CN1295313C - 含多取代蛋白酶变体的清洗组合物 - Google Patents
含多取代蛋白酶变体的清洗组合物 Download PDFInfo
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- CN1295313C CN1295313C CNB2004100399770A CN200410039977A CN1295313C CN 1295313 C CN1295313 C CN 1295313C CN B2004100399770 A CNB2004100399770 A CN B2004100399770A CN 200410039977 A CN200410039977 A CN 200410039977A CN 1295313 C CN1295313 C CN 1295313C
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Abstract
含蛋白酶变体的清洗组合物及其用法,该组合物所含蛋白酶变体包括解淀粉芽胞杆菌枯草杆菌蛋白酶的相应103位点的氨基酸残基被另一种天然氨基酸残基取代,同时取代的还包括如说明书所述位点处的氨基酸残基,其中若所述蛋白酶变体包括相应位点103和76的氨基酸残基取代时,也同时包括相应下述位点以外的说明书所列位点的氨基酸残基的取代:27,99,101,104,107,109,123,128,166,204,206,210,216,217,218,222,260,265和274;该清洗组合物还含清洗添加剂。另一种清洗组合物含解淀粉芽胞杆菌枯草杆菌蛋白酶的相应62、212、230、232、252和257位点处被另一种天然氨基酸残基取代的蛋白酶变体,并含清洗添加剂。
Description
本申请是申请日为1998年10月23日、申请号为98812610.9的专利申请的分案申请。
本发明领域
本发明涉及包含1种或多种多取代蛋白酶变体和1种或多种清洁添加剂材料的清洗组合物。更具体地说,本发明涉及包含1种或多种多取代蛋白酶变体和1种或多种清洁添加剂材料的衣物洗涤剂组合物、餐具洗涤剂组合物、硬表面清洗组合物及个人清洗组合物。
本发明背景
各种类型的酶被用于衣物洗涤剂中以促进某些污渍从织物上的脱除。每一类酶(淀粉酶、蛋白酶等)通常能催化特定的化学反应。比如,蛋白酶以其水解(使化合物分解为2种或更多种更简单的化合物)其他蛋白(质)的能力而著称。此种能力一直是通过将天然存在或制造的蛋白酶加入到衣物洗涤剂组合物中而加以利用的。
近年来,人们还研究了各种酶在自动餐具洗涤组合物中的应用。遗憾的是,许多酶,例如许多传统蛋白酶,不能很好地转译为洗涤环境。具体地说,需要对热稳定性、pH稳定性、氧化稳定性及底物特异性进行优化才能获得满意的效果。
授予Estell等人的美国专利RE34,606公开了枯草杆菌蛋白酶氨基酸残基的改性,这些残基对应于解淀粉芽胞杆菌(Bacillusamyloliguefaciens)枯草杆菌蛋白酶中的下列位置:酪氨酸-1、天冬氨酸+32、天冬酰胺+155、酪氨酸+104、甲硫氨酸+222、甘氨酸+166、组氨酸+64、甘氨酸+169、苯丙氨酸+189、丝氨酸+33、丝氨酸+221、酪氨酸+217、谷氨酸+156及丙氨酸+152。
美国专利5,182,204公开了对解淀粉芽胞杆菌枯草杆菌蛋白酶中氨基酸+224残基的改性,以及对可通过取代、插入或缺失实现其他枯草杆菌蛋白酶中相同位置(残基)上的改性,此种改性物还可配合着实施对美国专利RE34,606中指出的各种残基的改性,最终形成有用的枯草杆菌蛋白酶突变体或变体。美国专利5,182,204还公开了对枯草杆菌蛋白酶中多种氨基酸残基的改性,这些残基具体地包括+99、+101、+103、107、+126、+128、+135、+197和+204。
美国专利5,155,033公开了类似的突变体枯草杆菌蛋白酶,它们在与解淀粉芽胞杆菌枯草杆菌蛋白酶的+225等同的位置进行了改性。
美国专利5,185,258和5,204,015公开了类似的突变体枯草杆菌蛋白酶,它们在位点+123和/或+274进行了改性。
美国专利4,914,031公开了某种枯草杆菌蛋白酶类似物,包括在位点+76改性的枯草杆菌蛋白酶。
授予Baeck等人的美国专利5,679,630公开了一种包含蛋白酶变体的清洗组合物,该变体包括在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的位点76并结合在1个或多个下列位点用其他氨基酸残基对(原来)氨基酸残基进行的取代:99、101、103、104、107、123、27、105、109、126、128、135、156、166、195、197、204、206、210、216、217、218、222、260、265和/或274;以及1种或多种清洗组合物材料。
然而,目前依然存在着对于当用于洗涤剂及清洗组合物中时能改善和强化清洁能力的蛋白酶,特别是丝氨酸蛋白酶。
而且,本申请中要求保护的特异性组合乃是任何先有技术文献中从未提到的。
本发明概述
本发明满足了上述要求:现已惊奇地发现,本发明的多取代蛋白酶变体当用于清洗组合物中时可改善并强化清洁能力,包括但不限于,在污渍和/或污垢的脱除和/或减少,和/或保持白度和/或污迹的清除和/或色斑和/或膜状物的脱除和/或减少上,均优于传统含蛋白酶清洗组合物。
本发明的多取代蛋白酶变体适用于高及低密度粒状、重垢型及轻垢型液态、片剂以及合成洗涤剂条(块)状组合物,乃至其他清洗组合物。
本发明一个方面提供一种清洗组合物,包含:
(a)蛋白酶变体,优选有效数量蛋白酶变体,更优选占清洗组合物重量的约0.0001~约10%的蛋白酶变体,其中所述蛋白酶变体包括在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的氨基酸残基位点103用另一种天然存在的氨基酸残基取代其氨基酸残基,并结合在解淀粉芽胞杆菌枯草杆菌蛋白酶1个或多个下列氨基酸残基位点用另一天然存在的氨基酸残基取代其氨基酸残基:
1,3,4,8,9,10,12,13,16,17,18,19,20,21,22,24,27,33,37,38,42,43,48,55,57,58,61,62,68,72,75,76,77,78,79,86,87,89,97,98,99,101,102,104,106,107,109,111,114,116,117,119,121,123,126,128,130,131,133,134,137,140,141,142,146,147,158,159,160,166,167,170,173,174,177,181,182,183,184,185,188,192,194,198,203,204,205,206,209,210,211,212,213,214,215,216,217,218,222,224,227,228,230,232,236,237,238,240,242,243,244,245,246,247,248,249,251,252,253,254,255,256,257,258,259,260,261,262,263,265,268,269,270,271,272,274及275
其中当所述蛋白酶变体包括对应于位点103及76上的氨基酸残基取代时,则还存在1个或多个除对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的氨基酸残基位点27、99、101、104、107、109、123、128、166、204、206、210、216、217、218、222、260、265或274以外的氨基酸残基位点上的氨基酸残基取代;以及
(b)1种或多种清洁添加剂材料。
本发明另一个方面提供一种织物清洗组合物,包含:
(a)蛋白酶变体,优选有效数量蛋白酶变体,更优选占织物清洗组合物的约0.0001~约10wt%蛋白酶变体,其中所述蛋白酶变体如同上面所述;以及
(b)占织物清洗组合物的至少约5wt%表面活性剂;以及
(c)占织物清洗组合物的至少约5wt%助洗剂。
本发明又一个方面提供一种清洁需要清洁的织物的方法,包括令织物与本发明的织物清洗组合物进行接触。
本发明又一个方面提供一种餐具洗涤组合物,包含:
(a)蛋白酶变体,优选有效数量蛋白酶变体,更优选占餐具洗涤组合物的约0.0001~约10wt%蛋白酶变体,其中所述蛋白酶变体如同上面所述;以及
(b)约0.1%~约10wt%表面活性剂。
本发明又一个方面提供一种清洁需要清洁的餐具的方法,包括令餐具与本发明的餐具洗涤组合物进行接触。
本发明又一个方面提供一种个人清洗组合物,包括:
(a)蛋白酶变体,优选有效数量蛋白酶变体,更优选占个人清洗组合物的约0.001~约5wt%蛋白酶变体,其中所述蛋白酶变体如同上面所述;以及
(b)占个人清洗组合物的约0.1%~约95wt%表面活性剂体系;以及
(c)任选地,占个人清洗组合物的约0.05%~约50wt%酶稳定剂。
本发明又一个方面提供一种清洁人体或较低级动物身体需清洁部分的方法,包括令该部分与本发明的个人清洗组合物进行接触。
本发明又一个方面提供一种清洗组合物,包含:
(a)蛋白酶变体,优选有效数量蛋白酶变体,更优选占清洗组合物的约0.0001~约10wt%蛋白酶变体,其中所述蛋白酶变体包括在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的位点62、212、230、232、252及257中1个或多个氨基酸残基位点用另一种天然存在的氨基酸残基取代氨基酸残基;以及
(b)1种或多种清洁添加剂材料。
本发明另一个方面提供一种织物清洗组合物,包含:
(a)蛋白酶变体,优选有效数量蛋白酶变体,更优选占织物清洗组合物的约0.0001~约10wt%蛋白酶变体,其中所述蛋白酶变体包括在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的位点62、212、230、232、252及257中1个或多个氨基酸残基位点用另一种天然存在的氨基酸残基取代氨基酸残基;以及
(b)占织物清洗组合物的至少约5wt%表面活性剂;以及
(c)占织物清洗组合物的至少约5wt%助洗剂。
本发明又一个方面提供一种清洁需要清洁的织物的方法,包括令织物与本发明的织物清洗组合物进行接触。
本发明又一个方面提供一种餐具洗涤组合物,包含:
(a)蛋白酶变体,优选有效数量蛋白酶变体,更优选占餐具洗涤组合物的约0.0001~约10wt%蛋白酶变体,其中所述蛋白酶变体包括在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的位点62、212、230、232、252及257中1个或多个氨基酸残基位点用另一种天然存在的氨基酸残基取代氨基酸残基;以及
(b)占餐具洗涤组合物的约0.1%~约10wt%表面活性剂,
本发明又一个方面提供一种清洁需要清洁的餐具的方法,包括令餐具与本发明的餐具洗涤组合物进行接触。
本发明又一个方面提供一种个人洗漱组合物,包括:
(a)蛋白酶变体,优选有效数量蛋白酶变体,更优选占个人洗漱组合物的约0.001~约5wt%蛋白酶变体,其中所述蛋白酶变体包括在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的位点62、212、230、232、252及257中1个或多个氨基酸残基位点用另一种天然存在的氨基酸残基取代氨基酸残基;以及
(b)占个人洗漱组合物的约0.1%~约95wt%表面活性剂体系;以及
(c)任选地,占个人洗漱组合物的约0.05%~约50wt%任选的酶稳定剂。
本发明又一个方面提供一种清洁人体或较低级动物身体需洗漱部分的方法,包括令该部分与本发明的个人洗漱组合物进行接触。
综上所述,本发明的一个目的是提供清洗组合物,它包含能改善并强化织物、餐具、餐桌用具、厨房用具、炊具及其他硬表面清洁效果的蛋白酶变体。本发明另一个目的是提供采用本发明含蛋白酶变体的清洗组合物来清洗织物、餐具、餐桌用具、厨房用具、炊具及其他硬表面的方法。
以上以及其他目的、特征及优点在研读了下面的详细描述、实施例及所附权利要求之后将变得更加清楚。
本文所使用的所有百分率、比值和比例均以重量为基准,除非另行指明。
附图简述
图1A~C描绘出解淀粉芽胞杆菌枯草杆菌蛋白酶的DNA和氨基酸序列以及此种基因的局部限制图。
图2描绘出来自解淀粉芽胞杆菌(BPN)’及迟缓芽胞杆菌(Bacillus lentus)(野生型)的枯草杆菌蛋白酶之间的保守氨基酸残基。
图3A和3B描绘出4种枯草杆菌蛋白酶的氨基酸序列。顶部的一行代表来自解淀粉芽胞杆菌的枯草杆菌蛋白酶(有时亦称作枯草杆菌蛋白酶BPN’)的氨基酸序列。第2行描绘出来自枯草芽胞杆菌(Bacillus subtilis)的枯草杆菌蛋白酶的氨基酸序列。第3行描绘出来自地衣芽胞杆菌(B.Licheniformis)的枯草杆菌蛋白酶的氨基酸序列。第4行描绘出来自迟缓芽胞杆菌的枯草杆菌蛋白酶(在PCTWO89/06276中亦称作枯草杆菌蛋白酶309)的氨基酸序列。符号*表示与枯草杆菌蛋白酶BPN’相比,不存在的特异氨基酸残基。
本发明详述
蛋白酶-蛋白酶是通常会导致蛋白或肽的肽键发生断裂的羰基水解酶。本文所使用的术语“蛋白酶”是指天然存在的蛋白酶或重组蛋白酶。天然存在的蛋白酶包括α-氨酰基肽水解酶、肽基氨基酸水解酶、酰氨基水解酶、丝氨酸羧肽酶、金属羧肽酶、硫醇蛋白酶、羧基蛋白酶及金属蛋白酶。丝氨酸、金属、硫醇及酸型蛋白酶,乃至内切及外切-蛋白酶,一律包括在内。
本发明包括的非天然存在的蛋白酶是这样一类羰基水解酶变体(蛋白酶变体),它具有的蛋白水解活性、稳定性、底物特异性、pH特性曲线和/或性能特征不同于衍生出该变体氨基酸序列的前体羰基水解酶。具体地说,此类蛋白酶变体所具有的氨基酸序列在自然界中不存在,它是通过以不同的氨基酸取代前体蛋白酶的多个氨基酸残基而衍生的。前体蛋白酶可以是天然存在的或者是重组蛋白酶。前面已经提到,这些蛋白酶变体被设计成具备类似胰蛋白酶的特异性并优选对漂白保持稳定。
这里有用的蛋白酶变体涵盖:19种天然存在的L-氨基酸中任何一种在指定氨基酸残基位点发生了取代。
此种取代可在任何前体枯草杆菌蛋白酶(原核/真核/哺乳动物类等)中实施。在本申请全文中,各种氨基酸采用其常用的1-和3-字母代码来称呼。此种代码可见诸于Dale.M.W.(1989),《细菌分子遗传学》,John Wiley&Sons公司,附录B。
这里有用的蛋白酶变体优选由芽胞杆菌属枯草杆菌蛋白酶衍生而来。更优选的是,这些蛋白酶变体由迟缓芽胞杆菌枯草杆菌蛋白酶和/或枯草杆菌蛋白酶309衍生而来。
羰基水解酶-羰基水解酶是能水解含
键的化合物的蛋白酶,其中X是氧或氮。它们包括天然存在的羰基水解酶和重组羰基水解酶。天然存在的羰基水解酶主要包括诸如肽水解酶之类的水解酶,如枯草杆菌蛋白酶或金属蛋白酶。肽水解酶包括α-氨酰基肽水解酶、酰氨基水解酶、丝氨酸羧肽酶、金属羧肽酶、硫醇蛋白酶、羧基蛋白酶及金属蛋白酶。丝氨酸、金属、硫醇及酸型蛋白酶的,乃至内切及肽外切蛋白酶均包括在内。
枯草杆菌蛋白酶-枯草杆菌蛋白酶是通常能使蛋白或肽的肽基断裂的细菌和真菌类蛋白酶。本文所使用的术语“枯草杆菌蛋白酶”是指天然存在的枯草杆菌蛋白酶或重组枯草杆菌蛋白酶。已知有一系列天然存在的枯草杆菌蛋白酶可由各种各样微生物种产生,常常是分泌出来。这一系列当中,各个成员的氨基酸序列不完全是同源的。然而,这系列中的枯草杆菌蛋白酶却表现出相同或相近类型的蛋白水解活性。这类丝氨酸蛋白酶具有相同的氨基酸序列,该序列确定了使其区别于与胰凝乳蛋白酶相关的一类丝氨酸蛋白酶的催化三分体。与枯草杆菌蛋白酶和胰凝乳蛋白酶相关的丝氨酸蛋白酶都具有包含天冬氨酸、组氨酸及丝氨酸的催化三分体。在枯草杆菌蛋白酶相关的蛋白酶中,这些氨基酸的相对序列,当从氨基末端读到羧基末端时,为天冬氨酸-组氨酸-丝氨酸。然而在胰凝乳蛋白酶相关的蛋白酶中,该相对序列则是组氨酸-天冬氨酸-丝氨酸。因此本文的枯草杆菌蛋白酶是指具有枯草杆菌蛋白酶系催化三分体的丝氨酸蛋白酶。例子包括但不限于,本文图3中给出的枯草杆菌蛋白酶。一般地且就本发明目的而言,蛋白酶中的氨基酸编号与图1中给出的成熟解淀粉芽胞杆菌枯草杆菌蛋白酶序列的指定代号相对应。
蛋白酶变体-蛋白酶变体具有由其“前体蛋白酶”氨基酸序列衍生的氨基酸序列。前体蛋白酶包括天然存在和重组的蛋白酶。蛋白酶变体的氨基酸序列是通过对1个或多个前体氨基酸序列的氨基酸实施取代、缺失或插入,由前体蛋白酶氨基酸序列“衍生”而来。这样的改性是针对编码前体蛋白酶氨基酸序列的“前体NDA序列”所做的改变,而不是对前体蛋白酶本身所做的改变。对前体DNA序列实施此种改变的适当方法包括本文所公开的方法,以及本领域技术人员熟知的方法(例如参见,EP 0328 299、WO 89/06279及本文已援引的美国专利和申请)。
在优选的实施方案中,可用在本发明清洗组合物中作为蛋白酶的蛋白酶变体包含:一种蛋白酶变体,它涵盖在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶位点103的氨基酸残基位点用另一种天然存在的氨基酸残基取代氨基酸残基,并另外在1个或多个对应于解淀粉芽胞杆菌下列位点的氨基酸残基位点以另一种天然存在的氨基酸残基取代氨基酸残基:
1,3,4,8,9,10,12,13,16,17,18,19,20,21,22,24,27,33,37,38,42,43,48,55,57,58,61,62,68,72,75,76,77,78,79,86,87,89,97,98,99,101,102,104,106,107,109,111,114,116,117,119,121,123,126,128,130,131,133,134,137,140,141,142,146,147,158,159,160,166,167,170,173,174,177,181,182,183,184,185,188,192,194,198,203,204,205,206,209,210,211,212,213,214,215,216,217,218,222,224,227,228,230,232,236,237,238,240,242,243,244,245,246,247,248,249,251,252,253,254,255,256,257,258,259,260,261,262,263,265,268,269,270,271,272,274及275,
其中当所述蛋白酶变体包括对应于位点103及76的氨基酸残基的取代时,则还存在在1个或多个除对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的氨基酸残基位点27、99、101、104、107、109、123、128、166、204、206、210、216、217、218、222、260、265或274以外的氨基酸残基位点上的氨基酸残基取代;以及1种或多种清洁添加剂材料。
虽然可采取以上所列举的氨基酸取代的任意组合,但用作本发明的优选蛋白酶变体酶则包括按下列组合的氨基酸残基取代、缺失或插入:
(1)包括对位点103以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:236及245;
(2)包括对位点103及236以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体;
12,61,62,68,76,97,98,101,102,104,109,130,131,159,183,185,205,209,210,211,212,213,215,217,230,232,248,252,257,260,270及275;
(3)包括对位点103及245以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,76,97,98,101,102,104,109,130,131,159,170,183,185,205,209,210,211,212,213,215,217,222,230,232,248,252,257,260,261,270及275;
以及
(4)包括对位点103及236及245以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,76,97,98,101,102,104,109,130,131,159,183,185,205,209,210,211,212,213,215,217,230,232,243,248,252,257,260,270及275。
可用于本发明清洗组合物的更优选的蛋白酶变体包括选自下表的取代组(在下表I中每一行对应于一个取代组)的取代:
表1
76 | 98 | 103 | 104 | |||||||||
76 | 78 | 103 | 104 | |||||||||
76 | 103 | 104 | 107 | |||||||||
4 | 76 | 103 | 104 |
76 | 103 | 104 | 246 | |||||||||
76 | 77 | 103 | 104 | |||||||||
76 | 103 | 104 | 183 | 218 | ||||||||
16 | 76 | 103 | 104 | 248 | ||||||||
76 | 103 | 104 | ||||||||||
76 | 103 | 104 | 261 | |||||||||
76 | 103 | 104 | 160 | |||||||||
76 | 103 | 104 | 216 | |||||||||
17 | 76 | 103 | 104 | |||||||||
37 | 76 | 103 | 104 | |||||||||
76 | 77 | 103 | 104 | 174 | ||||||||
38 | 76 | 103 | 104 | |||||||||
38 | 76 | 103 | 104 | 237 | ||||||||
8 | 76 | 103 | 104 | |||||||||
76 | 103 | 104 | 183 | |||||||||
19 | 76 | 103 | 104 | |||||||||
13 | 76 | 103 | 104 | |||||||||
19 | 76 | 103 | 104 | |||||||||
76 | 103 | 104 | 184 | |||||||||
76 | 103 | 104 | 252 | |||||||||
76 | 103 | 104 | 259 | |||||||||
76 | 103 | 104 | 251 | |||||||||
76 | 86 | 103 | 104 | |||||||||
72 | 76 | 103 | 104 | 185 | ||||||||
76 | 103 | 104 | 237 | 274 | ||||||||
76 | 103 | 104 | 160 | |||||||||
76 | 103 | 104 | 228 | |||||||||
55 | 76 | 103 | 104 | 240 | ||||||||
76 | 103 | 104 | 254 | |||||||||
76 | 103 | 104 | 204 | |||||||||
76 | 103 | 104 | 204 | |||||||||
43 | 76 | 103 | 104 |
76 | 103 | 104 | 159 | |||||||||
10 | 76 | 103 | 104 | 177 | ||||||||
58 | 76 | 103 | 104 | |||||||||
76 | 103 | 104 | 270 | |||||||||
76 | 103 | 104 | 185 | |||||||||
27 | 76 | 103 | 104 | |||||||||
76 | 103 | 104 | 262 | |||||||||
76 | 78 | 103 | 104 | |||||||||
24 | 76 | 103 | 104 | |||||||||
76 | 103 | 104 | 166 | 236 | 251 | |||||||
17 | 76 | 103 | 104 | 237 | ||||||||
76 | 103 | 104 | 130 | |||||||||
76 | 103 | 104 | 109 | |||||||||
76 | 99 | 103 | 104 | 204 | ||||||||
76 | 103 | 104 | 181 | |||||||||
12 | 76 | 103 | 104 | |||||||||
76 | 103 | 104 | 212 | 271 | ||||||||
76 | 103 | 104 | 252 | 261 | ||||||||
76 | 103 | 104 | 242 | |||||||||
76 | 103 | 104 | 271 | |||||||||
12 | 76 | 103 | 104 | 242 | ||||||||
43 | 76 | 103 | 104 | 116 | 183 | |||||||
76 | 103 | 104 | 258 | |||||||||
76 | 103 | 104 | 271 | |||||||||
61 | 76 | 103 | 104 | |||||||||
38 | 76 | 103 | 104 | 182 | 263 | |||||||
76 | 103 | 104 | 182 | 272 | ||||||||
76 | 103 | 104 | 109 | 246 | ||||||||
76 | 87 | 103 | 104 | 206 | 249 | 265 | ||||||
76 | 103 | 104 | 137 | 238 | 271 | |||||||
103 | 104 | 228 | ||||||||||
76 | 103 | 104 | 182 | 198 |
21 | 76 | 103 | 104 | 182 | ||||||||
76 | 103 | 104 | 119 | 137 | ||||||||
76 | 103 | 104 | 137 | 248 | ||||||||
13 | 76 | 103 | 104 | 206 | ||||||||
76 | 103 | 104 | 206 | |||||||||
76 | 103 | 104 | 212 | 258 | ||||||||
58 | 76 | 103 | 104 | 271 | ||||||||
76 | 103 | 104 | 206 | 261 | ||||||||
4 | 76 | 103 | 104 | 206 | ||||||||
76 | 77 | 103 | 104 | 206 | ||||||||
76 | 103 | 104 | 158 | |||||||||
76 | 103 | 104 | 206 | |||||||||
4 | 76 | 103 | 104 | 159 | 217 | 251 | ||||||
4 | 76 | 103 | 104 | 159 | 217 | 252 | ||||||
76 | 77 | 103 | 104 | 133 | 185 | 251 | ||||||
76 | 103 | 104 | 159 | 206 | 244 | |||||||
4 | 76 | 103 | 104 | 188 | ||||||||
4 | 76 | 103 | 104 | 158 | ||||||||
76 | 77 | 103 | 104 | 185 | ||||||||
76 | 103 | 104 | 206 | 251 | ||||||||
48 | 76 | 103 | 104 | 111 | 159 | |||||||
68 | 76 | 103 | 104 | 159 | 236 | |||||||
42 | 76 | 103 | 104 | 159 | ||||||||
12 | 62 | 76 | 103 | 104 | 159 | |||||||
42 | 76 | 103 | 104 | 159 | ||||||||
76 | 103 | 104 | 146 | 159 | ||||||||
76 | 103 | 104 | 159 | 238 | ||||||||
76 | 103 | 104 | 159 | 224 | ||||||||
76 | 103 | 104 | 212 | 268 | 271 | |||||||
76 | 89 | 103 | 104 | |||||||||
76 | 87 | 103 | 104 | 212 | 271 | |||||||
76 | 103 | 104 | 212 | 245 | 271 |
76 | 103 | 104 | 134 | 141 | 212 | 271 | ||||||
76 | 103 | 104 | 212 | 236 | 243 | 271 | ||||||
76 | 103 | 104 | 109 | 245 | ||||||||
76 | 103 | 104 | 109 | 210 | ||||||||
20 | 62 | 76 | 103 | 104 | ||||||||
68 | 76 | 103 | 104 | 236 | ||||||||
68 | 76 | 103 | 104 | 159 | 236 | 271 | ||||||
68 | 76 | 103 | 104 | 159 | 236 | 245 | ||||||
68 | 76 | 103 | 104 | 159 | 217 | 236 | 271 | |||||
17 | 68 | 76 | 103 | 104 | ||||||||
68 | 76 | 103 | 104 | |||||||||
68 | 76 | 103 | 104 | 159 | 236 | |||||||
68 | 75 | 76 | 103 | 104 | 159 | 236 | ||||||
68 | 76 | 76 | 103 | 114 | 121 | 159 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 236 | ||||||
68 | 76 | 103 | 104 | 159 | 209 | 236 | 253 | |||||
68 | 76 | 103 | 104 | 117 | 159 | 184 | 236 | |||||
68 | 76 | 103 | 104 | 159 | 236 | 243 | ||||||
68 | 76 | 103 | 104 | 159 | 236 | 245 | ||||||
68 | 76 | 103 | 104 | 142 | 159 | |||||||
68 | 76 | 103 | 104 | 123 | 159 | 236 | 249 | |||||
68 | 76 | 103 | 104 | 159 | 236 | 249 | ||||||
76 | 103 | 104 | 222 | 245 | ||||||||
12 | 76 | 103 | 104 | 222 | 249 | |||||||
76 | 103 | 104 | 173 | 222 | ||||||||
76 | 103 | 104 | 222 | 263 | ||||||||
21 | 76 | 103 | 104 | 222 | 237 | 263 | ||||||
76 | 103 | 104 | 109 | 222 | ||||||||
76 | 103 | 104 | 109 | 222 | 271 | |||||||
61 | 76 | 103 | 104 | 222 | ||||||||
76 | 103 | 104 | 137 | 222 | ||||||||
76 | 103 | 104 | 109 | 222 | 248 |
76 | 103 | 104 | 222 | 249 | ||||||||
68 | 76 | 103 | 104 | 159 | 236 | 245 | 261 | |||||
68 | 76 | 103 | 104 | 141 | 159 | 236 | 245 | 255 | ||||
68 | 76 | 103 | 104 | 159 | 236 | 245 | 247 | |||||
68 | 76 | 103 | 104 | 159 | 174 | 204 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 159 | 204 | 236 | 245 | |||||
68 | 76 | 103 | 104 | 133 | 159 | 218 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 76 | 103 | 104 | 159 | 194 | 203 | 236 | 245 | ||||
12 | 76 | 103 | 104 | 222 | 245 | |||||||
76 | 103 | 104 | 232 | 245 | ||||||||
24 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | |||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
12 | 76 | 103 | 104 | 222 | 244 | 245 | ||||||
12 | 76 | 103 | 222 | 210 | 245 | |||||||
12 | 76 | 103 | 104 | 130 | 222 | 245 | ||||||
22 | 68 | 76 | 103 | 104 | ||||||||
68 | 76 | 103 | 104 | 184 | ||||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | ||||||
68 | 103 | 104 | 140 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
68 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | 275 | |||
12 | 76 | 103 | 104 | 130 | 222 | 245 | 248 | 262 | ||||
12 | 76 | 103 | 104 | 130 | 215 | 222 | 245 | |||||
12 | 76 | 103 | 104 | 130 | 222 | 227 | 245 | 262 | ||||
12 | 76 | 103 | 104 | 130 | 222 | 245 | 261 | |||||
76 | 103 | 104 | 130 | 222 | 245 | |||||||
12 | 76 | 103 | 104 | 130 | 218 | 222 | 245 | 262 | 269 |
12 | 57 | 76 | 103 | 104 | 130 | 222 | 245 | 251 | ||||
12 | 76 | 103 | 104 | 130 | 170 | 185 | 222 | 243 | 245 | |||
12 | 76 | 103 | 104 | 130 | 222 | 245 | 268 | |||||
12 | 76 | 103 | 104 | 130 | 222 | 210 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 103 | 104 | 116 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | |||||
10 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 203 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 237 | 245 | |||||
68 | 76 | 79 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 183 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 174 | 206 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 188 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | |||||
68 | 98 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | |||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | ||||
76 | 103 | 104 | 232 | 236 | 245 | 257 | ||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 275 | ||||
76 | 103 | 104 | 257 | 275 | ||||||||
68 | 103 | 104 | 159 | 224 | 232 | 236 | 245 | 257 | ||||
76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 76 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 159 | 211 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 214 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
20 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 259 |
68 | 76 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 261 | ||||
76 | 103 | 104 | 232 | 236 | 242 | 245 | ||||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
12 | 48 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
76 | 103 | 104 | 232 | 236 | 245 | |||||||
76 | 103 | 104 | 159 | 192 | 232 | 236 | 245 | |||||
76 | 103 | 104 | 147 | 159 | 232 | 236 | 245 | 248 | 251 | |||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 272 | |||
68 | 76 | 103 | 104 | 159 | 183 | 206 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 256 | ||||
68 | 76 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | ||||
27 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 116 | 159 | 170 | 185 | 232 | 236 | 245 | ||
61 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 99 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | |||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
68 | 103 | 104 | 159 | 185 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 185 | 210 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 213 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 216 | 232 | 236 | 245 | 248 | 252 |
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 173 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 251 | 252 | |||
68 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
55 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 255 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 256 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 257 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 258 | |||
8 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 269 | ||
68 | 103 | 104 | 116 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 232 | 236 | 245 | 248 | 252 | |||||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
18 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 76 | 101 | 103 | 104 | 159 | 213 | 218 | 232 | 236 | 245 | 260 | |
68 | 103 | 104 | 159 | 228 | 232 | 236 | 245 | 248 | 252 | |||
33 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
68 | 76 | 89 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | |
61 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
101 | 104 | 159 | 205 | 210 | 232 | 236 | 245 | |||||
61 | 68 | 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | ||
61 | 68 | 103 | 104 | 133 | 137 | 159 | 232 | 236 | 245 | 248 | 252 | |
61 | 103 | 104 | 133 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 218 | 232 | 236 | 245 | 248 | 252 |
61 | 68 | 103 | 104 | 159 | 160 | 232 | 236 | 245 | 248 | 252 | ||
3 | 61 | 68 | 76 | 103 | 104 | 232 | 236 | 245 | 248 | 252 | ||
61 | 68 | 103 | 104 | 159 | 167 | 232 | 236 | 245 | 248 | 252 | ||
97 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
98 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
99 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 106 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | ||||
62 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 166 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 217 | 232 | 236 | 245 | 248 | 252 | ||||
20 | 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 159 | 206 | 217 | 232 | 236 | 245 | 248 | 252 | |||
62 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
27 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
38 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
38 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | 271 | ||
68 | 76 | 103 | 104 | 159 | 209 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 205 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 260 | |||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||
76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 |
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | |||||||
68 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | |||||||
68 | 103 | 104 | 159 | 126 | 232 | 236 | 245 | |||||||
68 | 103 | 104 | 159 | 205 | 232 | 236 | 245 | |||||||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | |||||||
103 | 104 | 159 | 230 | 236 | 245 | |||||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||||||
103 | 104 | 159 | 232 | 236 | 245 | |||||||||
68 | 103 | 104 | 159 | 174 | 232 | 236 | 245 | 257 | ||||||
68 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 257 | ||||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 257 | ||||||
103 | 104 | 159 | 232 | 236 | 245 | 257 | ||||||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | 261 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 261 | ||||||
103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||||||
103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | ||||||
103 | 104 | 159 | 209 | 232 | 236 | 245 | 257 | |||||||
68 | 76 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | ||||
12 | 103 | 104 | 159 | 209 | 213 | 232 | 236 | 245 | 260 | |||||
103 | 104 | 209 | 232 | 236 | 245 | 257 | ||||||||
103 | 104 | 159 | 205 | 210 | 213 | 232 | 236 | 245 | 260 | |||||
103 | 104 | 159 | 205 | 209 | 232 | 236 | 245 | 260 | ||||||
68 | 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | |||||
103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | 257 | |||||
103 | 104 | 159 | 205 | 209 | 232 | 236 | 245 | 257 | ||||||
68 | 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | 260 | ||||
103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | ||||||
103 | 104 | 159 | 209 | 210 | 232 | 236 | 245 | |||||||
103 | 104 | 159 | 205 | 210 | 232 | 236 | 245 | |||||||
68 | 103 | 104 | 128 | 159 | 232 | 236 | 245 | |||||||
48 | 103 | 104 | 159 | 230 | 236 | 245 | ||||||||
48 | 68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 |
48 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
48 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 261 | |||
102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
12 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
101 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
102 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 232 | 236 | 244 | 245 | 248 | 252 | ||||
62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | 256 | ||
12 | 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 159 | 185 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
98 | 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 248 | 252 | |||
62 | 103 | 104 | 109 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 159 | 212 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 101 | 103 | 104 | 159 | 212 | 213 | 232 | 236 | 245 | 248 | 252 | |
103 | 104 | 159 | 232 | 245 | 248 | 252 | ||||||
103 | 104 | 159 | 230 | 245 | ||||||||
62 | 103 | 104 | 130 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 128 | 159 | 232 | 236 | 245 | 248 | 252 | |||
62 | 101 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 128 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 128 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
101 | 103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 |
98 | 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
99 | 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
76 | 103 | 104 | 167 | 170 | 194 | |||||||
101 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 205 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 230 | 236 | 245 | ||||||
101 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 248 | 252 | |||
76 | 101 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | 248 | 252 | |||
62 | 103 | 104 | 159 | 185 | 206 | 213 | 232 | 236 | 245 | 248 | 252 | 271 |
可用于本发明清洗组合物的进一步优选的蛋白酶变体包括选自下表的取代组(在下表II中每一行对应于一个取代组)的取代:
表II
N76D | A98E | S103A | V104I | |||||||||
N76D | S78T | S103A | V104I | |||||||||
N76D | S103A | V104I | 1107V | |||||||||
V4E | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | 1246V | |||||||||
N76D | N77D | S103A | V104I | |||||||||
N76D | S103A | V104I | N183D | N218I | ||||||||
A16T | N76D | S103A | V104I | N248D | ||||||||
A1E | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | N261D | |||||||||
N76D | S103A | V104I | S160T | |||||||||
N76D | S103A | V104I | S216C | |||||||||
H17Q | N76D | S103A | V104I | |||||||||
S37T | N76D | S103A | V104I | |||||||||
N76D | N77D | S103A | V104I | A174V | ||||||||
T38S | N76D | S103A | V104I |
T38S | N76D | S103A | V104I | K237Q | ||||||||
18V | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | N183D | |||||||||
R19L | N76D | S103A | V104I | |||||||||
A13V | N76D | S103A | V104I | |||||||||
R19C | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | N184D | |||||||||
N76D | S103A | V104I | N252D | |||||||||
N76D | S103A | V104I | S259C | |||||||||
N76D | S103A | V104I | K251T | |||||||||
N76D | P86S | S103A | V104I | |||||||||
172V | N76D | S103A | V104I | N185D | ||||||||
N76D | S103A | V104I | K237E | T274A | ||||||||
N76D | S103A | V104I | S160L | |||||||||
N76D | S103A | V104I | A228V | |||||||||
P55S | N76D | S103A | V104I | S240T | ||||||||
N76D | S103A | V104I | A254T | |||||||||
N76D | S103A | I104N | N204T | |||||||||
N76D | S103A | V104I | N204D | |||||||||
N43S | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | G159D | |||||||||
R10H | N76D | S103A | V104I | V177A | ||||||||
T58S | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | A270V | |||||||||
N76D | S103A | V104I | N185D | |||||||||
K27N | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | L262M | |||||||||
N76D | S78P | S103A | V104I | |||||||||
S24P | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | S166G | Q236R | K251R | |||||||
H17L | N76D | S103A | V104I | K237E | ||||||||
N76D | S103A | V104I | S130L | |||||||||
N76D | S103A | V104I | O109R | |||||||||
N76D | S99R | S103A | V104I | N204T | ||||||||
N76D | S103A | V104N | D181N |
Q12R | N76D | S103A | V104I | |||||||||
N76D | S103A | V104I | S212P | E271V | ||||||||
N76D | S103A | V104I | N252K | N261Y | ||||||||
N76D | S103A | V104I | S242T | |||||||||
N76D | S103A | V104I | E271Q | |||||||||
Q12R | N76D | S103A | V104I | S242T | ||||||||
N43S | N76D | S103A | V104I | N116K | N183I | |||||||
N76D | S103A | V104I | G258R | |||||||||
N76D | S103A | V104I | E271G | |||||||||
G61R | N76D | S103A | V104I | |||||||||
T38S | N76D | S103A | V104I | Q182R | Y263H | |||||||
N76D | S103A | V104I | Q182R | A272S | ||||||||
N76D | S103A | V104I | Q109R | I246V | ||||||||
N76D | S87G | S103A | V104I | Q206R | H249Q | S265G | ||||||
N76D | S103A | V104I | Q137R | N238Y | E271V | |||||||
S103A | V104I | A228T | ||||||||||
N76D | S103A | V104I | Q182R | I198V | ||||||||
L21M | N76D | S103A | V104I | Q182R | ||||||||
N76D | S103A | V104I | M119I | Q137R | ||||||||
N76D | S103A | V104I | Q137R | N248S | ||||||||
A13T | N76D | S103A | V104I | Q206R | ||||||||
N76D | S103A | V104I | Q206R | |||||||||
N76D | S103A | V104I | S212P | G258R | ||||||||
T58S | N76D | S103A | V104I | E271G | ||||||||
N76D | S103A | V104I | Q206E | N261D | ||||||||
V4E | N76D | S103A | V104I | Q206E | ||||||||
N76D | N77D | S103A | V104I | Q206E | ||||||||
N76D | S103A | V104I | A158E | |||||||||
N76D | S103A | V104I | Q206E | |||||||||
V4E | N76D | S103A | V104I | G159D | L217E | K251Q | ||||||
V4E | N76D | S103A | V104I | G159D | L217E | N252D | ||||||
N76D | N77D | S103A | V104I | A133T | N185D | K251T | ||||||
N76D | S103A | V104I | G159D | Q206E | V244A | |||||||
V4E | N76D | S103A | V104I | S188E | ||||||||
V4E | N76D | S103A | V104I | A158E | ||||||||
N76D | N77D | S103A | V104I | N185D |
N76D | S103A | V104I | Q206E | K251T | ||||||||||||||||
A48T | N76D | S103A | V104I | L111M | G159D | |||||||||||||||
V68A | N76D | S103A | V104I | G159D | Q236H | |||||||||||||||
L42V | N76D | S103A | V104I | G159D | ||||||||||||||||
Q12H | N62H | N76D | S103A | V104I | G159D | |||||||||||||||
L42I | N76D | S103A | V104I | G159D | ||||||||||||||||
N76D | S103A | V104I | G146S | G159D | ||||||||||||||||
N76D | S103A | V104I | G159D | N238S | ||||||||||||||||
N76D | S103A | V104I | G159D | T224A | ||||||||||||||||
N76D | S103A | V104I | S212P | V268F | E271V | |||||||||||||||
N76D | E89A | S103A | V104I | |||||||||||||||||
N76D | S87R | S103A | V104I | S212P | E271V | |||||||||||||||
N76D | S103A | V104I | S212P | Q245L | E271V | |||||||||||||||
N76D | S103A | V104I | T134S | S141N | S212P | E271V | ||||||||||||||
N76D | S103A | V104I | S212P | Q236L | N243S | E271V | ||||||||||||||
N76D | S103A | V104I | Q109R | Q245R | ||||||||||||||||
N76D | S103A | V104I | Q109R | P210L | ||||||||||||||||
G20V | N62S | N76D | S103A | V104I | ||||||||||||||||
V68A | N76D | S103A | V104I | Q236H | ||||||||||||||||
V68A | N76D | S103A | V104I | G159D | Q236H | E271V | ||||||||||||||
V68A | N76D | S103A | V104I | Q159D | Q236H | Q245R | ||||||||||||||
V68A | N76D | S103A | V104I | G159D | L217I | Q236H | E271V | |||||||||||||
H17Q | V68A | N76D | S103A | V104I | ||||||||||||||||
V68A | N76D | S103A | V104I | |||||||||||||||||
V68A | N76D | S103A | V104I | G159D | Q236R | |||||||||||||||
V68A | L75R | N76D | S103A | V104I | G159D | Q236H | ||||||||||||||
V68A | N76D | N76D | S103A | A114V | V121I | G159D | Q236H | Q245R | ||||||||||||
Q12R | V68A | N76D | S103A | V104I | G159D | Q236H | ||||||||||||||
V68A | N76D | S103A | V104I | G159D | Y209S | Q236H | T253K | |||||||||||||
V68A | N76D | S103A | V104I | N117K | G159D | N184S | Q236H | |||||||||||||
V68A | N76D | S103A | V104I | G159D | Q236H | N243I | ||||||||||||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245L | ||||||||||||||
V68A | N76D | S103A | V104I | A142V | G159D | |||||||||||||||
V68A | N76D | S103A | V104I | N123S | Q159D | Q236H | H249Y | |||||||||||||
V68A | N76D | S103A | V104I | G159D | Q236H | H249Q | ||||||||||||||
N76D | S103A | V104I | M222S | Q245R |
Q12R | N76D | S103A | V104I | M222S | H249R | |||||||||
N76D | S103A | V104I | N173R | M222S | ||||||||||
N76D | S103A | V104I | M222S | Y263F | ||||||||||
L21M | N76D | S103A | V104I | M222S | K237R | Y263F | ||||||||
N76D | S103A | V104I | Q109R | M222S | ||||||||||
N76D | S103A | V104I | Q109R | M222S | E271D | |||||||||
G61R | N76D | S103A | V104I | M222S | ||||||||||
N76D | S103A | V104I | Q137R | M222S | ||||||||||
N76D | S103A | V104I | Q109R | M222S | N248S | |||||||||
N76D | S103A | V104I | M222S | H249R | ||||||||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245R | N261D | |||||||
V68A | N76D | S103A | V104I | S141N | G159D | Q236H | Q245R | T255S | ||||||
V68A | N76D | S103A | V104I | G159D | Q236H | Q245R | R247H | |||||||
V68A | N76D | S103A | V104I | G159D | A174V | N204D | Q236H | Q245R | ||||||
V68A | N76D | S103A | V104I | G159D | N204D | Q236H | Q245R | |||||||
V68A | N76D | S103A | V104I | A133V | G159D | N218D | Q236H | Q245R | ||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||
V68A | N76D | S103A | V104I | G159D | A194I | V203A | Q236H | Q245R | ||||||
Q12R | N76D | S103A | V104I | M222S | Q245R | |||||||||
N76D | S103A | V104I | A232V | Q245R | ||||||||||
S24T | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | |||||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||
Q12R | N76D | S103A | I104T | M222S | V244I | Q245R | ||||||||
Q12R | N76D | S103A | M222S | P210T | Q245R | |||||||||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | ||||||||
T22K | V68A | N76D | S103A | V104I | ||||||||||
V68A | N76D | S103A | V104I | N184D | ||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||||
V68A | S103A | V104I | N140D | G159D | A232V | Q236H | Q245R | N252K | ||||||
N43S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||||
N43K | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||||
V68A | S87G | S103A | V104I | G159D | A232V | O236H | Q245R | N252K | R275S | |||||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N248S | L262M |
Q12R | N76D | S103A | I104T | S130T | A215V | M222S | Q245R | |||||||||||||
Q12R | N76D | S103A | I104T | S130T | M222S | V227A | Q245R | L262S | ||||||||||||
Q12R | N76D | S103A | I104T | S130T | A215T | M222S | Q245R | |||||||||||||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N261D | |||||||||||||
N76D | S103A | I104T | S130T | M222S | Q245R | |||||||||||||||
Q12R | N76D | S103A | 1104T | S130T | N218D | M222S | Q245R | L262S | N269D | |||||||||||
Q12R | S57P | N76D | S103A | I104T | S130T | M222S | Q245R | K251Q | ||||||||||||
Q12R | N76D | S103A | I104T | S130T | R170S | N185D | M222S | N243D | Q245R | |||||||||||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | V268A | |||||||||||||
Q12R | N76D | S103A | I104T | S130T | M222S | P210S | Q245R | |||||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||||||||||
V68A | S103A | V104I | N116D | G159D | A232V | Q236H | Q245R | |||||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | |||||||||||||
R10C | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||||||||
V68A | S103A | V104I | G159D | V203E | A232V | Q236H | Q245R | |||||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | K237E | Q245R | |||||||||||||
V68A | N76D | I79N | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||||||||
V68A | S103A | V104I | G159D | N183D | A232V | Q236H | Q245R | |||||||||||||
V68A | S103A | V104I | G159D | A174V | Q206L | A232V | Q236H | Q245R | ||||||||||||
V68A | S103A | V104I | G159D | S188C | A232V | Q236H | Q245R | |||||||||||||
V68A | S103A | V104I | G159D | A230T | A232V | Q236H | Q245R | |||||||||||||
V68A | A98T | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||||||||
V68A | S103A | V104I | G159D | A215T | A232V | Q236H | Q245R | |||||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248S | |||||||||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||||||||
V68A | N76D | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | ||||||||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | ||||||||||||
N76D | S103A | V104I | A232V | Q236H | Q245R | L257V | ||||||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | R275H | ||||||||||||
N76D | S103A | V104I | L257V | R275H | ||||||||||||||||
V68A | S103A | V104I | G159D | T224A | A232V | Q236H | Q245R | L257V | ||||||||||||
N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||||||||||
V68A | N76D | S103A | V104I | G159D | Y209W | A232V | Q236H | O245R | ||||||||||||
V68A | N76D | S103A | V104I | G159D | G211R | A232V | Q236H | Q245R | ||||||||||||
V68A | N76D | S103A | V104I | G159D | G211V | A232V | O236H | O245R | ||||||||||||
O12R | V68A | N76D | S103A | V104I | G159D | Y214L | A232V | O236H | O245R |
V68A | N76D | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | ||||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
G20R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S259G | |||
V68A | N76D | S87R | S103A | V104I | G159D | A232V | Q236H | Q245R | T260V | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261G | ||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261W | ||||
N76D | S103A | V104I | A232V | Q236H | S242P | Q245R | ||||||
V68A | N76D | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | ||||
Q12R | A48V | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||
N76D | S103A | V104I | A232V | Q236H | Q245R | |||||||
N76D | S103A | V104I | G159D | Y192F | A232V | Q236H | Q245R | |||||
N76D | S103A | V104I | V147I | G159D | A232V | Q236H | Q245R | N248S | K251R | |||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | A272S | |||
V68A | N76D | S103A | V104I | G159D | N183K | Q206L | A232V | Q236H | Q245R | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S256R | ||||
V68A | N76D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | ||||
K27R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | N116T | G159D | R170S | N185S | A232V | Q236H | Q245R | ||
G61E | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | S99N | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | Y209F | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V1041 | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D | |||
V68A | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210T | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210S | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | N185D | P210L | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212A | A232V | O236H | O245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212G | A232V | O236H | O245R | N248D | N252K |
V68A | S103A | V104I | G159D | S212E | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | T213S | A232V | Q236H | Q245R | N248D | N252K | |||||||
V68A | A103V | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | T213G | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | A215V | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | S216T | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | S216V | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | S216C | A232V | Q236H | Q245R | N248D | N252K | ||||||
G20A | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | N173D | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | K251V | N252K | ||||||
V68A | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F | |||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252L | |||||||
P55S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T255V | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256N | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256E | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256R | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260R | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | L257R | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | G258D | ||||||
I8V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N269D | |||||
V68A | S103A | V104I | N116S | G159D | A232V | Q236H | Q245R | N248D | N252K | T260E | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D | ||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||||
V68A | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | ||||||||
S103A | V104I | G159D | A232S | Q236H | O245R | N248D | N252K | ||||||||
V68A | S103A | V104I | G159D | A232V | Q236R | O245R | N248D | N252K | |||||||
N18S | V68A | S103A | V1041 | G159D | A232V | O236H | O245R | N248D | N252K | ||||||
V68A | S103A | V104I | G159D | A232V | Q236H | O245V | N248D | N252K | |||||||
V68A | N76D | S101T | S103A | V104I | G159D | T213R | N218S | A232V | O236H | O245R | T260A |
V68A | S103A | V104I | G159D | A228V | A232V | Q236H | Q245R | N248D | N252K | |||
T33S | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | N76D | E89D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | |
G61E | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | V205I | P210I | A232V | Q236H | Q245R | |||||
G61E | V68A | S103A | V104I | S130A | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
G61E | V68A | S103A | V104I | A133S | Q137R | G159D | A232V | Q236H | Q245R | N248D | N252K | |
G61E | S103A | V104I | A133V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248G | N252K | ||||
V68A | S103A | V104I | G159D | N218S | A232V | Q236H | Q245R | N248D | N252K | |||
G61E | V68A | S103A | V104I | G159D | S160V | A232V | Q236H | Q245R | N248D | N252K | ||
S3L | G61E | V68A | N76D | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | ||
G61E | V68A | S103A | V104I | G159D | S167F | A232V | Q236H | Q245R | N248D | N252K | ||
G97E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
A98D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S99E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | S106E | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | Q109E | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | ||||
S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
N62D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | S166D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | L217E | A232V | Q236H | Q245R | N248D | N252K | ||||
G20R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
S103A | V104I | G159D | Q206R | L217E | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | |||
S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
K27N | S103A | V104I | G159D | A232V | O236H | O245R | N248D | N252K | ||||
T38G | S103A | V104I | G159D | A232V | O236H | O245R | N248D | N252K | ||||
T38A | N76D | S103A | V104I | G159D | T213R | A232V | O236H | O245R | T260A |
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | E271G | |||||||||||
V68A | N76D | S103A | V1041 | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | |||||||||||
V68A | N76D | S103A | V104I | G159D | P2101 | T213R | A232V | Q236H | Q245R | T260A | |||||||||||
V68A | N76D | S103A | V104I | G159D | V205I | T213R | A232V | Q236H | Q245R | T260A | |||||||||||
V68A | N76D | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | T260A | ||||||||||||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||||||||||
N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||||||||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | ||||||||||||||
V68A | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | ||||||||||||||
V68A | S103A | V104I | G159D | A230V | A332V | Q236H | Q245R | ||||||||||||||
V68A | S103A | V104I | G159D | L126F | A232V | Q236H | Q245R | ||||||||||||||
V68A | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | ||||||||||||||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | ||||||||||||||
S103A | V104I | G159D | A230V | Q236H | Q245R | ||||||||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | T260A | ||||||||||||||
S103A | V1041 | G159D | A232V | Q236H | Q245R | ||||||||||||||||
V68A | S103A | V104I | G159D | A174V | A232V | Q236H | Q245R | L257V | |||||||||||||
V68A | S103A | V104I | G159D | A194S | A232V | Q236H | Q245R | L257V | |||||||||||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | |||||||||||||
S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||||||||||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | N261W | |||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | N261W | |||||||||||||
S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | ||||||||||||||
S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | |||||||||||||
S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | ||||||||||||||
V68A | N76D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | |||||||||||
Q12R | S103A | V104I | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | ||||||||||||
S103A | V104I | Y209W | A232V | Q236H | Q245R | L257V | |||||||||||||||
S103A | V104I | G159D | V205I | P210I | T213R | A232V | Q236H | Q245R | T260A | ||||||||||||
S103A | V104I | G159D | V205I | Y209W | A232V | Q236H | Q245R | T260A | |||||||||||||
V68A | S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | ||||||||||||
S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | L257V | ||||||||||||
S103A | V104I | G159D | V205I | Y209W | A232V | O236H | Q245R | L257V | |||||||||||||
V68A | S103A | V104I | G159D | Y205I | Y209W | P210 | A232V | Q236H | O245R | 7260A | |||||||||||
S103A | V104I | G159D | V205I | Y209W | P210I | A232V | O236H | O245R | |||||||||||||
S103A | V104I | G159D | Y209W | P210I | A232V | O236H | O245R |
S103A | V104I | G159D | V205I | P210I | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | ||||||
A48V | S103A | V104I | G159D | A230V | Q236H | Q245R | |||||||
A48V | V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | |||||
A48V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
A48V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | N261W | ||||
G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||||
Q12R | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | G102A | S103A | V104I | G156D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
A98L | Q102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
G102A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | N184S | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | N184G | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | A232V | Q236H | V244T | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | A232V | Q236H | V244A | Q245R | N248D | N252K | |||||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | S256R | |||
Q12R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | Q159D | N185D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101G | S103A | V104I | G159D | Q206E | A232V | Q236H | Q245R | N248D | N252K | ||||
S101G | S103A | V104I | G159D | T213Q | A232V | Q236H | Q245R | N248D | N252K | ||||
A98L | G102A | S103A | V104I | G159D | A332V | Q236H | Q245R | N248D | N252K | ||||
S101G | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | N248D | N252K | ||||
N62D | S103A | V104I | Q109R | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S103A | V104I | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S101G | S103A | V104I | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | A232V | Q245R | N248D | N252K | |||||||
S103A | V104I | G159D | A230V | Q245R | |||||||||
N62D | S103A | V104I | S130G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101G | S103A | V104I | S128G | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S101G | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
N62D | S101G | S103A | V104I | G159D | T213R | A232V | O236H | O245R | N248D | N252K | |||
N62D | S103A | V104I | S128G | G159D | T213R | A232V | O236H | O245R | N248D | N252K |
N62D | S103A | V104I | S128L | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260A | |||
S101G | S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
A98V | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S99G | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | N248D | N252K | |||
S101G | S103A | V104I | G159D | A230V | Q236H | Q245R | ||||||
S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | |||
N76D | S101G | S103A | V104I | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | S103A | V104I | G159D | A230V | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S103A | V104I | G159D | N185D | Q206E | T213R | A232V | Q236H | Q245R | N248D | N252K | E271Q |
可用于本发明清洗组合物的更进一步优选的蛋白酶变体包括选自表I的取代组的取代,但下列表III的取代组除外:
表III
76 | 103 | 104 | 259 | |||||||
76 | 86 | 103 | 104 | |||||||
76 | 103 | 104 | 130 | |||||||
76 | 99 | 103 | 104 | 204 | ||||||
76 | 103 | 104 | 242 | |||||||
76 | 103 | 104 | 104 | 182 | 198 | |||||
21 | 76 | 103 | 104 | 182 | ||||||
76 | 103 | 104 | 119 | 137 | ||||||
76 | 103 | 104 | 173 | 222 | ||||||
61 | 76 | 103 | 104 | 222 | ||||||
68 | 76 | 103 | 104 | 116 | 159 | 170 | 185 | 232 | 236 | 245 |
可用于本发明清洗组合物的更进一步优选的蛋白酶变体包括选自表IV的取代组的取代:
表IV
76 | 103 | 104 | 222 | 245 | ||||||||
76 | 103 | 104 | 222 | 249 | ||||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | |||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
22 | 68 | 76 | 103 | 104 | ||||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | ||||||
68 | 103 | 104 | 140 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
12 | 76 | 103 | 104 | 130 | 222 | 245 | 261 | |||||
76 | 103 | 104 | 130 | 222 | 245 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 224 | 232 | 236 | 245 | 257 | ||||
76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 76 | 103 | 104 | 159 | 211 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 214 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
20 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 259 | |||
68 | 76 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 261 | ||||
12 | 48 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
76 | 103 | 104 | 159 | 192 | 232 | 236 | 245 | |||||
76 | 103 | 104 | 147 | 159 | 232 | 236 | 245 | 248 | 251 | |||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 272 | |||
68 | 76 | 103 | 104 | 159 | 183 | 206 | 232 | 236 | 245 |
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 256 | |||
68 | 76 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | |||
27 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | ||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 213 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 216 | 232 | 236 | 245 | 248 | 252 | ||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 255 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 256 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | ||
68 | 103 | 104 | 159 | 228 | 232 | 236 | 245 | 248 | 252 | ||
68 | 76 | 89 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 |
68 | 103 | 104 | 159 | 218 | 232 | 236 | 245 | 248 | 252 |
可用于本发明清洗组合物的更进一步优选的蛋白酶变体包括选自表V的取代组的取代:
表V
V68A | S103A | V104I | G159D | A228V | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | N218S | A232V | Q236H | Q245R | N248D | N252K | ||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | N76D | E89D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A |
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256R | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260R | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T255V | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256N |
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252L | |||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | A215V | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | N248D | N242K | ||
V68A | S103A | V104I | G159D | S216T | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | S216V | A232V | Q236N | Q245R | N248D | N252K | ||
V68A | S103A | V104I | T213S | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | S212C | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | Y209F | A232V | Q236H | Q245R | N248D | N252K | ||
Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N261D | ||||
N76D | S103A | I104T | S130T | M222S | Q245R | ||||||
N76D | S103A | V104I | M222S | H249R | |||||||
N76D | S103A | V104I | M222S | Q245R | |||||||
N76D | S103A | V104I | G159D | Y192F | A232V | Q236H | Q245R | ||||
N76D | S103A | V104I | V147I | G159D | A232V | Q236H | Q245R | N248S | K251R | ||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | A272S | ||
V68A | N76D | S103A | V104I | G159D | N183K | Q206L | A232V | Q236H | Q245R | ||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S256R | |||
V68A | N76D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | |||
K27R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||
Q12R | A48V | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261W | |||
V68A | N76D | S103A | V104I | G159D | G211R | A232V | Q236H | Q245R | |||
V68A | N76D | S103A | V104I | G159D | G211V | A232V | Q236H | Q245R | |||
Q12R | V68A | N76D | S103A | V104I | G159D | Y214L | A232V | Q236H | Q245R | ||
V68A | N76D | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | |||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||
G20R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S259G | ||
V68A | N76D | S87R | S103A | V104I | G159D | A232V | Q236H | Q245R | T260V | ||
N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | ||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | ||
T22K | V68A | N76D | S103A | V104I | |||||||
V68A | N76D | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | |||
V68A | S103A | V104I | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | T224A | A232V | Q236H | Q245R | L257V | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252S | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | N140D | G159D | A232V | Q236H | Q245R | N252K |
N43S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | |||
N43K | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V |
可用于本发明清洗组合物的高度优选的蛋白酶变体包括选自下列取代组的取代:
12/102/103/104/159/212/232/236/245/248/252;12/76/103/104/130/170/185/222/243/245;
12/76/103/104/130/222/245/261; 12/76/103/104/130/222/245;
12/76/103/104/222/245;
61/68/103/104/159/232/236/245/248/252; 62/103/104/159/213/232/236/245/248/252;
62/103/104/109/159/213/232/236/245/248/252;62/103/104/159/232/236/245/248/252;
62/101/103/104/159/212/213/232/236/245/248/252;
62/103/104/130/159/213/232/236/245/248/252;68/103/104/159/232/236/245/248/252/270;
68/103/104/159/185/232/236/245/248/252; 68/103/104/159/210/232/236/245/248/252;
68/103/104/159/185/210/232/236/245/248/252;68/103/104/159/213/232/236/245/248/252;
68/103/104/159/230/232/236/245; 68/76/103/104/159/209/232/236/245;
68/103/104/232/236/245/248/257/275; 68/103/104/213/232/236/245/248/252;
68/103/104/159/232/236/245/248/252; 68/103/104/159/209/232/236/245;
68/76/103/104/159/236; 68/76/103/104/159/236/245;
68/76/103/104/159/232/236/245; 68/103/104/159/232/236/245/252;
68/103/104/159/232/236/245; 68/103/104/159/232/236/245/257;
68/76/103/104/159/211/232/236/245; 68/76/103/104/159/215/232/236/245;
68/103/104/159/210/232/236/245; 68/103/104/159/213/232/236/245/260;
68/76/103/104/159/213/232/236/245/260; 68/103/104/159/236;
68/76/103/104/159/210/232/236/245/260; 68/103/104/159/236/245;
68/103/104/159/183/232/236/245/248/252; 68/76/103/104/159/236/245;
68/103/104/232/236/245/257/275; 68/103/104/159/213/232/236/245;
76/103/222/245; 76/103/104/222/245;
76/103/104/159/232/236/245;
76/103/104/159/213/232/236/245/260; 76/103/104/159;
76/103/104/131/159/232/236/245/248/252; 97/103/104/159/232/236/245/248/252;
98/102/103/104/159/212/232/236/245/248/252;98/103/104/159/232/236/245/248/252;
101/103/104/159/232/236/245/248/252; 102/103/104/159/232/236/245/248/252;
103/104/159/232/236/245; 103/104/159/232/236/245/248/252;
103/104/159/205/209/232/236/245/257 103/104/159/232/245/248/252;
103/104/159/205/209/210/232/236/245/257; 103/104/159/213/232/236/245/248/252;
103/104/159/217/232/236/245/248/252; 103/104/130/159/232/236/245/248/252;
103/104/159/230/236/245; 103/104/159/236/245;
103/104/159/248/252/270; 103/104/131/159/232/236/245/248/252;
103/104/159/205/209/232/236/245;及 103/104/159/232/236/245/257。
可用于本发明清洗组合物的更高度优选的蛋白酶变体包括选自下列取代组的取代:
12R/76D/103A/104T/130T/222S/245R;
12R/76D/103A/104I/222S/245R;
12R/102A/103A/104I/159D/212G/232V/236H/245R/248D/252K;
12R/76D/103A/104T/130G/222S/245R/261D;
12R/76D/103A/104T/130G/170S/185D/222S/243D/245R;
61E/68A/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/109R/159D/213R/232V/236H/245R/248D/252K;
62D/103A/104I/159D/213R/232V/236H/245R/248D/252K;
62D/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/130G/159D/213R/232V/236H/245R/248D/252K;
62D/101G/103A/104I/159D/212G/213R/232V/236H/245R/248D/252K;
68A/103A/104I/159D/232V/236H/245R/248D/252K/270A;
68A/76D/103A/104I/159D/213R/232V/236H/245R/260A;
68A/103A/104I/159D/236H;
68A/103A/104I/159D/236H/245R;
68A/76D/103A/104I/159D/210I/232V/236H/245R/260A;
68A/103A/104I/159D/183D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/209W/232V/236H/245R;
68A/76D/103A/104I/159D/211R/232V/236H/245R;
68A/76D/103A/104I/159D/215R/232V/236H/245R;
68A/103A/104I/159D/213R/232V/236H/245R/260A;
68A/76D/103A/104I/159D/236H;
68A/76D/103A/104I/159D/236H/245R;
68A/76D/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/252K;
68A/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/257V;
68A/103A/104I/159D/185D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/185D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/230V/232V/236H/245R;
68A/76D/103A/104I/159D/209W/232V/236H/245R;
68A/103A/104I/232V/236H/245R/248D/257V/275H;
68A/103A/104I/232V/236H/245R/257V/275H;
68A/103A/104I/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210I/232V/236H/245R;
68A/103A/104I/159D/210L/232V/236H/245R;
68A/103A/104I/159D/213G/232V/236H/245R;
76D/103A/222S/245R;
76D/103A/104I/222S/245R;
76D/103A/104I/159D/232V/236H/245R;
76D/103A/104I/159D;
76D/103A/104I/131V/159D/232V/236H/245R/248D/252K;
76D/103A/104I/159D/213R/232V/236H/245R/260A;
97E/103A/104I/159D/232V/236H/245R/248D/252K;
98L/103A/104I/159D/232V/236H/245R/248D/252K;
98L/102A/103A/104I/159D/212G/232V/236H/245R/248D/252K;
101G/103A/104I/159D/232V/236H/245R/248D/252K;
102A/103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/213R/232V/236H/245R/248D/252K;
103A/104I/130G/159D/232V/236H/245R/248D/252K;
103A/104I/159D/230V/236H/245R;
103A/104I/159D/217E/232V/236H/245R/248D/252K;
103A/104I/159D/236H/245R;
103A/104I/159D/248D/252K/270V;
103A/104I/159D/232V/236H/245R;
103A/104I/159D/205I/209W/232V/236H/245R;
103A/104I/159D/232V/236H/245R/257V;
103A/104I/159D/205I/209W/232V/236H/245R/257V;
103A/104I/131V/159D/232V/236H/245R/248D/252K;
103A/104I/159D/205I/209W/210I/232V/236H/245R/257V;及
103A/104I/159D/232V/245R/248D/252K。
可用于本发明清洗组合物的进一步高度优选的蛋白酶变体包括选自下列取代组的取代:
12/76/103/104/130/222/245/261;
62/103/104/159/232/236/245/248/252;
62/103/104/159/213/232/236/245/248/252;
62/101/103/104/159/212/213/232/236/245/248/252;
68/103/104/159/232/236/245;
68/103/104/159/230/232/236/245;
68/103/104/159/209/232/236/245;
68/103/104/159/232/236/245/257;
68/76/103/104/159/213/232/236/245/260;
68/103/104/159/213/232/236/245/248/252;
68/103/104/159/183/232/236/245/248/252;
68/103/104/159/185/232/236/245/248/252;
68/103/104/159/185/210/232/236/245/248/252;
68/103/104/159/210/232/236/245/248/252;
68/103/104/159/213/232/236/245;
98/103/104/159/232/236/245/248/252;
98/102/103/104/159/212/232/236/245/248/252;
101/103/104/159/232/236/245/248/252;
102/103/104/159/232/236/245/248/252;
103/104/159/230/236/245;
103/104/159/232/236/245/248/252;
103/104/159/217/232/236/245/248/252;
103/104/130/159/232/236/245/248/252;
103/104/131/159/232/236/245/248/252;
103/104/159/213/232/236/245/248/252;及
103/104/159/232/236/245
可用于本发明清洗组合物的最高度优选的蛋白酶变体包括选自下列取代组的取代:
12R/76D/103A/104T/130T/222S/245R/261D;
62D/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/159D/213R/232V/236H/245R/248D/252K;
68A/103A/104I/159D/209W/232V/236H/245R;
68A/76D/103A/104I/159D/213R/232V/236H/245R/260A;
68A/103A/104I/159D/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/183D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/230V/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/257V;
68A/103A/104I/159D/213G/232V/236H/245R/248D/252K;
68A/103A/104I/159D/185D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/185D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/213G/232V/236H/245R;
98L/103A/104I/159D/232V/236H/245R/248D/252K;
98L/102A/103A/104I/159D/212G/232V/236H/245R/248D/252K;
101G/103A/104I/159D/232V/236H/245R/248D/252K;
102A/103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/230V/236H/245R;
103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/217E/232V/236H/245R/248D/252K;
103A/104I/130G/159D/232V/236H/245R/248D/252K;
103A/104I/131V/159D/232V/236H/245R/248D/252K;
103A/104I/159D/213R/232V/236H/245R/248D/252K;及
103A/104I/159D/232V/236H/245R。
在另一种优选实施方案中,可用在本发明清洗组合物中作为蛋白酶的蛋白酶变体包含这样一类蛋白酶变体,它涵盖在1个或多个对应于解淀粉芽胞杆菌枯草杆菌蛋白酶位点62、212、230、232、252和257的氨基酸残基位点上取代氨基酸残基。
虽然可采取以上所列举的氨基酸取代的任意组合,但用作本发明的优选蛋白酶变体酶包括对下列组合的氨基酸残基取代、缺失或插入:
(1)包括对位点62以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:103,104,109,159,213,232,236,245,248及252;
(2)包括对位点212以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:12,98,102,103,104,159,232,236,245,248及252;
(3)包括对位点230以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:68,103,104,159,232,236和245;
(4)包括对位点232以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270及275;
(5)包括对位点232以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:103,104,236及245;
(6)包括对位点232及103以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270及275;
(7)包括对位点232及104以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270及275;
(8)包括对位点232及236以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270及275;
(9)包括对位点232及245以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270及275;
(10)包括对位点232、103、104、236及245以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270及275;
(11)包括对位点252以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248及270;
(12)包括对位点252以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:103、104、236及245;
(13)包括对位点252及103以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248及270;
(14)包括对位点252及104以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248及270;
(15)包括对位点252及236以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248及270;
(16)包括对位点252及245以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248及270;
(17)包括对位点252、103、104、236及245以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248及270;
以及
(18)包括对位点257以及下列中1个或多个位点氨基酸残基取代的蛋白酶变体:
68,103,104,205,209,210,232,236,
245及275。
可用于本发明清洗组合物的更优选的蛋白酶变体包括选自表VI的取代组(在下表VI中每一行对应于一个取代组)的取代:
表VI
76 | 103 | 104 | 212 | 271 | ||||||||
76 | 103 | 104 | 252 | 261 | ||||||||
76 | 103 | 104 | 212 | 258 | ||||||||
4 | 76 | 103 | 104 | 159 | 217 | 252 |
12 | 62 | 76 | 103 | 104 | 159 | |||||||
76 | 103 | 104 | 212 | 268 | 271 | |||||||
76 | 87 | 103 | 104 | 212 | 271 | |||||||
76 | 103 | 104 | 212 | 245 | 271 | |||||||
76 | 103 | 104 | 134 | 141 | 212 | 271 | ||||||
76 | 103 | 104 | 212 | 236 | 243 | 271 | ||||||
20 | 62 | 76 | 103 | 104 | ||||||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
76 | 103 | 104 | 232 | 245 | ||||||||
24 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | |||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | ||||||
68 | 103 | 104 | 140 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
68 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | 275 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 103 | 104 | 116 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | |||||
10 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 203 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 237 | 245 | |||||
68 | 76 | 79 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 183 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 174 | 206 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 188 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | |||||
68 | 98 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | |||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 |
76 | 103 | 104 | 232 | 236 | 245 | 257 | ||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 275 | ||||
76 | 103 | 104 | 257 | 275 | ||||||||
68 | 103 | 104 | 159 | 224 | 232 | 236 | 245 | 257 | ||||
76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
68 | 76 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 159 | 211 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 214 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | ||||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
20 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 259 | |||
68 | 76 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 261 | ||||
76 | 103 | 104 | 232 | 236 | 242 | 245 | ||||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
12 | 48 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
76 | 103 | 104 | 232 | 236 | 245 | |||||||
76 | 103 | 104 | 159 | 192 | 232 | 236 | 245 | |||||
76 | 103 | 104 | 147 | 159 | 232 | 236 | 245 | 248 | 251 | |||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 272 | |||
68 | 76 | 103 | 104 | 159 | 183 | 206 | 232 | 236 | 245 | |||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 256 | ||||
68 | 76 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | ||||
27 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 76 | 103 | 104 | 116 | 159 | 170 | 185 | 232 | 236 | 245 | ||
61 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 99 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | |||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
68 | 103 | 104 | 159 | 185 | 232 | 236 | 245 | 248 | 252 |
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 159 | 185 | 210 | 232 | 236 | 245 | 248 | 252 | ||||||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 213 | 232 | 236 | 245 | 248 | 252 | ||||||||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 159 | 216 | 232 | 236 | 245 | 248 | 252 | |||||||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 159 | 173 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 251 | 252 | |||||||
68 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||||||
55 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 03 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 255 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 256 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 257 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 258 | |||||||
8 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 269 | ||||||
68 | 103 | 104 | 116 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | ||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||||||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 232 | 236 | 245 | 248 | 252 | |||||||||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||||||
18 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||||||
68 | 76 | 101 | 103 | 104 | 159 | 213 | 218 | 232 | 236 | 245 | 260 | |||||
68 | 103 | 104 | 159 | 228 | 232 | 236 | 245 | 248 | 252 | |||||||
33 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||||
68 | 76 | 89 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | |||||
61 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||||
103 | 104 | 159 | 205 | 210 | 232 | 236 | 245 | |||||||||
61 | 68 | 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | ||||||
61 | 68 | 103 | 104 | 133 | 137 | 159 | 232 | 236 | 245 | 248 | 252 |
61 | 103 | 104 | 133 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 218 | 232 | 236 | 245 | 248 | 252 | |||
61 | 68 | 103 | 104 | 159 | 160 | 232 | 236 | 245 | 248 | 252 | ||
3 | 61 | 68 | 76 | 103 | 104 | 232 | 236 | 245 | 248 | 252 | ||
61 | 68 | 103 | 104 | 159 | 167 | 232 | 236 | 245 | 248 | 252 | ||
97 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
98 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
99 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 106 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | ||||
62 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 166 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 217 | 232 | 236 | 245 | 248 | 252 | ||||
20 | 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 159 | 206 | 217 | 232 | 236 | 245 | 248 | 252 | |||
62 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
27 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
38 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
38 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | 271 | ||
68 | 76 | 103 | 104 | 159 | 209 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 205 | 213 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 260 | |||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||
76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 |
68 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | ||||||||
68 | 103 | 104 | 159 | 126 | 232 | 236 | 245 | ||||||||
68 | 103 | 104 | 159 | 205 | 232 | 236 | 245 | ||||||||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||||||
103 | 104 | 159 | 230 | 236 | 245 | ||||||||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | ||||||||
103 | 104 | 159 | 232 | 236 | 245 | ||||||||||
68 | 103 | 104 | 159 | 174 | 232 | 236 | 245 | 257 | |||||||
68 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 257 | |||||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 257 | |||||||
103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | 261 | |||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 261 | |||||||
103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||||||
103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||||||
103 | 104 | 159 | 209 | 232 | 236 | 245 | 257 | ||||||||
68 | 76 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | |||||
12 | 103 | 104 | 159 | 209 | 213 | 232 | 236 | 245 | 260 | ||||||
103 | 104 | 209 | 232 | 236 | 245 | 257 | |||||||||
103 | 104 | 159 | 205 | 210 | 213 | 232 | 236 | 245 | 260 | ||||||
103 | 104 | 159 | 205 | 209 | 232 | 236 | 245 | 260 | |||||||
68 | 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | ||||||
103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | 257 | ||||||
103 | 104 | 159 | 205 | 209 | 232 | 236 | 245 | 257 | |||||||
68 | 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | 260 | |||||
103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | |||||||
103 | 104 | 159 | 209 | 210 | 232 | 236 | 245 | ||||||||
103 | 104 | 159 | 205 | 210 | 232 | 236 | 245 | ||||||||
68 | 103 | 104 | 128 | 159 | 232 | 236 | 245 | ||||||||
48 | 103 | 104 | 159 | 230 | 236 | 245 | |||||||||
48 | 68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | |||||||
48 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||||
48 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 261 | ||||||
102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||||||
12 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||||
101 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 |
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
102 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||||
103 | 104 | 159 | 232 | 236 | 244 | 245 | 248 | 252 | ||||
62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | 256 | ||
12 | 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 159 | 185 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
98 | 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 248 | 252 | |||
62 | 103 | 104 | 109 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 159 | 212 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 101 | 103 | 104 | 159 | 212 | 213 | 232 | 236 | 245 | 248 | 252 | |
103 | 104 | 159 | 232 | 245 | 248 | 252 | ||||||
103 | 104 | 159 | 230 | 245 | ||||||||
62 | 103 | 104 | 130 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 128 | 159 | 232 | 236 | 245 | 248 | 252 | |||
62 | 101 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 128 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
62 | 103 | 104 | 128 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
101 | 103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | |||
98 | 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
99 | 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 205 | 232 | 236 | 245 | 248 | 252 | |||
101 | 103 | 104 | 159 | 230 | 236 | 245 | ||||||
101 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 248 | 252 | |||
76 | 101 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 248 | 252 |
101 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | 248 | 252 | |||
62 | 103 | 104 | 159 | 185 | 206 | 213 | 232 | 236 | 245 | 248 | 252 | 271 |
可用于本发明清洗组合物的进一步优选的蛋白酶变体包括选自表VII的取代组(在下表VII中每一行对应于一个取代组)的取代:
表VII
N76D | S103A | V104I | S212P | E271V | ||||||||
N76D | S103A | V104I | N252K | N261Y | ||||||||
N76D | S103A | V104I | S212P | G258R | ||||||||
V4E | N76D | S103A | V104I | G159D | L217E | N252D | ||||||
Q12H | N62H | N76D | S103A | V104I | G159D | |||||||
N76D | S103A | V104I | S212P | V268F | E271V | |||||||
N76D | S87R | S103A | V104I | S212P | E271V | |||||||
N76D | S103A | V104I | S212P | Q245L | E271V | |||||||
N76D | S103A | V104I | T134S | S141N | S212P | E271V | ||||||
N76D | S103A | V104I | S212P | Q236L | N243S | E271V | ||||||
G20V | N62S | N76D | S103A | V104I | ||||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
N76D | S103A | V104I | A232V | Q245R | ||||||||
S24T | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | |||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | N140D | G159D | A232V | Q236H | Q245R | N252K | ||||
N43S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||
N43K | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||
V68A | S87G | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | R275S | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||
V68A | S103A | V104I | N116D | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | |||||
R10C | V68A | S103A | V104I | G159D | A232V | O236H | O245R |
V68A | S103A | V104I | G159D | V203E | A232V | Q236H | Q245R | ||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | K237E | Q245R | ||||||||||
V68A | N76D | I79N | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||||
V68A | S103A | V104I | G159D | N183D | A232V | Q236H | Q245R | ||||||||||
V68A | S103A | V104I | G159D | A174V | Q206L | A232V | Q236H | Q245R | |||||||||
V68A | S103A | V104I | G159D | S188C | A232V | Q236H | Q245R | ||||||||||
V68A | S103A | V104I | G159D | A230T | A232V | Q236H | Q245R | ||||||||||
V68A | A98T | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||||||
V68A | S103A | V104I | G159D | A215T | A232V | Q236H | Q245R | ||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248S | ||||||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||||||
V68A | N76D | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | |||||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | |||||||||
N76D | S103A | V104I | A232V | Q236H | Q245R | L257V | |||||||||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | R275H | |||||||||
N76D | S103A | V104I | L257V | R275H | |||||||||||||
V68A | S103A | V104I | G159D | T224A | A232V | Q236H | Q245R | L257V | |||||||||
N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | ||||||||||
V68A | N76D | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | |||||||||
V68A | N76D | S103A | V104I | G159D | G211R | A232V | Q236H | Q245R | |||||||||
V68A | N76D | S103A | V104I | G159D | G211V | A232V | Q236H | Q245R | |||||||||
Q12R | V68A | N76D | S103A | V104I | G159D | Y214L | A232V | Q236H | Q245R | ||||||||
V68A | N76D | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | |||||||||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||||
G20R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S259G | ||||||||
V68A | N76D | S87R | S103A | V104I | G159D | A232V | Q236H | Q245R | T260V | ||||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261G | |||||||||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261W | |||||||||
N76D | S103A | V104 | A232V | Q236H | S242P | Q245R | |||||||||||
V68A | N76D | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | |||||||||
Q12R | A48V | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||||||
N76D | S103A | V104I | A232V | Q236H | Q245R | ||||||||||||
N76D | S103A | V104I | G159D | Y192F | A232V | Q236H | Q245R | ||||||||||
N76D | S103A | V104I | V147I | G159D | A232V | Q236H | Q245R | N248S | K251R | ||||||||
D12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | A272S | ||||||||
V68A | N76D | S103A | V104I | G159D | N183K | Q206L | A232V | Q236H | Q245R |
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S256R | ||||
V68A | N76D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | ||||
K27R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | N116T | G159D | R170S | N185S | A232V | Q236H | Q245R | ||
G61E | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | S99N | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | Y209F | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D | |||
V68A | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | P210T | A232V | Q236H | Q245R | N248D | N252K | |||
V684 | S103A | V104I | G159D | P210S | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | N185D | P210L | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212A | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | T213S | A232V | Q236H | Q245R | N248D | N252K | ||||
V68A | A103V | V104I | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | T213G | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A215V | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216T | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216V | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216C | A232V | Q236H | Q245R | N248D | N252K | |||
G20A | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | N173D | A232V | Q236H | O245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | O236H | O245R | N248D | K251V | N252K |
V68A | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q235H | Q245R | N248D | N252F | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252L | ||||
P55S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252F | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T255V | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256N | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256E | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | L257R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | G258D | |||
I8V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N269D | ||
V68A | S103A | V104I | N116S | G159D | A232V | Q236H | Q245R | N248D | N252K | T260E | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | A232S | Q236H | Q245R | N248D | N252K | |||||
V68A | S103A | V104I | G159D | A232V | Q236R | Q245R | N248D | N252K | ||||
N18S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245V | N248D | N252K | ||||
V68A | N76D | S101T | S103A | V104I | G159D | T213R | N218S | A232V | Q236H | Q245R | T260A | |
V68A | S103A | V104I | G159D | A228V | A232V | Q236H | Q245R | N248D | N252K | |||
T33S | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | N76D | E89D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | |
G61E | V68A | N76D | S103A | V104I | Q159D | A232V | Q236H | Q245R | N248D | N252K | ||
S103A | V104I | G159D | V205I | P210I | A232V | Q236H | Q245R | |||||
G61E | V68A | S103A | V104I | S130A | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
G61E | V68A | S103A | V104I | A133S | Q137R | G159D | A232V | Q236H | Q245R | N248D | N252K | |
G61E | S103A | V104I | A133V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248G | N252K | ||||
V68A | S103A | V104I | G159D | N218S | A232V | Q236H | Q245R | N248D | N252K | |||
G61E | V68A | S103A | V104I | G159D | S160V | A232V | Q236H | Q245R | N248D | N252K | ||
S3L | G61E | V68A | N76D | S103A | V104I | A232V | Q236H | Q245R | N248D | N252K | ||
G61E | V68A | S103A | V104I | Q159D | S167F | A232V | Q236H | Q245R | N248D | N252K | ||
G97E | S103A | V104I | G159D | A232V | Q236H | Q245R | N242D | N252K |
A98D | S103A | V104I | Q159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S99E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S101E | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | S106E | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | Q109E | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | |||||
S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
N62D | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | S166D | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | L217E | A232V | Q236H | Q245R | N248D | N252K | |||||
G20R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | Q206R | L217E | A232V | Q236H | Q245R | N248D | N252K | ||||
N62D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
K27N | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
T38G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
T38A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | ||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | E271G | |||
V68A | N76D | S103A | V104I | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | |||
V68A | N76D | S103A | V104I | G159D | P210I | T213R | A232V | Q236H | Q245R | T260A | |||
V68A | N76D | S103A | V104I | G159D | V205I | T213R | A232V | Q236H | Q245R | T260A | |||
V68A | N76D | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | T260A | ||||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||
N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | G159D | A230V | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | G159D | L126F | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | ||||||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | ||||||
S103A | V104I | G159D | A230V | O236H | O245R |
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | T260A | |||||
S103A | V104I | G159D | A232V | Q236H | Q245R | |||||||
V68A | S103A | V104I | G159D | A174V | A232V | Q236H | Q245R | L257V | ||||
V68A | S103A | V104I | G159D | A194S | A232V | Q236H | Q245R | L257V | ||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | ||||
S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | ||||||
V68A | N76D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | N261W | ||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | N261W | ||||
S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||
S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | |||||
V68A | N76D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | ||
Q12R | S103A | V104I | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | |||
S103A | V104I | Y209W | A232V | Q236H | Q245R | L257V | ||||||
S103A | V104I | G159D | V205I | P210I | T213R | A232V | Q236H | Q245R | T260A | |||
S103A | V104I | G159D | V205I | Y209W | A232V | Q236H | Q245R | T260A | ||||
V68A | S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | |||
S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | L257V | |||
S103A | V104I | G159D | V205I | Y209W | A232V | Q236H | Q245R | L257V | ||||
V68A | S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | T260A | ||
S103A | V104I | G159D | V205I | Y209W | P210I | A232V | Q236H | Q245R | ||||
S103A | V104I | G159D | Y209W | P210I | A232V | Q236H | Q245R | |||||
S103A | V104I | G159D | V205I | P210I | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | S128L | G156D | A232V | Q236H | Q245R | |||||
A48V | S103A | V104I | G159D | A230V | Q236H | Q245R | ||||||
A48V | V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | ||||
A48V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
A48V | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V | N261W | |||
G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
Q12R | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
S101G | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
A98L | G102A | S103A | V104I | G159D | S212G | A232W | Q236H | Q245R | N248D | N252K | ||
G102A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
S102A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
S103A | V104I | G159D | N184S | A232V | O236H | O245R | N248D | N252K |
S103A | V104I | G159D | N184G | A232V | Q236H | Q245R | N248D | N252K | ||||||||
S103A | V104I | G159D | A232V | Q236H | V244T | Q245R | N248D | N252K | ||||||||
S103A | V104I | G159D | A232V | Q236H | V244A | Q245R | N248D | N252K | ||||||||
N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | S256R | ||||||
Q12R | N62D | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||||
S101G | S103A | V104I | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | G159D | G206E | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | G159D | T213Q | A232V | Q236H | Q245R | N248D | N252K | |||||||
A98L | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | G102A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||||||
A98L | G102A | S103A | V104I | G159D | S212G | A232V | Q236H | N248D | N252K | |||||||
N62D | S103A | V104I | Q109R | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||||
N62D | S103A | V104I | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||||
N62D | S101G | S103A | V104I | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | |||||
S103A | V104I | G159D | A232V | Q245R | N248D | N252K | ||||||||||
S103A | V104I | G159D | A230V | Q245R | ||||||||||||
N62D | S103A | V104I | S130G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||||
S101G | S103A | V104I | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | S128G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | S128L | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||||
N62D | S101G | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||||
N62D | S103A | V104I | S128G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||||
N62D | S103A | V104I | S128L | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||||||
S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260A | |||||||
S101G | S103A | V104I | P131V | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||||
A98V | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||||
S99G | S101G | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | G159D | V205I | A232V | Q236H | Q245R | N248D | N252K | |||||||
S101G | S103A | V104I | G159D | A230V | Q236H | Q245R | ||||||||||
S101G | S103A | V104I | G159D | A194P | A232V | O236H | Q245R | N248D | N252K | |||||||
N76D | S101G | S103A | V104I | G159D | A194P | A232V | O236H | O245R | N248D | N252K | ||||||
S101G | S103A | V104I | G159D | A230V | A232V | O236H | O245R | N248D | N252K |
N62D | S103A | V104I | G159D | N185D | Q206E | T213R | A232V | Q236H | Q245R | N248D | N252K | E271Q |
可用于本发明清洗组合物的更进一步优选的蛋白酶变体包括选自表VI的取代组的取代,但下面的表VIII中的取代组除外:
表VIII
68 | 76 | 103 | 104 | 116 | 159 | 170 | 185 | 232 | 236 | 245 |
可用于本发明清洗组合物的更进一步优选的蛋白酶变体包括选自表IX的取代组的取代:
表IX
68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
68 | 103 | 104 | 140 | 159 | 232 | 236 | 245 | 252 | |||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | |||
43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | ||||
68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | |||
68 | 103 | 104 | 159 | 224 | 232 | 236 | 245 | 257 | |||
76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | ||||
68 | 76 | 103 | 104 | 159 | 211 | 232 | 236 | 245 | |||
12 | 68 | 76 | 103 | 104 | 159 | 214 | 232 | 236 | 245 | ||
68 | 76 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | |||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
20 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 259 | ||
68 | 76 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | ||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 261 | |||
12 | 48 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||
76 | 103 | 104 | 159 | 192 | 232 | 236 | 245 |
76 | 103 | 104 | 147 | 159 | 232 | 236 | 245 | 248 | 251 | ||
12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 272 | ||
68 | 76 | 103 | 104 | 159 | 183 | 206 | 232 | 236 | 245 | ||
68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 256 | |||
68 | 76 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | |||
27 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | ||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 213 | 232 | 236 | 245 | 248 | 252 | |||
68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 216 | 232 | 236 | 245 | 248 | 252 | ||
20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 255 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 256 | ||
68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | ||
68 | 103 | 104 | 159 | 228 | 232 | 236 | 245 | 248 | 252 | ||
68 | 76 | 89 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 |
68 | 103 | 104 | 159 | 218 | 232 | 236 | 245 | 248 | 252 |
可用于本发明清洗组合物的更进一步优选的蛋白酶变体包括选自表X的取代组的取代:
表X
V68A | S103A | V104I | G159D | A228V | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | N218S | A232V | Q236H | Q245R | N248D | N252K | ||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | N76D | E89D | S103A | V104I | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A |
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256R |
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T260R | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | T255V | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | S256N | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252L | ||||
V68A | S103A | V104I | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A215V | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216T | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S216V | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | T213S | A232V | Q236H | Q245R | N248D | N252K | ||||
V68A | S103A | V104I | G159D | P210L | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212C | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||||
V68A | S103A | V104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
G20R | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | Y209F | A232V | Q236H | Q245R | N248D | N252K | |||
N76D | S103A | V104I | G159D | Y192F | A232V | Q236H | Q245R | |||||
N76D | S103A | V104I | V147I | G159D | A232V | Q236H | Q245R | N248S | K251R | |||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | A272S | |||
V68A | N76D | S103A | V104I | G159D | N183K | Q206L | A232V | Q236H | Q245R | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S256R | ||||
V68A | N76D | S103A | V104I | G159D | Q206R | A232V | Q236H | Q245R | ||||
K27R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
Q12R | A48V | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | |||
V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | N261W | ||||
V68A | N76D | S103A | V104I | G159D | G211R | A232V | Q236H | Q245R | ||||
V68A | N76D | S103A | V104I | G159D | G211V | A232V | Q236H | Q245R | ||||
Q12R | V68A | N76D | S103A | V104I | G159D | Y214L | A232V | Q236H | Q245R | |||
V68A | N76D | S103A | V104I | G159D | A215R | A232V | Q236H | Q245R | ||||
Q12R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
G20R | V68A | N76D | S103A | V104I | G159D | A232V | Q236H | Q245R | S259G | |||
V68A | N76D | S87R | S103A | V104I | G159D | A232V | O236H | O245R | T260V | |||
N76D | S103A | V104 | G159D | A232V | Q236H | Q245R | L257V | |||||
V68A | N76D | S103A | V104 | G159D | T213R | A232V | Q236H | Q245R | T260A |
V68A | N76D | S103A | V104I | G159D | P210R | A232V | Q236H | Q245R | |||
V68A | S103A | V104I | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | ||
V68A | S103A | V104I | G159D | T224A | A232V | Q236H | Q245R | L257V | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252S | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | ||||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | |||||
V68A | S103A | V104I | N140D | G159D | A232V | Q236H | Q245R | N252K | |||
N43S | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | |||
N43K | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | ||||
N43D | V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | N252K | |||
V68A | S103A | V104I | G159D | A232V | Q236H | Q245R | L257V |
可用于本发明清洗组合物的高度优选的蛋白酶变体包括选自下列取代组的取代:
12/102/103/104/159/212/232/236/245/248/252;61/68/103/104/159/232/236/245/248/252;
62/103/104/130/159/213/232/236/245/248/252;62/103/104/159/213/232/236/245/248/252;
62/103/104/109/159/213/232/236/245/248/252;62/103/104/159/232/236/245/248/252;
62/101/103/104/159/212/213/232/236/245/248/252;
68/103/104/159/232/236/245/248/252/270;
68/103/104/159/185/232/236/245/248/252; 68/103/104/159/210/232/236/245/248/252;
68/103/104/159/185/210/232/236/245/248/252;68/103/104/159/213/232/236/245/248/252;
68/103/104/159/230/232/236/245; 68/76/103/104/159/209/232/236/245;
68/103/104/232/236/245/248/257/275; 68/103/104/213/232/236/245/248/252;
68/103/104/159/232/236/245/248/252; 68/103/104/159/209/232/236/245;
68/76/103/104/159/232/236/245; 68/103/104/159/232/236/245/252;
68/103/104/159/232/236/245; 68/103/104/159/232/236/245/257;
68/76/103/104/159/211/232/236/245; 68/76/103/104/159/215/232/236/245;
68/103/104/159/210/232/236/245; 68/103/104/159/213/232/236/245/260;
68/76/103/104/159/213/232/236/245/260; 68/76/103/104/159/210/232/236/245/260;
68/103/104/159/183/232/236/245/248/252; 68/103/104/232/236/245/257/275;
68/103/104/159/213/232/236/245; 76/103/104/159/232/236/245;
76/103/104/159/213/232/236/245/260; 76/103/104/131/159/232/236/245/248/252;
97/103/104/159/232/236/245/248/252; 98/103/104/159/232/236/245/248/252;
98/102/103/104/159/212/232/236/245/248/252;101/103/104/159/232/236/245/248/252;
102/103/104/159/232/236/245/248/252; 103/104/159/232/236/245;
103/104/159/248/252/270; 103/104/159/232/236/245/248/252;
103/104/159/205/209/232/236/245/257 103/104/159/232/245/248/252;
103/104/159/205/209/210/232/236/245/257; 103/104/159/213/232/236/245/248/252;
103/104/159/217/232/236/245/248/252; 103/104/130/159/232/236/245/248/252;
103/104/131/159/232/236/245/248/252; 103/104/159/205/209/232/236/245;及
103/104/159/232/236/245/257。
可用于本发明清洗组合物的更高度优选的蛋白酶变体包括选自下列取代组的取代:
12R/102A/103A/104I/159D/212G/232V/236H/245R/248D/252K;
61E/68A/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/109R/159D/213R/232V/236H/245R/248D/252K;
62D/103A/104I/159D/213R/232V/236H/245R/248D/252K;
62D/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/130G/159D/213R/232V/236H/245R/248D/252K;
62D/101G/103A/104I/159D/212G/213R/232V/236H/245R/248D/252K;
68A/76D/103A/104I/159D/213R/232V/236H/245R/260A;
68A/76D/103A/104I/159D/210I/232V/236H/245R/260A;
68A/103A/104I/159D/183D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/209W/232V/236H/245R;
68A/76D/103A/104I/159D/211R/232V/236H/245R;
68A/76D/103A/104I/159D/215R/232V/236H/245R;
68A/103A/104I/159D/213R/232V/236H/245R/260A;
68A/76D/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/252K;
68A/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/257V;
68A/103A/104I/159D/185D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/185D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/230V/232V/236H/245R;
68A/76D/103A/104I/159D/209W/232V/236H/245R;
68A/103A/104I/232V/236H/245R/248D/257V/275H;
68A/103A/104I/232V/236H/245R/257V/275H;
68A/103A/104I/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210I/232V/236H/245R;
68A/103A/104I/159D/210L/232V/236H/245R;
68A/103A/104I/159D/213G/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/248D/252K/270A;
76D/103A/104I/159D/232V/236H/245R;
76D/103A/104I/131V/159D/232V/236H/245R/248D/252K;
76D/103A/104I/159D/213R/232V/236H/245R/260A;
97E/103A/104I/159D/232V/236H/245R/248D/252K;
98L/103A/1041/159D/232V/236H/245R/248D/252K;
98L/102A/103A/104I/159D/212G/232V/236H/245R/248D/252K;
101G/103A/104I/159D/232V/236H/245R/248D/252K;
102A/103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/213R/232V/236H/245R/248D/252K;
103A/104I/130G/159D/232V/236H/245R/248D/252K;
103A/104I/159D/217E/232V/236H/245R/248D/252K;
103A/104I/159D/248D/252K/270V;
103A/104I/159D/232V/236H/245R;
103A/104I/159D/205I/209W/232V/236H/245R;
103A/104I/159D/232V/236H/245R/257V;
103A/104I/159D/205I/209W/232V/236H/245R/257V;
103A/104I/131V/159D/232V/236H/245R/248D/252K;
103A/104I/159D/205I/209W/210I/232V/236H/245R/257V;及
103A/104I/159D/232V/245R/248D/252K。
重组蛋白酶/重组枯草杆菌蛋白酶-“重组蛋白酶”或“重组枯草杆菌蛋白酶”是指这样的蛋白酶或枯草杆菌蛋白酶,其中编码天然存在的蛋白酶或枯草杆菌蛋白酶的DNA序列分别经过了修饰,从而形成突变体DNA序列,该序列编码蛋白酶或枯草杆菌蛋白酶氨基酸序列中1个或多个氨基酸的“取代、插入或缺失”的编码。合适的改性方法公开在本文以及美国专利RE 34,606、5,204,015及5,185,258中。
非人体蛋白酶/非人体枯草杆菌蛋白酶-“非人体蛋白酶”或“非人体枯草杆菌蛋白酶”及编码它们的DNA编码可从许多原核及真核生物体中获得。原核生物的合适例子包括革兰氏阴性生物,如大肠杆菌E.coli或假单胞菌属;以及革兰氏阳性细菌如微球菌属或芽胞杆菌属。可由其获得羰基水解酶及其基因的真核生物的例子包括例如酿酒酵母(Saccharomyces cerevisiae),真菌如曲霉菌及非人体哺乳动物源,例如牛,由它可获得编码蛋白酶凝乳酶或枯草杆菌蛋白酶凝乳酶的基因。有一系列蛋白酶和/或枯草杆菌蛋白酶可由各种各样相关的品种获得,这些品种具有的氨基酸序列虽在该系列成员之间不完全同源但却表现出相同或相近类型的生物活性。因此,本发明使用的非人体蛋白酶或非人体枯草杆菌蛋白酶具有分别涉及蛋白酶或枯草杆菌蛋白酶的功能定义,它们又直接或间接与原核和真核源相联系。
变体DNA序列-编码这些蛋白酶或枯草杆菌蛋白酶变体的变体DNA序列衍生自编码天然存在或重组体前体酶的前体DNA序列。变体DNA序列是通过改变前体DNA序列以便编码对应于解淀粉芽胞杆菌位点103并配合1个或多个下列位点上,前体DNA序列编码的1个或多个特定氨基酸残基的取代:
1,3,4,8,9,10,12,13,16,17,18,19,20,21,22,24,27,33,37,38,42,43,48,55,57,58,61,62,68,72,75,76,77,78,79,86,87,89,97,98,99,101,102,104,106,107,109,111,114,116,117,119,121,123,126,128,130,131,133,134,137,140,141,142,146,147,158,159,160,166,167,170,173,174,177,181,182,183,184,185,188,192,194,198,203,204,205,206,209,210,211,212,213,214,215,216,217,218,222,224,227,228,230,232,236,237,238,240,242,243,244,245,246,247,248,249,251,252,253,254,255,256,257,258,259,260,261,262,263,265,268,269,270,271,272,274及275
其中当所述蛋白酶变体包括在对应于位点103和76的氨基酸残基的取代时,则还存在对应于1个或多个除解淀粉芽胞杆菌枯草杆菌蛋白酶位点27、99、101、104、107、109、123、128、166、204、206、210、216、217、218、222、260、265或274氨基酸残基位点以外的氨基酸残基位点上的氨基酸残基取代。虽然这里所指出的氨基酸残基改性位点是按照适用于解淀粉芽胞杆菌的编号法给出的(该方法已成为确定所有枯草杆菌蛋白酶中的残基位点的惯用方法),但是可用于本发明的优选前体DNA序列则是如图3所示的迟缓芽胞杆菌的DNA序列。
在优选的实施方案中,这些变体DNA序列编码对应于解淀粉芽胞杆菌枯草杆菌蛋白酶位点103以及配合着在1个或多个对应于下列解淀粉芽胞杆菌位点上的氨基酸残基的取代、插入或缺失:
1,3,4,8,9,10,12,13,16,17,18,19,20,21,22,24,27,33,37,38,42,43,48,55,57,58,61,62,68,72,75,76,77,78,79,86,87,89,97,98,99,101,102,104,106,107,109,111,114,116,117,119,121,123,126,128,130,131,133,134,137,140,141,142,146,147,158,159,160,166,167,170,173,174,177,181,182,183,184,185,188,192,194,198,203,204,205,206,209,210,211,212,213,214,215,216,217,218,222,224,227,228,230,232,236,237,238,240,242,243,244,245,246,247,248,249,251,252,253,254,255,256,257,258,259,260,261,262,263,265,268,269,270,271,272,274及275
其中当所述蛋白酶变体包括在对应于位点103和76的位点氨基酸残基的取代时,则还存在对应于1个或多个除解淀粉芽胞杆菌枯草杆菌蛋白酶位点27、99、101、104、107、109、123、128、166、204、206、210、216、217、218、222、260、265或274氨基酸残基位点以外的氨基酸残基位点上的氨基酸残基取代。更优选的是,这些变体DNA序列编码这里所描述的蛋白酶变体。
在另一种优选的实施方案中,这些变体DNA序列编码对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的位点62、212、230、232、252及257上的氨基酸残基的取代、插入或缺失。更优选的是,这些变体DNA序列编码这里所描述的蛋白酶变体。
虽然这里所指出的氨基酸残基是按照适用于解淀粉芽胞杆菌的编号法给出的(该方法已成为确定所有枯草杆菌蛋白酶中的残基位点的惯用方法),但是可用于本发明的优选前体DNA序列则是如图3所示的迟缓芽胞杆菌的DNA序列。
这些重组体DNA序列编码具有新颖氨基酸序列的蛋白酶变体,且通常该变体具有至少1种性质显著不同于前体蛋白酶DNA序列编码的酶的对应性质。这些性质包括在蛋白水解活性、底物特异性、稳定性、pH曲线发生改变和/或性能特性上得到强化。
特异性取代对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的位点103并配合着1个或多个下列位点:
1,3,4,8,9,10,12,13,16,17,18,19,20,21,22,24,27,33,37,38,42,43,48,55,57,58,61,62,68,72,75,76,77,78,79,86,87,89,97,98,99,101,102,104,106,107,109,111,114,116,117,119,121,123,126,128,130,131,133,134,137,140,141,142,146,147,158,159,160,166,167,170,173,174,177,181,182,183,184,185,188,192,194,198,203,204,205,206,209,210,211,212,213,214,215,216,217,218,222,224,227,228,230,232,236,237,238,240,242,243,244,245,246,247,248,249,251,252,253,254,255,256,257,258,259,260,261,262,263,265,268,269,270,271,272,274及275
其中当所述蛋白酶变体包括在对应于位点103和76的氨基酸残基取代时,则还存在对应于1个或多个除解淀粉芽胞杆菌枯草杆菌蛋白酶位点27、99、101、104、107、109、123、128、166、204、206、210、216、217、218、222、260、265或274氨基酸残基位点以外的氨基酸残基位点上的氨基酸残基取代,其中这里所描述的编号位点对应于来自解淀粉芽胞杆菌的天然存在的枯草杆菌蛋白酶,或者对应于其他羰基水解酶或枯草杆菌蛋白酶(如迟缓芽胞杆菌枯草杆菌蛋白酶)中的等价氨基酸残基。再有,特异性取代对应于位点62、212、230、232、252和257中的1个或多个,其中这里所描述的编号位点对应于来自解淀粉芽胞杆菌的天然存在的枯草杆菌蛋白酶,或者对应于其他羰基水解酶或枯草杆菌蛋白酶(如迟缓芽胞杆菌枯草杆菌蛋白酶)中的等价氨基酸残基。这些氨基酸位点代号可参见图1中给出的成熟解淀粉芽胞杆菌枯草杆菌蛋白酶序列。然而,本发明不限于此种特殊枯草杆菌蛋白酶的突变体,它可延伸至含有与前面具体指出的解淀粉芽胞杆菌枯草杆菌蛋白酶残基等价的位点上的氨基酸残基的前体蛋白酶。在本发明的优选实施方案中,前体蛋白酶是迟缓芽胞杆菌枯草杆菌蛋白酶,而取代、缺失或插入则发生在对应于上面列举的迟缓芽胞杆菌的等价氨基酸残基上。
某种前体蛋白酶的残基(氨基酸)等价于解淀粉芽胞杆菌枯草杆菌蛋白酶残基的条件是,它与解淀粉芽胞杆菌枯草杆菌蛋白酶中的某一特定残基或该残基的部分同源(即,一级或三级结构中的位置上的对应)或者类似(即,具有相同或相近的结合、反应或化学相互作用的功能)。
为了确认与一级结构为同源性,应直接将该前体蛋白酶的氨基酸序列与解淀粉芽胞杆菌枯草杆菌蛋白酶一级序列,特别是与已知序列的枯草杆菌蛋白酶中已知未变异的一组残基进行对比。例如,本文的图2给出了解淀粉芽胞杆菌枯草杆菌蛋白酶与迟缓芽胞杆菌枯草杆菌蛋白酶之间保守的残基。将这些保守残基对齐,同时做一些必要的插入和缺失以便维持对齐(即避免由于随意的缺失和插入而导致保留残基被消除)之后,便可确定与解淀粉芽胞杆菌枯草杆菌蛋白酶的一级序列中特定氨基酸等价的残基。保守残基的对齐结果,优选应保留100%此种残基。然而,大于75%或低至50%保守残基的对齐也就足以确定等价残基了。催化三分体--Asp32/His64/Ser221--的保守性应当保持。
例如,在图3中,将来自解淀粉芽胞杆菌、枯草芽胞杆菌、地衣芽胞杆菌(carlsbergensis)及迟缓芽胞杆菌的枯草杆菌蛋白酶的氨基酸序列对齐,便可提供各种氨基酸序列之间最大程度的同源性。这些序列之间的对比表明,在每种序列中均包含大量保守残基。这些保守残基(例如BPN’与B.lentus之间)在图2中标出。
于是,可利用这些保守残基来确定迟缓芽胞杆菌(PCT公开号WO89/0629,1989-07-13发表),即本发明优选的蛋白酶前体酶或者被称之为PB92(EP 0 328 299)的枯草杆菌蛋白酶,的对应等价氨基酸残基,这是一种与优选的迟缓芽胞杆菌枯草杆菌蛋白酶高度同源的蛋白酶的等价残基。这些枯草杆菌蛋白酶中某些的氨基酸序列在图3A和3B中与解淀粉芽胞杆菌枯草杆菌蛋白酶对齐排列在一起,对齐原则是使它们的保守残基之间达到最大限度的对应。从图中可见,与解淀粉芽胞杆菌枯草杆菌蛋白酶相比,在迟缓芽胞杆菌的序列中存在许多缺失。譬如,在解淀粉芽胞杆菌枯草杆菌蛋白酶中的等价于Val165的氨基酸,在其他枯草杆菌蛋白酶中则为对应于迟缓芽胞杆菌和地衣芽胞杆菌的异亮氨酸。于是,例如+76位点上的氨基酸在解淀粉芽胞杆菌和迟缓芽胞杆菌中均为天冬酰胺(N)。然而在本发明蛋白酶变体中,等价于解淀粉芽胞杆菌中+76的氨基酸被取代为天冬氨酸(D)了。本发明针对所有氨基酸所使用的缩写和一字母代号均与《patentin使用者手册》一致(GenBank.Mountain View.CA)1990.p.101)。
“等价残基”还可以通过在前体蛋白酶的三级结构水平上确定同源性而定义,其三级结构是通过x射性结晶学方法确定的。等价残基的定义是前体蛋白酶与解淀粉芽胞杆菌枯草杆菌蛋白酶的2或更多个特定氨基酸残基主链原子的原子坐标(之差)(N对N,CA对CA、C对C,O对O),对齐之后在0.13nm范围内,优选0.1nm之内。达到对齐的标准是,将最佳模型取向并定位,使待测蛋白酶的非氢蛋白原子的原子坐标与解淀粉芽胞杆菌枯草杆菌蛋白酶达到最大重合的程度。最佳模型是,在能达到的最高分辨率条件下能产生最低用于衍射实验数据的R系数值的结晶学模型。
在功能上与解淀粉芽胞杆菌枯草杆菌蛋白酶特定残基等价的残基被定义为这样的前体蛋白酶氨基酸,即,它能采取某种构象,以致使其能经过改变、改性或促进而获得如同针对解淀粉芽胞杆菌枯草杆菌蛋白酶特定残基所规定和特有的蛋白结构、底物结合或催化的行为。而且,它们又是这样的前体蛋白酶(其三级结构已用X射线结晶学方法获得)残基,它占据的位置是如此近似,以致虽然该给定残基的主链原子可能不满足按照占据同源位置来衡量的等价标准,但是该残基的至少2个侧链原子的位置与对应的解淀粉芽胞杆菌枯草杆菌蛋白酶侧链原子之间相差在0.13nm范围内。解淀粉芽胞杆菌枯草杆菌蛋白酶的三维结构坐标载于EPO公开号0 251 446(等价于美国专利5,182,204,其公开内容并入本文作为参考),可被用来按照上面概述的那样在三级结构水平上确定等价残基。
所指出的可进行取代、插入或缺失的残基中,有些是保守残基,而有些则不是。在不是保守残基的情况下,1个或多个氨基酸的取代应限制在所产生的变体氨基酸序列乃是自然界中不存在的那些取代。在保守残基的情况下,此种取代不应导致天然存在序列的生成。本发明的蛋白酶变体包括蛋白酶变体的各种成熟形式,以及此种蛋白酶变体的原-及前-原-形式。前原-形式是优选的,因为这将有利于蛋白酶变体的表达、分泌及成熟。
“原序列”是指一种结合在蛋白酶成熟形式的N-末端部分上的氨基酸序列,当它被去掉时,将导致该蛋白质“成熟”形式的出现。许多蛋白水解酶是以转译酶原酶产物的形式存在于自然界的,在没有转译后加工的情况下,总是以此种方式来表达。产生蛋白酶变体的优选原序列是解淀粉芽胞杆菌枯草杆菌蛋白酶的这种推断原序列,虽然也可使用其他蛋白酶原序列。
“信号序列”或“前序列”是指任何结合在可参与该蛋白酶成熟或原形式的分泌的蛋白酶N末端部分,或原蛋白酶N-末端部分上的氨基酸序列。信号序列的这一定义是功能定义,旨在涵盖所有编码在天然条件下参与实现蛋白酶分泌过程的氨基酸序列的蛋白酶基因N-末端部分。本发明利用此种序列来实现这里所规定的蛋白酶变体的分泌。一种可能的信号序列包含:来自枯草芽胞杆菌枯草杆菌蛋白酶的该信号序列的头7个氨基酸残基融合到来自迟缓芽胞杆菌的枯草杆菌蛋白酶其余信号序列上(ATCC 21536)。
蛋白酶变体的“前原”形式由这样的蛋白酶成熟形式组成,它具有一个可操作地连接到蛋白酶的氨基末端的原序列,以及一个可操作地连接到该原序列氨基末端上的“前”或“信号”序列。
“表达载体”是指一种DNA结构,它所包含的DNA序列可操作地连接到一个能实现所述DNA在适当宿主中表达的适当控制序列上。此种控制序列包括一个能实现转录的启动子、一个任选的控制此种转录过程的操纵子序列、一个载有适当mRNA核糖体结合位点编码的序列以及能控制转录或转译终止的序列。该载体可以是质粒、噬菌体颗粒或者简单地是一种潜在的基因组插入物。一旦转移到适当的宿主中,载体可复制并独立于该宿主基因组地发挥作用,或者在某些情况下结合到基因组本身中。在本说明中,“质粒”和“载体”有时可彼此通用,因为质粒是目前最普遍使用的载体形式。然而,本发明还意在包括那些能起到等价功能且在技术上已知或将变为已知的其他表达载体形式。
本发明所使用的术语“宿主细胞”通常是原核或真核宿主,它优选地按照美国专利RE 34,606公开的方法操作过,从而使之不能分泌具有酶促活性的内切蛋白酶。用于表达蛋白酶的优选宿主细胞是缺乏“具有酶促活性的中性蛋白酶及碱性蛋白酶(枯草杆菌蛋白酶)”的Bacillus菌株BG2036。菌株BG2036的结构详细描述于美国专利5,264,366中。用于表达蛋白酶的其他宿主细胞包括枯草芽胞杆菌168(还见诸于美国专利RE34,606和美国专利5,264,366中,在此将其公开内容并入本文作为参考),以及任何合适的杆菌菌株如地衣芽胞杆菌、迟缓芽胞杆菌等)。
宿主细胞由采用重组DNA技术所构造的载体予以转化或转染。此种经转化的宿主细胞能复制编码蛋白酶变体的载体或者能表达所要求的蛋白酶变体。在该载体编码蛋白酶变体的前-或前原-形式的情况下,在表达此种变体时,通常是将其从宿主细胞内分泌到宿主细胞介质中。
“可操作地连接”,当描述2个DNA区域之间的关系时,简单地表示它们在功能上彼此相关。例如,若某一原序列起到信号序列的功能,参与蛋白成熟形式的分泌过程,最可能涉及该信号序列的分裂,则该原序列便可操作地连接在相应的肽上了。“某启动子可操作地连接在某一编码序列上”,是指它能控制该序列的转录;核糖体结合位点可操作地连接在一个编码序列上,是指它所处的位置使得转译得以发生。
载有天然存在的前体蛋白酶的基因可按照本领域技术人员普遍已知的方法获得。这些方法一般包括合成具有目标蛋白酶推断序列编码区的标记探针,由表达该蛋白酶的生物制备基因组文库,以及通过与探针杂交,从文库中筛选出该目标基因。然后,对阳性杂交克隆进行作图和测序。
然后用该克隆的蛋白酶来转化宿主细胞,以表达该蛋白酶。随后,将该蛋白酶基因连接到高拷贝数的质粒上。该质粒在宿主中进行复制的结果应使它包含为实现质粒复制所需要的熟知要素:一种可操作地连接在目标基因上的启动子(如果它被识别,即被宿主转录的话,它可以该基因本身的同源启动子的形式提供给宿主)、转录终止及多腺苷酸化区域(为使宿主从某些真核宿主细胞中转录的蛋白酶基因mRNA达到稳定化所需要的),它可以是外来的或者是由该蛋白基因的内源终止子区提供的,以及优选地,一种选择基因,例如抗生素抗性基因,它能通过在含抗生素介质中的生长使被质粒感染的宿主细胞维持连续培养。高拷贝数质粒还包含针对宿主的复制起点,从而使大量质粒得以在细胞质中产生,而不受染色体限制。然而,将多拷贝的蛋白酶基因结合到宿主基因组中的做法也属于本发明的范围。这一方法可通过特别容易发生同源重组的原核及真核生物的采用而得到加速。这种基因可以是天然迟缓芽胞杆菌基因。作为替代的方案,可制备一种编码天然存在或突变体前体蛋白酶的合成基因。按此种方式实施时,测定该前体蛋白酶的DNA或/或氨基酸序列。随后,合成多个重叠、合成的、单一链DNA片断,它们在杂交并连接之后,将生成编码前体蛋白酶编码的合成DNA。合成基因制备的例子载于美国专利5,204,105,实施例3中,在此将其公开内容并入本文作为参考。
一旦克隆了天然存在或合成的前体蛋白酶基因之后,可采取多种改性措施,以便使该基因的用途超出天然前体蛋白酶“合成”的范畴。这类改性包括:重组蛋白酶的制备,如公开在美国专利RE34,606及EPO公开号0 251 446中;以及本文所描述的蛋白酶变体的制备。
下面的盒式诱变方法可能有助于本发明蛋白酶变体的制备,尽管其他方法也可使用。首先,获得编码该蛋白酶的天然存在的基因并全部或部分地测序。然后,扫描该序列以找出希望对编码酶中1个或多个氨基酸实施突变(缺失、插入或取代)的点。评估该点两侧的序列,找出用于以某种当表现出来时将编码各种不同突变体的寡核苷酸库取代该基因某一短片断的限制位点。此种限制位点优选是蛋白酶基因中唯一的位点,以便有利于该基因片断的取代。然而,任何方便的限制位点,只要在蛋白酶基因中不是过度冗余的,均可使用,但条件是,通过限制消化所产生的基因片断能够按恰当序列进行重组。倘若限制位点不是位于距所选点一段方便的距离内(10~15个核苷酸),则通过对基因中核苷酸的取代来生成此种位点,此间,最终构造中的读框及其编码的氨基酸均不得发生改变。为将基因序列改变为符合所要求序列的基因突变,是通过熟知的方法的M13引物延伸技术实现的。找出合适的两侧区域及评估为获得2个方便的限制位点序列所需进行的改变--这一任务已成为一种由遗传密码的丰度、基因限制酶图谱和大量不同的限制性酶所组成的常规操作程序。要注意的是,如果现成就存在某种方便的两侧限制位点,则上述方法只需针对不含此种位点的两侧区域使用。
一旦克隆了天然存在的DNA或合成DNA,就用“同源限制酶”消化掉待诱发突变位点两侧的限制位点,并在该基因上连接大量末端互补的寡核苷酸框。采用这一方法可简化诱变过程,因为所有的寡核苷酸都可合成,从而具有相同的限制位点,而且不需要合成接头来创造限制位点。本文所使用的术语“蛋白水解活性”的定义是,每毫克活性酶的肽键水解速率。现有许多熟知的程序,可用来测定蛋白水解活性(K.M.Kalisz,“微生物蛋白酶”《生物化学工程/生物技术进展》,A.Fiechter主编,1988)。与改性后蛋白水解活性并存或作为替代的性质,本发明变体酶还可具有其他改性性质,例如Km、k催化、k催化/Km比值和/或改性底物特异性和/或改性pH活性曲线。此类酶还可专为特定底物定制,这些底物例如是存在于肽的制备中的,或者针对水解过程如用于洗衣的。
本发明的一个目的是制取与前体蛋白酶相比,蛋白水解活性改变的变体蛋白酶,因为提高此种活性(数值增大),将使得该酶能更有效地作用于目标底物。同样重要的是,该变体酶,与前体相比,在热稳定性上发生了改变和/或底物特异性发生了改变。在某些情况下,可能希望较低的蛋白水解活性,例如,蛋白水解活性的降低在希望获得蛋白酶“合成活性”的场合是有用的(譬如用于肽的合成)。人们之所以希望降低此种活性,可能因为它能破坏此种合成的产物。相反,在某些情况下,可能希望提高变体酶相对于其前体的蛋白水解活性。另外,可能希望提高或降低变体稳定性,不论对碱或对热的稳定性。提高或降低Km、k催化或k催化/Km都是针对测定这些动力学参数所使用的底物而言的。
本发明另一个方面是确定了如下事实:在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶位点103并配合在1个或多个下列位点实施取代:
1,3,4,8,9,10,12,13,16,17,18,19,20,21,22,24,27,33,37,38,42,43,48,55,57,58,61,62,68,72,75,76,77,78,79,86,87,89,97,98,99,101,102,104,106,107,109,111,114,116,117,119,121,123,126,128,130,131,133,134,137,140,141,142,146,147,158,159,160,166,167,170,173,174,177,181,182,183,184,185,188,192,194,198,203,204,205,206,209,210,211,212,213,214,215,216,217,218,222,224,227,228,230、232,236,237,238,240,242,243,44,245,246,247,248,249,251,252,253,254,255,256,257,258,259,260,261,262,263,265,268,269,270,271,272,274及275,对调节酶的全面稳定性和/或蛋白水解活性是重要的。
本发明又一个方面是确定了:在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶位点62、212、230、232、252及257中1个或多个上的取代,对调节酶的全面稳定性和/或蛋白水解活性也是重要的。
这种取代优选在迟缓芽胞杆菌(重组或天然型)枯草杆菌蛋白酶上实施,虽然该取代可在任何芽胞杆菌属蛋白酶上实施。
根据对由这些变体蛋白酶所获结果的筛选,解淀粉芽胞杆菌枯草杆菌蛋白酶所发生的显著突变对于此种酶的蛋白水解活性和/或稳定性乃至此种变体酶的清洁或洗涤性能都是重要的。
制备用于本发明洗涤剂和清洗组合物中的酶的方法和程序是已知的,且公开在PCT公开号WO 95/10615中。
本发明的酶具备类似胰蛋白酶的特异性。就是说,本发明的酶通过择优裂解带电荷的氨基酸残基肽键来水解蛋白质,更具体地讲,诸如精氨酸和赖氨酸之类的氨基酸残基,而不是择优裂解疏水氨基酸残基,更具体地说苯丙氨酸、色氨酸及酪氨酸的肽键。具有后一种特征的酶具备类似胰凝乳蛋白酶的特异性。上面所讨论的底物特异性可利用酶对2种合成底物的作用来说明。具备类似胰蛋白酶特异性的蛋白酶优先于合成底物sucAAPF-pNA地水解合成底物bVGR-pNA。而类似胰凝乳蛋白酶的蛋白酶则与此相反,它水解sucAAPF-pNA的速率比水解bVGR-pNA快。为本发明的目的,采用下面的程序来定义本发明蛋白酶的似胰蛋白酶特异性:
固定数量的pH等于10、温度25℃的甘氨酸缓冲液被加入到标准10mL试管中。在该试管中加入0.5ppm待测活性酶。在该试管中加入约1.25mg合成底物每mL缓冲液。让混合物在25℃温育15分钟。温育时间结束之后,向混合物中按0.5mg每毫升缓冲液的用量加入酶抑制剂PMSF。读取混合物在410nm的吸收率或OD值。如此获得的吸收值表示酶在合成底物上的活性。吸收值越大,对该底物的活性越高。
为随后确定个别酶的特异性,可将这2种合成底物上的吸收值换算为特异性比。为本发明的目的,该比值由如下公式的特异性数值来确定:
[sAAPF-pNA上的活性]/[bVGR-pNA上的活性]
若酶的该比值小于约10,更优选小于约5,最优选小于约2.5,则认为该酶表现出似胰蛋白酶活性。
此种变体一般至少有一种性质不同于衍生出该变体氨基酸序列的蛋白酶前体的同一性质。
本发明的一个方面是组合物,如洗涤剂和清洗组合物,用于处理纺织品、餐具、餐桌用具、厨房用具、炊具以及其他硬表面底物,该组合物包含1种或多种本发明变体蛋白酶。该含蛋白酶组合物可用于处理例如:丝、毛乃至其他类型织物,正如RD 216,034、EP134,267、美国专利4,533,359及EP 344,259中所描述的;以及餐具、餐桌用具、厨房用具、炊具和其他硬表面底物,如美国专利5,478,742、美国专利5,346,822、美国专利5,679,630及美国专利5,677,272中所描述的。
清洗组合物
本发明清洗组合物除了包含以上所描述的1种或多种蛋白酶变体之外,还包含1种或多种清洁添加剂材料,优选是与这些蛋白酶变体相容的。本文所使用的术语“清洁添加剂材料”是指针对所要求的清洗组合物具体类型和产品形式(液体、粒状、粉末、条块、膏体、喷雾剂、片剂、凝胶、泡沫组合物)来选择的任何液体、固体或气体材料,该材料还优选与组合物中所用蛋白酶相容。粒状组合还可呈“紧凑”形式;液体组合物还可呈“浓缩”形式。
清洁添加剂材料的具体选择可根据待清洁表面、物品或织物以及清洗组合物使用时的要求形式(例如,根据洗涤剂用途)等因素的考虑方便地确定。本文所使用的术语“相容”是指这些清洗组合物材料不使蛋白酶的蛋白水解活性降低到在正常使用条件下达不到对其所要求的有效程度。合适的清洁添加剂材料的例子包括但不限于表面活性剂、助洗剂、漂白剂、漂白活化剂、漂白催化剂、其他酶酶稳定体系、螯合剂、荧光增白剂、去污聚合物、染料转移剂、分散剂,抑泡剂、染料、香料、着色剂、填料盐、水溶助长剂、光活化剂、荧光剂、织物调理剂、可水解表面活性剂、防腐剂、抗氧剂、防缩剂、防皱剂、灭菌剂、杀真菌剂、彩色斑、银器清洁剂、防晦暗剂和/或防腐蚀剂、碱性源、增溶剂、载体、加工助剂、颜料及pH控制剂,正如美国专利5,705,464、5,710,115、5,698,504、5,695,679、5,686,014和5,646,101中所描述的。下面将详细说明具体的清洗组合物材料的例子。
倘若清洁添加剂材料与清洗组合物中的蛋白酶变体不相容,则可采用的适宜方法是将清洁添加剂材料与蛋白酶变体分开保存(保持不接触),直至适合将这2种组分合在一起时。合适的方法可以是技术上已知的任何方法,例如凝胶盖子(gelcaps)、胶囊、片剂、物理分隔等。
优选将有效数量1种或多种上面所描述的蛋白酶变体包括在用于清除各种各样需要清除蛋白类污渍的表面的组合物中。此种清洗组合物包括清洁硬表面的、形式不限的(例如液体和粒状)洗涤剂组合物;用于清洁织物的、形式不限(例如粒状、液体及条块配制物)的洗涤剂组合物;餐具洗涤组合物(形式不限的,既包括粒状也包括液体的自动洗碗机用);口腔清洗组合物,形式不限的(例如牙粉、牙膏及漱口制剂);以及牙托清洗组合物,形式不限的(例如,液体、片剂)。
本文所使用的术语“有效数量蛋白酶变体”是指为使特定清洗组合物达到所需酶活性所需上述蛋白酶变体的数量。该有效数量可由本领域技术人员很容易地确定,要考虑多种因素,例如所用的具体变体、清洁用途、清洗组合物的具体组成,以及需要液态抑或干燥的(例如粒状、条块)组合物等因素。
优选的是,该清洗组合物包含以清洗组合物重量为基准,从约0.0001%,优选从约0.001%,更优选从约0.01%1种或多种本发明蛋白酶变体,到约10%,优选到约1%,更优选到约0.1%。还优选的是,本发明蛋白酶变体在组合物中的含量足以提供一定的每100克组合物的活性蛋白酶毫克数与洗涤液中任何过氧酸提供可用O2(“AvO2”)理论ppm数值之间的比值,本文称之为酶/漂白剂比(E/B比),它应介于约1∶1~约20∶1。下面将讨论可使用本发明蛋白酶变体的各种清洗组合物的几个例子。还有,该清洁织物还可包括占组合物重量的约1%~约99%清洁添加剂材料。
本发明清洗组合物可以“织物清洗组合物”或“非织物清洗组合物”形式使用。
本文所使用的术语“织物清洗组合物”包括手洗及机洗衣物洗涤剂组合物,包括含有洗衣添加剂组合物,及适用于污渍织物浸泡和/或预处理使用的组合物。
本文所使用的术语“非织物清洗组合物”包括硬表面清洗组合物、餐具洗涤剂组合物、口腔清洗组合物、牙托清洗组合物及个人清洗组合物。
当本发明清洗组合物配制成适合洗衣机洗涤方法用的组合物时,本发明组合物优选包含表面活性剂及助洗剂化合物,以及另外1种或多种清洁添加剂材料,优选地选自有机聚合物化合物、漂白剂、附加酶、抑泡剂、分散剂、钙皂分散剂、污垢悬浮剂及抗再沉积剂和缓蚀剂。洗衣组合物还可包含柔软剂,作为外加的清洁添加剂材料。
本发明组合物还可当作固体或液体形式的洗涤剂添加剂产品使用。此种添加剂产品的作用在于弥补或强化传统洗涤剂组合物的性能,且可在清洁过程的任何阶段加入。
当配制成用于手工餐具洗涤法的组合物时,本发明组合物优选包含表面活性剂,并优选包含其他清洁添加剂材料,选自有机聚合物化合物、增泡剂、族II金属离子、溶剂、水溶助长剂及附加酶。
需要的话,本发明衣物洗涤剂组合物在20℃测定的密度介于400~1200g/L,优选500~950g/L组合物。
这里所说的清洗组合物的“坚实”形式最好地反映在密度上,而就组成而言,则反映在无机填料盐的含量上;无机填料盐是粉末形式洗涤剂组合物中的惯用成分;在传统洗涤剂组合物中,填料盐占相当大的比例,典型含量占整个组合物的17~35wt%。在坚实组合物中,填料盐含量不超过整个组合物的15wt%,优选不超过10%,最优选不超过5wt%。无机填料盐,例如本发明组合物中所指的,选自碱金属和碱土金属的硫酸盐和氯化物盐。优选的填料盐是硫酸钠。
本发明的液体清洗组合物还可是“浓缩形式”的,在这种情况下,本发明液体清洗组合物将具有比传统液体洗涤剂低的含水量。就典型而言,浓缩液体清洗组合物的含水量优选小于清洗组合物重量的40%,更优选小于30%,最优选小于20%。
清洁添加剂材料
表面活性剂体系-含于本发明的全配方清洗组合物中的洗涤表面活性剂占到清洗组合物重量的至少0.01%,优选至少约0.1%,更优选至少约0.5%,最优选至少约1%,直至约60%,更优选至约35%,最优选至约30%,视所用具体表面活性剂及所要求的效果而定。
洗涤表面活性剂可以是非离子、阴离子、两性、两性离子、阳离子、半极性非离子类型的及其混合型,其非限制性例子公开在美国专利5,707,950及5,576,282中。优选的洗涤剂及清洗组合物包含阴离子洗涤表面活性剂,或阴离子表面活性剂与其他表面活性剂,尤其与非离子表面活性剂的混合物。
可用于本发明的表面活性剂非限制性例子包括传统的C11~C18烷基苯磺酸盐和伯、仲及无规烷基硫酸盐、C10~C18烷基烷氧基硫酸盐、C10~C18烷基聚糖苷及其相应的硫酸化聚糖苷、C12~C18α-磺化脂肪酸酯、C12~C18烷基及烷基酚的烷氧基化物(尤其是乙氧基化物和乙氧基/丙氧基混合物)、C12~C18甜菜碱及磺基甜菜碱(“sultaine”)、C10~C18氧化胺等。其他传统使用的表面活性剂可参见各种标准中的规定。
表面活性剂优选配制成与组合物中存在的酶组分彼此相容的。在液体或凝胶组合物中,表面活性剂最优选配制成能提高,或者至少不恶化这些组合物中任何酶的稳定性的形式。
非离子表面活性剂-聚环氧乙烷、聚环氧丙烷及聚环氧丁烷与烷基酚的缩合产物适合作为本发明表面活性剂体系的非离子表面活性剂使用,其中以聚环氧乙烷缩合产物为优选。此种类型市售非离子表面活性剂包括IgepalTM CO-630,由GAF公司销售;及TritonTMX-45、X-114、X-100及X-102,全部由Rohm & Haas公司销售。这些表面活性剂统称烷基酚烷氧基化物(例如,烷基酚乙氧基化物)。
伯及仲脂族醇与约1~约25mol环氧乙烷的缩合产物适合用作本发明非离子表面活性剂体系的非离子表面活性剂。这类型市售非离子表面活性剂的例子包括TergitolTM 15-S-9(C11~C15线型醇与9mol环氧乙烷的缩合产物)、TergitolTM 24-L-6NMW(C12~C14伯醇与6mol环氧乙烷的缩合产物,具有窄分子量分布),这2种均由联合碳化物公司销售;NeodolTM45-9(C14~C15线型醇与9mol环氧乙烷的缩合产物)、NeodolTM 23-3(C12~C13线型醇与3.0mol环氧乙烷的缩合产物)、NeodolTM45-7(C14~C15线型醇与7mol环氧乙烷的缩合产物)、NeodolTM45-5(C14~C15线型醇与5mol环氧乙烷的缩合产物),由壳牌化学公司销售;KyroTM EOB(C13~C15醇与3或5mol环氧乙烷的缩合产物),由procter & Gamble公司销售;以及Genapol LAO3O或O5O(C12~C14线型醇与3或5mol环氧乙烷的缩合产物),由Hoechst销售。这些产品的优选HLB值介于8~11,最优选8~10。
还可用作本发明表面活性剂体系的非离子表面活性剂的是美国专利4,565,647中所公开的烷基聚糖。
优选的烷基聚糖苷具有通式:R2O(CnH2nO)t(糖基)x,其中R2选自烷基、烷基苯基、羟烷基、羟烷基苯基及其混合物,其中烷基基团包含约10~约18,优选约12~约14个碳原子;n是2或3,优选2;t是0~约10,优选0;x为约1.3~约10,优选约1.3~约3,最优选约1.3~2.7。
环氧乙烷与由环氧丙烷-丙二醇缩合生成的疏水基之间的缩合产物也适合作为本发明的附加非离子表面活性剂。这类型化合物的例子包括某些市售品PlurafacTM LF404和PluronicTM表面活性剂,由BASF销售。
也适合作为本发明非离子表面活性剂体系的非离子表面活性剂的是环氧乙烷与,环氧丙烷-乙二胺反应产物生成的缩合产物。这类型非离子表面活性剂的例子包括某些市售TetronicTM化合物,由BASF销售。
优选作为本发明表面活性剂体系的非离子表面活性剂的是烷基酚的聚环氧乙烷缩合产物、伯和仲脂族醇与约1~约25mol环氧乙烷的缩合产物、烷基聚糖及其混合物。最优选的是具有3~15个乙氧基基团的C8~C14烷基酚乙氧基化物及具有2~10个乙氧基基团的C8~C18醇乙氧基化物(优选平均C10),及其混合物。
高度优选的非离子表面活性剂是如下通式的多羟基脂肪酸酰胺表面活性剂:R2-C(O)-N(R1)-Z,其中R1是H,或R1是C1-4烃基、2-羟乙基、2-羟丙基或其混合物,R2是C5-31烃基,Z是具有链上直接连接着至少3个羟基的线型烃基链的多羟基烃基或其烷氧基化衍生物。优选的是,R1是甲基,R2是直链C11-15烷基或C16-18烷基或链烯基链,如椰子烷基或其混合物,Z是在还原胺化反应中由还原性糖,如由葡糖、果糖、麦芽糖、乳糖衍生而来的。
阴离子表面活性剂-适合使用的阴离子表面活性剂是线型烷基苯磺酸盐、烷基酯磺酸盐表面活性剂,包括,按照《美国石油化学家协会会志》,52(1975),pp.323~329,以气态三氧化硫实施磺化的C8~C20羧酸(即脂肪酸)-线型(烷基)酯。合适的原料包括天然脂肪物质,如由牛脂、棕榈油等衍生而来。
优选的烷基酯磺酸盐表面活性剂,尤其用于洗衣用途的,包含下列结构通式的烷基酯磺酸盐表面活性剂:
其中R3是C8~C20烃基,优选烷基或其组合,R4是C1~C6烃基,优选烷基,或其组合,M是与烷基酯磺酸形成水溶性盐的阳离子。合适的成盐阳离子包括诸如钠、钾及锂之类金属,以及取代或未取代的铵阳离子如单乙醇胺、二乙醇胺及三乙醇胺。优选的是,R3是C10~C16烷基,R4是甲基、乙基或异丙基。尤其优选式中R3是C10~C16烷基的甲基酯磺酸盐。
其他合适的阴离子表面活性剂包括烷基硫酸(盐)表面活性剂,它是通式ROSO3M的水溶性盐或酸,其中R优选是C10~C24烃基,优选为具有C10~C20烷基成分的烷基或羟烷基,更优选C12~C18烷基或羟烷基,M是H或阳离子。就典型而言,C12~C16烷基链优选用于较低温度的洗涤(例如,低于约50℃),而C16~C18烷基链则优选用于较高温度的洗涤(如高于约50℃)。
其他洗涤目的用阴离子表面活性剂包括下列化合物的盐:皂类、C8~C22伯或仲链烷磺酸盐、C8~C24链烯磺酸盐、通过对柠檬酸碱土金属盐热解产物进行的磺化制备的磺化多羧酸,例如描述于英国专利说明书1,082,179中,C8~C24烷基聚乙二醇醚硫酸盐(含最多10mol环氧乙烷);烷基甘油磺酸盐、脂族酰基甘油磺酸盐、脂族油基甘油硫酸盐、烷基酚环氧乙烷醚硫酸盐、石蜡磺酸盐、烷基磷酸盐,羟乙磺酸盐如酰基羟乙磺酸盐、N-酰氨基乙磺酸盐、烷基琥珀酸及磺基琥珀酸盐;磺基琥珀酸单酯(尤其是饱和及不饱和C12~C18单酯)盐及磺基琥珀酸二酯(尤其是饱和及不饱和C6~C12二酯)盐、酰基肌氨酸盐、烷基聚糖硫酸盐,如烷基聚葡糖苷硫酸盐(其非离子非硫酸化的化合物见下文)、支链伯烷基硫酸盐以及烷基聚乙氧基羧酸盐如通式RO(CH2CH2O)k-CH2COO-M+,其中R是C8~C22烷基,k是1~10的整数,M是可溶性成盐阳离子。树脂酸及氢化树脂酸也是合适的,例如松香、氢化松香以及以妥尔油或其衍生物形式存在的树脂酸及氢化树脂酸。
进一步的实施例描述于《表面活性剂及洗涤剂》(卷I及II,由Schwartz、Perry及Berch主编)。各种各样此类表面活性剂还一般性地公开在美国专利3,929,678中,1975-12-30授予Laughlin等人,23栏58行-29栏,23行(在此并入本文作为参考)。
高度优选的阴离子表面活性剂包括通式RO(A)mSO3M的烷基烷氧基化硫酸盐表面活性剂,可以是水溶性盐或其酸,其中R是未取代的C10~C24烷基或具有C10~C24烷基部分的羟烷基基团,优选C12~C20烷基或羟烷基,更优选C12~C18烷基或羟烷基,A是乙氧基或丙氧基单元,m大于0,典型值介于约0.5~约6之间,更优选介于约0.5~约3,M是H或阳离子,例如可以是金属阳离子(例如钠、钾、锂、钙、镁等),铵或取代的铵阳离子。烷基乙氧基化硫酸盐乃至烷基丙氧基化硫酸盐也包括在内。取代的铵阳离子的具体例子包括甲基-、二甲基-、三甲基-铵阳离子及季铵盐阳离子如四甲基-铵及二甲基吡啶阳离子及由烷基胺如乙胺、二乙胺、三乙胺衍生的那些,及其混合物等。范例表面活性剂是C12~C18烷基聚乙氧基(1.0)化硫酸盐(C12~C18E(1.0)M),C12~C18烷基聚乙氧基(2.25)化硫酸盐(C12~C18E(3.0)M),以及C12~C18烷基聚乙氧基(4.0)化硫酸盐(C12~C18E(4.0)M),其中M可方便地选自钠和钾。
当加入其中时,本发明清洗组合物一般地包含重量百分比约1%,优选约3%~约40%,更优选约20wt%此类阴离子表面活性剂。
阳离子表面活性剂-适用于本发明清洗组合物中的阳离子洗涤表面活性剂是具有1个长链烃基基团的那些。此类阳离子表面活性剂的例子包括铵表面活性剂,如卤化烷基三甲基铵,以及具有通式[R2(OR3)y]R4(OR3)y]2R5N+X-的表面活性剂,其中R2是烷基链中有约8~约18个碳原子的烷基或烷基苄基基团,每个R3选自-CH2CH2-、-CH2CH(CH3)-、-CH2CH(CH2OH)-、-CH2CH2CH2-及其混合物;每个R4选自C1~C4烷基、C1~C4羟烷基、由2个R4基团连接形成的苄基环结构、-CH2CHOH-CHOHCOR6CHOHCH2OH,其中R6是任何分子量小于约1000的己糖或己糖聚合物,以及当y不是0时,是氢;R5与R4相同或者是烷基链,其中R2+R5的碳原子总数不大于约18;每个y是0~约10,且y值的总和是0~约15;X是任何相容的阴离子。
高度优选的阳离子表面活性剂是对本发明有用的水溶性季铵化合物,具有通式(i):R1R2R3R4N+X-,其中R1是C8~C16烷基,R2、R3及R4中每一个独立地是C1~C4烷基、C1~C4羟烷基、苄基及-(C2H40)xH,其中x是2~5的数值,X是阴离子。R2、R3或R4中为苄基的应不超过1个。R1的优选烷基链长是C12~C15,尤其是当该烷基基团是由椰子或棕榈仁脂肪或者按合成方法由烯烃聚合或者由OXO醇合成衍生的不同链长的混合物时。优选作为R2、R3及R4的基团是甲基及羟乙基基团,阴离子X可选自卤素、甲基硫酸、乙酸及磷酸(根)离子。
适合本发明的通式(i)的季铵化合物的例子包括但不限于:氯化或溴化椰子(基)三甲基铵;氯化或溴化椰子甲基二羟乙基铵;氯化癸基三乙基铵;氯化或溴化癸基二甲基羟乙基铵;氯化或溴化C12-15二甲基羟乙基铵;氯化或溴化椰子二甲基羟乙基铵;甲基硫酸肉豆蔻基三甲基铵;氯化或溴化月桂基二甲基苄基铵;氯化或溴化月桂基二甲基(乙氧基)4铵;胆碱酯(通式(i)的化合物,但其中R1是烷基且R2R3R4是甲基者);
以及二烷基咪唑啉[(i)]。
其他可用于本发明的阳离子表面活性剂还可见诸于美国专利4,228,044,1980-10-14授予Cambre,以及欧洲专利申请EP 000,224。
当加入其中时,本发明的清洗组合物一般包含从约0.2%,优选从约1%到约25%,优选至约8wt%此类阳离子表面活性剂。
两性表面活性剂-两性表面活性剂,其例子描述于美国专利3,929,678中,也适用于本发明的清洗组合物。
当加入其中时,本发明清洗组合物一般包含从约0.2%,优选从约1%,至约15%,优选至约10wt%此类两性表面活性剂。
两性离子表面活性剂-两性离子表面活性剂,其例子描述于美国专利3,929,678中,也适用于本发明的清洗组合物。
当加入其中时,本发明清洗组合物一般包含从约0.2%,优选从约1%,至约15%,优选至约10wt%此类两性离子表面活性剂。
半极性非离子表面活性剂-半极性非离子表面活性剂是一类特殊的非离子表面活性剂,包括如下通式的水溶性氧化胺:
其中R3是烷基、羟烷基或烷基苯基基团或其混合物,它们包含约8~约22个碳原子;R4是包含约2~约3个碳原子的亚烷基或羟亚烷基基团或其混合物;x是0~约3;每个R5是包含约1~约3个碳原子的烷基或羟烷基基团或者包含约1~约3个氧乙烯基团的聚环氧乙烷基团(R5基团可彼此连接,例如通过氧或氮原子,形成一个环状结构);含一个约10~18个碳原子的烷基部分及2个选自含约1~约3个碳原子烷基基团和羟烷基基团部分的水溶性氧化膦;含1个约10~约18个碳原子烷基部分及1个选自约1~约3个碳原子烷基及羟烷基部分的水溶性亚砜。
该氧化胺表面活性剂尤其包括C10~C18烷基二甲基氧化胺和C8~C12烷氧基乙基二羟基乙基氧化胺。
当加入其中时,本发明的清洗组合物一般包含从约0.2%,优选从约1%到约15%,优选至约10wt%此类半极性非离于表面活性剂。
助表面活性剂-本发明清洗组合物还可包含助表面活性剂。选自伯或叔胺。适合本发明使用的伯胺包括符合通式R1NH2的胺,其中R1是C6~C12,优选C6~C10烷基链或R4X(CH2)n,X是-O-、-C(O)NH-或-NH-,R4是C6~C12烷基链,n介于1~5,优选3。R1烷基链可以是直链或支链且中间可嵌入最多12,优选少于5个氧乙烯部分。
优选的以上通式的胺是正烷基胺。适合用于此处的胺选自1-己胺、1-辛胺、1-癸胺及月桂胺。其他优选的伯胺包括C8~C10氧丙基胺、辛基氧基丙基胺、2-乙基己基-氧丙基胺、月桂酰胺基丙胺及酰胺基丙胺(amido propylamine)。最优选用于本发明组合物的胺是1-己胺、1-辛胺、1-癸胺、1-十二烷基胺。尤其可人的是正十二烷基二甲胺和二羟乙基椰子基烷基胺及油基胺的7倍乙氧基化物。月桂酰胺基丙胺及椰子酰胺基丙胺。
LFNI-本发明的自动餐具洗涤组合物(ADD)中的尤其优选的表面活性剂是低泡沫非离子表面活性剂(LFNI),描述于美国专利5,705,464及5,710,115中。LFNI的含量在0.01~约10wt%,优选约0.1%~约10%,最优选约0.25%~约4%。LFNI最典型的用途是用于ADD产品,这是因为它们能改善ADD产品成水膜作用(water-sheeting),(尤其是从玻璃上流过时)。它们还涵盖下面将进一步说明且据知具有对自动洗碗中遇到的食物污垢的消泡作用的非硅氧烷、非磷酸酯聚合物材料。
优选的LFNI包括非离子烷氧基化表面活性剂,尤其是由伯醇衍生的乙氧基化物,及其与更复杂的表面活性剂的共混物,如美国专利5,705,464和5,710,115描述的聚环氧丙烷/聚环氧乙烷/聚环氧丙烷(PO/EO/PO)反向嵌段聚合物。
还可使用的LFNI包括Olin公司供应的那种POLY-TERGENT_SLF-18非离子表面活性剂以及具有上面所描述的熔点性质的任何可生物降解LFNI。
这些以及其他非离子表面活性剂在技术上是熟知的,较详细地描述在Kirk Othmer的《化学技术大全》,第3版,卷22,pp.260~379,“表面活性剂及洗涤剂体系”,在此并入本文作为参考。
漂白体系-本发明的清洗组合物优选包含漂白体系。漂白体系通常包含“漂白剂”(过氧化氢源)及“引发剂”或“催化剂”。当存在时。漂白剂的典型含量为组合物重量的从约1%。优选约5%~约30%,更优选至约20%。存在的话,漂白活化剂的典型含量为含该漂白剂加漂白活化剂组成的漂白组合物的从约0.1%,优选约0.5~约60%,更优选至约40wt%。
漂白剂-过氧化氢源详细描述在收作本文参考的Kirk Othmer的《化学技术大全》,第4版(1992,John Wiley & Sons),卷4,pp.271~300,“漂白剂(综述)”,它包括各种形式的过硼酸钠和过碳酸钠,包括其各种涂层的及改性的形式。
优选用于本发明的过氧化氢源可以是任何方便的来源,包括过氧化氢本身。例如可使用,过硼酸盐,如过硼酸钠(任何水合物,但优选一-或四水合物)、碳酸钠过氧水合物(双氧水合物)或等价的过碳酸盐、焦磷酸钠过氧水合物、尿素过氧水合物或过氧化钠。市售氧源也可使用,例如过硫酸盐漂白剂(如,OXONE,杜邦公司制造)。过硼酸钠一水合物及过碳酸钠是尤其优选的。任何方便的过氧化氢源的混合物也可使用。
优选的过碳酸盐漂白剂包含约平均粒度在约500μm~约1,000μm的干颗粒,所述颗粒中小于约200μm者不超过约10wt%,且所述颗粒中大于约1,250μm者不超过约10wt%。任选地,过碳酸盐可涂以硅酸盐、硼酸盐或水溶性表面活性剂涂层。过碳酸盐可由各种商业来源获得,例如FMC、Solvay及Tokai Denka等公司。
本发明清洗组合物还可包含氯型漂白材料作为漂白剂。此种剂在技术上是熟知的,例如包括二氯代异氰尿酸钠(“NaDCC”)。然而,氯型漂白剂对于包含酶的组合物来说是较次优选的。
(a)漂白活化剂-该组合物中的过氧化氢漂白成分优选与活化剂(过氧酸前体)配合使用。活化剂的含量为组合物重量的从约0.01%,优选从约0.5%,更优选从约1%,至约15%,优选至约10%,更优选至约8%。优选的活化剂选自四乙酰乙二胺(TAED)、苯甲酰己内酰胺(BzCL)、4-硝基苯甲酰己内酰胺、3-氯苯甲酰已内酰胺、苯甲酰氧基苯磺酸盐(BOBS)、壬酰氧基苯磺酸盐(NOBS)、苯甲酸苯酯(PhBz)、癸酰氧基苯磺酸盐(C10-OBS)、苯甲酰戊内酰胺(BZVL)、辛酰氧基苯磺酸盐(C8-OBS)、全水解酯及其混合物,最优选苯甲酰己内酰胺及苯甲酰戊内酰胺。在pH约8~约9.5范围尤其优选使用的漂白活化剂选自具有OBS或VL离去基团的那些。
优选的疏水漂白活化剂包括(但不限于),壬酰氧基苯磺酸盐(NOBS)、4-[N-(壬酰氧基)氨基己酰氧基]-苯磺酸钠盐(NACA-OBS),其一例描述于美国专利5,523,434中,十二烷酰氧基苯磺酸盐(LOBS或C12-OBS)、10-十一烷酰氧基苯磺酸盐(UDOBS或C11-OBS,其不饱和键位于10位),以及癸酰氧基苯甲酸(DOBA)。
优选的漂白活化剂描述于美国专利5,698,504,1997-12-16授予Christie等人;美国专利5,695,679,1997-12-09授予Christie等人;美国专利5,686,401,1997-11-11授予Willey等人;美国专利5,686,014,1997-11-11授予Hartshorn等人;美国专利5,405,412,1995-04-11授予Willey等人;美国专利5,405,413,1995-04-11授予Willey等人;美国专利5,130,045,1992-07-14授予Mitchel等人;以及美国专利4,412,934,1983-11-01授予Chung等人,以及共同未决美国专利申请序列号08/709,072、08/064,564,一律并入本文作为参考。
在本发明中,过氧漂白化合物(记作AvO)对漂白活化剂的摩尔比一般至少从1∶1,优选从约20∶1,更优选从约10∶1,至约1∶1,优选至约3∶1。
季铵取代的漂白活化剂也可包括在内。本发明清洗组合物优选包含季铵取代的漂白活化剂(QSBA)或季铵取代的过氧酸(QSP);前者更为优选。优选的QSBA结构进一步描述于美国专利5,686,015,1997-11-11授予Willey等人;美国专利5,654,421,1997-08-05授予Taylor等人;美国专利5,460,747,1995-10-24授予Gosselink等人;美国专利5,584,888,1996-12-17授予Miracle等人;以及美国专利5,578,136,1996-11-26授予Taylor等人;一律并入本文作为参考。
本发明高度优选的漂白活化剂是酰胺取代的,如描述于美国专利5,698,504、美国专利5,695,679及美国专利5,686,014中,这些均已在上面举出过。此类漂白活化剂的优选实施例包括:(6-辛酰胺己酰)氧基苯磺酸盐、(6-壬酰胺己酰)氧基苯磺酸盐、(6-癸酰胺己酰)氧基苯磺酸盐及其混合物。
其他有用的活化剂,公开在美国专利5,698,504,美国专利5,695,679,美国专利5,686,014。均在上面援引过,以及美国专利4,966,723,1990-10-30授予Hodge等人的,包括苯并_嗪型活化剂,如C6H4环,其1,2-位上稠合了一个-C(O)OC(R1)=N-部分。
依活化剂及具体用途而定,良好的漂白结果可由使用时pH值约6~约13,优选约9.0~约10.5的漂白体系来获得。就典型而言,例如具有吸电子部分的活化剂可用于接近中性或亚中性pH范围。可采用碱或缓冲剂来获得此种pH。
酰基内酰胺活化剂,如描述于美国专利5,698,504、美国专利5,695,679及美国专利5,686,014中,均在上面提到过,非常适用于本发明,尤其是酰基己内酰胺(例如参见WO94-28102A)及酰基戊内酰胺(参见美国专利5,503,639,1996-04-02授予Willey等人,并入本文作为参考)。
(b)有机过氧化物,尤其是二酰基过氧化物-这些均全面描述于Kirk Othmer的《化学技术大全》,卷17,John Wiley & Sons 1982,pp.27-90,尤其是pp.63~72中,全部内容并入本文作为参考。若使用二酰基过氧化物,优选它对成斑/成膜的不利影响应尽量小。
(c)含金属漂白催化剂-本发明组合物及方法可利用在漂白组合物中使用有效的含金属漂白催化剂。优选含锰及钴的漂白催化剂。
一种类型含金属漂白催化剂是包含:有规定漂白催化活性的过渡金属阳离子的催化剂体系,例如含铜、铁、钛、钌、钨、钼或锰阳离子;有很小或没有漂白催化活性的辅助金属阳离子,如锌或铝阳离子;以及对催化及辅助金属阳离子有规定稳定常数的螯合剂,特别是乙二胺四乙酸、乙二胺四(亚甲基膦酸)及其水溶性盐。这类催化剂公开在美国专利4,430,243中,1982-02-02授予Bragg。
锰金属配合物-需要的话,本发明组合物可借助锰化合物来催化。这类化合物及其用量乃是技术上熟知的,例如包括:锰基催化剂,公开在美国专利5,576,282、5,246,621、5,244,594、5,194,416及5,114,606;以及欧洲专利申请公开号549,271 A1、549,272 A1、544,440 A2及544,490 A1;这些催化剂的优选实施例包括MnIV 2(u-O)3(1,4,7-三甲基-1,4,7-三氮杂环壬烷)2(PF6)2、MnIII 2(u-O)3(1,4,7-三甲基-1,4,7-三氮杂环壬烷)2(ClO4)2、MnIV 2(u-O)3(1,4,7-三氮杂环壬烷)2(ClO4)4、MnIIIMnIV 4(u-O)1(u-OAc)2-(1,4,7-三甲基-1,4,7-三氮杂环壬烷)2(ClO4)3、MnIV(1,4,7-三甲基-1,4,7-三氮杂环壬烷-(OCH3)3(PF6),及其混合物。其他金属基漂白催化剂包括公开在美国专利4,430,243及美国专利5,114,611中的那些。还有,以锰与各种配位体来改善漂白效果的用法也见诸于下列文献的报道:美国专利4,728,455、5,284,944、5,246,612、5,256,779、5,280,117、5,274,147、5,153,161以及5,227,084。
钴金属配合物-可用于本发明的钴漂白催化剂是已知的,例如描述在美国专利5,597,936、5,595,967及5,703,030及M.L.Tobe,“过渡金属配合物的碱性水解”《Adv.Inorg.Bioinorg.Mech.》(1983),2,pp.1~94。最优选用于本发明的钴催化剂是通式为[Co(NH3)5OAc]Ty的乙酸五氨合钴的盐,其中“OAc”代表乙酸根部分,“Ty”是阴离子;尤其是:氯化乙酸五氨合钴,即[Co(NH3)5OAc]Cl2;以及[Co(NH3)5OAc](OAc)2;[Co(NH3)5OAc](PF6)2;[Co(NH3)5OAc](SO4);[Co(NH3)5OAc](BF4)2;及;[Co(NH3)5OAc](NO3)2(本文称“PAC”)。
这些钴催化剂可采用已知的程序方便地制备,例如按美国专利5,597,936、5,595,967和5,703,030的公开;Tobe的文章及其援引的参考文献中的;以及美国专利4,810,410;《J.Chem.Ed.》(1989),66(12),1043~45;《无机化合物的合成及鉴定》,W.L.Jolly(Printice-Hall,1970),pp.461~3;《无机化学》,18,1497~1502(1979);《无机化学》21,2881~2885(1982);《无机化学》,18,2023~2025(1979);《无机合成》,173~176(1960);以及《物理化学杂志》,56,22~25(1952)中的方法。
“大多环刚性配位体”的过渡金属配合物-本发明组合物也宜于包含大多环配位体的过渡金属配合物,作为漂白催化剂。术语“大多环刚性配位体”在下面的讨论中有时简写为“MRL”。其用量为催化有效的数量,合适的数值为约1ppb或更多,最高例如至约99.9%,更典型的约0.001ppm或更多,优选约0.05ppm~约500ppm(其中“ppb”代表每十亿重量份的份数;“ppm”代表每百万重量份的份数)。
下面将举例说明合适的过渡金属,如Mn。“大多环”意思是,一种MRL既是大环,又是多环。“多环”是指至少2个环。术语“刚性”在这里涵盖“具有超结构”和“横越桥连”。“刚性”曾被定义为“柔性”的受限的反面:参见D.H.Busch,《化学评论》(1993),93,847~860,收作参考。更具体地说,本文所使用的术语“刚性”是指,该MRL,与其他均相同(具有相同环尺寸及类型以及主环中的原子数)但缺乏MRL中具备的超结构(连接部分,或优选地,横越桥连部分)的大环(“前体大环”)相比,必须可测定地更为僵硬。在测定具有或不具有超结构的大环之间的对比刚性时,业内人士将采用自由形式(不是金属键合的形式)的大环。众所周知,刚性,在比较不同大环时是有用的参数;适合确定、测量或比较刚性的工具包括计算法(例如参见,Zimmer,《化学评论》(1995),95(38),2629~2648或Hancock等人,《无机化学学报(Inorganica Chimica Acta)》(1989),164,73~84。
本发明优选的MRL是特定类型的横越桥连、超刚性配位体。“横越桥连”在下面的1.11中给出了非限制性例子。在1.11中,该横越桥连是-CH2CH2-部分。它将示意性结构中的N1和N8彼此桥连。相比之下,譬如“同侧”桥,倘若被引入到1.11中的N1与N12之间,将不足以构成一种“横越桥连”,并因此将不可取。
刚性配位体配合物中的合适金属包括Mn(II)、Mn(III)、Mn(IV)、Mn(V)、Fe(II)、Fe(III)、Fe(IV)、Co(I)、Co(II)、Co(III)、Ni(I)、Ni(II)、Ni(III)、Cu(I)、Cu(II)、Cu(III)、Cr(II)、Cr(III)、Cr(IV)、Cr(V)、Cr(VI)、V(III)、V(IV)、V(V)、Mo(IV)、Mo(V)、Mo(VI)、W(IV)、W(V)、W(VI)、Pd(II)、Ru(II)、Ru(III)及Ru(IV)。优选的本发明过渡金属漂白催化剂中的过渡金属包括锰、铁和铬。
较一般地,该MRL(及其对应的过渡金属催化剂)宜于包含:
(a)至少1种含4或更多个杂原子的大环主环;以及
(b)能提高该大环刚性的共价连接非金属超结构,优选地选自
(i)桥连超结构,例如连接部分;
(ii)横越桥连超结构,如横越桥连连接部分;以及
(iii)二者的组合。
本文所使用的术语“超结构”符合Busch等人的文献的定义,例如参见Busch在《化学评论》中的文章。
这里优选的超结构不仅将增强前体大环的刚性。而且有利于大环的折叠,以便于它配位到裂口中的金属上。合适的超结构可相当地简单,譬如,诸如下面式1和式2所画出的任何一种连接部分,均可使用。
式1
其中,n是整数,例如介于2~8,优选小于6,典型值2~4,或者
式2
其中m是约1~8的整数,更优选1~3;Z是N或CH;T是相容的取代基,例如H、烷基、三烷基铵、卤素、硝基、磺基(磺酸根)或诸如此类的。在1.10中的芳环可换成饱和环,其中连接到环中的Z原子可包含N、O、S或C。
下面再举出另一种合适的MRL非限制性化合物例子:
式3
这是一种本发明高度优选的、横越桥连、甲基取代的(全部在叔氮原子上)的环状内酰胺(cyclam)衍生物。正规地说,该配位体叫做5,12-二甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷,按扩展的von Baeyer系统(命名法)。参见,《IUPAC有机化合物命名法指南:建议意见1993》,R.Panico,W.H.Powell及J-C Richer(主编),Blackwell科学出版社,波士顿,1993;尤其参见R-2.4.2.1节。
适用于本发明组合物的大环刚性配位体的过渡金属漂白催化剂,-般地可包括符合上述定义的已知化合物,乃至更优选地,专门为本发明洗衣或清洁用途明确设计的大量新型化合物中的任何一种,其非限制性例子可举出下列化合物中任何一种:
二氯-5,12-二甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷(合)锰(II)
六氟磷酸二水合-5,12-二甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(II)
六氟磷酸水合-羟基-5,12-二甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(III)
四氟硼酸(根)二水合-5,12-二甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(II)
六氟磷酸二氯-5,12-二甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(III)
二氯-5,12-二正丁基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(II)
二氯-5,12-二苄基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(II)
二氯-5-二正丁基-12-甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(II)
二氯-5-二正辛基-12-甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(II)
二氯-5-二正丁基-12-甲基-1,5,8,12-四氮杂双环[6.6.2]十六烷锰(II)。
在实际使用中且不具限制性地说,可对本发明组合物和清洁方法进行调节以在洗涤水介质中提供约每一亿份中有1份活性漂白催化剂种,优选在洗涤水中提供从约0.01ppm~约25ppm。更优选约0.05ppm~10ppm,最优选约0.1ppm~约5ppm漂白催化剂种。为了在自动洗涤过程的洗涤液中提供这样水平的含量,典型的本发明组合物将包含以漂白组合物重量为基准,约0.0005%~约0.2%,更优选约0.004%~约0.08%漂白催化剂,尤其是锰或钴催化剂。
(d)其他漂白催化剂-本发明组合物可包含1种或多种其他漂白催化剂。优选的漂白催化剂是两性离子漂白催化剂,它们描述于美国专利5,576,282中(尤其是丙烷磺酸3-(3,4-二氢异喹啉_)。其他漂白催化剂包括阳离子漂白催化剂,它们描述于美国专利5,360,569、5,442,066、5,478,357、5,370,826、5,482,515、5,550,256及WO95/13351、WO95/13352及WO95/13353中。
适合用作漂白剂的还有预生成的过氧酸,例如邻苯二甲酰亚胺-过氧-己酸(“PAP”)。参见美国专利5,487,818、5,310,934、5,246,620、5,279,757及5,132,431中。
任选的洗涤酶-本发明洗涤剂及清洗组合物还可任选地包含1种或多种类型的洗涤剂酶。这些酶可包括其他蛋白酶、淀粉酶、纤维素酶及脂酶。这些物质在技术上是已知的且以各种商品名在市场上销售。它们掺入到本发明非水液体洗涤剂组合物中的形式可以是悬浮体、“圆球”或“小球”。另一种适宜类型的酶包含以酶在非离子表面活性剂中的淤浆形式的那些,例如由NovoNordisk按商品名“SL”销售的酶或者由Novo Nordisk按商品名“LDP”销售的微胶囊化的酶。合适的酶及其用量描述于美国专利5,576,282、5,705,464及5,710,115中。
本发明尤其优选以传统酶小球形式加入到这里的组合物中的酶。合适的小球粒度一般在约100~1,000μm,更优选约200~800μm,并被悬浮在组合物的整个非水液相中。本发明组合物中的小球,据发现,与其他酶形式相比,表现出尤其可人的酶活性随时间保持恒定的酶稳定性。因此,采用酶小球的组合物不需要包含诸如当将酶掺入到含水液体洗涤剂中时常常必须采用的传统酶稳定剂。
然而,加入到本发明组合物中的酶也可呈粒状,优选T字形颗粒。
“洗涤酶”,在这里是指在洗衣、硬表面清洁或个人洗漱洗涤剂组合物中具有清洁、除渍或其他有益效果的任何酶。优选的洗涤酶可以是水解酶,如蛋白酶、淀粉酶和脂酶。洗衣目的优选的酶包括但不限于,蛋白酶、纤维素酶、脂酶和过氧化物酶。自动餐具洗涤高度优选的是淀粉酶和/或蛋白酶,既包括目前市售的类型,也包括各种改进类型,后者,尽管通过不断的努力已变得越来越与漂白剂相容,但依然具有残余程度的易使漂白而失活的特性。
合适酶的例子包括但不限于,半纤维素酶、过氧化物酶、蛋白酶、纤维素酶、木聚糖酶、脂酶、磷脂酶、酯酶、角质酶、果胶酶、角蛋白酶、还原酶、氧化物酶、酚氧化酶、脂氧合酶、木素酶、支链淀粉酶、单宁酶、戊聚糖酶、马兰酶(malanases)、β-葡聚糖酶、阿拉伯糖酶、透明质酸酶、软骨素酶、漆酶及各种已知的淀粉酶,或其混合物。
此种适宜酶的例子公开在美国专利5,705,464、5,710,115、5,576,282、5,728,671及5,707,950中。
可用于本发明的纤维素酶既包括细菌源的也包括真菌源的纤维素酶。优选的是,它们具有的pH最佳值介于5~12且“比活性”大于50CEVU/mg(纤维素粘度单位)。合适的纤维素酶公开在美国专利4,435,307、J61078384及WO96/02653中,后者公开了分别由Humicola insolens、木霉菌属(Trichoderma)、棱孢菌属(Thielavia)及S-侧孢霉属(Sporotrichum)制备的真菌纤维素酶。EP 739 982描述了由新型芽胞杆菌中分离出的纤维素酶。合适的纤维素酶还公开在GB-A-2.075.028、GB-A-2.095.275、DE-OS-2.247.832及WO95/26398中。
这类纤维素酶的例子是由菌株Humicola insolens(Humicolagrisea var.thermoidea),尤其是由腐殖霉属菌株DSM 1800所生产的纤维素酶。其他合适的纤维素酶是由分子量约50KDa、等电点5.5并包含415个氨基酸的Humicola insolens产生的纤维素酶;以及由Humicola insolens,DSM 1800产生、表现出纤维素活性的内切葡聚糖酶;优选的内切葡聚糖酶成分具有WO 91/17243所公开的氨基酸序列。合适的纤维素酶还有EGIII纤维素酶,由授予Genencor的WO94/21801所描述的长臂木霉菌属(Trichodermalongibrachiatum)产生。尤其合适的纤维素酶是具有颜色护理效用的纤维素酶。此类纤维素酶的例子是欧洲专利申请号91202879.2所描述的纤维素酶,1991-11-06提交(Novo)。Carezyme和Celluzyme(Novo Nordisk A/S)尤其有用。还可参见WO91/17244及WO91/21801。其他适合织物护理和/或有清洁性能的纤维素酶描述在WO96/24092、WO96/17994及WO95/24471中。
当存在时,纤维素酶在清洗组合物中的加入量为清洗组合物重量的0.0001%~2%纯酶。
过氧化物酶应与氧源配合使用,例如与过碳酸盐、过硼酸盐、过硫酸盐、过氧化氢等并与酚底物配合使用,后者作为漂白增强分子。它们用于“溶液漂白”,即,防止染料在洗涤操作期间由一种底物转移到洗涤溶液中其他底物上。过氧化物酶在技术上是已知的,例如包括辣根过氧化物酶、木素酶及卤代过氧化物酶如氯-及溴-过氧化物酶。合适的过氧化物及含过氧化物酶组合物,例如公开在美国专利5,705,464、5,710,115、5,576,282、5,728,671及5,707,950、PCT国际申请WO 89/099813、WO89/09813及欧洲专利申请EP号91202882.6,1991-11-06提交,以及EP号96870013.8,1996-02-20提交。适合的还有漆酶。
增效剂的加入量为整个组合物重量的0.1%~5%。优选的增效剂是取代的吩噻嗪和吩_嗪10-吩噻嗪丙酸(PPT)、10-乙基吩噻嗪-4-羧酸(EPC)、10-吩_嗪丙酸(POP)及10-甲基吩_嗪(描述于WO94/12621)以及取代的丁香酸酯(C3~C5取代的丁香酸烷基酯)及酚类。过碳酸钠或过硼酸钠是优选的过氧化氢源。所述过氧化物酶在清洗组合物中的加入量一般介于:清洗组合物重量的0.0001%~2%纯酶的范围。
酶体系可用作漂白剂。过氧化氢的存在,也可通过在洗涤和/或清洗的开始或过程中加入能产生过氧化氢的酶体系(即,酶与其底物)来实现。此种酶体系公开在EP专利申请91202655.6中,1991-10-09提交。
其他可包括在本发明清洗组合物中的优选酶包括脂酶。合适的洗涤剂用脂酶包括由假单胞菌类微生物,如英国专利1,372,034中所公开的施氏假单胞菌ATCC 19.154产生的脂酶。合适的脂酶包括对脂酶抗体表现出阳性免疫交叉反应的那些,可通过微生物荧光假单胞菌IAM 1057产生。该脂酶由Amano制药公司(Nagoya,日本)按商品名脂酶P“Amano”供应,在下文中称之为“Amano-P”。其他合适的商品脂酶包括Amano-CES、源于粘稠色杆菌(Chromabacter viscosum)的脂酶,例如粘稠色杆菌解脂变种(Chromabacter viscosum var.Lipolyticum)NRRLB 3673,由美国生物化学公司和荷兰Disoynth公司供应,以及源于唐昌蒲假单胞菌的脂酶。尤其合适的脂酶是下列脂酶,诸如M1 Lipase_及Lipomax_(Gist-Brocades)及Lipolase_及LipolaseUltra_(Novo),据发现,它们与本发明组合物配合使用时非常有效。合适的还有脂解酶,描述于EP 258 068、WO92/05249及WO95/22615,由Novo Nordisk公开,以及WO 94/03578、WO 95/35381及WO 96/00292,由Unilever公开。
合适的还有角质酶[EC 3.1.1.50],可视为一种特殊脂酶,即,不要求界面活性的脂酶。角质酶在清洗组合物重量中的添加,公开在例如WO-A-88/09367(Genencor)、WO 90/09446(植物基因系统公司)及WO 94/14963及WO 94/14964(Unilever)中。
脂酶和/或角质酶,当存在时,在清洗组合物重量中的加入量一般为,清洗组合物重量的0.0001%~2%纯酶。
除了以上援引的脂酶之外,磷脂酶也可被加入到本发明清洗组合物中。合适的磷脂酶的非限制性实施例包括:EC 3.1.1.32磷脂酶A1、EC 3.1.1.4磷脂酶A2、EC 3.1.1.5溶血磷脂酶、EC 3.1.4.3磷脂酶C、EC 3.1.4.4磷脂酶D。市售的磷脂酶包括LECITASE_,由丹麦的Novo Nordisk公司供应;以及磷脂酶A2,由Sigma公司供应。当在本发明组合物中加入磷脂酶时,优选的是,还加入淀粉酶。不拟囿于理论,但据信,磷脂酶与淀粉酶的联合作用可提供显著改善的污渍去除效果,尤其是对油脂类、淀粉类以及高度着色色斑及污垢。优选的是,磷脂酶与淀粉酶,当存在时,在本发明组合物中的加入量按纯酶重量比计,介于4500∶1~1∶5,更优选介于50∶1~1∶1。
合适的蛋白酶是由特殊枯草芽胞杆菌和地衣芽胞杆菌菌株获得的枯草杆菌蛋白酶(枯草杆菌蛋白酶BPN及BPN’),一种合适的蛋白酶是由在8~14整个pH范围内具有最大活性的芽胞杆菌菌株获得,由丹麦Novo工业公司(以下称“Novo”)以商品名ESPERASE_研制并销售。此种酶及其类似酶的制备方法描述在授予Novo的GB 1,243,784中。蛋白水解酶还涵盖改性细菌丝氨酸蛋白酶,例如描述在欧洲专利申请序列号87 303761.8中,1987-04-28提交(尤其是pp.17、24及98),在本文中称其为“蛋白酶B”,以及欧洲专利申请199,404,Venegas,1986-10-29发表,它涉及在本文中称作“蛋白酶A”的改性细菌丝氨酸蛋白水解酶。合适的是叫做“蛋白酶C”的,它是来自芽胞杆菌的碱性丝氨酸蛋白酶的变体,其中赖氨酸取代了位点27的精氨酸;酪氨酸取代了位点104的缬氨酸;丝氨酸取代了位点123的天冬酰胺;丙氨酸取代了位点274的苏氨酸。蛋白酶C描述在EP90915958:4,对应于WO 91/06637,1991-05-16发表。各种遗传改性变体,特别是蛋白酶C的,也包括在内。
被称为“蛋白酶D”的优选蛋白酶是如美国专利5,677,272及WO95/10591中所描述的羰基水解酶。合适的还有WO95/10591中所描述的羰基水解酶变种,它们具有通过对前体酶中多个氨基酸残基的取代而衍生的氨基酸序列,这些位点对应于+210以及配合着1个或多个下列位点:
+33,+62,+67,+76,+100,+101,+103,+104,+107,+128,+129,+130,+132,+135,+156,+158,+164,+166,+167,+170,+209,+215,+217,+218,及+222,
其中数字代号位点对应于来自解淀粉芽胞杆菌枯草杆菌蛋白酶的天然存在枯草杆菌蛋白酶以及其他羰基水解酶或枯草杆菌蛋白酶,例如迟缓芽胞杆菌枯草杆菌蛋白酶(共同未决美国专利申请序列号60/048.550,1997-06-04提交;以及PCT国际申请序列号PCT/IB98/00853)。
适合本发明的还有EP251 4446和WO 91/06637中所描述的蛋白酶、E091/02792中所描述的蛋白酶BLAP_以及WO 95/23221中所描述的其变种。
还可参见授予Novo的WO 93/18140所描述的来自芽胞杆菌NCIMB 40338的高pH蛋白酶。一种包含蛋白酶、1种或多种其他酶及可逆蛋白酶抑制剂的加酶洗涤剂,公开在授予Novo的WO92/03529 A中。需要的话,具有降低吸附和提高水解能力的蛋白酶也已研制出来,如公开在授予Procter & Gamble的WO95/07791中。适合本发明洗涤剂使用的重组类胰蛋白酶的蛋白酶公开在授予Novo的WO 94/25583中。其他合适的蛋白酶描述于授予Unilever的EP 516 200中。
尤其有用的蛋白酶描述在下列PCT出版物中:WO 95/30010、WO95/30011及WO 95/29979。合适的蛋白酶可按下列商品名购得:ESPERASE_、ALCALASE_、DURAZYM_、SAVINASE_、EVERLASE_及KANNASE_,均由Novo Nordisk公司(丹麦)供应,以及MAXATASE_、MAXACAL_、PROPERASE_及MAXAPEM_,均由Genencor国际公司(原来的Gist-Brocades(荷兰)供应。
这些蛋白水解酶,当在本发明清洗组合物中存在时,其加入量为组合物重量的0.0001%~2%,优选0.001%~0.2%,更优选0.005%~0.1%纯酶。
可加入淀粉酶(α-或β-),以去除碳水化合物为主的污渍。WO94/02597描述了包含突变体淀粉酶的清洗组合物。还可参见WO95/10603。其他已知用于清洗组合物的淀粉酶包括α-及β-淀粉酶。α-淀粉酶在技术上是已知的,包括下列文献中公开的:美国专利5,003,257、EP252,666、WO 91/00353、FR 2.676.456、EP285,123、EP525,610、EP 368,341及英国专利说明书号1,296,839(Novo)。另一类合适的淀粉酶是稳定性增强的淀粉酶,描述在WO 94/18314及WO 96/05295,Genencor,以及在直接前体上进行附加改性的淀粉酶变体,由Novo Nordisk公司公开在WO95/10603中。合适的还有EP 277216中所描述的淀粉酶。
市售α-淀粉酶产品的例子是Purafect Ox Am_,由Genencor供应,以及Termamyl_、Ban_、Fungamyl_及Duramyl_,由丹麦NovoNordisk公司供应。WO95/26397描述了另一类合适的淀粉酶:一种α-淀粉酶,特征是,在25℃~55℃的温度和pH值8~10范围的比活性比Termamyl_的比活性至少高出25%,该数值是按Phadebas_α-淀粉酶活性试验测定的。上面WO96/23873(Novo Nordisk)中所述酶的各种变体是合适的。在活性水平及热稳定性与较高活性的综合方面改善的另一些淀粉水解酶公开在WO95/35382中。
此种淀粉水解酶,当存在时,在本发明清洗组合物中的加入量为组合物重量的0.0001%~2%,优选0.00018%~0.06%,更优选0.00024%~0.048%纯酶。
上述酶可取自任何一种合适来源,例如植物、动物、细菌、真菌及酵母源。来源还可是嗜温或嗜极端(extremophilic)(嗜冷、嗜寒(psychrotrophic)、嗜热、嗜压、嗜碱、嗜酸、嗜盐等)的。可使用这些酶的提纯及非提纯形式。如今,野生类型酶改性的通行做法是采用蛋白/基因工程技术以优化它们在本发明衣物洗涤剂和/或织物护理组合物中的性能效力。譬如,可通过对各种变体的设计,来提高酶与此类组合物中经常遇到的成分之间的相容性。或者,可通过变体设计来调节酶变体的最佳pH、漂白或螯合剂稳定性、催化活性之类,使之适合特定的清洁用途。
重点尤其应集中在:在追求漂白剂稳定性的情况下,对氧化敏感的氨基酸上;而为达到表面活性剂相容性--则在表面电荷上。此种酶的等电点可通过对某些带电氨基酸的取代来改变,例如提高等电点有助于改善与阴离子表面活性剂的相容性。酶的稳定性还可通过例如附加盐桥及强钙结合位点的生成以提高螯合剂稳定性,从而进一步提高。
这些任选的酶,当存在时,在清洗组合物重量中的加入量一般在清洗组合物重量的0.0001%~2%纯酶。这些酶可以分开的单一成分形式(含1种酶的小球、粒状、稳定化液体等),或者以2种或更多种酶的混合物形式(例如复合粒料)加入。
另一种适合加入的洗涤剂成分是酶氧化(剂)清除剂。此种酶氧化清除剂的例子是乙氧基化四亚乙基多胺。
一系列酶物质及其加入到合成洗涤剂组合物中的方式还公开在授予Genencor国际公司的WO 9307263及WO 9307260、美国专利3,553,139中,1971-01-05授予McCarty等人。进一步的酶公开在美国专利4,101,457和美国专利4,507,219中。用于液体洗涤剂组合物中的酶物质及其加入到此种制剂中的方法公开在美国专利4,261,868中。
酶稳定剂-用于洗涤剂中的酶可通过各种技术获得稳定。酶稳定技术公开在例如美国专利3,600,319、EP 199,405及EP 200,586中。酶稳定体系还描述于,例如美国专利3,519,570中。一种产生蛋白酶、木聚糖酶及纤维素酶的有用芽胞杆菌AC13,公开在WO9401532中。这里使用的酶可通过某种水溶性钙离子和/镁离子源在成品组合物中的存在并从而向其中的酶提供这些离子而获得稳定。合适的酶稳定剂及其使用方法公开在美国专利5,705,464、5,710,115及5,576,282中。
助洗剂-本文所描述的洗涤剂及清洗组合物优选包含1种或多种洗涤助洗剂或助洗剂体系。当存在时,组合物将通常包含至少约1%助洗剂,优选至少约5%,更优选至少约10%,至约80%,优选至约50%,更优选至约30wt%洗涤助洗剂。然而,更低或更高的助洗剂用量也不拟排除在外。
优选用于洗涤剂和清洗组合物,尤其是本文所描述的餐具洗涤组合物中的优选助洗剂包括但不限于,如公开在美国专利5,695,679、5,705,464及5,710,115中的水溶性助洗剂化合物(例如,多羧化物)。其他合适的多羧化物公开在美国专利4,144,226、3,308,067及3,723,322中。优选的多羧化物是每分子含最多3个羧基--更具体地说,被滴定液中的--多羧化物。
无机或含磷洗涤助洗剂包括但不限于,聚磷酸酯(例如三聚磷酸酯、焦磷酸酯及玻璃态聚合物偏磷酸酯)膦酸酯的碱金属、铵及醇胺的盐(例如参见美国专利3,159,581、3,213,030、3,422,021、3,400,148及3,422,137)、肌醇六磷酸、硅酸盐、碳酸盐(包括碳酸氢盐、倍半碳酸盐)、硫酸盐及硅铝酸盐。
然而,某些情况要求非磷酸盐助洗剂。重要的是,本发明组合物即便在所谓“弱”助洗剂(与磷酸盐相比)如柠檬酸盐,或者在所谓“低级助洗剂”的情况下,即,采用沸石或层状硅酸盐助洗剂时,依然令人惊奇地发挥很好的功能。
合适的硅酸盐包括水溶性硅酸钠,其SiO2∶Na2O比例介于约1.0~2.8,优选该比例在约1.6~2.4,最优选约2.0的比例。硅酸盐可以是无水盐或者是水合盐的形式。SiO2∶Na2O比例等于2.0的硅酸钠是最优选的。硅酸盐,当存在时,优选在本文所描述的洗涤剂及清洗组合物中的用量为组合物重量的约5%~约50wt%,更优选约10%~约40wt%。
适合用于该洗涤剂及清洗组合物,尤其是粒状洗涤剂组合物中的部分溶解或不溶的助洗剂化合物,包括但不限于结晶、层状硅酸盐,优选结晶层状硅酸钠(部分地溶解于水),如公开在美国专利4,664,839中,以及硅铝酸钠(不溶于水)。当存在于洗涤剂及清洗组合物中时,这些助洗剂的典型含量为组合物重量的约1%~80wt%,优选约10%~70wt%,最优选约20~60wt%。
可在本文所描述的的组合物中使用具有通式NaMSixO2x+1.yH2O的结晶层状硅酸钠,其中M是钠或氢,x是约1.9~约4,优选约2~约4,最优选2的数值,y是约0~约20,优选是0的数值。此种类型结晶层状硅酸钠公开在EP-A-0164514中,其制备方法公开在DE-A-3417649及DE-A-3742043中。最优选的材料是δ-Na2SiO5,由Hoechst公司按商品名NaSKS-6供应(通常在本文中简称“SKS-6”)。与沸石助洗剂不同,Na SKS-6硅酸盐助洗剂不包含铝。Na SKS-6具有层状硅酸盐的δ-Na2SiO5的形态。SKS-6是高度优选用于本文所描述的组合物中的层状硅酸盐,然而其他此类层状硅酸盐如具有通式NaMSixO2x+1.yH2O的,其中M是钠或氢,x是约1.9~约4,优选2,y是0~20,优选是数值0的,均可用于本文所描述的的组合物中。由Hoechst供应的各种各样其他层状硅酸盐包括NaSKS-5、NaSKS-7及NaSKS-11,呈α、β-及γ形式者。如上所述,δ-Na2SiO5(NaSKS-6型)是这里最优选的。其他硅酸盐也是有用的,例如硅酸镁,可在粒状制剂中作为酥脆剂、作为氧漂白的稳定剂以及作为泡沫控制体系的成分。
结晶层状硅酸钠材料优选以与固体、水溶性可电离材料的紧密混合物的颗粒形式存在于粒状洗涤剂组合物中。固体、水溶性可电离材料优选地选自有机酸、有机及无机酸盐及其混合物。
硅铝酸盐助洗剂乃是目前市场上供应的重垢型粒状洗涤剂组合物中非常重要的成分,且还可作为液体洗涤剂制剂中的重要助洗剂成分。硅铝酸盐助洗剂具有经验式:
[Mz(AlO2)y].xH2O
其中z和y是至少6的整数,z/y摩尔比介于1.0~约0.5,x为约15~约264的整数。优选的是,硅铝酸盐助洗剂是单元晶格如下式所示的硅铝酸盐沸石:
Naz[(AlO2)z(SiO2)y].xH2O
其中z和y至少是6;z/y摩尔比介于1.0~约0.5,x至少是5,优选7.5~276,最优选10~264。硅铝酸盐助洗剂优选为水合形式且优选是结晶的,含有约10%~约28%,更优选约18%~约22%结合水。
这些硅铝酸盐离子交换材料的结构可以是结晶或无定形的且可以是天然存在的硅铝酸盐或合成的。一种生产硅铝酸盐离子交换材料的方法公开在美国专利3,985,669中。这里优选的合成结晶硅铝酸盐离子交换材料在市场上以商品名沸石A、沸石B、沸石P、沸石X、沸石MAP和沸石HS及其混合物供应。在尤其优选的实施方案中,结晶硅铝酸盐离子交换材料具有通式:
Na12[(AlO2)12(SiO2)12].xH2O
其中x为约20~约30,尤其约27。该材料叫做沸石A。脱水沸石(x=0~10)也可在此使用。优选的是,该硅铝酸盐的粒度(直径)介于约0.1~10μm。沸石X具有通式:
Na86[(AlO2)86(SiO2)106]·276H2O
柠檬酸助洗剂,例如柠檬酸及其可溶性盐(尤其是钠盐),是高效液体洗涤剂制剂用的特别重要的多羧酸类助洗剂,因为它们易从可再生资源获得且具有可生物降解性。
柠檬酸类也可用于粒状组合物中,尤其是与沸石和/或层状硅酸盐助洗剂配合使用。氧联二琥珀酸类也是特别适用于此类组合物及其组合中的。
适合本文所描述的的洗涤剂组合物中的还有3,3-二羧基-4-氧杂-1,6-己二酸酯及其相关化合物,公开在美国专利4,566,984中。有用的琥珀酸助洗剂包括C5~C20烷基及链烯基琥珀酸及其盐。特别优选的此类化合物是十二碳烯基琥珀酸。琥珀酸助洗剂的具体例子包括:月桂基琥珀酸、肉豆蔻基琥珀酸、棕榈基琥珀酸、2-十二碳烯基琥珀酸(优选的)、2-十五碳烯基琥珀酸等。月桂基琥珀酸是这类型中优选的助洗剂,公开在欧洲专利申请86200690.5/0.200.263中,1986-11-05发表。
脂肪酸,如C12~C18单羧酸也可单独或与上述助洗剂,尤其是柠檬酸和/或琥珀酸助洗剂组合起来加入到组合物中,以提供附加的助洗剂活性。脂肪酸的此种用法一般将导致减少起泡,配制者应考虑这一点。
分散剂-1种或多种合适的聚亚烷基亚胺分散剂可加入到本发明的清洗组合物中。此类合适分散剂的例子可参见欧洲专利申请号111,965、111,984及112,592;美国专利4,597,898、4,548,744及5,565,145。然而,任何适当的粘土/污垢分散剂或抗再沉积剂均可用于本发明洗衣组合物中。
另外,包括聚合物多羧化物和聚乙二醇的聚合物分散剂也适用于本发明。可聚合生成合适的聚合物多羧化物的不饱和单体酸包括丙烯酸、马来酸(或马来酐)、富马酸、衣康酸、阿康酸、中康酸、柠康酸及亚甲基丙二酸。特别合适的聚合物多羧酸(多羧化物)可由丙烯酸衍生而来。这里有用的此类以丙烯酸为基础的聚合物是聚合丙烯酸的水溶性盐。酸性形式的此种聚合物的平均分子量优选在约2,000~10,000,更优选约4,000~7,000,最优选约4,000~5,000。此种丙烯酸聚合物的水溶性盐例如可包括碱金属、铵及取代的铵盐。此类型可溶性聚合物是已知材料。此类型丙烯酸盐在洗涤剂组合物中的应用已公开在例如美国专利3,308,067中。
以丙烯酸/马来酸为基础的共聚物也可用作优选的分散/抗再沉积剂的成分。此类材料包括丙烯酸与马来酸共聚物的水溶性盐。此种丙烯酸-马来酸共聚物的平均分子量,以酸性形式计,优选约2,000~100,000,更优选约5,000~75,000,最优选约7,000~65,000。丙烯酸对马来酸链段在此种共聚物中的比例一般为约30∶1~约1∶1,更优选约10∶1~2∶1。此种丙烯酸/马来酸共聚物的水溶性盐可包括,例如碱金属、铵及取代的铵盐。此种丙烯酸/马来酸共聚物的水溶性盐是已知材料,公开在欧洲专利申请66915中,1982-12-15发表,以及EP 193,360,1986-09-03发表,其中还描述了包含丙烯酸羟丙基酯的此类聚合物。另一些有用的其他分散剂包括马来酸/丙烯酸/乙烯醇三元共聚物。此种材料也公开在EP193,360中,包括,例如马来酸/丙烯酸/乙烯醇按45/45/10比例的三元共聚物。
可包括在内的另一种聚合物材料是聚乙二醇(PEG)。PEG可表现出分散剂性能,以及作为粘土污垢脱除-抗再沉积剂。为此目的的典型分子量介于约500~约100,000,优选约1,000~约50,000,更优选约1,500~约10,000。
聚天冬氨酸和聚谷氨酸分散剂也可使用,尤其与沸石助洗剂联合使用。诸如聚天冬氨酸之类的分散剂优选具有约10.00的分子量(平均)。
去污剂-本发明组合物可任选地包含1种或多种去污剂。使用的话,去污剂一般将占到组合物重量的约0.01%,优选约0.1%,更优选约0.2~约10%,优选约5%,更优选约3%wt%。适宜去污的聚合物的非限制性例子公开在:美国专利5,728,671、5,691,298、5,599,782、5,415,807、5,182,043、4,956,447、4,976,879、4,968,451、4,925,577、4,861,512、4,877,896、4,771,730、4,711,730、4,721,580、4,000,093、3,959,230及3,893,929;以及欧洲专利申请0 219 048中。
另一些合适的去污剂公开在美国专利4,201,824、4,240,918、4,525,524、4,579,681、4,220,018及4,787,989;EP 279,134 A、EP457,205 A,以及DE 2,335,044中。
螯合剂-本发明组合物还可任选地包含螯合剂,它起到螯合金属离子及金属杂质的作用,否则这些金属往往导致漂白剂失活。有用的螯合剂可包括氨基羧酸、磷酸、氨基磷酸、多官能取代的芳族螯合剂及其混合物。另一些螯合剂的例子及其用量描述在美国专利5,705,464、5,710,115、5,728,671及5,576,282中。
本发明组合物还可包含水溶性甲基甘氨酸二乙酸(MGDA)盐(或酸形式)作为螯合剂,或者与例如不溶性助洗剂如沸石、层状硅酸盐之类一起使用作为共助洗剂。
使用的话,这些螯合剂一般将占到该洗涤剂组合物重量的约0.1%~约15%,更优选约0.1%~约3.0wt%。
抑泡剂-另一种任选的成分是抑泡剂,例如硅氧烷以及二氧化硅-硅氧烷混合物。合适的抑泡剂的例子公开在美国专利5,707,950及5,728,671中。这些抑泡剂的用量一般为组合物重量的0.001~2wt%,优选0.01%~1wt%。
柔软剂-织物柔软剂也可加入到本发明衣物洗涤剂组合物中。元机柔软剂的例子是公开在GB-A-1 400 898及美国专利5,019,292中的绿土(粘土)。有机柔软剂包括如公开在GB-A-1 514 276及EP-B-011 340中的水不可溶叔胺,及其与单C12~C14季铵盐的组合,如公开在GB-B-026 527及EP-B-026 528中,以及二长链酰胺,如公开在EP-B-0 242 919中。另一些用于织物柔软剂体系的有机成分包括高分子量聚环氧乙烷材料,如公开在EP-A-0 299 575及0 313 140中。
特别合适的织物柔软剂公开在美国专利5,707,950及5,728,673中。
绿土的用量一般介于2%~20%,更优选5%~15wt%,该材料以干混合成分加入到配制物的其余成分中。诸如不溶于水的叔胺或二长链酰胺材料之类的有机织物柔软剂的加入量,介于0.5~5wt%,通常在1%~3wt%之间,而高分子量聚环氧乙烷材料及水溶性阳离子材料则按0.1~2%,一般在0.15%~1.5wt%的水平加入。这些材料一般加入到组合物的喷雾干燥部分中,虽然某些情况下它也可更方便地以干混合粒料形式加入,或者将它们以熔融液体形式喷涂到组合物的其他固体成分上。
可生物降解季铵化合物,如EP-A-040 562及EP-A-239 910中所描述的,已被提出作为传统上使用的氯化及甲基硫酸二长链烷基铵的替代物。
用于季铵盐化合物及胺前体的柔软剂-相容阴离子的非限制性例子包括氯根及甲基硫酸根。
染料转移抑制-本发明洗涤剂组合物还可包含用于在涉及有色织物的织物洗涤及调理操作期间抑制被增溶和悬浮的染料从一种织物转移到另一种织物上的化合物。
聚合物染料转移抑制剂
本发明洗涤剂组合物还可包含0.001%~10%,优选0.01%~2%,更优选0.05%~1wt%聚合物染料转移抑制剂。将所述聚合物染料转移抑制剂加入到洗涤剂组合物中的目的通常是为了抑制染料从有色织物转移到一起洗涤的织物上。这种聚合物能够将从染色织物上洗脱下来变为游离的这些染料在尚未来得及附着到洗涤中其他物品上之前络合或吸收。
尤其合适的聚合物染料转移抑制剂是N-氧化多胺聚合物、N-乙烯基吡咯烷酮与N-乙烯基咪唑的共聚物、聚乙烯基吡咯烷酮聚合物、聚_唑烷酮及聚乙烯基咪唑,或其混合物。此类染料转移抑制剂的例子公开在美国专利5,707,950及5,707,951中。
另一类合适的染料转移抑制剂包括但不限于交联的聚合物。交联聚合物是其主链间一定程度上互连的聚合物:这些连接的性质可以是化学的或者是物理的,可能与主链上或者支链上的活性基团连接;交联聚合物描述于《聚合物科学杂志》卷22,pp.1035~1039中。
在一种实施方案中,交联聚合物被制成具有三维刚性结构的,可将染料俘获在该三维结构所形成的孔中。在另一种实施方案中,交联聚合物借助溶胀来俘获染料。此类交联聚合物描述于共同未决欧洲专利申请94870213.9中。
此种聚合物的加入还可增强本发明酶的效果。
pH及缓冲范围-许多本文所描述的洗涤剂及清洗组合物是采取了缓冲措施的,即,它们对在酸性污垢存在下的pH降低具有相对的承受能力。然而,本文中另一些组合物则可能具有异常低的缓冲能力,或者可能是基本上没有加缓冲措施的。将pH值控制或改变到建议使用水平的技术,较一般地说不仅包括使用缓冲剂,而且可还可加入附加碱、酸、pH-突变体系、双室容器等,这些乃是本领域技术人员熟知的。
本发明优选的ADD组合物包含pH-调节成分,选自水溶性碱性无机盐及水溶性有机或无机助洗剂,如美国专利5,705,464和5,710,115中所述。
材料护理剂-优选的ADD组合物可包含1种或多种可有效地起到缓蚀剂和/或防晦暗酸之类作用的材料护理剂,如美国专利5,705,464、5,710,115及5,646,101中所描述的。
当存在时,此种保护材料优选少量地加入,例如为ADD组合物的约0.01%~约5%。
其他材料-本发明组合物中任选包括的洗涤成分或添加剂可包括1种或多种用于辅助或强化清洁能力、被清洁底物的清洁处理效果或者旨在改善组合物外观的材料。可按传统技术的习惯用量(一般地,添加剂材料总共占到组合物重量的约30%~约99.9%,优选约70~约95%wt%)加入到本发明组合物中的添加剂,还包括其他活性成分,如非磷酸(盐)助洗剂、彩色斑点、银器清洁剂、防晦暗剂和/或防腐蚀剂、染料、填料、灭菌剂、碱性源、水溶助长剂、抗氧剂、香料、增溶剂、载体、加工助剂、颜料及pH控制剂,正如美国专利5,705,464、5,710,115、5,698,504,5,695,679、5,686,014和5,646,101中所描述的。
清洁方法-除了本文已描述过的清洁织物、餐具及其他硬表面以及清洁个人身体某些位点等方法之外,本发明还涵盖对已沾污或染斑织物的洗涤“预处理”方法,包括让所述色斑和/或污垢直接与高度浓缩形式的以上所描述清洗组合物进行接触,然后再采用传统水洗溶液洗涤该织物。优选的是,清洗组合物与污垢/色斑保持接触达约30s~24h,然后再按传统方式洗涤经预处理的沾污/染斑底物。更优选的是,预处理的时间介于约1~180min。
下面的实施例旨在举例说明本发明组合物,不一定要限制或以其他方式界定本发明的范围。
在下面的实施例中,蛋白酶1是指一种蛋白酶变体,它包含在对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的位点101G/103A/104I/159D/232V/236H/245R/248D/252K上,以另一种天然存在的氨基酸残基取代其氨基酸残基。蛋白酶1还可被替换为任何其他本发明蛋白酶变体,其结果与下面的实施例相当接近。
在本发明的清洗组合物实施例中,蛋白酶1的用量以纯酶对整个组合物重量关系表示;其他酶的用量以原料对整个组合物重量表示;除非另行规定,其他成分一律以整个组合物重量为基准表示。
再有,在下面的实施例中,使用了某些与本文公开内容相一致并为本领域技术人员已知的某些缩写。
硬表面、餐具及织物清洗组合物实施例
1.硬表面清洗组合物
本文所使用的术语“硬表面清洗组合物”是指用于清洁诸如地板、墙壁、浴室砖之类硬表面的液体或粒状洗涤剂组合物。本发明硬表面清洗组合物包含有效数量1种或多种蛋白酶,优选占组合物的约0.0001%~约10%,更优选约0.001%~约5%,进一步优选约0.001%~约1wt%活性蛋白酶。除了包含1种或多种蛋白酶之外,此种硬表面清洗组合物一般还包含表面活性剂及水溶性螯合助洗剂。然而在某些专用产品中,例如玻璃窗喷雾清洁剂,有时不使用表面活性剂,因为它们将在玻璃表面形成膜状/条纹状残余物。(参见美国专利5,679,630实施例)。
表面活性剂成分。当存在时可占到少至组合物的0.1%但在典型情况下,组合物将包含约0.25%~约10%,更优选约1%~约5%表面活性剂。
就典型而言,组合物将包含约0.5%~约50%洗涤助洗剂,优选约1%~约10%。优选的是,pH应介于约8~12的范围。若需要调节pH值的话,可使用传统pH调节剂,如氢氧化钠、碳酸钠或盐酸。
组合物中可包含溶剂。有用的溶剂包括但不限于,二醇醚,如二甘醇一己基醚、二甘醇一丁基醚、乙二醇一丁基醚,以及二醇一己基醚、聚乙二醇一丁基醚、二丙二醇一丁基醚,以及二醇类,如2,2,4-三甲基-1,3-戊二醇及2-乙基-1,3-己二醇。当使用时,这些溶剂的典型用量为约0.5%~约15%,优选约3%~约11%。
另外,当在表面上施涂“全效”组合物之后不清洗表面时,本发明组合物也可使用高度挥发性溶剂如异丙醇或乙醇,以加速组合物从表面上的蒸发。当使用时,挥发性溶剂的典型含量为组合物的约2%~约12%。
本发明硬表面清洗组合物的实施方案体现在下列非限制性实施例上。
实施例1~7
液体硬表面清洗组合物
成分 | 实施例号 | ||||||
1 | 2 | 3 | 4 | 5 | 6 | 7 | |
蛋白酶1 | 0.05 | 0.05 | 0.20 | 0.02 | 0.03 | 0.10 | 0.03 |
蛋白酶2 | - | - | - | - | - | 0.20 | 0.1 |
螯合剂** | - | - | - | 2.90 | 2.90 | - | - |
柠檬酸 | - | - | - | - | - | 2.90 | 2.90 |
LAS | - | 1.95 | - | 1.95 | - | 1.95 | - |
AS | 2.00 | - | 2.20 | - | 2.20 | - | 2.20 |
AES | 2.00 | - | 2.20 | - | 2.20 | - | 2.20 |
氧化胺 | 0.40 | - | 0.50 | - | 0.50 | - | 0.50 |
水溶助长剂 | - | 1.30 | - | 1.30 | - | 1.30 | - |
溶剂*** | - | 6.30 | 6.30 | 6.30 | 6.30 | 6.30 | 6.30 |
水及微量成分 | 凑足100% |
2蛋白酶,除蛋白酶1以外的,包括但不限于本文所述可用于本发明的附加蛋白酶。
**Na4乙二胺二乙酸
***二甘醇一己基醚
****全部配制物调节到pH7
在实施例6和7中,尤其要指出,本文所引可用于本发明的蛋白酶的任意组合均可用来替代蛋白酶1和蛋白酶2,并获得类似的结果。
实施例8~13
清洁硬表面及家庭除霉用喷雾组合物
成分 | 实施例号 | |||||
8 | 9 | 10 | 11 | 12 | 13 | |
蛋白酶1 | 0.20 | 0.05 | 0.10 | 0.30 | 0.20 | 0.30 |
蛋白酶2 | - | - | - | - | 0.30 | 0.10 |
C8AS | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 |
C12AS | 4.00 | 4.00 | 4.00 | 4.00 | 4.00 | 4.00 |
碱 | 0.80 | 0.80 | 0.80 | 0.80 | 0.80 | 0.80 |
硅酸盐 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 |
香料 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
水及微量成分 | 凑足100% |
2蛋白酶,除蛋白酶1以外的,包括但不限于本文所述可用于本发明的附加蛋白酶。
****产品pH为约7
在实施例12和13中,尤其要指出,本文所引可用于本发明的蛋白酶的任意组合均可用来替代蛋白酶1和蛋白酶2,并获得类似的结果。
2.餐具洗涤组合物
实施例14~19
餐具洗涤组合物
成分 | 实施例号 | |||||
14 | 15 | 16 | 17 | 18 | 19 | |
蛋白酶1 | 0.05 | 0.50 | 0.02 | 0.40 | 0.10 | 0.03 |
蛋白酶2 | - | - | - | - | 0.40 | 0.1 |
TFAAI | 0.90 | 0.90 | 0.90 | 0.90 | 0.90 | 0.90 |
AES | 12.00 | 12.00 | 12.00 | 12.00 | 12.00 | 12.00 |
2-甲基十二烷酸 | 4.50 | 4.50 | - | 4.50 | 4.50 | - |
C12醇的乙氧基化物(4) | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 |
氧化胺 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 |
水溶助长剂 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 |
乙醇 | 4.00 | 4.00 | 4.00 | 4.00 | 4.00 | 4.00 |
Mg++(按氯化镁计) | 0.20 | 0.20 | 0.20 | 0.20 | 0.20 | 0.20 |
Ca++(按氯化钙计) | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 |
水及微量成分**** | 凑足100% |
2蛋白酶,除蛋白酶1以外的,包括但不限于本文所述可用于本发明的附加蛋白酶。
****产物pH值调节至7。
在实施例18和19中,尤其要指出,本文所引可用于本发明的蛋白酶的任意组合均可用来替代蛋白酶1和蛋白酶2,并获得类似的结果。
实施例20
餐具洗涤组合物
成分 | A(ADW) | B(ADW) | C(LDL) |
STPP | 17.5 | - | - |
柠檬酸 | - | 15.0 | - |
聚丙烯酸钠(MW4500) | 0.80 | - | - |
Acusol 480N | - | 5.10 | - |
碳酸钾 | 8.30 | - | - |
碳酸钠 | - | 8.50 | - |
2.1比例硅酸钾 | 3.99 | - | - |
2.0比例硅酸钠 | 2.00 | - | - |
3.2比例硅酸钠 | 5.18 | - | - |
三硬脂酸铝 | 0.10 | - | - |
非离子表面活性剂 | - | 2.50 | - |
NaAE0.6S | - | - | 24.70 |
葡糖酰胺 | - | - | 3.09 |
C10E8 | - | - | 4.11 |
甜菜碱 | - | - | 2.06 |
氧化胺 | - | - | 2.06 |
镁(按氧化物计) | - | - | 0.49 |
水溶助长剂 | - | - | 4.47 |
次氯酸钠,按活性氯计 | 1.15 | - | - |
蛋白酶1 | 0.01 | 0.43 | 0.05 |
其余凑足100% |
实施例21
液体餐具洗涤组合物(尤其适合日本条件下使用)
成分 | A | B |
AE1.4S | 24.69 | 24.69 |
N-椰子基-N-甲基葡糖胺 | 3.09 | 3.09 |
氧化胺 | 2.06 | 2.06 |
甜菜碱 | 2.06 | 2.06 |
非离子表面活性剂 | 4.11 | 4.11 |
水溶助长剂 | 4.47 | 4.47 |
镁 | 0.49 | 0.49 |
乙醇 | 7.2 | 7.2 |
LemonEase | 0.45 | 0.45 |
Geraniol/BHT | - | 0.60/0.02 |
淀粉酶 | 0.03 | 0.005 |
蛋白酶1 | 0.01 | 0.43 |
其余凑足100% |
实施例22
粒状自动餐具洗涤组合物
成分 | A | B | C |
柠檬酸 | 15.0 | - | - |
柠檬酸盐 | 4.0 | 29.0 | 15.0 |
丙烯酸酯/甲基丙烯酸酯共聚物 | 6.0 | - | 6.0 |
丙烯酸/马来酸共聚物 | - | 3.7 | - |
干添加碳酸盐 | 9.0 | - | 20.0 |
碱金属硅酸盐 | 8.5 | 17.0 | 9.0 |
石蜡 | - | 0.5 | - |
苯并三唑 | - | 0.3 | - |
Termamyl 60T | 1.6 | 1.6 | 1.6 |
蛋白酶1 | 0.2 | 0.1 | 0.06 |
过碳酸盐(活性氧) | 1.5 | - | - |
过硼酸一水合物 | - | 0.3 | 1.5 |
过硼酸四水合物 | - | 0.9 | - |
四乙酰乙二胺 | 3.8 | 4.4 | - |
二亚乙基三胺五甲基膦酸(镁盐) | 0.13 | 0.13 | 0.13 |
烷基乙氧基硫酸酯-3倍乙氧基化物 | 3.0 | - | - |
烷基乙氧基丙氧基非离子表面活性剂 | - | 1.5 | - |
抑泡剂 | 2.0 | - | - |
Olin SLF18非离子表面活性剂 | - | - | 2.0 |
硫酸盐 | 凑足100% |
实施例23
坚实高密度(0.96kg/l)本发明餐具洗涤剂组合物A~F:
成分 | A | B | C | D | E | F |
STPP | - | 51.4 | 51.4 | - | - | 44.3 |
柠檬酸盐 | 17.05 | - | - | 49.6 | 40.2 | - |
碳酸盐 | 17.50 | 14.0 | 20.0 | 8.0 | 33.6 | |
碳酸氢盐 | - | - | - | 26.0 | - | - |
硅酸盐 | 14.81 | 15.0 | 8.0 | - | 25.0 | 3.6 |
偏硅酸盐 | 2.50 | 4.5 | 4.5 | - | - | - |
PB1 | 9.74 | 7.79 | 7.79 | - | - | - |
PB4 | - | - | - | 9.6 | - | - |
过碳酸盐 | - | - | - | - | 11.8 | 4.8 |
非离子 | 2.00 | 1.50 | 1.50 | 2.6 | 1.9 | 5.9 |
TAED | 2.39 | - | - | 3.8 | - | 1.4 |
HEDP | 1.00 | - | - | - | - | - |
DETPMP | 0.65 | - | - | - | - | - |
Mn TACN | - | - | - | - | 0.008 | - |
PAAC | - | 0.008 | 0.008 | - | - | - |
石蜡 | 0.50 | 0.38 | 0.38 | 0.6 | - | - |
蛋白酶1 | 0.1 | 0.06 | 0.05 | 0.03 | 0.07 | 0.01 |
淀粉酶 | 1.5 | 1.5 | 1.5 | 2.6 | 2.1 | 0.8 |
BTA | 0.30 | 0.22 | 0.22 | 0.3 | 0.3 | 0.3 |
多羧化物 | 6.0 | - | - | - | 4.2 | 0.9 |
香料 | 0.2 | 0.12 | 0.12 | 0.2 | 0.2 | 0.2 |
硫酸盐/水 | 20.57 | 1.97 | 2.97 | 3.6 | 4.5 | 3.9 |
pH(1%溶液) | 11.0 | 11.0 | 11.3 | 9.6 | 10.8 | 10.9 |
实施例24
堆积密度1.02kg/l的本发明粒状餐具洗涤剂组合物例A~F:
成分 | A | B | C | D | E | F |
STPP | 30.00 | 33.5 | 27.9 | 29.62 | 33.8 | 22.0 |
碳酸盐 | 30.50 | 30.50 | 30.5 | 23.00 | 34.5 | 45.0 |
硅酸盐 | 7.40 | 7.50 | 12.6 | 13.3 | 3.2 | 6.2 |
偏硅酸盐 | - | 4.5 | ||||
过碳酸盐 | - | - | - | 4.0 | ||
PB1 | 4.4 | 4.5 | 4.3 | - | - | |
NaDCC | - | - | 2.00 | - | 0.9 | |
非离子 | 1.0 | 0.75 | 1.0 | 1.90 | 0.7 | 0.5 |
TAED | 1.00 | - | - | 0.9 | ||
PAAC | - | 0.004 | - | - | ||
石蜡 | 0.25 | 0.25 | - | - | ||
蛋白酶1 | 0.05 | 0.06 | 0.025 | 0.1 | 0.02 | 0.7 |
淀粉酶 | 0.38 | 0.64 | 0.46 | - | 0.6 | |
BTA | 0.15 | 0.15 | - | 0.2 | ||
香料 | 0.2 | 0.2 | 0.05 | 0.1 | 0.2 | |
硫酸盐/水 | 23.45 | 16.87 | 22.26 | 30.08 | 21.7 | 25.4 |
PH(1%溶液) | 10.80 | 11.3 | 11.0 | 10.70 | 11.5 | 10.9 |
实施例25
制备了本发明片剂洗涤剂组合物例A~H,它是通过利用标准12头旋转压机在13KN/cm2压力下由粒状餐具洗涤剂组合物压制而成的:
成分 | A | B | C | D | E | F | G | H |
STPP | - | 48.8 | 54.7 | 38.2 | - | 52.4 | 56.1 | 36.0 |
柠檬酸盐 | 20.0 | - | - | - | 35.9 | - | - | - |
碳酸盐 | 20.0 | 5.0 | 14.0 | 15.4 | 8.0 | 23.0 | 20.0 | 28.0 |
硅酸盐 | 15.0 | 14.8 | 15.0 | 12.6 | 23.4 | 2.9 | 4.3 | 4.2 |
蛋白酶1 | 0.05 | 0.09 | 0.05 | 0.03 | 0.06 | 0.03 | 0.03 | 0.1 |
淀粉酶 | 1.5 | 1.5 | 1.5 | 0.85 | 1.9 | 0.4 | 2.1 | 0.3 |
PB1 | 14.3 | 7.8 | 11.7 | 12.2 | - | - | 6.7 | 8.5 |
PB4 | - | - | - | - | 22.8 | - | 3.4 | - |
过碳酸盐 | - | - | - | - | - | 10.4 | - | - |
非离子 | 1.5 | 2.0 | 2.0 | 2.2 | 1.0 | 4.2 | 4.0 | 6.5 |
PAAC | - | - | 0.016 | 0.009 | - | - | - | - |
Mn TACN | - | - | - | - | 0.007 | - | - | - |
TAED | 2.7 | 2.4 | - | - | - | 2.1 | 0.7 | 1.6 |
HEDP | 1.0 | - | - | 0.93 | - | 0.4 | 0.2 | - |
DETPMP | 0.7 | - | - | - | - | - | - | - |
石蜡 | 0.4 | 0.5 | 0.5 | 0.55 | - | - | 0.5 | - |
BTA | 0.2 | 0.3 | 0.3 | 0.33 | 0.3 | 0.3 | 0.3 | - |
多羧化物 | 4.0 | - | - | - | 4.9 | 0.6 | 0.8 | - |
PEG | - | - | - | - | - | 2.0 | - | 2.0 |
甘油 | - | - | - | - | - | 0.4 | - | 0.5 |
香料 | - | - | - | 0.05 | 0.20 | 0.2 | 0.2 | 0.2 |
硫酸盐/水 | 17.4 | 14.7 | - | 15.74 | - | - | - | 11.3 |
片剂重量 | 20g | 25g | 20g | 30g | 18g | 20g | 25g | 24.0 |
pH(1%溶液) | 10.7 | 10.6 | 10.7 | 10.7 | 10.9 | 11.2 | 11.0 | 10.8 |
实施例26
酒窝状片型自动餐具洗涤组合物
成分 | A(%R.M.) | B(gR.M.) | C(gR.M.) |
片剂本体 | |||
碳酸钠 | 15.348 | 3.500 | 5.25 |
STPP(12%水) | 46.482 | 10.600 | 9.93 |
粒状HEDP | 0.789 | 0.180 | 0.28 |
SKS 6 | 6.578 | 1.500 | 2.25 |
2倍硅酸盐 | 7.016 | 1.600 | 1.65 |
PB1 | 10.743 | 2.450 | 3.68 |
Termamyl 2xPCA | 0.491 | 0.112 | 0.17 |
Savinase | 0.526 | 0.120 | 0.18 |
Plurafac | 3.508 | 0.800 | 0.9 |
BTA | 0.263 | 0.060 | 0.09 |
PEG | 1.140 | 0.260 | - |
PEG 4000 | - | - | 0.39 |
Winog | 0.439 | 0.100 | 0.15 |
香料 | 0.101 | 0.023 | 0.01 |
酒窝充填物 | |||
柠檬酸 | 0.987 | 0.225 | 0.23 |
碳酸氢盐 | 2.600 | 0.593 | 0.59 |
Sandolan EHRL染料 | 0.007 | 0.0017 | 0.0017 |
PEG 400/4000 | 0.395 | 0.090 | |
PEG 400 | - | - | 0.02 |
PEG 4000 | - | - | 0.08 |
淀粉酶 | 1.412 | 0.322 | 0.32 |
蛋白酶1 | 0.05 | 0.268 | 0.27 |
3.织物清洗组合物
粒状织物清洗组合物
本发明粒状织物清洗组合物包含有效数量1种或多种蛋白酶,优选用量为组合物的约0.001~约10%,更优选约0.005%~约5%,更优选0.01%~约1wt%活性蛋白酶。(参见美国专利5,679,630实施例)。
实施例27
粒状织物清洗组合物
成分 | 实施例号 | |||
A | B | C | D | |
蛋白酶1 | 0.10 | 0.20 | 0.03 | 0.05 |
蛋白酶2 | - | - | 0.2 | 0.15 |
C13线型烷基苯磺酸酯 | 22.00 | 22.00 | 22.00 | 22.00 |
磷酸盐(三聚磷酸钠形式) | 23.00 | 23.00 | 23.00 | 23.00 |
碳酸钠 | 23.00 | 23.00 | 23.00 | 23.00 |
硅酸钠 | 14.00 | 14.00 | 14.00 | 14.00 |
沸石 | 8.20 | 8.20 | 8.20 | 8.20 |
螯合剂(二亚乙基三胺-五乙酸) | 0.40 | 0.40 | 0.40 | 0.40 |
硫酸钠 | 5.50 | 5.50 | 5.50 | 5.50 |
水 | 凑足100% |
2蛋白酶,除蛋白酶1以外的,包括(但不限于)本文所述用于本发明的附加蛋白酶。
在实施例27C和D中,尤其要指出,本文所引可用于本发明的蛋白酶的任意组合均可用来替代蛋白酶1和蛋白酶2,并获得类似的结果。
实施例28
粒状织物清洗组合物
成分 | 实施例号 | |||
A | B | C | D | |
蛋白酶1 | 0.10 | 0.20 | 0.03 | 0.05 |
蛋白酶2 | - | - | 0.2 | 0.1 |
C12烷基苯磺酸酯 | 12.00 | 12.00 | 12.00 | 12.00 |
沸石A(1-10μm) | 26.00 | 26.00 | 26.00 | 26.00 |
C12-C14仲(2,3)烷基硫酸酯,钠盐 | 5.00 | 5.00 | 5.00 | 5.00 |
柠檬酸钠 | 5.00 | 5.00 | 5.00 | 5.00 |
荧光增白剂 | 0.10 | 0.10 | 0.10 | 0.10 |
硫酸钠 | 17.00 | 17.00 | 17.00 | 17.00 |
填料、水、微量成分 | 凑足100% |
2蛋白酶,除蛋白酶1以外的,包括(但不限于)本文所述用于本发明的附加蛋白酶。
在实施例28C和D中,尤其要指出,本文所引可用于本发明的蛋白酶的任意组合均可用来替代蛋白酶1和蛋白酶2,并获得类似的结果。
实施例29
粒状织物清洗组合物
成分 | 实施例号 | |
A | B | |
线型烷基苯磺酸酯 | 11.4 | 10.70 |
牛脂烷基硫酸酯 | 1.80 | 2.40 |
C14~15烷基硫酸酯 | 3.00 | 3.10 |
C14~15醇-7倍乙氧基化物 | 4.00 | 4.00 |
牛脂醇11倍乙氧基化物 | 1.80 | 1.80 |
分散剂 | 0.07 | 0.1 |
硅氧烷流体 | 0.80 | 0.80 |
柠檬酸三钠 | 14.00 | 15.00 |
柠檬酸 | 3.00 | 2.50 |
沸石 | 32.50 | 32.10 |
马来酸丙烯酸共聚物 | 5.00 | 5.00 |
二亚乙基三胺五亚甲基膦酸 | 1.00 | 0.20 |
膦酸 | ||
蛋白酶1 | 0.1 | 0.01 |
脂酶 | 0.36 | 0.40 |
淀粉酶 | 0.30 | 0.30 |
硅酸钠 | 2.00 | 2.50 |
硫酸钠 | 3.50 | 5.20 |
聚乙烯基吡咯烷酮 | 0.30 | 0.50 |
过硼酸盐 | 0.5 | 1 |
苯酚磺酸酯 | 0.1 | 0.2 |
过氧化物酶 | 0.1 | 0.1 |
微量成分 | 凑足100% | 凑足100% |
实施例30
粒状织物清洗组合物
成分 | 实施例号 | |
A | B | |
线型C12烷基苯磺酸钠 | 6.5 | 8.0 |
硫酸钠 | 15.0 | 18.0 |
沸石A | 26.0 | 22.0 |
次氮基三乙酸钠 | 5.0 | 5.0 |
聚乙烯基吡咯烷酮 | 0.5 | 0.7 |
四乙酰乙二胺 | 3.0 | 3.0 |
硼酸 | 4.0 | - |
过硼酸盐 | 0.5 | 1 |
苯酚磺酸酯 | 0.1 | 0.2 |
蛋白酶1 | 0.2 | 0.05 |
填料(如,硅酸盐、碳酸盐、香料、水) | 凑足100% | 凑足100% |
实施例31
紧凑粒状织物清洗组合物
成分 | wt% |
烷基硫酸酯 | 8.0 |
烷基乙氧基硫酸酯 | 2.0 |
C25与C45醇的3和7倍乙氧基化物的混合物 | 6.0 |
多羟基脂肪酸酰胺 | 2.5 |
沸石 | 17.0 |
层状硅酸盐/柠檬酸盐 | 16.0 |
碳酸盐 | 7.0 |
马来酸丙烯酸共聚物 | 5.0 |
去污聚合物 | 0.4 |
羧甲基纤维素 | 0.4 |
N-氧化聚(4-乙烯基吡啶) | 0.1 |
乙烯基咪唑与乙烯基吡咯烷酮共聚物 | 0.1 |
PEG 2000 | 0.2 |
蛋白酶1 | 0.03 |
脂酶 | 0.2 |
纤维素酶 | 0.2 |
四乙酰乙二胺 | 6.0 |
过碳酸盐 | 22.0 |
乙二胺二琥珀酸 | 0.3 |
抑泡剂 | 3.5 |
4,4’-双(2-马啉基-4-苯胺基-s-三嗪-6-基-氨基)均 | 0.25 |
二苯代乙烯-2,2’-二磺酸二钠 | |
二钠-4,4’-双(2-磺基苯乙烯基)联苯 | 0.05 |
水、香料及微量成分 | 凑足100% |
实施例32
粒状织物清洗组合物
成分 | wt% |
线型烷基苯磺酸酯 | 7.6 |
C16~C18烷基硫酸酯 | 1.3 |
C14~15醇的7倍乙氧基化物 | 4.0 |
椰子-烷基-二甲基羟乙基氯化铵 | 1.4 |
分散剂 | 0.07 |
硅氧烷流体 | 0.8 |
柠檬酸三钠 | 5.0 |
沸石4A | 15.0 |
马来酸丙烯酸共聚物 | 4.0 |
二亚乙基三胺五亚甲基磺酸 | 0.4 |
过硼酸盐 | 15.0 |
四乙酰乙二胺 | 5.0 |
绿土 | 10.0 |
聚(氧乙烯)(MW 300,000) | 0.3 |
蛋白酶1 | 0.02 |
脂酶 | 0.2 |
淀粉酶 | 0.3 |
纤维素酶 | 0.2 |
硅酸钠 | 3.0 |
碳酸钠 | 10.0 |
羧甲基纤维素 | 0.2 |
增白剂 | 0.2 |
水、香料及微量成分 | 凑足100% |
实施例33
粒状织物清洗组合物
成分 | Wt% |
线型烷基苯磺酸酯 | 6.92 |
牛脂烷基硫酸酯 | 2.05 |
C14~15醇的7倍乙氧基化物 | 4.4 |
C12~15烷基乙氧基硫酸酯-3倍乙氧基化物 | 0.16 |
沸石 | 20.2 |
柠檬酸盐 | 5.5 |
碳酸盐 | 15.4 |
硅酸盐 | 3.0 |
马来酸丙烯酸共聚物 | 4.0 |
羧甲基纤维素 | 0.31 |
去污聚合物 | 0.30 |
蛋白酶1 | 0.1 |
脂酶 | 0.36 |
纤维素酶 | 0.13 |
过硼酸盐四水合物 | 11.64 |
过硼酸盐一水合物 | 8.7 |
四乙酰乙二胺 | 5.0 |
二亚乙基三胺五甲基膦酸 | 0.38 |
硫酸镁 | 0.40 |
增白剂 | 0.19 |
香料、硅氧烷、抑泡剂 | 0.85 |
微量成分 | 凑足100% |
实施例34
粒状织物清洗组合物
成分 | A | B | C |
基础颗粒成分LAS/AS/AES(65/35)LAS/AS/AES(70/30)硅铝酸盐碳酸钠硅酸钠NaPAA固体PEG固体增白剂DTPA硫酸盐DC-1400脱气剂水份微量成分 | 9.95-14.0611.860.582.261.010.17-5.460.023.730.31 | -12.0515.7412.740.582.261.120.17-6.640.023.980.49 | -7.7017.1013.070.581.470.660.110.704.250.024.330.31 |
B.O.T.喷涂物非离子表面活性剂附聚物成分LAS/AS(25/75)硅铝酸盐碳酸盐PEG 4000水份/微量成分 | 0.5011.7013.738.110.594.88 | 0.509.6011.266.660.484.00 | 0.5010.4712.287.260.524.36 |
功能添加剂碳酸钠过硼酸盐AC基础涂层NOBS去污聚合物纤维素酶 | 7.371.03--0.410.33 | 6.981.031.00-0.410.33 | 7.452.56-2.400.310.24 |
蛋白酶1AE-碎片 | 0.10.40 | 0.050.40 | 0.150.29 |
液体喷涂物香料非离子喷涂物 | 0.421.00 | 0.421.00 | 0.420.50 |
微量成分 | 凑足100% |
实施例35
粒状织物清洗组合物
A | B | ||
表面活性剂-Na LAS-KLAS-AS/AE3S-TAS-C24AE5-Genagen-N-椰子基N-甲基葡糖胺(线型)-C8~10二甲基羟乙基氯化铵 | 6.40-6.400.083.48-1.141.00 | -9.904.390.11-1.882.821.40 | |
助洗剂-沸石-SKS-6-柠檬酸 | 20.5910.842.00 | 13.3910.78- | |
缓冲剂-碳酸盐-碳酸氢盐-硫酸盐-硅酸盐 | 9.602.002.640.61 | 12.072.00-0.16 | |
聚合物-丙烯酸/马来酸共聚物(Na)-羧甲基纤维素-聚合物 | 1.170.450.34 | 1.120.240.18 |
-六亚甲基二胺四-E24乙氧基化物,以氯代甲烷进行季铵化的产物 | 1.00 | 1.00 |
酶-蛋白酶1(纯酶百分含量)-纤维素酶-淀粉酶-脂酶 | 0.030.260.650.27 | 0.030.260.730.15 |
漂白剂-TAED(100%)-N-壬酰-6-氨基己酸的苯酚磺酸酯-过碳酸盐-HEDP-EDDS | 3.85-16.200.480.30 | 3.502.7518.300.480.30 |
其余-苹果酸颗粒-增白剂15/49-酞菁磺酸锌-聚二甲基硅氧烷,带有三甲基甲硅烷封端单元-皂类-香料 | 0.077/0.0140.00260.25-0.45 | 2.20+bicarb0.07/0.0140.00260.241.000.55 |
合计 | 100 | 100 |
实施例36
粒状织物清洗组合物
A | B | |
表面活性剂NaLASKLASFASASC25AE3SAE5 | 6.8-0.90.60.14.2 | 0.410.90.11.5-- |
N-椰子基-N-甲基葡糖胺GenagenC8~10二甲基羟乙基氯化铵 | --- | 1.81.21.0 |
助洗剂SKS-6沸石柠檬酸 | 3.317.21.5 | 9.018.9- |
缓冲剂碳酸盐碳酸氢钠硫酸盐苹果酸硅酸盐 | 21.1-15.2-0.1 | 15.02.65.52.9- |
聚合物丙烯酸/马来酸共聚物(Na)六亚甲基二胺四-E24乙氧基化物,以氯代甲烷进行二季铵化的产物聚合物CMC | 2.20.50.10.2 | 0.90.70.10.1 |
酶蛋白酶1(纯酶百分含量)脂酶淀粉酶纤维素酶 | 0.020.180.640.13 | 0.050.140.730.26 |
漂白剂TAEDN-壬酰-6-氨基己酸的苯酚磺酸酯过碳酸钠PB4EDDSMgSO4HEDP | 2.2--15.60.170.350.15 | 2.51.9613.1-0.210.470.34 |
其余增白剂-酞菁磺酸锌-聚二甲基硅氧烷,带有三甲基甲硅烷封端单元皂类香料彩斑剂 | 0.060.00150.040.50.350.5 | 0.040.00200.140.70.450.6 |
实施例37
下面的粒状衣物洗涤剂组合物37A~C是本发明专门为适合欧洲机器洗涤条件制备的:
成分 | A | B | C |
LAS | 7.0 | 5.61 | 4.76 |
TAS | - | - | 1.57 |
C45AS | 6.0 | 2.24 | 3.89 |
C25E3S | 1.0 | 0.76 | 1.18 |
C45E7 | - | 2.0 | |
C25E3 | 4.0 | 5.5 | - |
QAS | 0.8 | 2.0 | 2.0 |
STPP | - | - | |
沸石A | 25.0 | 19.5 | 19.5 |
柠檬酸 | 2.0 | 2.0 | 2.0 |
NaSKS-6 | 8.0 | 10.6 | 10.6 |
碳酸盐I | 8.0 | 10.0 | 8.6 |
MA/AA | 1.0 | 2.6 | 1.6 |
CMC | 0.5 | 0.4 | 0.4 |
PB4 | - | 12.7 | - |
过碳酸盐 | - | - | 19.7 |
TAED | 3.1 | 5.0 | |
柠檬酸盐 | 7.0 | - | - |
DTPMP | 0.25 | 0.2 | 0.2 |
HEDP | 0.3 | 0.3 | 0.3 |
QEA 1 | 0.9 | 1.2 | 1.0 |
蛋白酶1 | 0.02 | 0.05 | 0.035 |
脂酶 | 0.15 | 0.25 | 0.15 |
纤维素酶 | 0.28 | 0.28 | 0.28 |
淀粉酶 | 0.4 | 0.7 | 0.3 |
PVPI/PVNO | 0.4 | - | 0.1 |
光活化漂白剂(ppm) | 15ppm | 27ppm | 27ppm |
增白剂1 | 0.08 | 0.19 | 0.19 |
增白剂2 | - | 0.04 | 0.04 |
香料 | 0.3 | 0.3 | 0.3 |
泡腾颗粒(苹果酸40%、碳酸氢钠40%,碳酸钠20%) | 15 | 15 | 5 |
硅氧烷消泡剂 | 0.5 | 2.4 | 2.4 |
微量/惰性物凑足100% |
实施例38
下列配方是本发明组合物的一个实施例,它可以是颗粒形式或者是片剂形式。
成分 | 38 |
C45AS/TAS | 3.0 |
LAS | 8.0 |
C25AE3S | 1.0 |
NaSKS-6 | 9.0 |
C25AE5/AE3 | 5.0 |
沸石A | 10.0 |
SKS-(I)(干添加) | 2.0 |
MA/AA | 2.0 |
柠檬酸 | 1.5 |
EDDS | 0.5 |
HEDP | 0.2 |
PBI | 10.0 |
NACA OBS | 2.0 |
TAED | 2.0 |
碳酸盐 | 8.0 |
硫酸盐 | 2.0 |
淀粉酶 | 0.3 |
脂酶 | 0.2 |
酶 | 0.02 |
微量成分(增白剂/SRP1/CMC/光漂白剂/硫酸镁/PVPVI/抑泡剂/PEG) | 0.5 |
香料 | 0.5 |
实施例39
粒状衣物洗0.5涤剂组合物39A~E尤其适合日本机器洗涤条件,是按照本发明制备的:
成分 | A | B | C | D | E | |||
LAS | 23.57 | 23.57 | 21.67 | 21.68 | 21.68 | |||
FAS | 4.16 | 4.16 | 3.83 | 3.83 | 3.83 | |||
非离子表面活性剂 | 3.30 | 3.30 | 2.94 | 3.27 | 3.27 | |||
双(羟乙基)甲基烷基氯化铵 | 0.47 | 0.47 | 1.20 | 1.20 | 1.20 | |||
SKS-6 | 7.50 | 7.50 | 5.17 | 5.76 | 5.06 | |||
聚丙烯酸酯共聚物(MW11000)(马来酸/丙烯酸酯比例4∶6) | 7.03 | 7.03 | 14.36 | 14.36 | 14.36 | |||
沸石 | 11.90 | 11.40 | 10.69 | 11.34 | 11.34 | |||
碳酸盐 | 14.90 | 14.82 | 11.71 | 11.18 | 11.18 | |||
硅酸盐 | 12.00 | 12.00 | 12.37 | 12.38 | 12.38 | |||
蛋白酶1 | 0.016 | 0.016 | 0.046 | 0.046 | 0.046 | |||
脂酶 | - | - | 0.28 | - | - | |||
淀粉酶 | - | - | 0.62 | - | - | |||
纤维素酶 | - | - | 0.48 | - | 0.70 | |||
NOBS | 3.75 | 3.75 | 2.70 | 2.70 | 2.70 | |||
PB1 | 3.53 | - | 2.60 | - | - | |||
过碳酸钠 | - | 4.21 | - | 3.16 | 3.16 | |||
SRP | 0.52 | 0.52 | 0.70 | 0.70 | 0.70 | |||
增白剂 | 0.31 | 0.31 | 0.28 | 0.28 | 0.50 | |||
AE-复合碎片 | 0.17 | 0.20 | 0.17 | 0.17 | 0.17 | |||
聚二甲基硅氧烷 | - | - | 0.68 | 0.68 | 0.68 | |||
香料 | 0.06 | 0.06 | 0.08 | - | - | |||
香料 | - | - | - | 0.23 | 0.23 | |||
疏水、沉积二氧化硅 | 0.30 | 0.30 | 0.30 | 0.30 | 0.30 | |||
PEG4000 | 0.19 | 0.19 | 0.17 | 0.17 | 0.17 | |||
微量成分/惰性成分凑足100% |
液态织物清洗组合物
本发明液态织物清洗组合物优选包含有效数量1种或多种蛋白酶,优选为组合物重量的约0.0001%~约10,更优选约0.001%~约1%,最优选约0.001%~约0.1wt%活性蛋白酶。(参见美国专利5,679,630实施例)。
实施例40
液态织物清洗组合物
成分 | 实施例号 | ||||
A | B | C | D | E | |
蛋白酶1 | 0.05 | 0.03 | 0.03 | 0.03 | 0.10 |
蛋白酶2 | - | - | - | 0.1 | 0.20 |
C12~C14烷基硫酸酯,钠 | 20.00 | 20.00 | 20.00 | 20.00 | 20.00 |
2-丁基辛酸 | 5.00 | 5.00 | 5.00 | 5.00 | 5.00 |
柠檬酸钠 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
C10醇乙氧基化物(3) | 13.00 | 13.00 | 13.00 | 13.00 | 13.00 |
一乙醇胺 | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 |
水/丙二醇/乙醇(100∶1∶1) | 凑足100% |
2蛋白酶,除蛋白酶1以外的,包括但不限于本文所述可用于本发明的附加蛋白酶。
在实施例40D和E中,尤其要指出,本文所引可用于本发明的蛋白酶的任意组合均可用来替代蛋白酶1和蛋白酶2,并获得类似的结果。
实施例41
液态织物清洗组合物
成分 | 实施例号 | |
A | B | |
C12~C14链烯基琥珀酸 | 3.0 | 8.0 |
柠檬酸一水合物 | 10.0 | 15.0 |
C12-15烷基硫酸钠 | 8.0 | 8.0 |
C12~15醇2倍乙氧基化物的硫酸钠 | - | 3.0 |
C12~15醇7倍乙氧基化物 | - | 8.0 |
二亚乙基三胺五(亚甲基膦酸) | 0.2 | - |
油酸 | 1.8 | - |
乙醇 | 4.0 | 4.0 |
丙二醇 | 2.0 | 2.0 |
蛋白酶1 | 0.01 | 0.02 |
聚乙烯基吡咯烷酮 | 1.0 | 2.0 |
抑泡剂 | 0.15 | 0.15 |
氢氧化钠 | up to pH 7.5 | |
过硼酸盐 | 0.5 | 1 |
苯酚磺酸酯 | 0.1 | 0.2 |
过氧化物酶 | 0.4 | 0.1 |
水及微量成分 | 凑足100% |
实施例42
液态织物清洗组合物
成分 | 实施例号 |
40 | |
NaLAS(100%am) | 16 |
Neodol | 21.5 |
柠檬酸盐 | 6.8 |
EDDS | 1.2 |
分散剂 | 1.3 |
过硼酸盐 | 12 |
N-壬酰-6-氨基己酸的苯酚磺酸酯 | 6 |
蛋白酶1(%纯酶) | 0.03 |
淀粉酶 | 0.40 |
纤维素酶 | 0.03 |
溶剂(BPP) | 18.5 |
聚合物 | 0.1 |
碳酸盐 | 10 |
FWA 15 | 0.2 |
二氧化钛 | 0.5 |
PEG 8000 | 0.4 |
香料 | 1.0-1.2 |
抑泡剂 | 0.06 |
水及微量成分 | 凑足100% |
实施例43
液态织物清洗组合物
成分 | 实施例号 | |
A | B | |
DI H2O | 38.63 | - |
MEA | 0.48 | 9.0 |
氢氧化钠 | 4.40 | 1.0 |
聚二醇(pdiol) | 4.00 | 10.0 |
柠檬酸 | 2.50 | 2.0 |
硫酸钠 | 1.75 | - |
DTPA | 0.50 | 1.0 |
FWA预混合物(Br 15/MEA/NI 13-9) | 0.15 | 0.15 |
Na C25AE1.80S | 23.50 | - |
AE3S(H) | - | 4.0 |
C11.8HLAS | 3.00 | 14.0 |
Neodol | 2.00 | 6.0 |
乙醇 | 0.50 | 2.0 |
甲酸钙* | 0.10 | 0.1 |
Borax预混合物(Borax/MEA/聚二醇/柠檬酸) | 2.50 | - |
硼酸 | - | 1.0 |
C10 APA | 1.50 | - |
TEPA 105 | 1.20 | - |
FA C12-18 | 5.00 | - |
Neptune LC | 0.50 | - |
染料 | 0.0040 | 0.0015 |
纤维素酶 | 0.053 | 0.2 |
淀粉酶 | 0.15 | 0.2 |
蛋白酶1 | 0.1 | 0.1 |
DC 2-3597 | 0.12 | 0.2 |
油菜籽FA | 6.50 | 4.0 |
水及微量成分 | 凑足100% |
实施例44
液态织物清洗组合物
成分 | 44 |
氢氧化钠 | 5.50 |
聚二醇(pdiol) | 6.90 |
柠檬酸 | 1.50 |
DTPA | 1.50 |
FWA预混合物(Br15/MEA/NI 23-9) | 0.15 |
AE3S(H) | 2.50 |
LAS(H) | 13.0 |
Neodol | 2.00 |
乙醇 | 3.50 |
甲酸钙* | 0.10 |
硼酸 | 1.00 |
粘土 | 4.00 |
淀粉酶 | 0.15 |
蛋白酶1 | 0.02 |
脂肪酸 | 16.50 |
水及微量成分 | 凑足100% |
实施例45
液态织物清洗组合物
按本发明制备了下列特别适用于日本机洗洗涤条件的液态织物清洗组合物:
成分 | 45 |
AE2.5S | 15.00 |
AS | 5.50 |
N-椰子基N-甲基葡糖胺 | 5.00 |
非离子表面活性剂 | 4.50 |
柠檬酸 | 3.00 |
脂肪酸 | 5.00 |
碱 | 0.97 |
一乙醇胺 | 5.10 |
1,2-丙二醇 | 7.44 |
乙醇 | 5.50 |
HXS | 1.90 |
硼酸 | 3.50 |
乙氧基化四亚乙基五胺 | 3.00 |
SRP | 0.30 |
蛋白酶1 | 0.069 |
淀粉酶 | 0.06 |
纤维素酶 | 0.08 |
脂酶 | 0.18 |
增白剂 | 0.10 |
微量成分/惰性成分凑足100% |
实施例46
液态织物清洗组合物
按本发明制备了下列特别适用于日本机洗洗涤条件和细织物的液态织物清洗组合物:
成分 | 46 |
AE2.5S | 2.16 |
AS | 3.30 |
N-椰子基N-甲基葡糖胺 | 1.10 |
非离子表面活性剂 | 10.00 |
柠檬酸 | 0.40 |
脂肪酸 | 0.70 |
碱 | 0.85 |
一乙醇胺 | 1.01 |
1,2-丙二醇 | 1.92 |
乙醇 | 0.24 |
HXS | 2.09 |
蛋白酶1 | 0.01 |
淀粉酶 | 0.06 |
微量成分/惰性成分凑足100% |
条块状织物清洁剂组合物
适合手洗脏织物的本发明条块状织物清洁剂组合物典型地包含有效数量1种或多种蛋白酶,优选为组合物的约0.001~约10%,更优选约0.01~约1wt%活性蛋白酶。(参见美国专利5,679,630)。
实施例47
条块状织物清洁剂组合物
成分 | 实施例号 | |||
A | B | C | D | |
蛋白酶1 | 0.3 | - | 0.1 | 0.02 |
蛋白酶2 | - | - | 0.4 | 0.1 |
C12~C16烷基硫酸钠 | 20.0 | 20.0 | 20.0 | 20.00 |
C12~C14N-甲基葡糖酰胺 | 5.0 | 5.0 | 5.0 | 5.00 |
C11~C13烷基苯磺酸钠 | 10.0 | 10.0 | 10.0 | 10.00 |
焦磷酸钠 | 7.0 | 7.0 | 7.0 | 7.00 |
三聚磷酸钠 | 7.0 | 7.0 | 7.0 | 7.00 |
沸石A(0.1~.10μm) | 5.0 | 5.0 | 5.0 | 5.00 |
羧甲基纤维素 | 0.2 | 0.2 | 0.2 | 0.20 |
聚丙烯酸酯(MW 1400) | 0.2 | 0.2 | 0.2 | 0.20 |
椰子基一乙醇酰胺 | 5.0 | 5.0 | 5.0 | 5.00 |
增白剂、香料 | 0.2 | 0.2 | 0.2 | 0.20 |
硫酸钙 | 1.0 | 1.0 | 1.0 | 1.00 |
硫酸镁 | 1.0 | 1.0 | 1.0 | 1.00 |
水 | 4.0 | 4.0 | 4.0 | 4.00 |
填料* | 凑足100% |
*可选自传统材料如碳酸钙、滑石粉、粘土、硅酸盐等。
2蛋白酶,除蛋白酶1以外的,包括但不限于本文所述可用于本发明的附加蛋白酶。
在实施例47C和D中,尤其要指出,本文所引可用于本发明的蛋白酶的任意组合均可用来替代蛋白酶1和蛋白酶2,并获得类似的结果。
4.口腔清洗组合物
口腔清洗组合物(牙粉、牙膏、牙用凝胶、牙粉、漱口水、口腔喷雾剂、口腔凝胶、口香糖、锭剂、香囊、片剂、生物凝胶、预防膏、牙科处理液等)典型地包含药物学可接受数量1种或多种蛋白酶,优选约0.0001%~约20%,更优选约0.001%~约10%,最优选约0.01%~约5wt%用于从牙齿或牙托上清除蛋白类龋斑的活性蛋白酶。(参见美国专利5,679,630实施例)。
实施例48
牙粉组合物
成分 | 实施例号 | |||
A | B | C | D | |
蛋白酶1 | 0.4 | 0.35 | 0.15 | 0.2 |
山梨醇(70%水溶液) | 35.000 | 35.000 | 35.000 | 35.000 |
PEG-6* | 1.000 | 1.000 | 1.000 | 1.000 |
二氧化硅牙科磨料** | 20.000 | 20.000 | 20.000 | 20.000 |
氟化钠 | 0.243 | 0.243 | 0.243 | 0.243 |
二氧化钛 | 0.500 | 0.500 | 0.500 | 0.500 |
糖精钠 | 0.286 | 0.286 | 0.286 | 0.286 |
烷基硫酸钠(27.9%水溶液) | 4.000 | 4.000 | 4.000 | 4.000 |
香料 | 1.040 | 1.040 | 1.040 | 1.040 |
羧乙烯基聚合物*** | 0.300 | 0.300 | 0.300 | 0.300 |
角叉菜胶**** | 0.800 | 0.800 | 0.800 | 0.800 |
水 | 凑足100% |
*PEG-6=分子量600的聚乙二醇。
**沉淀法二氧化硅,商品名Zeodent 119,由J.M.Huber供应。
***Carbopol,由B.F.Goodrich化学公司供应。
****Iota角叉菜胶由Hercules化学公司供应。
实施例49
漱口组合物
成分 | 实施例号 | |||
A | B | C | D | |
蛋白酶1 | 0.3 | 0.75 | 0.5 | 1.00 |
SDA 40醇 | 8.00 | 8.00 | 8.00 | 8.00 |
香料 | 0.08 | 0.08 | 0.08 | 0.08 |
氟化钠 | 0.05 | 0.05 | 0.05 | 0.05 |
甘油 | 10.00 | 10.00 | 10.00 | 10.00 |
甜味剂 | 0.02 | 0.02 | 0.02 | 0.02 |
苯甲酸 | 0.05 | 0.05 | 0.05 | 0.05 |
氢氧化钠 | 0.20 | 0.20 | 0.20 | 0.20 |
染料 | 0.04 | 0.04 | 0.04 | 0.04 |
水 | 凑足100% |
实施例50
锭剂组合物
成分 | 实施例号 | |||
A | B | C | D | |
蛋白酶1 | 0.01 | 0.03 | 0.10 | 0.02 |
山梨醇 | 17.50 | 17.50 | 17.50 | 17.50 |
甘露糖醇 | 17.50 | 17.50 | 17.50 | 17.50 |
淀粉 | 13.60 | 13.60 | 13.60 | 13.60 |
甜味剂 | 1.20 | 1.20 | 1.20 | 1.20 |
香料 | 11.70 | 11.70 | 11.70 | 11.70 |
着色剂 | 0.10 | 0.10 | 0.10 | 0.10 |
玉米糖浆 | 凑足100% |
实施例51
口香糖组合物
成分 | 实施例号 | |||
A | B | C | D | |
蛋白酶1 | 0.03 | 0.02 | 0.10 | 0.05 |
山梨醇晶体 | 38.44 | 38.40 | 38.40 | 38.40 |
Paloja-T树胶基质* | 20.00 | 20.00 | 20.00 | 20.00 |
山梨醇(70%水溶液) | 22.00 | 22.00 | 22.00 | 22.00 |
甘露糖醇 | 10.00 | 10.00 | 10.00 | 10.00 |
甘油 | 7.56 | 7.56 | 7.56 | 7.56 |
香料 | 1.00 | 1.00 | 1.00 | 1.00 |
*由L.A.Dreyfus公司提供。
5.牙托清洗组合物
牙托清洗组合物典型地包含有效数量1种或多种蛋白酶,优选为组合物与牙托清洁载体的约0.0001%~约50%,更优选约0.001~约35%,最优选约0.01%~约20wt%活性蛋白酶。(参见美国专利5,679,630实施例)。
实施例52
双层泡腾牙托清洁片剂
成分 | 实施例号 | |||
A | B | C | D | |
酸性层 | ||||
蛋白酶1 | 1.0 | 1.5 | 0.01 | 0.05 |
酒石酸 | 24.0 | 24.0 | 24.00 | 24.00 |
碳酸钠 | 4.0 | 4.0 | 4.00 | 4.00 |
氨基磺酸 | 10.0 | 10.0 | 10.00 | 10.00 |
PEG 20,000 | 4.0 | 4.0 | 4.00 | 4.00 |
碳酸氢钠 | 24.5 | 24.5 | 24.50 | 24.50 |
过硫酸钾 | 15.0 | 15.0 | 15.00 | 15.00 |
酸性焦磷酸钠 | 7.0 | 7.0 | 7.00 | 7.00 |
高温法二氧化硅 | 2.0 | 2.0 | 2.00 | 2.00 |
四乙酰乙二胺 | 7.0 | 7.0 | 7.00 | 7.00 |
蓖麻油酰磺基琥珀酸酯 | 0.5 | 0.5 | 0.50 | 0.50 |
香料 | 1.0 | 1.0 | 1.00 | 1.00 |
碱性层 | ||||
过硼酸钠一水合物 | 32.0 | 32.0 | 32.00 | 32.00 |
碳酸氢钠 | 19.0 | 19.0 | 19.00 | 19.00 |
EDTA | 3.0 | 3.0 | 3.00 | 3.00 |
三聚磷酸钠 | 12.0 | 12.0 | 12.00 | 12.00 |
PEG 20,000 | 2.0 | 2.0 | 2.00 | 2.00 |
过硫酸钾 | 26.0 | 26.0 | 26.00 | 26.00 |
碳酸钠 | 2.0 | 2.0 | 2.00 | 2.00 |
高温法二氧化硅 | 2.0 | 2.0 | 2.00 | 2.00 |
染料/香料 | 2.0 | 2.0 | 2.00 | 2.00 |
虽然已就本发明具体实施方案做了描述,但本领域技术人员十分清楚,在不偏离本发明精神和范围的条件下还可针对本发明做出各种各样改变和修改。本发明人认为,所有此类属于本发明范围内的修改均涵盖在所附权利要求范围内。
本发明组合物适于采用配制人选择的任何方法来制备,其非限制性实施例载于美国专利5,691,297,1997-11-11授予Nassano等人;美国专利5,574,005,1996-11-12授予Welch等人;美国专利5,569,645,1996-10-29授予Dinniwell等人;美国专利5,565,422,1996-10-15授予Del Greco等人;美国专利5,516,448,1996-05-14授予Capeci等人;美国专利5,489,392,1996-02-06授予Capeci等人;美国专利5,486,303,1996-01-23授予Capeci等人,在此全部并入本文作为参考。
除了以上的实施例之外,本发明清洗组合物还可被配制到任何适当的洗衣洗涤剂组合物中,其非限制性实施例载于美国专利5,679,630,1997-10-21授予Baeck等人;美国专利5,565,145,1996-10-15授予Watson等人;美国专利5,478,489,1995-12-26授予Fredj等人;美国专利5,470,507,1995-11-28授予Fredj等人;美国专利5,466,802,1995-11-14授予Panandiker等人;美国专利5,460,752,1995-10-24授予Fredj等人;美国专利5,458,810,1995-10-17授予Fredj等人;美国专利5,458,809,1995-10-17授予Fredj等人;美国专利5,288,431,1994-02-22授予Huber等人,全部收作本文的参考。
在结合优选实施方案及实施例详细描述了本发明之后,本领域技术人员将十分清楚,在不偏离本发明范围的条件下还可做出各种各样的修改和改变,因此本发明不应视为仅局限于本说明书中所描述的内容。
Claims (38)
1.一种织物和/或餐具洗涤和/或硬表面清洗组合物,包含:
(a)有效量蛋白酶变体,其中所述蛋白酶变体包括对1个或多个对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的230和232位点处氨基酸残基以另一种天然存在的氨基酸残基进行取代;以及
(b)1种或多种清洗添加剂材料。
2.权利要求1的清洗组合物,其中所述蛋白酶变体由芽胞杆菌属枯草杆菌蛋白酶衍生而来。
3.权利要求1的清洗组合物,其中所述蛋白酶变体由迟缓芽胞杆菌枯草杆菌蛋白酶或枯草杆菌蛋白酶309衍生而来。
4.权利要求1的清洗组合物,其中所述蛋白酶变体包括对1个或多个选自下列位点的氨基酸残基进行取代:
(1)位点230以及下列中1个或多个位点:68、103、104、159、232、236和245;
(2)位点232以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(3)位点232以及下列中1个或多个位点:103、104、236和245;
(4)位点232和103以及下列中1个或多个位点:12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275
(5)位点232和104以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(6)位点232和236以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(7)位点232和245以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(8)位点232、103、104、236和245以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275.
。
5.权利要求1的清洗组合物,其中所述蛋白酶变体包括对选自下列中一组的位点的取代:
12/102/103/104/159/212/232/236/245/248/252;61/68/103/104/159/232/236/245/248/252;
62/103/104/130/159/213/232/236/245/248/252;62/103/104/159/213/232/236/245/248/252;
62/103/104/109/159/213/232/236/245/248/252;62/103/104/159/232/236/245/248/252;
62/101/103/104/159/212/213/232/236/245/248/252;
68/103/104/159/232/236/245/248/252/270;
68/103/104/159/185/232/236/245/248/252; 68/103/104/159/210/232/236/245/248/252;
68/103/104/159/185/210/232/236/245/248/252; 68/103/104/159/213/232/236/245/248/252;
68/103/104/159/230/232/236/245; 68/76/103/104/159/209/232/236/245;
68/103/104/232/236/245/248/257/275; 68/103/104/213/232/2236/245/248/252;
68/103/104/159/232/236/245/248/252; 68/103/104/159/209/232/236/245;
68/76/103/104/159/232/236/245; 68/103/104/159/232/236/245/252;
68/103/104/159/232/236/245; 68/103/104/159/232/236/245/257;
68/76/103/104/159/211/232/236/245; 68/76/103/104/159/215/232/236/245;
68/103/104/159/210/232/236/245; 68/103/104/159/213/232/236/245/260;
68/76/103/104/159/213/232/236/245/260; 68/76/103/104/159/210/232/236/245/260;
68/103/104/159/183/232/236/245/248/252; 68/103/104/232/236/245/257/275;
68/103/104/159/213/232/236/245; 76/103/104/159/232/236/245;
76/103/104/159/213/232/236/245/260; 76/103/104/131/159/232/236/245/248/252;
97/103/104/159/232/236/245/248/252; 98/103/104/159/232/236/245/248/252;
98/102/103/104/159/212/232/236/245/248/252; 101/103/104/159/232/236/245/248/252;
102/103/104/159/232/236/245/248/252; 103/104/159/232/236/245;
103/104/159/232/236/245/248/252;
103/104/159/205/209/232/236/245/257 103/104/159/232/245/248/252;
103/104/159/205/209/210/232/236/245/257; 103/104/159/213/232/236/245/248/252;
103/104/159/217/232/236/245/248/252; 103/104/130/159/232/236/245/248/252;
103/104/131/159/232/236/245/248/252; 103/104/159/205/209/232/236/245;和
103/104/159/232/236/245/257.
6.权利要求5的清洗组合物,其中所述蛋白酶变体包括对选自下列一组的位点的取代:
12R/102A/103A/104I/159D/212G/232V/236H/245R/248D/252K;
61E/68A/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/109R/159D/213R/232V/236H/245R/248D/252K;
62D/103A/104I/159D/213R/232V/236H/245R/248D/252K;
62D/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/130G/159D/213R/232V/236H/245R/248D/252K;62D/101G/103A/104I/159D/212G/213R/232V/236H/245R/248D/252K;
68A/76D/103A/104I/159D/213R/232V/236H/245R/260A;
68A/76D/103A/104I/159D/210I/232V/236H/245R/260A;
68A/103A/104I/159D/183D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/209W/232V/236H/245R;
68A/76D/103A/104I/159D/211R/232V/236H/245R;
68A/76D/103A/104I/159D/215R/232V/236H/245R;
68A/103A/104I/159D/213R/232V/236H/245R/260A;
68A/76D/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/252K;
68A/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/257V;
68A/103A/104I/159D/185D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/185D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/230V/232V/236H/245R;
68A/76D/103A/104I/159D/209W/232V/236H/245R;
68A/103A/104I/232V/236H/245R/248D/257V/275H;
68A/103A/104I/232V/236H/245R/257V/275H;
68A/103A/104I/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210I/232V/236H/245R;
68A/103A/104I/159D/210L/232V/236H/245R;
68A/103A/104I/159D/213G/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/248D/252K/270A;
76D/103A/104I/159D/232V/236H/245R;
76D/103A/104I/131V/159D/232V/236H/245R/248D/252K;
76D/103A/104I/159D/213R/232V/236H/245R/260A;
97E/103A/104I/159D/232V/236H/245R/248D/252K;
98L/103A/104I/159D/232V/236H/245R/248D/252K;
98L/102A/103A/104I/159D/212G/232V/236H/245R/248D/252K;
101G/103A/104I/59D/232V/236H/245R/248D/252K;
102A/103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/213R/232V/236H/245R/248D/252K;
103A/104I/130G/159D/232V/236H/245R/248D/252K;
103A/104I/159D/217E/232V/236H/245R/248D/252K;
103A/104I/159D/232V/236H/245R;
103A/104I/159D/205I/09W/232V/236H/245R;
103A/104I/159D/232V/236H/245R/257V;
103A/104I/159D/205I/209W/232V/236H/245R/257V;
103A/104I/131V/159D/232V/236H/245R/248D/252K;
103A/104I/159D/205I/209W/210I/232V/236H/245R/257V;和
103A/104I/159D/232V/245R/248D/252K.
7.权利要求1的清洗组合物,其中所述清洗添加剂材料选自下列材料:表面活性剂、溶剂、缓冲剂、酶、去污剂、去粘土污垢剂、分散剂、增白剂、抑泡剂、织物柔软剂、起泡助剂、酶稳定剂、助洗剂、其他漂白剂、染料、香料、螯合剂及其混合物。
8.权利要求7的清洗组合物,其中所述清洗添加剂材料包含至少1种洗涤表面活性剂。
9.权利要求8的清洗组合物,其中所述的洗涤表面活性剂是支链表面活性剂。
10.权利要求9的清洗组合物,其中所述的洗涤表面活性剂是中链支化的表面活性剂。
11.权利要求7的清洗组合物,其中清洗添加剂材料包含组合物重量的至少0.1wt%表面活性剂,所述表面活性剂包含选自下列的材料:烷基苯磺酸盐、伯烷基硫酸盐、仲烷基硫酸盐、烷基烷氧基硫酸盐、烷基烷氧基羧酸盐、烷基聚糖苷以及其相应的硫酸化聚糖苷、α-磺化脂肪酸酯、烷基及烷基酚的烷氧基化物、甜菜碱及磺基甜菜碱、氧化胺、N-甲基葡糖酰胺、非离子伯醇乙氧基化物、非离子伯醇混合乙氧基/丙氧基化物,以及上述的混合物。
12.权利要求11的清洗组合物,还包含至少5%助洗剂,选自沸石、多羧化物、层状硅酸盐、磷酸盐,及其混合物。
13.权利要求7的清洗组合物,其中所述清洗添加剂材料包含至少1种洗涤酶,选自纤维素酶、脂酶、淀粉酶、磷脂酶、其他蛋白酶、过氧化物酶,及其混合物。
14.权利要求7的清洗组合物,其中所述清洗添加剂材料包含至少1种漂白剂,以及视需要还包含至少1种漂白活化剂,还视需要包含至少1种漂白催化剂。
15.权利要求14的清洗组合物,其中所述的漂白剂选自过碳酸盐、过硼酸盐及其混合物;所述的漂白活化剂选自苯甲酰氧基苯磺酸盐、壬酰氧基苯磺酸盐、癸酰氧基苯磺酸盐、辛酰氧基苯磺酸盐、全水解酯、4-[N-(壬酰基)氨基己酰氧基]-苯磺酸钠盐、月桂酰氧基苯磺酸盐、10-十一碳烯酰氧基苯磺酸盐以及十二酰氧基苯甲酸及其混合物;所述的漂白催化剂是丙烷磺酸3-(3,4-二氢异喹啉_)。
16.权利要求1的清洗组合物,其中所述清洗组合物是织物清洗组合物,包含组合物重量的至少5%表面活性剂及至少5wt%助洗剂。
17.权利要求16的清洗组合物,其中所述的织物清洗组合物为液体、粒状、条块状、片剂、凝胶、粉末或泡沫形式。
18.权利要求1的清洗组合物,其中所述清洗组合物是织物清洗组合物,包含:
(a)0.0001%~10wt%所述蛋白酶变体;以及
(b)至少5wt%表面活性剂;而且其中助洗剂选自沸石、多羧化物、层状硅酸盐、磷酸盐,及其混合物;以及
(c)至少5wt%助洗剂。
19.权利要求18的清洗组合物,其中组分(b)的表面活性剂选自烷基苯磺酸盐、伯烷基硫酸盐、仲烷基硫酸盐、烷基烷氧基硫酸盐、烷基烷氧基羧酸盐、烷基聚糖苷以及它们相应的硫酸化聚糖苷、α-磺化脂肪酸酯、烷基及烷基酚烷氧基化物、甜菜碱及磺基甜菜碱、氧化胺、N-甲基葡糖酰胺、非离子伯醇乙氧基化物、非离子伯醇混合乙氧基/丙氧基化物,以及上述的混合物;组分(C)的助洗剂选自沸石、多羧化物、层状硅酸盐、磷酸盐,及其混合物。
20.权利要求18或19的清洗组合物,为浓缩粒状织物清洗组合物形式,包含至少15%表面活性剂。
21.一种清洁织物的方法,所述方法包括令需要清洁的织物与权利要求16-19中任一项的清洗组合物进行接触。
22.权利要求1的清洗组合物,其中所述清洗组合物是餐具洗涤组合物,包含:
(a)0.0001%~10wt%所述蛋白酶变体;以及
(b)0.1%~10wt%表面活性剂。
23.权利要求22的清洗组合物,其中所述的餐具洗涤组合物为液体、粒状、粉末,凝胶或片剂形式。
24.一种清洗餐具的方法,所述方法包括令需要清洗的餐具与权利要求22或23的清洗组合物进行接触。
25.一种个人清洗组合物,包含:
(a)有效量蛋白酶变体,其中所述蛋白酶变体包括以另一种天然存在的氨基酸残基对1个或多个对应于解淀粉芽胞杆菌枯草杆菌蛋白酶的230和232位点的氨基酸残基进行取代;以及
(b)1种或多种清洗添加剂材料。
26.权利要求25的个人清洗组合物,其中所述个人清洗组合物包含:
(a)0.001%~5wt%所述蛋白酶变体;和
(b)0.1%~95wt%表面活性剂体系;以及
(c)视需要含有0.05%~50wt%酶稳定剂。
27.权利要求26的个人清洗组合物,其中所述组分(a)的蛋白酶变体的含量为0.001~2wt%;所述组分(b)的表面活性剂体系选自阴离子羧酸盐、氧化胺、烷基葡糖苷、葡糖酰胺、烷基硫酸盐、烷基醚硫酸盐、酰基羟乙磺酸盐、烷基磺基琥珀酸盐、烷基磷酸酯、乙氧基化磷酸酯、烷基甘油醚磺酸盐,及其混合物。
28.权利要求27的个人清洗组合物,其中所述组分(a)的蛋白酶变体的含量为0.002%~0.8wt%,所述组分(b)的表面活性剂体系选自皂类、酰基谷氨酸盐、烷基肌氨酸盐、氧化月桂胺、氧化椰子胺、椰子酰氨基氧化丙胺、癸基葡糖苷、月桂基硫酸盐、月桂基乙基硫酸盐、C12-18酰基羟乙磺酸盐,及其混合物。
29.权利要求26-28中任一项的个人清洗组合物,其中所述表面活性剂是皂,其用量为清洗组合物重量的至少2%。
30.权利要求29的个人清洗组合物,其中所述表面活性剂的用量为清洗组合物重量的至少10%。
31.权利要求30的个人清洗组合物,其中所述表面活性剂的用量为清洗组合物重量的至少25wt%。
32.权利要求29的个人清洗组合物,其中皂与蛋白酶变体之间的比例为2,000∶1~8∶1。
33.权利要求32的个人清洗组合物,其中皂与蛋白酶变体之间的比例为400∶1~40∶1。
34.一种个人清洁方法,所述方法包括令人体或较低级动物身体需要清洗的部分与权利要求25的清洗组合物进行接触。
35.一种预处理需要清洗的织物的方法,所述方法包括在以含表面活性剂的水溶液洗涤所述织物之前,令所述织物与权利要求16-19中任一项的清洗组合物接触。
36.一种织物和/或洗餐具和/或硬表面清洗组合物,含有:
(a)有效量蛋白酶变体,其中所述蛋白酶变体包括以另一种天然氨基酸残基,对解淀粉芽胞杆菌枯草杆菌蛋白酶的相应62、212、230、232、252和257处一个或多个氨基酸位点的氨基酸残基进行取代;其中所述蛋白酶变体包括对选自下列的一个或多个位点处的氨基酸残基的取代:
(1)位点62以及下列中1个或多个位点:103,104,109,159,213,232,236,245,248和252;
(2)位点212以及下列中1个或多个位点:12,98,102,103,104,159,232,236,245,248和252;
(3)位点230以及下列中1个或多个位点:68,103,104,159,232,236和245;
(4)位点232以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(5)位点232以及下列中1个或多个位点:
103,104,236和245;
(6)位点232和103以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(7)位点232和104以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(8)位点232和236以及下列中1或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(9)位点232和245以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(10)位点232、103、104、236和245以及下列中1个或多个位点:
12,61,62,68,76,97,98,101,102,103,104,109,130,131,159,183,185,205,209,210,212,213,217,230,236,245,248,252,257,260,270和275;
(11)位点252以及下列中1个或多个位点:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248和270;
(12)位点252以及下列中1个或多个位点:103、104、236及245;
(13)位点252和103以及下列中1个或多个位点:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248和270;
(14)位点252和104以及下列中1个或多个位点:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248和270;
(15)位点252和236以及下列中1个或多个位点:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248和270;
(16)位点252和245以及下列中1个或多个位点:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248和270;
(17)位点252、103、104、236和245以及下列中1个或多个位点:
12,61,62,68,97,98,101,102,103,104,109,130,131,159,183,185,210,212,213,217,232,236,245,248和270;
(18)位点257以及下列中1个或多个位点:68,103,104,205,209,210,232,236,245和275。
37.一种织物和/或洗餐具和/或硬表面清洗组合物,含有:
(a)有效量蛋白酶变体,其中所述蛋白酶变体包括以另一种天然氨基酸残基,对解淀粉芽胞杆菌枯草杆菌蛋白酶的相应62、212、230、232、252和257处一个或多个氨基酸位点的氨基酸残基进行取代;其中所述蛋白酶变体包括选自下列的各组取代:
12/102/103/104/159/212/232/236/245/248/252;61/68/103/104/159/232/236/245/248/252;
62/103/104/130/159/213/232/336/245/248/252;62/103/104/159/213/232/236/245/248/252;
62/103/104/109/159/213/232/236/245/248/252;62/103/104/159/232/236/245/248/252;
62/101/103/104/159/212/213/232/236/245/248/252;
68/103/104/159/232/236/245/248/252/270;
68/103/104/159/185/232/236/245/248/252; 68/103/104/159/210/232/236/245/248/252;
68/103/104/159/185/210/232/236/245/248/252;68/103/104/159/213/232/236/245/248/252;
68/103/104/159/230/232/236/245; 68/76/103/104/159/209/232/236/245;
68/103/104/232/236/245/248/257/275; 68/103/104/213/232/236/245/248/252;
68/103/104/159/232/236/245/248/252; 68/103/104/159/209/232/236/245;
68/76/103/104/159/232/236/245; 68/103/104/159/232/236/245/252;
68/103/104/159/232/236/245; 68/103/104/159/232/236/245/257;
68/76/103/104/159/211/232/236/245; 68/76/103/104/159/215/232/236/245;
68/103/104/159/210/232/236/245; 68/103/104/159/213/232/236/245/260;
68/76/103/104/159/213/232/236/245/260; 68/76/103/104/159/210/232/236/245/260;
68/103/104/159/183/232/236/245/248/252; 68/103/104/232/236/245/257/275;
68/103/104/159/213/232/236/245; 76/103/104/159/232/236/245;
76/103/104/159/213/232/236/245/260; 76/103/104/131/159/232/236/245/248/252;
97/103/104/159/232/236/245/248/252; 98/103/104/159/232/236/245/248/252;
98/102/103/104/159/212/232/236/245/248/252;101/103/104/159/232/236/245/248/252;
102/103/104/159/232/236/245/248/252; 103/104/159/232/236/245;
103/104/159/248/252/270; 103/104/159/232/236/245/248/252;
103/104/159/205/209/232/236/245/257 103/104/159/232/245/248/252;
103/104/159/205/209/210/232/236/245/257; 103/104/159/213/232/236/245/248/252;
103/104/159/217/232/236/245/248/252; 103/104/130/159/232/236/245/248/252;
103/104/131/159/232/236/245/248/252; 103/104/159/205/209/232/236/245;和
103/104/159/232/236/245/257.
38.权利要求37的清洗组合物,其中所述蛋白酶变体包括选自下列的各组取代:
12R/102A/103A/104I/159D/212G/232V/236H/245R/248D/252K;
61E/68A/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/109R/159D/213R/232V/236H/245R/248D/252K;
62D/103A/104I/159D/213R/232V//236H/245R/248D/252K;
62D/103A/104I/159D/232V/236H/245R/248D/252K;
62D/103A/104I/130G/159D/213R/232V/236H/245R/248D/252K;
62D/101G/103A/104I/159D/212G/213R/232V/236H/245R/248D/252K;
68A/76D/103A/104I/159D/213R/232V/236H/245R/260A;
68A/76D/103A/104I/159D/210I/232V/236H/245R/260A;
68A/103A/104I/159D/183D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/209W/232V/236H/245R;
68A/76D/103A/104I/159D/211R/232V/236H//245R;
68A/76D/103A/104I/159D/215R/232V/236H/245R;
68A/103A/104I/159D/213R/232V/236H/245R/260A;
68A/76D/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/252K;
68A/103A/104I/159D/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/257V;
68A/103A/104I/159D/185D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/185D/210L/232V/236H/245R/248D/252K;
68A/103A/104I/159D/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/230V/232V/236H/245R;
68A/76D/103A/104I/159D/209W/232V/236H/245R;
68A/103A/104I/232V/236H/245R/248D/257V/275H;
68A/103A/104I/232V/236H/245R/257V/275H;
68A/103A/104I/213E/232V/236H/245R/248D/252K;
68A/103A/104I/159D/232V/236H/245R/248D/252K;
68A/103A/104I/159D/210I/232V/236H/245R;
68A/103A/104I/159D/210L/232V/236H/245R;
68A/103A/104I/159D/213G/232V/236H/245R;
68A/103A/104I/159D/232V/236H/245R/248D/252K/270A;
76D/103A/104I/159D/232V/236H/245R;
76D/103A/104I/131V/159D/232V/236H/245R/248D/252K;
76D/103A/104I/159D/213R/232V/236H/245R/260A;
97E/103A/104I/159D/232V/236H/245R/248D/252K;
98L/103A/104I/159D/232V/236H/245R/248D/252K;
98L/102A/103A/104L/159D/212G/232V/236H/245R/248D/252K;
101G/103A/104I/159D/232V/236H/45R/248D/252K;
102A/103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/232V/236H/245R/248D/252K;
103A/104I/159D/213R/232V/236H/245R/248D/252K;
103A/104I/130G/159D/232V/236H/245R/248D/252K;
103A/104I/159D/217E/232V/236H/245R/248D/252K;
103A/104I/159D/248D/252K/270V;
103A/104I/159D/232V/236H/245R;
103A/104I/159D/205I/209W/232V/236H/245R;
103A/104I/159D/232V/236H/245R/257V;
103A/104I/159D/205I/209W/232V/236H/245R/257V;
103A/104I/131V/159D/232V/236H/245R/248D/252K;
103A/104I/159D/205I/209W/210I/232V/236H/245R/257V;和
103A/104I/159D/232V/245R/248D/252K.
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CNB2004100399766A Expired - Fee Related CN100506963C (zh) | 1997-10-23 | 1998-10-23 | 包含多取代的蛋白酶变体的漂白组合物 |
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CN98811435A Pending CN1279721A (zh) | 1997-10-23 | 1998-10-23 | 具有改变的净电荷、在去污剂中使用的多点置换的蛋白酶变体 |
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CNB988126400A Expired - Fee Related CN100558871C (zh) | 1997-10-23 | 1998-10-23 | 含有多取代的蛋白酶变体和淀粉酶变体的清洗组合物 |
CNB988126109A Expired - Lifetime CN1191347C (zh) | 1997-10-23 | 1998-10-23 | 含多取代蛋白酶变体的清洗组合物 |
CN2010105436254A Pending CN102021159A (zh) | 1997-10-23 | 1998-10-23 | 具有改变的净电荷、在去污剂中使用的多点置换的蛋白酶变体 |
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EP (10) | EP1027418B2 (zh) |
JP (7) | JP2001520046A (zh) |
KR (6) | KR20010024555A (zh) |
CN (9) | CN1295313C (zh) |
AR (10) | AR015977A1 (zh) |
AT (9) | ATE292179T1 (zh) |
AU (7) | AU742632B2 (zh) |
BR (6) | BR9815230A (zh) |
CA (9) | CA2307638C (zh) |
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DE (7) | DE69839057T2 (zh) |
DK (9) | DK1398367T3 (zh) |
EG (2) | EG22075A (zh) |
ES (7) | ES2319470T5 (zh) |
HK (1) | HK1039159B (zh) |
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ID (4) | ID26501A (zh) |
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NO (3) | NO20001901L (zh) |
NZ (3) | NZ520771A (zh) |
PL (1) | PL191529B1 (zh) |
PT (8) | PT1027418E (zh) |
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