验中使用以下商业洗涤剂之一：例如Ariel HDP，P&G，Mexico；Omo Multi Acao HDP，Unilever，Brazil；Breeze HDP，UnileverThailand；Diao Pai，Nice，China；Tide HDL，P&G，US；Wisk HDL，Unilever，US；TOP HDP，Lion，Japan；Attack HDP，Kao，Japan；Ariel Regular HDP，P&G，Europe；Ariel Compact HDPC，P&G，Europe；Persil Megaperls，Henkel，Germanyand Persil，Unilever，UK。
此外，也能使用上述指定洗涤剂的品牌扩展(brand extension)和色彩/浓缩版本(color/compact version)。
如果洗涤剂含有酶，应该将所述洗涤剂在使用之前灭活，从而消除已经存在于该洗涤剂中的酶活性。这通过将洗涤剂原液(stock solution)在微波炉中加热至85℃ 5分钟来完成。微波炉中待灭活的洗涤剂原液的浓度是4-20g/l。
自动机械应力试验(Automatic Mechanical Stress Assay)
在下文实施例3中描述了自动机械应力试验(AMSA)。
小洗涤试验(mini wash assay)
在以下条件下进行毫升规模洗涤性能试验：
| 洗涤剂 | 拉丁美洲HDP |
| 洗涤剂剂量 | 1.5-4g/l |
| pH | 依照现状(As it is) |
| 洗涤时间 | 14-16分钟 |
| 温度 | 20-25℃ |
| 水硬度 | 6-12°dH，通过向milli-Q水添加CaCl2*2H2O、MgCl2*6H2O和NaHCO3来调节 |
| 酶浓度 | 5nM、10nM、30nM |
| 测试体系 | 125 ml玻璃烧杯。将织物浸入测试溶液中。在洗涤剂溶液中不断提起和放下，50次每分钟。 |
| 测试溶液体积 | 50ml |
在洗涤之后，将织物件在自来水中冲洗(flush)并且风干，使用Zeiss MCS521 VIS分光光度计在460nm测量测试材料的减退(remission)(R)。根据制造商的规程进行所述测量。
将新变体的性能与Savinase的性能通过如下计算相对性能进行比较：
RP＝(R变体-R空白)/(RSAVINASE-R空白)
如果变体在至少一种洗涤剂组合物中表现的好于参照，则认为该变体显示改进的洗涤性能。
实施例1
酶变体的构建和表达：
定点诱变：
本发明的包含特异性插入/缺失/取代的枯草溶菌素309(Savinase
)定点诱变变体通过传统DNA片段的克隆(Sambrook et al.，Molecular Cloning：ALaboratory Manual，2nd Ed.，Cold Spring Harbor，1989)制备，所述DNA片段使用含有期望突变的寡核苷酸通过PCR产生。
简而言之，将携带枯草溶菌素309wt或枯草溶菌素309变体基因的质粒DNApSX222(大肠杆菌/枯草芽孢杆菌穿梭载体，包括适当的选择标记、用于芽孢杆菌属和大肠杆菌的复制起点、消化位点等，公开于WO 96/34946)用作PCR反应中的模板。在第一PCR中，使用含有期望突变的寡核苷酸(反义)和合适的反向寡核苷酸(有义)。将所得DNA片段作为有义寡核苷酸，与合适的反义寡核苷酸一起用于第二PCR。将所得DNA片段用合适的限制酶消化并且连接到用相同酶消化的合适的大肠杆菌/芽孢杆菌属穿梭载体(例如pSX222)中。
将连接产物转化到感受态大肠杆菌中，并且涂布在含有适当选择标记的固体琼脂上。对从单克隆纯化的DNA测序以确认设计的突变。从携带质粒的大肠杆菌细胞分离质粒DNA，所述质粒包含具有设计突变的枯草溶菌素309基因，并且将所述质粒DNA转化至合适的感受态枯草芽孢杆菌菌株中，例如枯草芽孢杆菌DN1885：公开于WO 01/16285(例如，如Dubnau et al.，1971，J.Mol.Biol.56，pp.209-221所述)，并且涂布于含有适当选择标记的固体琼脂上。
从显示出蛋白酶活性的芽孢杆菌属单菌落分离质粒DNA，并且对其测序以确认设计的突变。将携带质粒DNA的芽孢杆菌属菌落在带挡板摇瓶中的合适培养基中发酵，所述质粒DNA包括具有期望突变的枯草溶菌素309基因。
实施例2
酶浓度(enzym concentration)的纯化和评估
发酵之后，使用疏水电荷诱导层析(Hydrophobic Charge InductionChromatography(HCIC))和后续的真空过滤来完成枯草溶菌素变体的纯化。为了捕获所述酶，HCIC使用与4-巯基-乙基-吡啶(4-Mercapto-Ethyl-Pyridine)(4-MEP)结合的纤维素基质。
将大小80-100μm的纤维素基质小珠与含有酵母提取物和能够分泌枯草溶菌素变体的转化的枯草芽孢杆菌的培养基混合，并且在Unifilter
微量培养板中于pH9.5温育。由于4-MEP在pH＞7是疏水的，并且枯草溶菌素变体在pH9.5是疏水的，所以在分泌的酶和小珠上的4-MEP之间形成疏水缔合(hydrophobic association)。温育之后，将培养基和细胞碎片通过真空过滤去除，同时将小珠和酶保留在滤器上。为了将酶从小珠上洗脱，此时通过用洗脱缓冲液(pH5)洗涤滤器将pH降低。由此酶从小珠上脱离并且能够从缓冲液中回收。
通过活性位点滴定(active site titration)(AST)来评估纯化的枯草溶菌素变体的浓度。将纯化的酶与不同浓度的高亲和性抑制剂CI-2A一起温育以抑制不同量的活性位点。蛋白酶和抑制剂以1∶1的比率相互结合，因此酶浓度能够与将全部蛋白酶失活的抑制剂浓度正相关。为了测量残余蛋白酶活性，在与抑制剂温育之后添加底物(Tris/HCl缓冲液中的0.6mMSuc-Ala-Ala-Pro-Phe-pNA)，在接下来的4分钟内，在Elisa Reader上于405nm周期性测量降解产物pNA(对硝基酚)的显色(development)。根据上述方法纯化本文表1所列的各种本发明的变体，接着测定酶浓度。
如下所述测试已知浓度的表1中变体在洗涤剂中的洗涤性能。
实施例3
包含枯草蛋白酶变体的洗涤剂组合物的洗涤性能
使用自动机械应力试验(AMSA)测试本申请的酶变体。使用AMSA试验，能够考察大量小体积酶洗涤剂溶液的洗涤性能。AMSA平板具有许多用于测试溶液的狭缝(slot)和将待洗涤的织物样品紧紧挤靠所有狭缝开度(slotopening)的盖子。在洗涤过程中，剧烈地摇动平板、测试溶液、织物和盖以使测试溶液与织物接触并且施加机械应力。进一步的描述参见WO 02/42740，特别是23-24页的段落“Special method embodiments(特殊方法实施方式)”。试验在如下的指定实验条件下进行。
洗涤剂 拉丁美洲型HDP
洗涤剂剂量 2.2g/l
测试溶液体积 160微升
pH 用NaHCO3调节至pH 9.5-10.5
洗涤时间 14分钟
温度 20℃
水硬度 9°dH*
测试溶液中的酶浓度 5nM、10nM和30nM
测试材料 C-10
*°dH：通过向milli-Q水添加CaCl2*2H2O；MgCl2*6H2O(比率Ca2+∶Mg2+-＝2∶1)来调节。
根据本文19页的洗涤剂实例1中的规定组成拉丁美洲型洗涤剂。洗涤之后，将织物件在自来水中冲洗并且风干。
将酶变体的性能测定为用特定酶变体洗涤的织物样品的颜色亮度(brightness of colour)。亮度也可表示为在用白光照射时从织物样品反射的光的强度。当织物受到沾污时，反射光的强度低于清洁织物的反射光的强度。因此，能够使用反射光的强度测量酶变体的洗涤性能。
用专业平板扫描仪(PFUDL2400pro)进行颜色测量，所述扫描仪用于捕获经洗涤织物样品的图像。使用200dpi的分辨率(resolution)和24bit的输出颜色深度(output colour dept)来进行扫描。为了获得精确的结果，经常性地使用Kodak reflective IT8 target校准扫描仪。为了从扫描图像中提取光强度值，使用特别设计的软件应用程序(Novozymes Color Vector Analyzer)。所述程序从图像还原24 bit像素值并且将它们转化成红(r)、绿(g)和蓝(b)值。通过将(r)、(g)和(b)值作为矢量加在一起，再取所得矢量的长度来计算强度值(Int)：
将变体的洗涤性能定义为用酶变体洗涤的织物表面的光强度值：
P＝Int(v)
结果示于表2，其中将性能作为在10nM蛋白酶浓度新变体的性能与Savinase性能相比的相对性能给出：
RP是(P变体-P空白)/(PSAVINASE-P空白)
表2.相对于Savinase性能的枯草蛋白酶变体洗涤性能测试结果
| 变体中的突变 | 相对性能 |
| T143K，Y167A，R170S，A194P | 1，8 |
| Y167A，R170S，A194P，K251R | 1，6 |
| Y167A，R170S，A194P，S265K | 2，0 |
| Y167A，R170S，A194P，V244R | 1，9 |
| S141E，Y167A，R170S，A194P | 1，1 |
| Y167A，R170S，M175I | 1，2 |
| Y167A，R170S，A172T | 1，2 |
| Y167A，R170S，A174V，M175F | 1，4 |
| Y167A，R170S，A172V，A174V | 1，5 |
| Y167A，R170S，A172E | 1，3 |
| Y167A，R170S，M175L | 1，5 |
| Y167A，R170S，A174T | 1，2 |
| Y167A，R170S，A174T，M175L | 1，4 |
| G53C，G61E | 1，3 |
| A98S，S99D，G100S | 1，4 |
| S9R，T22A，V68A，S99A，*99aD | 1，7 |
| S9R，P14H，R19L，N62D | 1，8 |
| G61P，*99aS | 1，7 |
| N43S，N62D | 1，7 |
| *96aG，P131S，V203A，A228T | 1，8 |
| N62D，A232C，Q236L，Q245N | 2，0 |
| *96aA，A98T，R247K | 2，0 |
| S99D，S101R，S103A，V104I，G160S，A194P，L217D | 1，4 |
| *61aD | 1，3 |
| N62D，S106A | 2，2 |
| V68A，S106M，N184D | 1，6 |
| S9R，A15T，*97aV，H120N | 1，4 |
| A15M，A16P，*99aD | 1，6 |
| *99aE，G160S，S163T，G195S，G211S，K237R，G258A，T260L | 1，2 |
| G23S，*99aD，A194P，S242T，Q245R | 1，5 |
| G100S，N173D | 1，4 |
| Y167A，R170S，A172E | 1，1 |
| A98T，Q137L，Y167A，R170S，M175L | 1，1 |
| *98aA，S99D | 1，8 |
| S99A，*99aD，V203A | 1，8 |
| N62D，K237R | 2，1 |
| V11M，N76D，L126F，K251R | 1，4 |
| S9F，A15L，A16P，T22I，*98aA，S99D，R170H | 1，2 |
| *96aA，*130aG，P131H | 1，5 |
| E54D，N62D | 2，0 |
| *98aA，*98bS，S99G，S101T | 1，8 |
| S9R，A15T，V68A，179T，G102S，P131H，Q137H | 1，7 |
| *100aA，*100bG，*100cS，*100dG | 1，7 |
| V68A，L111I | 1，8 |
| *98aA，R170H，Q245R | 1，8 |
| I35V，N62D，N183D，T224S | 1，2 |
| *97aG，P131S，V203A，A228T | 1，4 |
| S9R，R10K，P14Q，T22A，Y167A，R170S | 1，6 |
| S9R，*22aL，S57A，G61E，*98aA，V139L，N173S | 1，3 |
| P14T，N18K，Y167A，R170S | 2，0 |
| S9R，Q12E，P14Q，K27R，Y167A，R170S | 1，7 |
| N62D，R170L | 1，7 |
| N62D，R170S，Q245R | 1，4 |
| Y167A，R170S，A194P，K251R，S265K | 2，0 |
| P14T，N18K，Y167A，R170S，A194P | 2，0 |
| N62D，A151G，K237R | 2，0 |
| N62D，A151G，Q245R | 1，9 |
| N62D，A151G，K237R，Q245R | 2，0 |
实施例4
包含枯草蛋白酶变体的洗涤剂组合物的洗涤性能
在以下条件下进行毫升规模洗涤性能试验：
小洗涤试验
| 洗涤剂 | Persil，Lever，UK，HDP |
| 洗涤剂剂量 | 6g/l |
| pH | 依照现状 |
| 洗涤时间 | 20分钟 |
| 温度 | 30℃ |
| 水硬度 | 15°dH，通过向milli-Q水添加CaCl2*2H2O、MgCl2*6H2O和NaHCO3(4∶1∶7.5)来调节 |
| 酶浓度 | 2.5nM、5nM、10nM、30nM、60nM |
| 测试体系 | 125ml玻璃烧杯。将织物浸入测试溶液中。在洗涤剂溶液中不断提起和放下，每分钟50次。使用的样品：EMPA 116(2.5cm×7cm) |
| 测试溶液体积 | 50ml |
洗涤之后，将织物件在自来水中冲洗并且风干，使用Zeiss MCS 521 VIS分光光度计在460nm测量测试材料的减退(R)。根据制造商的规程进行所述测量。
通过如下计算相对性能，在10nM蛋白酶浓度将新变体的性能与Savinase的性能进行比较：
RP＝(R变体-R空白)/(RSAVINASE-R空白)
如果变体在至少一种洗涤剂组合物中的表现好于参照，则认为该变体显示出改进的洗涤性能。
记分方式：
对于具有等于或好于1.1的改进洗涤性能的变体，给予分数＝1。
| 突变 | 得分 |
| S103A，V104I，G159D，A232V，Q236H，Q245R | 1 |
| S9R，A15T，T22A，V139L | 1 |
| S9R，A15T，G61E，A85T，E89Q，P239L，Q245C | 1 |
| S9R，A15T，V68A，H120N，Q245R | 1 |
| N248R | 1 |
| S9R，A15T，*22aL，V139L，N204D，Q245L | 1 |
| N218S | 1 |
| S9R，A15T，V68A，Q245R，N252K | 1 |
| S9R，A15T，V68A，Q245R，H120N | 1 |
| V68A，S106A，H120N | 1 |
| V68A，S106A，N252K | 1 |
| A15T，V68A，S99G，Q245R，N261D | 1 |
| S9R，V68A，S99G，Q245R，N261D | 1 |
| V68A，S99G，Q245R，N261D | 1 |
| S9R，A15T，V68A，S99G，N261D | 1 |
| S9R，A15T，V68A，Q245R，N261D | 1 |
| S9R，A15T，*22aL，V139L，S163G，N204D，Q245L | 1 |
| Q245R，N252H | 1 |
| S9R，*22aL，G61E，*97aA，M119I，Q137H，N173S | 1 |
| V68A，S106A，T213A | 1 |
| S9R，A15T，V68A，H120N，P131S，Q137H，Q245M | 1 |
| S9R，A15T，V68A，I72F，S99G，Q245R，N261D | 1 |
| S9R，A15T，V68A，S99D，Q245R，N261D | 1 |
| S9R，A15T，V68A，S99G，A194P，Q245R，N261D | 1 |
| S9R，A15T，V68A，N76I，S99G，Q245R，N261D | 1 |
| S9R，A15T，V68A，S99G，A228V，Q245R，N261D | 1 |
序列表
<110>诺维信公司(Novozymes A/S)
<120>枯草蛋白酶变体
<130>10826.204-WO
<160>2
<170>PatentIn version 3.2
<210>1
<211>275
<212>PRT
<213>解淀粉芽孢杆菌(B.amyloliquefaciens)(枯草溶菌素BPN’)
<400>1
Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu
1 5 10 15
His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp
20 25 30
Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala
35 40 45
Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His
50 55 60
Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly
65 70 75 80
Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu
85 90 95
Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu
100 105 110
Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly
115 120 125
Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala
130 135 140
Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly
145 150 155 160
Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala
165 170 175
Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val
180 185 190
Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr
195 200 205
Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser
210 215 220
Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn
225 230 235 240
Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys
245 250 255
Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala
260 265 270
Ala Ala Gln
275
<210>2
<211>269
<212>PRT
<213>迟缓芽孢杆菌(B.lentus)(枯草溶菌素309)
<400>2
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala
1 5 10 15
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
20 25 30
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser
35 40 45
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu
65 70 75 80
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala
85 90 95
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala
100 105 110
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser
115 120 125
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly
130 135 140
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser
145 150 155 160
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln
165 170 175
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile
180 185 190
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr
195 200 205
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
210 215 220
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile
225 230 235 240
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu
245 250 255
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg
260 265
Claims (18)
1.亲本枯草溶菌素309的变体，其中所述变体选自下组的修饰：
S9R，A15T，V68A，S99G，A194P，Q245R，和N261D；或
S9R，A15T，V68A，S99G，A228V，Q245R，和N261D；
其中所述变体具有蛋白酶活性且每个位置相应于SEQ ID NO：1的氨基酸序列的位置。
2.分离的DNA序列，其编码权利要求1中定义的变体。
3.重组表达载体，其携带包含权利要求2的分离的DNA序列的DNA构建体。
4.微生物宿主细胞，其已用权利要求3的重组表达载体转化。
5.根据权利要求4的微生物宿主细胞，所述微生物宿主细胞是细菌。
6.根据权利要求5的微生物宿主细胞，所述微生物宿主细胞是芽孢杆菌属。
7.根据权利要求6的微生物宿主细胞，所述微生物宿主细胞是迟缓芽孢杆菌。
8.根据权利要求4的微生物宿主细胞，所述微生物宿主细胞是真菌。
9.根据权利要求8的微生物宿主细胞，所述微生物宿主细胞是丝状真菌。
10.根据权利要求9的微生物宿主细胞，所述微生物宿主细胞是曲霉属。
11.用于产生权利要求1中定义的变体的方法，其中在有益于表达和分泌所述变体的条件下培养权利要求4-10中任一项定义的宿主细胞，并且回收该变体。
12.包含权利要求1中定义的变体的清洁或洗涤剂组合物。
13.根据权利要求12的组合物，所述组合物是洗衣或洗碗组合物。
14.根据权利要求12或13的组合物，所述组合物额外地包含纤维素酶、淀粉酶、蛋白酶、半纤维素酶、酯酶、和/或木质酶。
15.根据权利要求14的组合物，其中所述淀粉酶为淀粉糖化酶。
16.根据权利要求14的组合物，其中所述半纤维素酶为乳糖酶或β-半乳糖苷酶。
17.根据权利要求12或13的组合物，所述组合物额外地包含脂肪酶、角质酶或多聚半乳糖醛酸酶。
18.权利要求1中定义的变体在洗衣和/或洗碗洗涤剂中的用途。
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| Application Number | Priority Date | Filing Date | Title |
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| DKPA200501007 | 2005-07-08 | ||
| DKPA200501007 | 2005-07-08 | ||
| PCT/DK2006/000399 WO2007006305A1 (en) | 2005-07-08 | 2006-07-06 | Subtilase variants |
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| CN201310544773.1A Division CN103555697A (zh) | 2005-07-08 | 2006-07-06 | 枯草蛋白酶变体 |
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| JP (3) | JP2009500023A (zh) |
| CN (2) | CN103555697A (zh) |
| AT (1) | ATE529505T1 (zh) |
| MX (1) | MX2007016045A (zh) |
| WO (1) | WO2007006305A1 (zh) |
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2006
- 2006-07-06 JP JP2008519797A patent/JP2009500023A/ja active Pending
- 2006-07-06 EP EP10180164A patent/EP2290061A3/en not_active Withdrawn
- 2006-07-06 EP EP10183130.3A patent/EP2385111B1/en not_active Not-in-force
- 2006-07-06 WO PCT/DK2006/000399 patent/WO2007006305A1/en not_active Application Discontinuation
- 2006-07-06 CN CN201310544773.1A patent/CN103555697A/zh active Pending
- 2006-07-06 AT AT06753336T patent/ATE529505T1/de not_active IP Right Cessation
- 2006-07-06 CN CN2006800247046A patent/CN101218343B/zh not_active Expired - Fee Related
- 2006-07-06 EP EP10183149.3A patent/EP2385112B1/en not_active Not-in-force
- 2006-07-06 EP EP06753336A patent/EP1904628B1/en not_active Not-in-force
- 2006-07-06 MX MX2007016045A patent/MX2007016045A/es active IP Right Grant
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- 2012-03-19 JP JP2012062381A patent/JP6054615B2/ja not_active Expired - Fee Related
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| CN1258745A (zh) * | 1998-12-31 | 2000-07-05 | 中国科学院上海生物工程研究中心 | 新型枯草杆菌蛋白酶及制法和用途 |
| CN1258744A (zh) * | 1998-12-31 | 2000-07-05 | 中国科学院上海生物工程研究中心 | 非水介质中稳定的多肽连接酶及制法和用途 |
Also Published As
| Publication number | Publication date |
|---|---|
| EP2385112A3 (en) | 2012-02-08 |
| JP6054615B2 (ja) | 2016-12-27 |
| CN103555697A (zh) | 2014-02-05 |
| ATE529505T1 (de) | 2011-11-15 |
| JP2009500023A (ja) | 2009-01-08 |
| WO2007006305A1 (en) | 2007-01-18 |
| MX2007016045A (es) | 2008-03-10 |
| JP2012125253A (ja) | 2012-07-05 |
| EP1904628A1 (en) | 2008-04-02 |
| EP2385111A3 (en) | 2012-02-08 |
| EP2385112B1 (en) | 2016-11-30 |
| EP2385111B1 (en) | 2016-09-07 |
| EP2290061A2 (en) | 2011-03-02 |
| EP2385112A2 (en) | 2011-11-09 |
| EP1904628B1 (en) | 2011-10-19 |
| EP2290061A3 (en) | 2011-07-06 |
| CN101218343A (zh) | 2008-07-09 |
| JP2016041077A (ja) | 2016-03-31 |
| EP2385111A2 (en) | 2011-11-09 |
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