CN1167503A - 一种具有脂解活性的酶 - Google Patents

一种具有脂解活性的酶 Download PDF

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CN1167503A
CN1167503A CN95196494A CN95196494A CN1167503A CN 1167503 A CN1167503 A CN 1167503A CN 95196494 A CN95196494 A CN 95196494A CN 95196494 A CN95196494 A CN 95196494A CN 1167503 A CN1167503 A CN 1167503A
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T·萨达勒
S·阔比内
L·V·果弗德
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Abstract

本发明特别涉及含有可编码表现出脂解活性的酶之DNA序列的DNA构建体，它包括：a)如SEQIDNo.1所示DNA的序列，或b)如SEQIDNo.1所示的DNA序列的类似物，其i)与SEQIDNo.1DNA所示序列同源，及/或ii)与和如SEQIDNo.1所示的DNA序列相同的寡核苷酸探针杂交，及/或iii)编码多肽，该多肽与由DNA序列(含如SEQIDNo.1所示的DNA序列)编码的多肽同源，及/或iv)编码多肽，该多肽可与抗纯化脂解酶(由如SEQIDNo.1所示的DNA序列所编码)的抗体发生免疫反应，及/或其衍生自HumicolainsolensDSM1800。本发明还公开了：由这种DNA构建体所编码的脂解酶；以及酶制剂、洗涤剂添加剂和含该脂解酶的洗涤剂组合物。

Description

一种具有脂解活性的酶
                            发明领域
本发明涉及一种具有脂解活性的酶、一种含有编码该酶的DNA序列的DNA构建体，以及一种产生该酶的方法。本发明尤其进一步涉及酶制剂、含有该酶的洗涤添加剂和洗涤剂组合物，以及把该酶用于多种工业和家庭用途。
                             发明背景
尽管在文献中对许多脂解酶[即可以催化脂质(如甘油三酯)水解的酶]已进行过描述和特性分析，人们仍然不断需要能提供新颖的脂解酶(尤其是单一成分形式的酶)，这些酶要具有能改善其多种工业或家庭用途的特性，例如在用于衣物洗涤或洗碟的洗涤剂组合物，或者用于硬表面清洗、脱脂等等的组合物方面。
                            发明综述
本发明现在已经令人惊奇地成功从Humicola·insolens菌株中分离出一种DNA序列并进行了特性分析，该序列编码表现出脂解活性的酶，因而有可能制备出单一成分的脂解酶。我们发现本发明的脂解酶尤其可具有更加适应上述多种用途的特性(参见下文)。
因此，本发明的第一方面涉及含有可编码表现出脂解活性之酶的DNA序列的DNA构建体，该DNA序列包括
a)如SEQ ID No.1所示的DNA序列，及/或可编码脂解酶的DNA序列，其可得自酿酒酵母DSM9975中的质粒，或
b)如SEQ ID No.1所示的DNA序列的类似物，及/或可编码脂解酶的DNA序列的类似物，其可得自酿酒酵母DSM9975中的质粒，其
i)与如SEQ ID No.1所示的DNA序列及/或可编码脂解酶的DNA序列同源，该DNA序列可得自酿酒酵母DSM9975中的质粒，及/或
ii)与和如SEQ ID No.1所示的DNA序列及/或可编码脂解酶的DNA序列相同的寡核苷酸杂交，该DNA序列可得自酿酒酵母DSM9975中的质粒，及/或
iii)可编码与由含如SEQ ID No.1所示的DNA序列的DNA序列及/或由可编码脂解酶的DNA序列所编码的多肽同源的多肽，该DNA序列可得自酿酒酵母DSM9975中的质粒，及/或
iv)可编码与抗纯化脂解酶(由如SEQ ID No.1所示的DNA序列及/或由可编码脂解酶的DNA序列所编码)的抗体进行免疫反应的多肽，该DNA序列由可得自酿酒酵母DSM9975中的质粒，及/或其来自Humicolainsolens DSM1800。
据信，如SEQ ID No.1所示的DNA序列与可编码脂解酶的DNA序列是完全相同的，该DNA序列可得自酿酒酵母DSM9975中的质粒。
如SEQ ID No.1所示的DNA序列编码具有其天然信号肽的成熟酶。相类似，如SEQ ID No.2所示的氨基酸序列构成具有其信号肽(氨基酸1-35)的成熟酶(氨基酸36-246)。
可以认为，当参考与本发明的酶或DNA构建体相关联的“如SEQ ID No.1所示的DNA序列”[如上述部分a)及b)中i)-iv)所使用的]时，所述序列可以是如SEQ ID No.1所示的整个序列，但较优选的为可编码成熟脂解酶的序列的部分。
同样，当参考与本发明的酶或DNA构建体相关联的“如SEQ ID No.2所示的氨基酸序列”时，可以认为所述序列可以是如SEQ ID No.2所示的整个序列，但较优选的为构成成熟脂解酶(即氨基酸残基36-246)的序列的部分。
在本发明的文本中，如SEQ ID No.1所示的DNA序列之“类似物”意指可编码表现出脂解活性的酶并且具有一种或多种或全部i)～iv)特性的任何DNA序列。这样的类似性DNA序列可以
-例如使用本文所述方法，依据如SEQ ID No.1所示的DNA序列或其适当的亚序列(如20-500bp)分离自另一种或相关的(如相同的)可产生具脂解活性的酶的有机体，因此其是含有本文所示DNA序列的DNA序列等位或种变异体，或
-依据如SEQ ID No.1所示的DNA序列进行构建，例如可通过引入并不产生脂解酶(由DNA序列所编码)的另一种氨基酸序列的核苷酸取代，但其对应于欲产生该酶的宿主有机体的密码子使用，或者可通过引入能产生不同氨基酸序列的核苷酸取代。然而，在后一种情况下，氨基酸变化优选为微小性质的变化，即并不明显影响蛋白质的折叠或活性的保守性氨基酸置换，尤其约30个氨基酸的小缺失；小氨基末端或羧基末端的延长，如氨基末端蛋氨酸残基，约达20-25个残基的小型衔接物肽，或可促进纯化的小型延长，如聚组氨酸段，抗原决基结合区域。通常参见Ford等人，蛋白质表达和纯化2：95-107，1991。保守性置换为碱性氨基酸(如精氨酸、赖氨酸、组氨酸)，酸性氨基酸(如谷氨酸和天冬氨酸)，极性氨基酸(如谷氨酰胺和天冬酰胺)，疏水氨基酸(如亮氨酸、异亮氨酸、缬氨酸)，芳香氨基酸(如苯丙氨酸、色氨酸、酪氨酸)以及小型氨基酸(如甘氨酸、丙氨酸、丝氨酸、苏氨酸、甲硫氨酸)基团内的置换。
显然，对于本领域的技术人员来说，这样的置换可以在对分子功能关键的区域之外进行，且仍然可产生出活性多肽。对由本发明DNA构建体所编码的多肽活性非常必要的以及因此最好不进行置换的氨基酸，可依据本技术领域熟知的方法进行鉴定，如位点针对性诱变或丙氨酸扫描诱变(Cunningham和Wells，科学，244，1081-1085，1989)。在后一技术中，突变被引入了分子的每一个残基处，对所生成的突变分子进行生物活性(即脂解活性)测试，以鉴定出对分子活性关键的氨基酸残基。也可以通过晶体结构的分析(由诸如核磁共振、结晶学或光亲和性标记等技术确定)来确定底物—酶的互作位点。例如可参见de Vos等人，科学255：306-312，1992；Smith等人，分子生物学杂志，224：899-904，1992；Wlodaver等人，欧洲生物化学学会联合会通讯，309：59-64，1992。
i)中的上述同源性是由能显示出第一序列从第二序列中衍生出的两个序列之间的同一程度来确定的。该同源性可以适当地通过本领域所熟知的计算机程序来确定，如在GCG程序包中提供的GAP(Needleman，S.B.和Wunsch，C.D.，分子生物学杂志，48：443-453，1970)。利用具有下列参数的GAP进行DNA序列比较：GAP创造罚值为5.0，而GAP延伸罚值为0.3，与如SEQ ID No.1所示的DNA序列或编码脂解酶的DNA序列的编码区域相比较，DNA序列的编码区域表现出优选至少为40％的同一性程度，较优选为至少50％，更优选至少为60％，再优选至少为70％，甚至更优选为至少80％，尤其至少为90％，该DNA序列可得自酿酒酵母DSM9975中的质粒。
ii)中的上述杂交欲表明类似性DNA序列与和在某些特殊条件(详见以下材料及方法部分)下编码脂解酶的DNA序列相同的探针杂交。通常，类似性DNA序列与DNA序列高度同源，例如与如SEQ ID No.1所示的编码本发明中脂解酶的DNA序列至少70％同源，与如SEQ ID No.1所示的DNA序列具同源性如至少为75％，至少80％，至少85％，至少90％或甚至至少95％。
iii)中的上述同源性程度是由能显示出第一序列从第二序列中衍生出的两个序列之间的同一程度来确定的。该同源性可以适当地通过本领域所熟知的计算机程序来确定，如在GCG程序包中提供的GAP(Needleman，S.B.和Wunsch，C.D.，分子生物学杂志，48：443-453，1970)。利用具有下列参数的GAP进行DNA序列比较：GAP创造罚值为3.0，而GAP延伸罚值为0.1，与由含有如SEQ ID No.1所示的DNA序列或编码脂解酶的DNA序列的DNA构建体所编码的酶相比较，由类似性DNA序列编码的多肽表现出优选至少为70％的同一性程度，较优选为至少80％，尤其至少为90％，该DNA序列可得自酿酒酵母DSM 9975中的质粒，该酶的氨基酸序列如SEQ ID No.2所示。
与上述特性iv)相关联的术语“衍生自”并不仅指由H.insolens菌株DSM1800所产生的脂解酶，而且还指由菌株DSM1800中分离的DNA序列编码以及在用上述DNA序列转化的宿主有机体中产生的脂解酶。可通过以下材料和方法部分中所述的方法来确定免疫反应性。
在进一步方面，本发明涉及包含本发明DNA构建体的表达载体，含有DNA构建体或表达载体的细胞，以及产生能表现出脂解活性的酶的方法，该方法包括在容许该酶生成的条件下培养所说的细胞以及从培养物中回收该酶。
本发明进而涉及能表现出脂解活性的酶，该酶
a)由本发明DNA构建体所编码，及/或
b)由本发明的方法所生产，及/或
c)可与抗纯化脂解酶的抗体进行免疫反应，该脂解酶由如SEQ ID No.1所示的DNA序列及/或编码脂解酶的DNA序列所编码，该DNA序列可得自酿酒酵母DSM9975中的质粒，及/或其衍生自Humicola insolens DSM1800。
本发明进一步涉及含有本发明酶的洗涤添加剂或洗涤剂组合物。
                      本发明详述
可以通过包括下列步骤的一般方法来分离出编码本发明中具有脂解活性的酶的DNA序列
-在适宜的载体中克隆来自H.insolens的DNA文库，
-用上述载体转化适宜的酵母宿主细胞，
-在适宜的条件下培养宿主细胞，以表达出由DNA文库中克隆所编码的任何令人感兴趣的酶，
-通过确定由该克隆所产生的酶的脂解活性来筛选阳性克隆，和
-从该克隆中分离出编码酶的DNA。
一般性方法进一步披露于WO94/14953中。对筛选方法的更详细描述见以下实施例1。
编码该酶的DNA序列例如可通过筛选H.insolens[如公众可得自DSM的DSM1800菌株(Deutsche Sammlung von Mikroorganismen undZellkulturen，Mascheroder Weg 1b，D-38124 Braunschweig，德国)]的cDNA文库或选择出能表达适当的酶活性(即脂解活性)的克隆来进行分离，或者可分离自(1995年5月11日)按照布达佩斯条约保藏于DSM的酿酒酵母DSM9975。随后可通过标准方法，例如，如实施例1所述从该克隆中分离出适当的DNA序列。
我们预计，编码同源酶的DNA序列(即类似性DNA序列)可以得自其它微生物。例如，可以类似地从其它微生物筛选cDNA文库而衍生DNA序列，特别是从真菌中，尤其是Sordariales目的真菌，如梭孢壳种的菌株(特别是土栖梭孢壳)、毛壳种的菌株(特别是高大毛壳或球毛壳)、麻孢壳种的菌株(特别是五谷麻孢壳)、脉孢菌种的菌株(特别是粗脉孢菌)、柄孢壳种的菌株(特别P.anseria)、粪壳种的菌株(特别是粪生粪壳或大孢粪壳)、或另一种腐殖霉种的菌株。其它令人感兴趣的真菌包括曲霉种的菌株(棘孢曲霉或黑曲霉)、木霉种的菌株(T.harzianum，T.reeseii，绿色木霉、T.longibrachiatum或康宁霉)、或镰孢种的菌株(如尖镰孢)。
另外，通过使用根据这里公开的DNA序列所制备的合成性寡核苷酸探针，可按照熟知的方法从适当来源(如上述任意有机物)的DNA方便地分离出编码本发明中脂解酶的DNA。例如，可依据如SEQ ID No.1所示的DNA序列或如SEQ ID No.2所示的氨基酸序列或这些序列中任意适当的亚序列来制备适当的寡核苷酸探针。
其后可把DNA序列***重组性表达载体中。载体可以是能方便接受重组性DNA程序处理的任意载体，载体的选择常常取决于其所引入的宿主细胞。这样，该载体可以是自主复制性载体，即作为染色体外实体而存在的载体，其复制与染色体复制无关，例如质粒。另外，载体可以是这样的载体，当引入至宿主细胞中时，它可以整合入宿主细胞的基因组并同其所整入的染色体一同复制。
在载体中，编码脂解酶的DNA序列应操作性地连接到适当的启动子和终止序列上。启动子可以是任意DNA序列，其在所选择的宿主细胞中显示出转录活性，可衍生自编码与宿主细胞同源或异源的蛋白质的基因。用于使编码脂解酶的DNA序列分别连接到启动子和终止子上和将其***适当的载体的方法是本领域的技术人员所熟悉的(例如可参见Sambrook等人，分子克隆，实验室手册，冷泉港，纽约，1989)。
用编码本发明酶的DNA序列转化的宿主细胞优选为真核细胞，特别是真菌细胞，如酵母或丝状真菌细胞。尤其是属于曲霉种的细胞，最优选的有米曲霉或黑曲霉。另外，还有属于木霉种的细胞(如T.reeseii)或镰孢种的细胞(如尖镰孢或禾本科镰孢)。可通过涉及原生质体形成及其转化的方法和随后以熟知的方法再生细胞壁来转化真菌细胞。使用曲霉属作为宿主微生物如欧洲专利0 238 023所述(Novo Nordisk A/S)。宿主细胞也可以是酵母细胞，如酵母属菌株(特别是酿酒酵母、克鲁弗酵母或葡萄汁酵母)、裂殖酵母种的菌株(如栗酒裂殖酵母)或汉逊酵母种的菌株、毕赤酵母种的、Yarrowia种的菌株(如Yarrowialipolytica)、或克鲁维酵母种菌株(如乳酸克鲁维酵母)。
在进一步方面，本发明涉及生产依据本发明之酶的方法，其中对用编码该酶的DNA序列所转化的适宜宿主细胞进行培养，培养条件为能生产出该酶来，并从培养物中回收所生成的酶。
用于培养转化的宿主细胞的培养基可以是任何适于该宿主细胞生长的常规培养基。所表达的脂解酶可以方便地分泌至培养基中，并可以通过熟知的方法进行回收，包括通过离心或过滤从培养基中分离细胞、利用盐(如硫酸铵)使培养基中的蛋白成分沉淀，随后进行色谱法(如离子交换色谱、亲和色谱等等)。
                          本发明的酶
本发明的酶是一种由本发明的DNA构建体所编码的酶。
本发明的优选酶可具有如下一种或多种特性：
-其分子量约为20-21kDa
-pl范围在7-9，例如约8
-pH最佳值范围约为6-10，如7-9，例如约8
-其对短链脂质底物具有特异性，或至少表现出最强的脂解活性。
本发明的酶优选地可得自腐殖霉属菌株，如H.insolens的菌株或来自作为编码同源酶的DNA序列之适宜来源所提到的任意多种有机体的菌株。
                          洗涤剂组合物
依据本发明，本发明的脂解酶可通常用作洗涤的液体组合物的成分(如用于衣物或织物洗涤的洗涤剂组合物)。因此，它可以无尘颗粒、稳定剂或被护酶的形式含于洗涤剂组合物中。可如美国专利4,106,991和4,661,452(均为Novo Industri A/S的)所公开的来生产出无尘颗粒，并可有选择地通过本领域所熟知的方法进行涂敷。蜡性涂敷材料的实例有聚(环氧乙烷)产物(聚乙二醇，PEG)，平均摩尔量为1000-20000；具有16-50个环氧乙烷单位的乙氧基化壬基酚；乙氧基化脂肪醇，其中醇含有12-20个碳原子，并有15-80个环氧乙烷单位；脂肪醇；脂肪酸；及脂肪酸的甘油一酯、甘油二酯和甘油三酯。适于通过流化床技术而应用的薄膜形成性涂敷材料的实例见英国专利1483591。例如，根据既成方法，可通过添加多羟基化合物(如丙二醇)、糖或糖醇、乳酸或硼酸来稳定液体酶制剂。其它酶稳定剂都是本领域所熟悉的。可根据欧洲专利238,216所述的方法来制备被护酶。
本发明的洗涤剂组合物可以是任何方便的形式，例如粉末、颗粒、糊剂或液体。液体洗涤剂可以是含水的，通常含有达70％的水和0-30％的有机溶剂，或是不含水的。
该洗涤剂组合物含有一或多种表面活性剂，其中每种可以是阴离子、非离子、阳离子或两性离子的。洗涤剂通常含有0-50％的阴离子表面活性剂，如直链烷基苯磺酸盐(LAS)、α-烯烃磺酸盐(AOS)、烷基硫酸盐(脂肪族醇硫酸酯)(AS)、脂肪醇乙氧基硫酸盐(AEOS或AES)、仲链烷磺酸盐(SAS)、α-磺基脂肪酸甲酯、烷基或链烯基丁二酸或肥皂。其还可含有0-40％的非离子表面活性剂，如脂肪醇乙氧基化物(AEO或AE)、羧基脂肪醇乙氧基化物、壬基酚乙氧基化物、烷基聚苷、烷基二甲基氧化胺、乙氧基化脂肪酸单乙醇酰胺、脂肪酸单乙醇酰胺、或多羟基烷基脂肪酸酰胺(如WO92/06154中所述)。
洗涤剂组合物还可另外含有一或多种其它的酶，如另一种脂解酶(脂酶)、淀粉酶、角质酶、蛋白酶、纤维素酶、过氧化物酶、或氧化酶(如漆酶)。
洗涤剂可含有1-65％的洗涤剂增效助剂或配位剂，如沸石、二磷酸盐、三磷酸盐、膦酸盐、柠檬酸盐、次氮基三乙酸(NTA)、乙二胺四乙酸(EDTA)、亚乙基三胺五乙酸(DTMPA)、烷基或链烯基丁二酸、可溶性硅酸盐或层叠式硅酸盐(如来自Hoechst的SKS-6)。该洗涤剂液也可以是未复配洗涤剂助剂的，即基本上无洗涤剂助剂。
该洗涤剂可含有一或多种聚合物。实例包括羧甲基纤维素(CMC)、聚(乙烯基吡咯烷酮)(PVP)、聚乙二醇(PEG)、聚(乙烯醇)(PVA)、聚羧酸盐(如聚丙烯酸盐)、马来酸/丙烯酸共聚物以及异丁烯酸月桂基酯/丙烯酸共聚物。
该洗涤剂可含有漂白系，其中含H2O2源，如过硼酸盐或过碳酸盐，它们可以与形成过酸的漂白活化剂，如四乙酰乙二胺(TAED)或壬酰氧基苯磺酸盐(NOBS)相结合。另外，漂白系可含有过氧酸，如酰胺、酰亚胺或砜类型。
可使用常规稳定剂来稳定本发明的洗涤剂组合物中的酶，例如多羟基化合物(如丙二醇或甘油)、糖或糖醇、乳酸、硼酸、或硼酸衍生物(如芳香硼酸酯)，可如WO92/19709和WO92/19708所述配制该组合物。
该洗涤剂还可含有其它常规洗涤成分，如织物调理剂，包括粘土、促泡剂、抑泡剂、防腐剂、污垢悬浮剂、防污垢再沉积剂、染料、杀菌剂、荧光增白剂或香料。
pH(在使用浓度下的水溶液中测得)通常为中性或碱性，如范围为7-11。
在本发明范围内的特定形式的洗涤剂组合物包括：
1)配制成堆积密度至少为600g/l的颗粒的洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 7-12％
脂肪醇乙氧基硫酸盐(如C12-18醇，1-2 EO)或烷基硫酸盐 1-4％
(如C16-18)
脂肪醇乙氧基化物(C14-15醇，7EO) 5-9％
碳酸钠(Na2CO3) 14-20％
可溶性硅酸盐(Na2O，2SiO2) 2-6％
沸石(NaAlSiO4) 15-22％
硫酸钠(Na2SO4) 0-6％
柠檬酸钠/柠檬酸(C6H5Na3O7/C6H8O7) 0-15％
过硼酸钠(NaBO3.H2O) 11-18％
四乙酰乙二胺 2-6％
羧甲基纤维素 0-2％
聚合物(如马来酸/丙烯酸共聚物，PVP，PEG) 0-3％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如抑泡剂、香料、荧光增白剂、光漂剂) 0-5％
2)配制成堆积密度至少为600g/l的颗粒的洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 6-11％
脂肪醇乙氧基硫酸盐(如C12-18醇，1-2EO)或烷基硫酸盐(如C16-18) 1-3％
脂肪醇乙氧基化物(C14-15醇，7EO) 5-9％
碳酸钠(Na2CO3) 15-21％
可溶性硅酸盐(Na2O，2SiO2) 1-4％
沸石(NaAlSiO4) 24-34％
硫酸钠(Na2SO4) 4-10％
柠檬酸钠/柠檬酸(C6H5Na3O7/C6H8O7) 0-15％
羧甲基纤维素 0-2％
聚合物(如马来酸/丙烯酸共聚物，PVP，PEG) 1-6％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如抑泡剂、香料) 0-5％
3)配制成堆积密度至少为600g/l的颗粒的洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 5-9％
脂肪醇乙氧基化物(C12-15醇，7EO) 7-14％
作为脂肪酸的肥皂(C16-22脂肪酸) 1-3％
碳酸钠(Na2CO3) 10-17％
可溶性硅酸盐(Na2O，2SiO2) 3-9％
沸石(NaAlSiO4) 23-33％
硫酸钠(Na2SO4) 0-4％
过硼酸钠(NaBO3，H2O) 8-16％
四乙酰乙二胺 2-8％
膦酸盐(例如EDTMPA) 0-1％
羧甲基纤维素 0-2％
聚合物(如马来酸/丙烯酸共聚物，PVP，PEG) 0-3％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如抑泡剂、香料、荧光增白剂) 0-5％
4)配制成堆积密度至少为600g/l的颗粒的洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 8-12％
脂肪醇乙氧基化物(C12-15醇，7EO) 10-25％
碳酸钠(Na2CO3) 14-22％
可溶性硅酸盐(Na2O，2SiO2) 1-5％
沸石(NaAlSiO4) 25-35％
硫酸钠(Na2SO4)   0-10％
羧甲基纤维素   0-2％
聚合物(如马来酸/丙烯酸共聚物，PVP，PEG)   1-3％
酶(以纯酶蛋白质计)   0.0001-0.1％
微量成分(如抑泡剂、香料)   0-5％
5)含水液体洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 15-21％
脂肪醇乙氧基化物(C12-15醇，7EO或C12-15醇，5EO) 12-18％
作为脂肪酸的肥皂(如油酸) 3-13％
链烯基丁二酸(C12-14) 0-13％
氨基乙醇 8-18％
柠檬酸 2-8％
膦酸盐 0-3％
聚合物(如PVP，PEG) 0-3％
硼酸盐(B4O7) 0-2％
乙醇 0-3％
丙二醇 8-14％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如分散剂、抑泡剂、香料、荧光增白剂) 0-5％
6)水结构的液体洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 15-21％
脂肪醇乙氧基化物(C12-15醇，7EO或C12-15醇，5EO) 3-9％
作为脂肪酸的肥皂(如油酸) 3-10％
沸石(NaAlSiO4) 14-22％
柠檬酸钾 9-18％
硼酸盐(B4O7) 0-2％
羧甲基纤维素 0-2％
聚合物(如PVP，PEG) 0-3％
结合性聚合物，如异丁烯酸月桂基酯/丙烯酸共聚物；摩尔比：25∶1；MW 3800 0-3％
甘油 0-5％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如分散剂、抑泡剂、香料、荧光增白剂) 0-5％
7)配制成堆积密度至少为600g/l的颗粒的洗涤剂组合物，含有
脂肪族醇硫酸酯 5-10％
乙氧基化脂肪酸单乙醇酰胺 3-9％
作为脂肪酸的肥皂 0-3％
碳酸钠(Na2CO3) 5-10％
可溶性硅酸盐(Na2O，2SiO2) 1-4％
沸石(NaAlSiO4) 20-40％
硫酸钠(Na2SO4) 2-8％
过硼酸钠(NaBO3.H2O) 12-18％
四乙酰乙二胺 2-7％
聚合物(如马来酸/丙烯酸共聚物，PEG) 1-5％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如荧光增白剂、抑泡剂、香料) 0-5％
8)配制成颗粒的洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 8-14％
乙氧基化脂肪酸单乙醇酰胺 5-11％
作为脂肪酸的肥皂 0-3％
碳酸钠(Na2CO3) 4-10％
可溶性硅酸盐(Na2O，2SiO2) 1-4％
沸石(NaAlSiO4) 30-50％
硫酸钠(Na2SO4) 3-11％
柠檬酸钠(C6H5Na3O7) 5-12％
聚合物(如PVP，马来酸/丙烯酸共聚物，PEG) 1-5％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如抑泡剂、香料) 0-5％
9)配制成颗粒的洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 6-12％
非离子表面活性剂 1-4％
作为脂肪酸的肥皂 2-6％
碳酸钠(Na2CO3) 14-22％
沸石(NaAlSiO4) 18-32％
硫酸钠(Na2SO4) 5-20％
柠檬酸钠(C6H5Na3O7) 3-8％
过硼酸钠(NaBO3.H2O) 4-9％
漂白活化剂(如NOBS或TAED) 1-5％
羧甲基纤维素 0-2％
聚合物(如聚羧酸酯或PEG) 1-5％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如香料、荧光增白剂) 0-5％
10)含水液体洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 15-23％
脂肪醇乙氧基硫酸盐(如C12-15C醇，2-3EO) 8-15％
脂肪醇乙氧基化物(C12-15醇，7EO或C12-15醇，5EO) 3-9％
作为脂肪酸的肥皂(月桂酸) 0-3％
氨基乙醇 1-5％
柠檬酸钠 5-10％
水溶助长剂(如甲苯磺酸钠) 2-6％
硼酸盐(B4O7) 0-2％
羧甲基纤维素 0-1％
乙醇 1-3％
丙二醇 2-5％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如聚合物、分散剂、香料、荧光增白剂) 0-5％
11)含水液体洗涤剂组合物，含有
直链烷基苯磺酸盐(以酸计) 20-32％
脂肪醇乙氧基化物(C12-15醇，7EO或C12-15醇，7EO) 6-12％
氨基乙醇 2-6％
柠檬酸 8-14％
硼酸盐(B4O7) 1-3％
聚合物(如马来酸/丙烯酸共聚物，结合性聚合物，如异丁烯酸月桂基酯/丙烯酸共聚 0-3％
物)
甘油 3-8％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如水溶助长剂、分散剂、香料、荧光增白剂) 0-5％
12)配制成堆积密度至少为600g/l的颗粒的洗涤剂组合物，含有
阴离子表面活性剂(直链烷基苯磺酸盐、烷基硫酸盐、α-烯烃磺酸盐、α-磺基脂肪酸甲酯、链烷磺酸盐、肥皂) 25-40％
非离子表面活性剂(如脂肪醇乙氧基化物) 1-10％
碳酸钠(Na2CO3) 8-25％
可溶性硅酸盐(Na2O，2SiO2) 5-15％
硫酸钠(Na2SO4) 0-5％
沸石(NaAlSiO4) 15-28％
过硼酸钠(NaBO3.4H2O) 0-20％
漂白活化剂(TAED或NOBS) 0-5％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如香料、荧光增白剂) 0-3％
13)如1)-12)所述的洗涤剂组合物，其中全部或部分直链烷基苯磺酸盐被(C12-18)烷基硫酸盐所代替。
14)配制成堆积密度至少为600g/l的颗粒的洗涤剂组合物，含有
C12-18烷基硫酸盐 9-15％
脂肪醇乙氧基化物 3-6％
聚羟基烷基脂肪酸酰胺 1-5％
沸石(NaAlSiO4) 10-20％
层状二硅酸盐(如Hoechst的SK56) 10-20％
碳酸钠(Na2CO3) 3-12％
可溶性硅酸盐(Na2O，2SiO2) 0-6％
柠檬酸钠 4-8％
过碳酸钠 13-22％
四乙酰乙二胺 3-8％
聚合物(如聚羧酸酯和PVP) 0-5％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如抑泡剂、香料、荧光增白剂、光漂剂) 0-5％
15)配制成堆积密度至少为600g/l的颗粒的洗涤剂组合物，含有
C12-18烷基硫酸盐 4-8％
脂肪醇乙氧基化物 11-15％
肥皂 1-4％
沸石MAP或沸石A 35-45％
碳酸钠(Na2CO3) 2-8％
可溶性硅酸盐(Na2O，2SiO2) 0-4％
过碳酸钠 13-22％
四乙酰乙二胺 1-8％
羧甲基纤维素 0-3％
聚合物(如聚羧酸酯和PVP) 0-3％
酶(以纯酶蛋白质计) 0.0001-0.1％
微量成分(如膦酸盐、香料、荧光增白剂) 0-3％
16)如1)-15)所述的洗涤剂配方，其中含有稳定的或包胶的过酸，既可以是附加成分也可以是已限定的漂白系的替代物。
17)如1)、3)、7)、9)和12)所述的洗涤剂组合物，其中过硼酸盐被过碳酸盐所替代。
18)如1)、3)、7)、9)、12)、14)和15)所述的洗涤剂组合物，其中还另外含有锰催化剂。锰催化剂可以是如自然，369，1994，pp.637-639“用于低温漂白的有效的锰催化剂”一文中所述的化合物之一。
19)配制成含液体非离子表面活性剂，例如直链烷氧基化伯醇、增效助剂系(如磷酸盐)、酶和碱的非水性洗涤液的洗涤剂组合物。该洗涤剂还可含有阴离子表面活性剂及/或漂白系。
                     洗碟组合物
本发明的脂解酶可适宜地作为洗碟洗涤剂组合物的成分。洗碟洗涤剂组合物可含有表面活性剂，可以是阴离子、非离子、阳离子、两性离子的或这些类型的混合物。该洗涤剂可含有0-90％的非离子表面活性剂，如低泡到无泡的乙氧基化丙氧基化直链醇。
该洗涤剂组合物可含有无机及/或有机类型的洗涤助剂盐。该洗涤助剂可再分成含磷或不含磷的类型。该洗涤剂组合物通常含有1-90％的洗涤增助效剂。
含磷的无机碱性洗涤助剂(当存在时)的实例包括水溶性盐，特别是碱金属焦磷酸盐、正磷酸盐、多磷酸盐和膦酸盐。不合磷的无机助洗剂的实例包括水溶性碱金属碳酸盐、硼酸盐和硅酸盐以及多种类型的水溶性结晶或非结晶硅铝酸盐，其中沸石是最有名的代表。
合适的有机助洗剂的实例包括碱金属、铵和取代铵、柠檬酸盐、琥珀酸盐、丙二酸盐、脂肪酸磺酸盐、羧基甲氧基琥珀酸盐、聚乙酸铵、羧酸盐、聚羧酸盐、氨基聚羧酸盐、聚乙酰羧酸盐和聚羟基磺酸盐。
其它适当的有机增效助剂包括高分子量聚合物和已知具有助洗剂特性的共聚物。例如合适的聚丙烯酸、聚马来酸和聚丙烯酸/聚马来酸共聚物及其盐。
洗碟洗涤剂组合物可含有氯/溴型或氧型的漂白剂。无机氯/溴型漂白剂的实例有次氯酸和次溴酸的锂盐、钠盐或钙盐，以及氯化磷酸三钠。有机氯/溴型漂白剂的实例有杂环N-溴及N-氯酰亚胺，如三氯异氰脲酸、三溴异氰脲酸、二溴异氰脲酸和二氯异氰脲酸及其含水溶性阳离子(如钾和钠)的盐。乙内酰脲化合物也是合适的。
优选应用氧漂白剂，例如无机过酸盐形式的，优选为具漂白前体或作为过氧酸化合物的。合适的过氧漂白化合物的典型实例有碱金属过硼酸盐(可以是一水合物也可以是四水合物)、碱金属过碳酸盐、过硅酸盐和过磷酸盐。优选的活化剂材料是四乙酰乙二胺和三乙酸甘油酯。
可使用酶的常规稳定剂来稳定本发明的洗涤剂组合物，例如多羟基化合物(如丙二醇)、糖或糖醇、乳酸、硼酸或硼酸衍生物(如芳香硼酸酯)。
洗碟洗涤剂组合物还可含有其它酶，特别是淀粉酶、蛋白酶和/或纤维素酶。
本发明的洗碟洗涤剂组合物还可含有其它常规洗涤剂成分，如抗絮凝剂材料、填充材料、抑泡剂、防腐剂、污垢悬浮剂、螯合剂、防污垢再沉积剂、脱水剂、染料、杀菌剂、荧光剂、增稠剂和香料。
以下举例说明特别优选的洗碟组合物：
1)粉状自动洗碟组合物
非离子表面活性剂 0.4-2.5％
硅酸钠 0-20％
二硅酸钠 3-20％
三磷酸钠 20-40％
碳酸钠 0-20％
过硼酸钠 2-9％
四乙酰乙二胺(TAED) 1-4％
硫酸钠 5-33％
酶 0.0001-0.1％
2)粉状自动洗碟组合物
非离子表面活性剂(如脂肪醇乙氧基化物) 1-2％
二硅酸钠 2-30％
碳酸钠 10-50％
膦酸钠 0-5％
柠檬酸三钠二水合物 9-30％
次氮基乙酸三钠(NTA) 0-20％
过硼酸钠一水化合物 5-10％
四乙酰乙二胺(TAED) 1-2％
聚丙烯酸酯聚合物(如马来酸/丙烯酸共聚物) 6-25％
酶 0.0001-0.1％
香料 0.1-0.5％
水 5-10
3)粉状自动洗碟组合物
非离子表面活性剂 0.5-2.0％
二硅酸钠 25-40％
柠檬酸钠 30-55％
碳酸钠 0-29％
二碳酸钠 0-20％
过硼酸钠一水合物 0-15％
四乙酰乙二胺(TAED) 0-6％
马来酸/丙烯酸共聚物 0-5％
粘土 1-3％
聚氨基酸 0-20％
聚丙烯酸钠 0-8％
酶 0.0001-0.1％
4)粉状自动洗碟组合物
非离子表面活性剂 1-2％
沸石MAP 15-42％
二硅酸钠 30-34％
柠檬酸钠 0-12％
碳酸钠 0-20％
过硼酸钠一水合物 7-15％
四乙酰乙二胺(TAED) 0-3％
聚合物 0-4％
马来酸/丙烯酸共聚物 0-5％
有机膦酸盐 0-4％
粘土 1-2％
酶 0.0001-0.1％
硫酸钠 余量
5)粉状自动洗碟组合物
非离子表面活性剂 1-7％
二硅酸钠 18-30％
柠檬酸三钠 10-24％
碳酸钠 12-20％
单过硫酸盐(2KHSO5.KHSO4.K2SO4) 15-21％
漂白稳定剂 0.1-2％
马来酸/丙烯酸共聚物 0-6％
二亚乙基三胺五乙酸盐，五钠盐 0-2.5％
酶 0.0001-0.1％
硫酸钠，水 余量
6)含清洗表面活性剂系的粉状和液体洗碟组合物
非离子表面活性剂 0-1.5％
十八烷基二甲胺N-氧化物二水合物 0-5％
十八烷基二甲胺N-氧化物二水合物和十六烷基二甲胺N-氧化物二水合物之80∶20wt.C18/C16掺合物 0-4％
十八烷基双(羟乙基)胺N-氧化物无水物和十六烷基双(羟乙基)胺N-氧化物无水物之70∶30wt.C18/C16掺合物 0-5％
平均乙氧基化程度为3的C13-15烷基乙氧基硫酸盐 0-10％
平均乙氧基化程度为3的C12-15烷基乙氧基硫酸盐 0-5％
平均乙氧基化程度为12的C13-15乙氧基化醇 0-5％
平均乙氧基化程度为9的C12-15乙氧基化醇的掺合物 0-6.5％
平均乙氧基化程度为30的C13-15乙氧基化醇的掺合物 0-4％
二硅酸钠 0-33％
三聚磷酸钠 0-46％
柠檬酸钠 0-28％
柠檬酸 0-29％
碳酸钠 0-20％
过硼酸钠一水合物 0-11.5％
四乙酰乙二胺(TAED) 0-4％
马来酸/丙烯酸共聚物 0-7.5％
硫酸钠 0-12.5％
酶 0.0001-0.1％
7)非水液体自动洗碟组合物
液体非离子表面活性剂(如脂肪醇乙氧基化物) 2.0-10.0％
碱金属硅酸盐 3.0-15.0％
碱金属磷酸盐 20.0-40.0％
选自高级乙二醇、聚乙二醇、多氧化物、乙二醇醚的液体载体 25.0-45.0％
稳定剂(如磷酸的偏酯和C16-C18链烷醇) 0.5-7.0％
抑泡剂(如硅氧烷) 0-1.5％
酶 0.0001-0.1％
8)非水液体洗碟组合物
液体非离子表面活性剂(如脂肪醇乙氧基化物) 2.0-10.0％
硅酸钠 3.0-15.0％
碱金属碳酸盐 7.0-20.0％
柠檬酸钠 0.0-1.5％
稳定系(如细碎硅氧烷和低分子量二烷基聚乙二醇醚混合物) 0.5-7.0％
低分子量聚丙烯酸酯聚合物 5.0-15.0％
粘土凝胶增稠剂(如皂土) 0.0-10.0％
羟丙基纤维素聚合物 0.0-0.6％
酶 0.0001-0.1％
选自高级乙二醇、聚乙二醇、多氧化物、乙二醇醚的液体载体 余量
9)触变性液体自动洗碟组合物
C12-C14脂肪酸 0-0.5％
嵌段共聚物表面活性剂 1.5-15.0％
柠檬酸钠 0-12％
三聚磷酸钠 0-15％
碳酸钠 0-8％
三硬脂酸铝 0-0.1％
枯烯磺酸钠 0-1.7％
聚丙烯酸酯增稠剂 1.32-2.5％
聚丙烯酸钠 2.4-6.0％
硼酸 0-4.0％
甲酸钠 0-0.45％
甲酸钙 0-0.2％
正癸基苯醚二磺酸钠 0-4.0％
单乙醇胺(MEA) 0-1.86％
氢氧化钠(50％) 1.9-9.3％
1，2-丙二醇 0-9.4％
酶 0.0001-0.1％
抑泡剂、染料、香料、水 余量
10)液体自动洗碟组合物
脂肪醇乙氧基化物 0-20％
脂肪酸酯磺酸盐 0-30％
十二烷基硫酸钠 0-20％
烷基聚苷 0-21％
油酸 0-10％
二硅酸钠一水合物 18-33％
柠檬酸钠二水合物 18-33％
硬脂酸钠 0-2.5％
过硼酸钠一水合物 0-13％
四乙酰乙二胺(TAED) 0-8％
马来酸/丙烯酸共聚物 4-8％
酶 0.0001-0.1％
11)含被护漂白颗粒的液体自动洗碟组合物
硅酸钠 5-10％
焦磷酸四钾 15-25％
三磷酸钠 0-2％
碳酸钾 4-8％
被护漂白颗粒，如氯 5-10％
聚合增稠剂 0.7-1.5％
氢氧化钾 0-2％
酶 0.0001-0.1％
水 余量
12)如1)、2)、3)、4)、6)和10)所述的自动洗碟组合物，其中过硼酸盐被过碳酸盐所代替。
13)如1)-6)所述的自动洗碟组合物，其中还含有锰催化剂。锰催化剂可以是例如，自然，369，1994，pp.637-639“用于低温漂白的有效锰催化剂”一文中所述的化合物之一。
另外，本发明的第一洗涤脂解酶可用于软化组合物中：
本发明的脂解酶可用于织物软化剂中，如“表面活性剂和消费者产品”中所述，由J.Falbe编辑，1987，pp295-296；“表面活性剂洗涤剂”，30(1993)，6，pp394-399；JAOCS，第61卷(1984)，2，pp367-376；欧洲专利517762；欧洲专利123400；WO92/19714；WO93/19147；美国专利5,082,578；欧洲专利494769；欧洲专利544,493；欧洲专利542,562；美国专利5,235,082；欧洲专利568297；欧洲专利570237。
本发明的脂解酶可以洗涤剂常规使用的浓度掺入。目前我们期望，掺入本发明洗涤剂组合物的本发明脂解酶之量为每升洗涤液0.00001-1mg(以纯酶蛋白质计)脂解酶。
我们期望，例如，本发明的脂解酶还可作为有机合成(如酯化、酯基转移或酯水解反应)中的催化剂用于烘烤业、造纸业(如去除树脂)及皮革、羊毛和相关行业(如动物皮革、羊皮或羊毛的脱脂)，以及其它涉及脱脂的应用。
再下列实施例中将进一步详细说明本发明，这些实施例绝不会限制本发明所要求的应用范围。
                   材料与方法
供体有机体：从生长在含玉米粒的发酵培养基(搅拌以保证充分通气)中的Humicola insolens DSM 1800中分离出mRNA。经3-5天生长之后，收获菌丝体，立刻冷冻在液态氮中并储藏在-80℃下。
酵母菌株：所用的酿酒酵母是yNG231(MATα，leu2，ura 3-52，his4-539，pep 4-δ1，cir+)  或JG169(MATα，ura 3-52；leu 2-3，112；his 3-D200；pep 4-1137；prc1∷HIS3；prb1∷LEU2；cir+)。
质粒：使用表达质粒pYES2.0(Invitrogen)。
使用曲霉属表达pHD414载体。pHD414是质粒p775(如欧洲专利0238023所述)的衍生物。pHD414的构建进一步如WO93/11249中所述。
利用硫氰酸鈲并而后通过5.7M CsCl垫进行超速离心而提取全部的RNA，利用WO94/14953中所述的步骤通过寡(dT)纤维素亲和色谱法来进行poly(A)+RNA的分离。
cDNA的合成与修饰：使用发夹修饰法通过RNase H法(Gubler和Hoffman 1983，Sambrook等人，1989)从5μg poly(A)+RNA中合成出双股cDNA。该步骤进一步如WO95/02043所述。在用绿豆核酸酶(Bethesda实验室)处理之后，依据厂商的指导，用T4 DNA聚合酶(Invitrogen)ds cDNA处理成平头，并把cDNA连接到非回文BstX1衔接头(1μg/μl，Invitrogen)上。
cDNA文库的构建：通过离心回收连接的ds cDNA，用70％的乙醇冲洗并重悬浮于25ml的H2O中。在大规模文库连接之前，进行四次试验连接反应，在10μl总体积中每次使用1μl ds cDNA(反应试管#1-#3)、2单位T4连接酶(Invitrogen)和50ng(试管#1)、100ng(试管#2)和200ng(试管#3及#4)Bst XI裂解酵母表达载体pYES 2.0(Invitrogen)。
利用最佳条件，在40μl的连接缓冲液中进行大规模的连接反应。把1ul等分试样转化入电感受态大肠杆菌1061细胞中，对所转化的细胞进行滴定，把文库涂布在LB+氨苄青霉素平板上[5000-7000菌落形成单位(c.f.u.)/平板]。在每一平板中加入3ml培养基。刮去细菌，加入1ml甘油，在-80℃下作为库储存。把剩余的2ml用于DNA的分离。进一步细节可参考WO94/14953。
酵母文库的构建：为了保证所有的细菌克隆在酵母中得到检测，把5倍于原始库中细菌克隆数量的许多酵母转化体设定为最大储额。
把来自各个库的纯化质粒DNA的1μl等分试样(100ng/μl)电极化(200，1.5kV，25μF)至40μl感受态酿酒酵母JG169细胞中(在500ml YPD中OD600＝1.5)，在冷去离子水中冲洗两次，在冷的1M山梨醇中冲洗一次，重悬浮在0.5ml的1M山梨醇中(Becker和Guarante，1991)。在加入1ml 1M的1ml冷山梨醇后，把80μl等分试样涂布在SC-URA+葡萄糖上，以得到250-400c.f.u./平板，并在30℃下温育3-5天。
阳性菌落的鉴定：经过3-5天生长之后，把琼脂平板复制铺板到SC-橄榄油/亮绿平板上，然后在30℃下温育2-4天以检测脂解活性。SC-URA橄榄油/亮绿平板为SC-URA+2％葡萄糖+0.6％橄榄油+1％亮绿溶液+0.036％聚乙烯醇(MW70,000-100,000σP-1763-尿嘧啶)。温育之后，以周围具绿色晕圈的白色菌落来鉴定脂解酶阳性菌落。
将脂解酶阳性菌落中的细胞涂布在琼脂上用于单个菌落分离，以每一个所鉴定的产脂解酶菌落来选择出产酶的单个菌落。
对阳性克隆的分析：作为单个菌落得到阳性克隆，利用生物素化多接头引物从酵母菌落中直接扩增cDNA***体，用磁珠(Dynabead M-280，Dynal)系提纯，使用链终止法(Sanger等人，1977)和测序酶系(美国生物化学公司)通过测定cDNA克隆5-末端序列来逐个进行表征。
分离用于在曲霉中表达的cDNA基因：
把一个或多个产脂解酶的酵母菌落接种到50ml玻璃试管中的20mlYPD肉汤中。在30℃下把试管摇动培养2天。以3000rpm离心10分钟来收获细胞。
依据WO94/14953分离DNA并溶解在50μl水中。如WO94/14953所述把DNA转化入大肠杆菌中。利用标准步骤从大肠杆菌中分离出质粒DNA，利用限制性内切酶分析方法进行分析。使用适当的限制性内切酶剪切cDNA***体，并连接到曲霉表达载体中。
米曲霉或黑曲霉的转化：
如WO95/02043所述(第16页，第21行-第17页，第12行)制备原生质体。
把100μl原生质体悬浮液同10μl STC(1.2M山梨醇，10mMTris-HCl，pH＝7.5，10mM CaCl2)中的5-25μg适当DNA相混合。将原生质体与p3SR2(无冠构巢曲霉amdS基因携带质粒)相混合。把混合物在室温下放置25分钟。加入0.2ml 60％的PEG4000(BDH29576)，10mM CaCl2和10mM Tris-HCl，pH7.5，仔细混合(两次)，最后加入0.85ml相同溶液并仔细混合。把混合液在室温下放置25分钟，以2500g旋转15分钟，再把丸粒重悬浮在2ml的1.2M山梨醇中。再次沉降后，把原生质体涂布在基本平板上[Cove，生物化学及生物生理Acta113(1966)51-56]，其含有1.0M蔗糖、pH7.0、10mM乙酰胺(作氮源)和20mM CsCl抑制本底的生长。在37℃下温育4-7天，挑选出孢子，涂布用于单个菌落。重复该步骤，把第二次重分离后单个菌落的孢子作为所定义的转化体储存。
米曲霉转化体的检测：
把每一个转化体接种到10ml YPM中并培育。经过在30℃下温育2-5天，除去10ml上清液。通过把10μl上清液加入琼脂平板(含有0.1MTris，pH9，0.1MCaCl2，1％Triton X-100，0.5％橄榄油)上开出的14mm直径的孔洞内来鉴定脂解活性。浑浊晕圈的形成就标志着脂解活性。
杂交条件[用于评估本发明DNA构建体的特性ii)]：
确定寡核苷酸探针和“类似性”DNA序列之间的杂交的合适条件涉及到预浸渍含DNA序列的填滤膜以在5xSSC中杂交，在～50℃下于含5xSSC，5xDenhardt溶液，50mM磷酸钠，pH6.8，和50μg变性声处理小牛胸腺DNA的溶液中预杂交该序列1小时，然后在～50℃下于补充了50μCi32P-dCTP标记探针的相同溶液中杂交18小时，然后在50℃下用2xSSC，0.2％十二烷基硫酸钠(SDS)冲洗三次(30分钟)。
可依据SEQ ID No.1所示的DNA序列或所说序列的适宜亚序列(如20个核苷酸片段)来制备杂交中所用的适当寡核苷酸探针。另外，可依据如SEQ ID No.2所示的氨基酸序列来制备适当的寡核苷酸探针。
免疫交叉反应性：可通过使用纯化脂解酶来制备用于确定免疫交叉反应性的抗体。尤其是可通过免疫兔子(或其它啮齿动物)来产生抗本发明酶的抗血清，依据N.Axelsen等人所述的方法，见：定量免疫电泳手册，Blackwell科学出版社，1973，第23章；或A.Johnstone和R.Thorpe，实用免疫化学，Blackwell科学出版社，1982(尤其参见第27-31页)。可从抗血清中得到纯化免疫球蛋白，例如可通过盐沉淀[(NH4)2SO4]，然后通过透析和离子交换层析，如在DEAE-SephadexTM上。可通过Outcherlony双扩散分析法[O.Ouchterlony，见：实验免疫学手册(D.M.Weir编辑)，Blackwell科学出版社，1967，655-706页]、交叉免疫电泳法(N.Axelsen等人，如上，第三章和第四章)或火箭免疫电泳法(N.Axelsen等人，如上第二章)来进行蛋白质的免疫化学表征。
培养基：
YPD：10g酵母膏，20g胨，加水到900ml。高压灭菌，加入100ml20％的葡萄糖(无菌过滤)。
YPM：10g酵母膏，20g胨，加水到900ml。高压灭菌，加入100ml20％的麦芽糖糊精(无菌过滤)。
10 x Basal盐：66.8g酵母氮碱。100g琥珀酸，60gNaOH，加水到1000ml，无菌过滤。
SC-URA：90ml 10 x Basal盐，22.5ml 20％的酪蛋白氨基酸，9ml 1％的色氨酸，加水到806ml，高压灭菌，加入3.6ml 5％的苏氨酸和90ml20％的葡萄糖或20％的半乳糖。
SC-URA琼脂：加入20g/l的SC-URA琼脂。
橄榄油：Sigma 0-1500。
亮绿溶液：水中4mg/l的亮绿(Sigma B-6756)。
                     实施例1
从50个库中约含106个单个克隆的H.insolens DSM1800中构建大肠杆菌文库。
从该文库的20个单个克隆中分离出DNA并进行cDNA***分析。发现***频率大于90％，平均***体大小约为1400bp。把来自某些库的DNA转化入酵母，从每个库中得到含200-500酵母菌落的50-100个平板。
鉴定了70多个阳性菌落并在琼脂平板上分离。从酵母菌落中直接扩增cDNA***体，并如上述材料与方法部分所述进行特性分析。编码脂解酶的cDNA序列如SEQ ID No.1中所示。
然后，如上所述分离编码脂解酶的cDNA用于在曲霉中表达，并使用标准方法转化入大肠杆菌中。从每个转化体中分离出两个大肠杆菌菌落，用可剪切DNA***体的限制性内切酶HindIII和XbaI进行分析。把来自这些克隆之一的DNA重转化入酵母菌株JG169中。
                     实施例2
为了在曲霉中表达基因，分离cDNA并用HindIII/XbaI消化，在凝胶上按分子大小进行分级分离并提纯，然后连接到pHD414上，产生质粒pA2L79。在大肠杆菌中扩增后，依据上述一般性步骤把该质粒转化入米曲霉或黑曲霉中。
对米曲霉转化体的检测
如上所述对每个转化体进行脂解活性的检测。一些转化体具有脂解活性，其明显高于米曲霉本底。这证明脂解酶在米曲霉中的有效表达。
选择出具有最高脂解活性的转化体，接种在装有YPM培养基的500ml摇瓶中。经过3-5天发酵(充分搅拌以保证良好通气)之后，以2000g对培养肉汤离心10分钟，回收上清液并进行分析。
如上所述对上清液中的脂解活性进行鉴定。
批量发酵
在一培养基(含作为碳源的麦芽糖浆、作为氮源的尿素和酵母膏)中进行批量发酵。通过把该米曲霉宿主细胞的摇瓶培养物接种到含3.0％碳源和0.4％氮源的培养基中实施批量发酵。经过24小时的培养(34℃，pH7.0)之后终止发酵，然后通过离心、超滤、澄清过滤和微生物过滤就能回收到酶。
                         实施例3
               H.insolens脂解酶的提纯和特性分析
离心批量发酵上清液并弃去含细胞碎片的沉淀物。用固体硫酸铵把该上清液调至60％的饱和度并搁置过夜。通过离心分离出含角质酶活性的沉淀物。把硫酸铵沉淀物溶解在水中，用稀乙酸调至pH为6。将含脂解活性的试样过Bacitracin琼脂糖柱以去除米曲霉中存在的碱性蛋白酶活性。以流出液来收集未结合到Bacitracin琼脂糖上的脂解活性。
把含活性的库调至pH为5.8，把离子强度调到2Milli Siemens。把试样加到阳离子交换剂SP-SepharoseTM高效PharmaciaTM柱上，该柱用25mM的乙酸钠缓冲液(pH5.8)进行平衡。
利用同一缓冲液以线性盐梯度来洗脱所结合的活性。
利用SDS-PAGE PHAST 8-25％梯度凝胶(PHAST systemTM，Pharmacia)来确定所提纯蛋白质的分子量。该分子量估计为20-21kD。
依据厂商的指导，利用AmpholineTM PAGE平板(pH3.5-9.5，Pharmacia LKB)来确定该蛋白质的等电点。发现pl为8。
发现脂解酶可受到苯基甲基磺酰氟的抑制，这表明了溶剂可及的活性位点以及酯酶的活性。
           对脂酶活性和脂解酶比活性的检测
利用***树胶作为乳化剂，通过由乳化三丁酸甘油酯(MERCK)制备的底物来确定脂酶的活性。
使用pH-stat法(利用辐射计VTT滴定计Tm)在pH为7下对脂酶活性进行检测。把一单位脂酶活性(LU)定义为每分钟释放一摩尔脂肪酸所需要的量。
在pH为7时所确定的比活性至少约为1200LU/mg。
底物特异性：为了把本发明的H.insolens脂解酶(具有如SEQ ID No.2所示的氨基酸序列)的脂解活性与长链底物(橄榄油)和短链底物(三丁酸甘油酯)进行比较，在pH为9时进行两次检测：
1)使用Sigma脂酶底物乳化剂(Sigma目录No.800-1)的Sigma橄榄油底物检测
2)利用***树胶作为乳化剂，用三丁精(三丁酸甘油酯)作为底物的检测。在pH为9时利用pH-stat法进行这两次检测。在pH为9时，在检测1和检测2中脂解酶活性分别约为500 Sigma脂酶单位/OD 280和1500LU/OD280，这表明与短链底物(如三丁精)相比该脂解酶具有更强的活性。
               H.insolens脂解酶的最佳pH值
使用pH-stat法，通过三丁酸甘油酯(作为底物)和***树胶(作为乳化剂)来研究不同pH值下酶的脂解活性。作为pH函数的酶活性(pH分别为6、7、8、9、10)如图1所示。最佳pH值似乎约为8，而最高活力的高百分比仍保持在pH为10时。
              N-末端序列测定
利用反相HPLC最终提纯本发明的H.insolens DSM1800脂解酶。利用实用生物***测序仪确定该酶的N末端氨基酸序列27个残基。所生成的序列如SEQ ID No.3所示，其中Xaa标明一个未指定的残基，其几乎肯定是半胱氨酸残基。在位置12处，发现Gly和Ala是等量的。
                       实施例4
在评估“初洗”脂解效果的检测中本发明之H.insolens DSM1800脂解酶的性能
在恒温Terg-O-Tometer(TOM)中进行一轮洗涤试验，随后晾干来测试本发明脂解酶的衣织物洗涤性能。
试验条件如下：
洗涤液：每烧杯1000ml
布样：每烧杯7块棉布样(9×9cm)
染色：用苏丹红(Sigma)将猪脂染色(每克猪脂0.75mg苏丹红)。把加热至70℃的50μl染色猪脂放置到每块布样的中心。在75℃下于烘箱中将布样加热25分钟，在第一次洗涤之前在室温下放置过夜。
水硬度：3.2mM Ca2+/Mg2+(比率为5∶1)
洗涤剂：5g/l的标准、商用欧洲压型粉末洗涤剂(ArielTM Futur)。不调整pH。
脂解酶浓度：0LU/l(对照)及12500LU/l
洗涤时间：20分钟
洗涤温度：30℃
漂洗：在流动自来水中洗15分钟
干燥：室温下放置过夜(约20℃，30-40％RH)
评价：洗涤、漂洗和干燥布样之后，在Soxhlet萃取设备中用石油醚提取残余的脂肪物质。将溶剂蒸馏掉，通过称重来确定提取自布样的残余脂肪物质的量。
结果：与用无酶洗涤液洗涤的布样中残余脂肪物质量相比，用含酶洗涤液洗涤布样去除掉了16％的脂肪物质。
从上述结果中可以明显看出，该脂解酶(即本发明的脂解酶)具有织物洗涤中“初洗”去除脂质的优异性能。
                      实施例5
        本发明的H.insolens DSM1800脂解酶的底物亲和性
下述步骤旨在评估脂解酶在脂质底物相(在本案即橄榄油)上/中积聚的能力，该相在非离子表面活性剂存在下与含酶的缓冲、碱性水相接触。在本实施例中，将本发明的上述脂解酶底物亲和性与商用性脂解酶LipolaseTM(购自Novo Nordisk A/S，Bagsvaerd，丹麦)的底物亲和性相比较。
步骤：
1.在两个同样的密封20ml管形瓶中各装入5ml缓冲液等分试样(100mM甘氨酸，pH9.0)；
2.把所选出的酶分别加入两个管形瓶中使得浓度范围为5-10LU/ml(两个瓶的浓度相同)；
3.把5ml的橄榄油加入一个管形瓶(“试样”瓶)中，猛烈摇动两个管形瓶并在4℃下温育24小时。
4.分别确定“试样”(s)瓶和“参照”(r)瓶中水相的残余脂解活性(以下标为Y，单位LU/ml)。
Ys/Yr比率给出了对该酶之底物亲和性的测度。
结果：本发明脂解酶及lipolaseTM的结果分别如下表所示。酶                             Ys/Yr x 100(％)本发明                         2LipolaseTM                    99
从上表中可明显看出，依据本发明的酶表现出对橄榄油的极高亲和力。
                               实施例6
在含脂肪醇乙氧基化物的混合单分子层中的本发明之H.insolens DSM1800脂解酶的脂解活性
使用KSV-5000单分子层设备(KSV仪器，芬兰)从涂布在水相(10mM甘氨酸，pH10.0，0.1mM EDTA，温度25℃)上的甘油三酯(dicaproin)和单组分脂肪醇乙氧基化物(七亚乙基二醇单十八烷基醚)中制备出混合单分子层。将表面压力调整到所需之值(在本案中为30mN/m)，把所选出的酶(10LU)注入水相中。
通过压缩单分子层的活动屏障的移动速度发生脂解作用，当水不溶性底物分子被水解而得到更多的水溶性产物时，该屏障靠这种速度移动以保持不变的表面压力。
利用该步骤，通过参数β来表征特殊的脂解酶，当脂解活性终止时，该参数标志着未被酶水解的剩余底物的最终面积百分比。
在本实施例中，将本发明的H.insolens DSM1800酶与商用性LipolaseTM相比较。
结果：结果总结于下表中。酶                                  β(％)本发明                               37LipolaseTM                          57
从后一结果中可明显看出，在脂肪醇乙氧基化物(即非离子表面活性剂)存在下，本发明的脂解酶在脂质水解方面性能极好。
参考资料
Becker，D.M.和Guarante，L.1991.酶学方法，194：182-187.
Gubler，U.和Hoffman，B.J.1983.基因25：263-269.
Sambrook，J.，Frisch，E.F.和Maniatis，T.1989.分子克隆化：实验室手册，冷泉港实验室，冷泉港，纽约
Sanger，F.，Nicklen，S.和Coulson，A.R.1977.国家学术科学汇编，美国.74：5463-5467.
                        序列表SEQ ID No.1编码H.insolens脂解酶的DNA序列(成熟酶+信号肽)
GCCACCACTTACCAACCAGCTTCCGCAAACAAAGTCGCCAACATGAAGTTCTTCACCACCATCCTCAGCACCGCCAGCCTTGTTGCTGCTCTCCCCGCCGCTGTTGACTCGAACCATACCCCGGCCGCTCCTGAACTTGTTGCCCGGCAGCTGGGAGCCATCGAGAACGGCCTTGAGAGCGGCAGCGCCAACGCCTGCCCCGACGCCATCCTGATCTTTGCTCGCGGCTCGACCGAGCCAGGCAACATGGGCATCACCGTCGGCCCTGCTCTCGCCAACGGCCTTGAGTCCCACATCCGGAACATCTGGATCCAGGGCGTCGGCGGCCCTTACGACGCCGCGCTGGCCACCAACTTCCTGCCGCGGGGCACCTCGCAGGCCAACATCGACGAGGGCAAGCGGCTGTTTGCGCTGGCCAACCAAAAGTGCCCCAACACGCCCGTCGTCGCCGGCGGGTACAGCCAGGGCGCGGCGCTCATCGCTGCCGCCGTCAGCGAGCTCAGCGGCGCCGTCAAGGAGCAGGTCAAGGGCGTCGCCCTCTTCGGATACACCCAAAACCTCCAGAACCGTGGCGGCATCGCCAACTACCCGCGCGAGCGCACCAAGGTGTTCTGCAACGTTGGCGACGCCGTCTGCACCGGCACGCTCATCATCACCCCGGCGCATCTGTCGTACACGATCGAGGCGCGCGGTGAGGCCGCGAGGTTCCTGCGGGATCGCATCCGTGCTTATATGGAATGGGTTATCAGAGGGAAAGATGGCTGGATAGGTAACAAAGGATGAGTCCGGGCGGGATTGGGTTCAGGAGTTGGGCAGGCGGATTGCTCGATGGCTGGATGGATGGATGGAAGCCGGGCTGGGACCGGAGGCTGATGACGGTGATGACCTTTTTCCTCAGTACATAGCATCATGATGTCTCCTGCACATATCTGTTTATGAATCGAGTTTTGGTTTGCGGCCGCTGCCTCAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAASEQ ID No.2H.insolens脂解酶的氨基酸序列(成熟酶+信号肽)  1 MKFFTTILST ASLVAALPAA VDSNHTPAAP ELVARQLGAI ENGLESGSAN ACPDAILIFA61 RGSTEPGNMG ITVGPALANG LESHIRNIWI QGVGGPYDAA LATNFLPRGT SQANIDEGKR121 LFALANQKCP NTPVVAGGYS QGAALIAAAV SELSGAVKEQ VKGVALFGYT QNLQNRGGIA181 NYPRERTKVF CNVGDAVCTG TLIITPAHLS YTIEARGEAA RFLRDRIRAY MEWVIRGKDG241 WIGNKG*SEQ ID No.3N末端氨基酸序列(成熟酶)Gln-Leu-Gly-Ala-Ile-Glu-Asn-Gly-Leu-Glu-Ser-Gly/Ala-Ser-Ala-Asn-Ala-Xaa-Pro-Asp-Ala-Ile-Leu-Ile-Phe-Ala-Arg-Gly-

Claims (17)

1.含有可编码表现出脂解活性之酶的DNA序列的DNA构建体，该DNA序列包括
a)如SEQ ID No.1所示的DNA序列，及/或可编码脂解酶的DNA序列，其可得自酿酒酵母DSM9975中的质粒，或
b)如SEQ ID No.1所示的DNA序列的类似物，及/或可编码脂解酶的DNA序列的类似物，其可得自酿酒酵母DSM9975中的质粒，其
i)与如SEQ ID No.1所示的DNA序列及/或可编码脂解酶的DNA序列同源，其可得自酿酒酵母DSM9975中的质粒，及/或
ii)与和如SEQ ID No.1所示的DNA序列及/或可编码脂解酶的DNA序列相同的寡核苷酸杂交，其可得自酿酒酵母DSM9975中的质粒，及/或
iii)可编码与由含如SEQ ID No.1所示的DNA序列的DNA序列及/或由可编码脂解酶的DNA序列所编码的多肽同源的多肽，其可得自酿酒酵母DSM9975中的质粒，及/或
iv)可编码与抗纯化脂解酶(由如SEQ ID No.1所示的DNA序列及/或由可编码脂解酶的DNA序列所编码)的抗体进行免疫反应的多肽，其可得自酿酒酵母DSM9975中的质粒，及/或来自Humicola insolens DSM1800。
2.依据权利要求1的DNA构建体，其中可编码表现出脂解活性的酶的DNA序列可得自微生物。
3.依据权利要求2的DNA构建体，其中DNA序列可得自丝状真菌或酵母。
4.依据权利要求3的DNA构建体，其中DNA序列可得自梭孢壳属、毛壳属、麻孢壳属、脉孢菌属、柄孢壳属、粪壳属或腐殖霉属的菌株。
5.依据权利要求4的DNA构建体，其中DNA序列可得自毛壳属或腐殖霉属的菌株，特别是H.insolens菌株。
6.依据权利要求5的DNA构建体，其中DNA序列分离自H.insolens DSM1800的DNA文库或在H.insolens DSM1800的DNA文库基础上产生。
7.含有依据权利要求1-6中任意一项的DNA构建体的重组表达载体。
8.含有依据权利要求1-6中任意一项的DNA构建体或依据权利要求7的重组表达载体的细胞。
9.依据权利要求8的细胞，其是真核细胞，特别是真菌细胞，如酵母细胞或丝状真菌细胞。
10.依据权利要求9的细胞，其中细胞属于曲霉属或木霉属菌株，特别是米曲霉或黑曲霉菌株。
11.生产能表现出脂解活性的酶的方法，该方法包括在有助于该酶生成的条件下培养依据权利要求8-10中任意一项的细胞以及从培养物中回收该酶。
12.能表现出脂解活性的酶，该酶
a)由依据权利要求1-6中任意一项的DNA构建体所编码，及/或
b)由依据权利要求11的方法所生产，及/或
c)与抗纯化脂解酶的抗体进行免疫反应，该纯化脂解酶由如SEQ IDNo.1所示的DNA序列及/或编码脂解酶的DNA序列所编码，该DNA序列可得自酿酒酵母DSM9975中的质粒，及/或衍生自Humicola insolens DSM1800。
13.含有依据权利要求12的脂解酶的酶制剂。
14.含有依据权利要求12的脂解酶的洗涤剂添加剂。
15.含有依据权利要求12的脂解酶的洗涤剂组合物。
16.依据权利要求15的用于洗衣物的洗涤剂组合物。
17.依据权利要求15的用于洗碟的洗涤剂组合物。
CN95196494A 1994-10-26 1995-10-26 一种具有脂解活性的酶 Pending CN1167503A (zh)

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