CN1072718C - 脂肪酶及其微生物、该脂肪酶的制造方法及用途 - Google Patents

脂肪酶及其微生物、该脂肪酶的制造方法及用途 Download PDF

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CN1072718C
CN1072718C CN94193220A CN94193220A CN1072718C CN 1072718 C CN1072718 C CN 1072718C CN 94193220 A CN94193220 A CN 94193220A CN 94193220 A CN94193220 A CN 94193220A CN 1072718 C CN1072718 C CN 1072718C
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石田礼子
铃木雅博
小塚隆司
崎元和范
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Abstract

一种属于假单胞菌属(Pseudomonas)的细菌、该细菌所产生的具有如下性质的脂肪酶、该脂肪酶的制造方法及含该脂肪酶的洗涤剂组合物。
以三油酸甘油酯乳液为基质，其作用pH为3.5-12，最佳pH为10-12；其作用温度为30-80℃，最佳温度为55-65℃；以SDS-聚丙烯酰胺凝胶电泳法所测得的分子量为31000±2000；由等电点聚丙烯酰胺凝胶电泳法所测得的等电点为5.2±0.5。本发明的脂肪酶在各种市售的洗涤剂溶液中，又在与表面活性剂、蛋白酶等的洗涤剂成分共存时，具有较高的稳定性和活性，可以在洗涤条件下有效地去除油脂污垢，可增加洗涤剂的洗净力。

Description

脂肪酶及其微生物、该脂肪酶的制造方法及用途
发明领域
本发明涉及一种新颖的脂肪酶、产生该脂肪酶的微生物、该脂肪酶的制造方法及用途。更详细地说，本发明涉及一种产生细菌的、在洗涤液中(碱性域)具有活性的脂肪酶，产生该脂肪酶的微生物，该脂肪酶的制造方法，以及含有在洗涤液中可分解脂质的酶的洗涤剂组合物。
为了提高洗涤时的洗净率，以往，已知有在洗涤剂中掺和使用酶的方法。例如，在洗涤剂组合物中掺用蛋白酶，以分解去除粘附于被洗物上的蛋白质及其它污垢。另外，也有掺用纤维素酶以去除粘附于纤维素纤维被洗物上的污垢，或掺用淀粉酶等多糖分解酶以去除粘附于被洗物上的多糖及其它污垢。
近年来，更有在洗涤剂中掺用脂肪酶以分解去除粘附于被洗物上的脂类，提高洗净率的方法。其用途记载于安得利(H.Andree)等人的题为“作为洗涤剂成分的脂肪酶”(Lipase as detergentcomponents)的报道(Journal of Applied Biochemistry,2,218-229(1980))上。
较理想的洗涤剂用的脂肪酶为在洗涤液中可充分发挥脂肪酶活性的脂肪酶。由于在通常的洗涤条件下，洗涤液pH处于碱性区域，因此，人们便要求在碱性pH域内具有功能脂肪酶。又，一般的脂质油污在高温/高碱的条件下比较容易去除，而在低温(60℃以下)下的洗涤则不容易充分洗净脂质油污。我国的洗涤方法从来都是以低温洗涤为主，近来，欧美各国也出现了洗涤温度低温化的倾向。因此，理想的洗涤剂用脂肪酶是在低温下也能充分发挥作用的脂肪酶。另外，理想的洗涤剂用脂肪酶在存在有表面活性剂等的洗涤剂成分，和许多种洗涤剂中含有的蛋白酶及漂白剂时，应也能充分发挥洗涤功能。而且，所述理想的洗涤剂用脂肪酶以掺和于洗涤剂中的状态保存时，对其余的洗涤剂的成分应保持稳定。人们希望开发掺有具有如上所述的特性的脂肪酶的、对脂质油污具有高洗净率的洗涤剂组合物。
已知，微生物产生的脂肪酶得自假单胞菌(Pseudomonas)属、产碱杆菌(Alcaligenes)属、消色杆菌(Achromobacter)属、毛霉菌(Mucor)属、假丝酵母菌(candida)属、腐植菌(Humicola)等菌属。
但得自这些菌株的脂肪酶的最合适的pH为中性至微碱性，在碱性洗涤剂溶液中不能充分发挥其性能，且在洗涤溶液中稳定性差。
再有，得自消色杆菌(Achromobacter)属、假丝酵母菌(candida)属、毛霉菌(Mucor)属、腐植菌(Humicola)属的各种脂肪酶在共存有阴离子表面活性剂时，其活性强烈受阻。
又，属于假单胞菌(Pseudomonas)属细菌的微生物产生脂肪酶，已广为人知。在产生脂肪酶的假单胞菌(Pseudomonas)属细菌中，有，Ps.(Pseu-domonas)fluorescens,Ps.cepacia,Ps.fragi,Ps.alcaligenes,Ps.pseudoal-caligenes,Ps,aeruginosa。但是，从上述菌株所得的已知的脂肪酶也不能满足前述特性。
                          发明目的
因此，本发明的目的是，提供一种脂肪酶、产生该脂肪酶的微生物、该脂肪酶的制造方法，所述脂肪酶在洗涤液中可充分发挥功能，其活性几乎不受洗涤剂中含有的成分所阻碍，且对如表面活性剂和蛋白酶等其它的洗涤剂成分呈现高的稳定性。
又，本发明的其它目的在于提供一种含有上述脂肪酶为洗涤辅助剂的洗涤剂组合物，及除了上述脂肪酶之外，再含有蛋白酶等其它的酶而组成的含酶洗涤剂组合物。
                      发明的揭示
本发明者们为得到具有上述性质的脂肪酶，对许多微生物进行了分离、培养并检索，其结果，发现由从东京都地下土壤中分离出来的菌株的假单胞菌属(Pseudomonas Sp.)SD705菌株所代表的假单胞菌属(Pseu-domonas)的菌株可产生出可有效地掺用于洗涤剂的新颖的脂肪酶，从而完成了本发明。
即，本发明涉及的是这样一种新的脂肪酶，该脂肪酶系由属于假单胞菌(Pseudomonas)属的微生物或其变异菌株产生，在pH3.5-12的整个反应pH范围内均有作用，其最适温度为55-65℃附近。
又，本发明涉及一种上述脂肪酶的制造方法，产生该脂肪酶的新颖微生物及含有该脂肪酶的洗涤剂组合物。
                   附图的简单说明
图1所示为以本发明的SD705菌株产生的脂肪酶的反应pH和相对活性之关系的图表。
图2所示为以本发明的SD705菌株产生的脂肪酶的反应温度和相对活性之关系的图表。
图3所示为将本发明的SD705菌株产生的脂肪酶在各种温度下，在pH7作一小时的处理后的残余活性的图表。
图4所示为将本发明的SD705菌株产生的脂肪酶在各种pH下，在37℃作一小时的处理后的残余活性的图表。
发明的详细说明(产生菌)
用于制造本发明的脂肪酶的微生物，可以产生出具有下述性质的脂肪酶，且，只要是具有下述分类学意义上的性质的假单胞菌(Pseudomonas)属细菌即可，并无特别限定。这种细菌可从保存的菌株中或从分离自自然界的微生物中选用。另外，这些细菌也可以是自然的或人工变异菌株，只要是可产生具有如下所述的性质的脂肪酶的细菌，皆在此范围。该此细菌菌株可按通常的方法，从土壤等其它的分离源分离而得。将被测微生物在通常的细菌用培养基中进行培养，按通常的方法，测定在高pH、常温的条件下的培养液的脂肪酶活性，由此选用目的菌株。
作为本发明的产生新颖的脂肪酶的、属于假单胞菌(Pseudomonas)属的菌株，可举出本发明者们从东京都的地下土壤中分离的SD705菌株。
该SD705菌株的菌学性质如表1所示。表1中，参照Bergey’s Manu-al of Systematic Bacteriology,1984，一并记载了与本菌具有比较类似的菌学性质的Ps.alcaligenes、Ps.pseudoalcaligenes的菌学性质。
表1
SD705菌株 Ps.alcaligenes  Ps.Pseudoalcaligenes
(1)形态     杆菌     杆菌     杆菌
(2)革兰氏染色性     阴性     阴性     阴性
(3)芽胞     无     无     无
(4)运动性     有     有     有
(5)鞭毛     极单毛     极单毛     极单毛
(6)氧化酶     阳性     阳性     阳性
(7)过氧化氢酶     阳性     阳性     阳性
(8)萤光色素的产生     无     无     无
(9)PHB的蓄积     阴性     阴性      d
(10)精氨酸二氢化酶     阴性     阳性      d
(11)41℃下的生长     可     可      可
(12)脱氮反应     阳性     阴性      d
(13)明胶液化     阴性     d      d
(14)淀粉分解     阴性     阴性      阴性
(15)葡萄糖的脂化性     阴性     阴性      阴性
(16)天冬氨酸盐的脂化性     阴性     阴性      阴性
(17)L谷氨酸盐的脂化性     阳性     阳性      阳性
(18)D-葡萄酸盐的脂化性     阴性     阴性      d
表1(续)
SD705菌株 Ps.alcaligenes   Ps.Pseudoalcaligenes
(19)L-组氨酸的脂化性     阴性     d      d
(20)乙醇胺的脂化性     阴性     阴性      阳性
(21)正丁醇的脂化性     阳性     d      阳性
(22)异丁醇的脂化性     阴性     d      阴性
(23)甘油丙三醇的脂化性     阴性     阴性      d
(24)山梨糖醇的脂化性     阴性     阴性      d
(25)衣康酸的脂化性     阴性     阴性      d
(26)中康酸的脂化性     阴性     阴性      阳性
(27)β-羟基丁酸盐的脂化性     阳性     阴性      阳性
(28)甜菜碱的脂化性      阴性  阴性  阳性
(29)果糖的脂化性        阴性  阴性  阳性
(30)丙三醇酸盐的脂化性  阴性  阴性  阳性
(31)GC含量(％)          60    64-68 62-64
d：属于该菌种菌株的11～89％为阳性。
如表1所示，SD705菌株在乙醇胺、中康酸、甜菜碱、果糖、甘油酸盐的脂化性上与假单胞菌(Pseudomonas)属、pseudoalcaligenes不同，在精氨酸脱羧酶的有无、β-二羟基丁酸盐的脂化性上与Ps.alcaligenes不同，且，其GC含量也较该二菌株为低。
又，根据日本细菌学杂志，45(5),1990所载，对Ps.alcaligenes的基准株ATCC909和Ps.pseudoalcaligenes的基准株ATCC17440进行定量DNA的杂种，这对于该二基准株都呈现了不到30％的相同性。由这些结果可以决定本菌株是属于假单胞菌属的Ps.alcaligenesde的近缘新菌种。
本菌株保存于通商产业省工业技术学院生命工程工业技术研究所(National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology)，其保存编号为FERM P-13781。以后，又根据国际上认可的有关微生物保存的专利手续上的布达佩斯条约(BUDAPEST TREATY ON THEINTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDURE)，转为国际保存，其保存编号为FEREM BP-4772。
又，以上述菌株为原菌株，通过自然变异或诱导变异所得到的变异菌株也可以用作本发明的脂肪酶的生产菌。
作为上述变异菌株的配制方法，有如下所述的通常的方法，即，不对原菌株进行紫外线辐射处理或用N-甲基-N’-硝基-N-亚硝基胍(NTG)等药剂的人工突变处理；或者是，对原菌株进行了如上的处理之后，将其扩展于含有橄榄油等的琼脂培养基中，从成长的菌株中选出其周围的净区范围较大的菌丛，以生产脂肪酶的培养基进行培养，选择出生产性最优异的菌株。
本发明的脂肪酶产生菌最好是SD705菌株和在DNA杂交中显示50％以上的同系现象的菌株，更好地，是显示70％以上的同系现象的菌株。
(制造方法)
本发明的脂肪酶通过培养属于假单胞菌(Pseudomonas)属的脂肪酶产生菌，主要地产生于菌体外(培养液中)。
作为培养基的营养源，可广泛使用通常用于培养的营养源。只要是可作为碳源同化的碳化合物或者是含有该碳化合物的即可，例如，可使用油脂、Tween系的表面活性剂等。作为氮源，只要是可作为氮源同化的氮化合物或者是含有该氮化合物的即可，例如，可用铵盐、硝酸盐、大豆粉、肉类提取物、玉米浸渍液、pharmamedia等。又，作为无机盐类，可适当添加磷酸氢氨、磷酸氢钾等的磷酸盐、镁盐、钙盐等的盐类。
培养条件依培养基的组份而多少不同，但只要是适合于产生目的的本发明中的脂肪酶的条件即可，通常，可选择以下的条件。即，培养温度为10-40℃，更好地，为20-37℃的范围；培养时间约为8-100小时，只要在本发明的脂肪酶的生产达到最高时结束培养即可。培养基的pH可在5-12的生产范围，特别是，7-10适于本发明的脂肪酶的生产。经如上所述的培养，主要在菌体外(培养液中)产生目的物的脂肪酶。
(分离精制法)
从如上所得的培养液中采集脂肪酶，可按照通常用于采集脂肪酶的方法，由分离、精制而进行。
即，可由下述方法获得本发明的脂肪酶：由过滤法或离心分离法从培养液中分离菌体及培养基固形物，得到上清液或滤液，对该些分离液进行或不进行浓缩，添加可溶性盐类，使酶沉淀的盐析法；添加亲水性的有机溶剂，使酶或夹杂物沉淀的有机溶剂沉淀法；使用离子交换树脂等的吸附脱离法；凝胶过滤法；添加稳定辅助剂或不添加稳定辅助剂的喷雾干燥法；单独或多个组合使用冷冻干燥法等的分离或精制方法。
(酶活性的测量方法)
在本发明中，使用了以三油酸甘油酯-聚乙烯醇(PVA)乳液为基质的测量方法进行对脂肪酶活性的测量。
将含酶液0.1ml、含1mM的氯化钙、由100mM的ε-氨基己酸、100mM双三(双(2-羟乙基)亚氨基三(羟甲基)甲烷)及100mM的TAPS(N-三(羟甲基)甲基-3-氨基丙磺酸)组成的混合液用氢氧化钠调节pH(pH10.0)的缓冲液0.4ml以及三油酸甘油酯乳液0.5ml组成的混合液在带毛玻璃塞的试管中，在37℃下加热10分钟，使其反应，用1N的盐酸0.2ml作为反应停止液使反应停止。这里，三油酸甘油酯乳液用的是在聚乙烯醇(PVA)2％的水溶液(Poval PVA 117(Kuraray公司商品名)：Poval PVA205(Kuraray公司商品名)=9∶1(w/w))10ml中加入2.5g的三油酸甘油酯并经过均化的乳液。反应停止后，加入正己烷2ml、异丙醇2ml、蒸馏水1ml，激烈搅拌，静置后，将己烷层取样，以TLC-FID方法(Minagawa等，Lipids,18,732(1983))测定油酸量。以一分钟内生成1毫摩尔的油酸的酶量作为活性单位的1单位(1U)。
又，在下面的实施例中，按特公昭60-55118号上所述的活性测定方法，测量在下面的实施例中掺用的蛋白酶的活性，其活性单位表示为纳卡他尔(nkatal=10-9katal，缩写为nkat)
(酶的性质)
本发明的脂肪酶具有下述性质。即，关于本发明的假单胞菌(Pseu-domonas)属的菌株SD705产生的脂肪酶，用下述性质加以记载。(1)作用作用于甘油三酸酯，水解该酯。(2)基质特异性可广范围地水解各种甘油三酸酯及酯类。
作为甘油三酸酯基质，使用的是各种甘油三酸酯-阿拉伯胶乳液。使用的乳液是在甘油三酸酯10g中加入阿拉伯胶10g和蒸馏水100g均化而成的乳液。
将含上述乳液5ml、100mM的氯化钠及25mM的氯化钙的10mM的三羟甲基氨基甲烷型缓冲液(pH10.0)5ml、蒸馏水4.5ml、酶液0.5ml的混合物在30℃、pH10的条件下反应，用0.05N氢氧化钠水溶液，pH起动测定法求得此时的脂肪酸生成速度。该脂肪酸的生成速度作为各基质的分解力。
如将三油酸甘油酯的分解力表示为100，则三丁酸甘油酯的相对活性显示为125，橄榄油的为55，大豆油的为70，棉籽油的为66。
另外，对于酯的分解力，曾以脂肪酸对硝基苯酯为基质，由在pH8.0、30℃下的水解反应生成的对硝基苯酚的比色(OD405)而求得。
如以pNPP(棕榈酸对硝基苯酯)的分解力为100，则pNPL(月桂酸对硝基苯酯)的相对活性显示为134,pNPV(戊酸对硝基苯酯)为34。(3)作用pH和最佳pH
以三油酸甘油酯乳液为基质，按前述的活性测试方法进行测定。测定的pH分别为3.5～12.0范围内的不同的pH值。但是，缓冲液使用了含有1.0mM的氯化钙，由将100mM的ε-氨基己酸、100mM的双三(双(2-羟乙基)亚氨基三(羟甲基)甲烷)以及100mM的TAPS(N-三(羟甲基)甲基-3-氨基丙磺酸)组成的混合缓冲液用盐酸或氢氧化钠调节pH的缓冲液。反应pH和相对活性的关系示于图1，在pH为3.5～12的范围内测定时，作用pH为3.5～12，最佳pH为10～11。(4)作用温度及最佳温度
以三油酸甘油酯乳液为基质，其它与前述的活性测定方法相同，在30-80℃的范围内的不同的反应温度下进行测试。反应温度和相对活性的关系示于图2，在30-80℃的温度范围内测定时，其作用温度为30-80℃，其最佳温度为55-65℃。在40及70℃时，表示了约为最佳温度时的活性的50％的相对活性。(5)温度稳定性
按上述活性测定法，测定在20-70℃的温度范围内的不同温度下，在pH7作一小时的保温处理后的剩余活性。此时的处理温度和残余活性的关系如图3所示，60℃的处理可具有80％以上的活性，处理时的缓冲液使用了将由50mM的ε-氨基己酸、50mM的双三(双(2-羟乙基)亚氨基三(羟甲基)甲烷)以及50mM的TAPS(N-三(羟甲基)甲基-3-氨基丙磺酸)组成的混合缓冲液用盐酸调节至7的缓冲液。(6)pH稳定性
按上述活性测定法，测定在pH4-12的范围内的不同pH下，在37℃的温度作一小时的保温处理后的剩余活性。此时的处理pH和残余活性的关系如图4所示，pH在4-10时具有50％以上的活性，处理时的缓冲液使用了将含有0.5mM的氯化钙、由50mM的ε-氨基己酸、50mM的双三(双(2-羟乙基)亚氨基三(羟甲基)甲烷)以及50mM的TAPS(N-三(羟甲基)甲基-3-氨基丙磺酸)组成的混合缓冲液用盐酸或氢氧化钠调节过pH的缓冲液。(7)分子量
以SDS-聚丙烯酰胺凝胶电泳法(分子量标准：细胞色素C(单体、二聚物、三聚物、四聚物、五聚物)所得到的分子量为31000±2000。(8)等电点
由等电点聚丙烯酰胺凝胶电泳法所测得的等电点为5.2±0.5。(9)洗涤剂成分的影响
以三油酸甘油酯乳液为基质，将下述的标准使用浓度的各种市售洗涤剂(4种)和代表性的洗涤剂用的碱性蛋白酶的API-21(特公昭60-55118号)以0.3nkat/ml的浓度添加其中，调节反应pH为10，反应时间为30分钟，其余如同前述的活性测定法，进行测量。以市售的洗涤剂及不添加API-21时的活性作为100时的相对活性示于表2，在含蛋白酶的各种洗涤剂中，具有高的活性。
具有上述的性质的脂肪酶在低温下(60℃以下)也具有足够的功能，在洗涤剂溶液中也可稳定地表现其活性，因此是很适合用于洗涤剂掺用的脂肪酶。(洗涤剂组合物)
根据本发明，可以提供具有上述性质的脂肪酶的洗涤剂组合物。掺用于本发明的洗涤剂组合物的脂肪酶的用量没有特别限制，通常是，对每g洗涤剂组合物掺用100-10000单位，更好地，对每g洗涤剂组合物掺用500-4000单位。其用量过少，则洗净效果不能充分提高；而在相反的情况下，如其用量过多，则该洗净效果并不随酶用量的增加而增加，其经济性并不理想。
按照本发明，上述脂肪酶可以在不改变洗涤剂的组成的情况下，掺用于已有公知的任一种洗涤剂组合物，对于本发明的洗涤剂组合物的成分并无特别的限制。这类洗涤剂组合物的代表性例子有：由分别占洗涤剂组合物重量的10-50％(重量)的表面活性剂，0-50％(重量)的助洗剂，1-50％(重量)的碱性剂或无机电解质，0.1-50％(重量)的选自再污染防止剂、酶、漂白剂、荧光染料、防结块剂以及抗氧化剂中的至少一种的掺用成分组成的洗涤剂组合物。
作为表面活性剂，可以使用肥皂；例如；具有直链或支链烷基或链烯基的硫酸盐、氨基硫酸盐；具有直链或支链烷基或链烯基、加合了如环氧乙烷、环氧丙烷及环氧丁烷中的单独或混合成分的烷基醚或链烯基醚那样的脂肪族硫酸化合物；如烷基磺酸盐、氨基磺酸盐、二烷基磺基琥珀酸酯、α-烯烃、亚乙烯基型烯烃及内烯烃的各种磺酸盐的脂肪族磺酸盐；具有直链或支链烷基或链烯基、加合了如环氧乙烷、环氧丙烷及环氧丁烷中的一个或多个成分的烷基或链烯基醚的羟酸盐或酰胺；α-磺基脂肪酸盐或酯；氨基酸型表面活性剂；烷基或链烯基酸性磷酸酯、烷基或链烯基磷酸酯盐的磷酸酯系表面活性剂；磺酸型两性表面活性剂；甜菜碱型两性表面活性剂；具有直链或支链烷基或链烯基、加合了如环氧乙烷、环氧丙烷及环氧丁烷中的一个或多个成分的烷基或链烯基醚或醇；具有直链或支链烷基或链烯基、加合了如环氧乙烷、环氧丙烷及环氧丁烷中的一个或多个成分的聚氧乙烯烷基苯基醚；高级脂肪酸烷醇酰胺或其环氧化物加合物；蔗糖脂肪酸酯；脂肪酸甘油单酯；烷基或链烯基胺的氧化物；四烷基铵盐型阳离子表面活性剂等，只要是通常可掺用于洗涤剂组合物的表面活性剂即可。在阴离子型的表面活性剂的场合，作为其抗衡离子最好是钠离子或钙离子。上述表面活性剂可单独或组合二种以上使用。
作为助洗剂及碱性剂或无机电解质可以使用：正磷酸盐、焦磷酸盐、三聚磷酸盐、偏磷酸盐、六偏磷酸盐、肌醇六磷酸盐等的磷酸盐；如乙烷-1,1-二膦酸、乙烷-1,1,2-三膦酸、乙烷-1-羟基-1,1-二膦酸及其衍生物；如乙烷羟基-1,1,2-三膦酸、乙烷-1,2-二羧酸-1,2-二膦酸、甲羟基膦酸等膦酸的盐；2-膦酰基丁烷-1,2-二羧酸、1-膦酰基丁烷-2,3,4-三羧酸、α-甲基膦酰基琥珀酸等膦酰基羧酸的盐；如天冬氨酸、谷氨酸等的氨基酸；如氮三乙酸盐、乙二氨四乙酸盐、二乙三氨五乙酸盐等的氨基多乙酸；如聚丙烯酸、聚衣康酸、聚马来酸、马来酸酐共聚物、羧甲基纤维素盐等的高分子电解质；如聚乙二醇、聚乙烯醇等的非离解性高分子；如二醇酸、羟基丁二酸、羧基甲氧基丁二酸、柠檬酸、乳酸、酒石酸、蔗糖、乳糖等的羧甲基化合物；如季戊四醇的羟甲基化合物；葡糖酸的羧甲基化合物；如苯多甲酸、草酸、苹果酸、羟基二琥珀酸、葡糖酸等的有机酸盐；如沸石等的铝硅酸盐；碳酸盐、倍半碳酸盐、硫酸盐、硅酸盐等作为碱金属盐的无机盐。又，也可使用如淀粉、脲等的有机物质及氯化钠、膨润土等的无机化合物。再有，作为有机碱剂，可以使用三乙醇胺、二乙醇胺、单乙醇胺、三异丙醇胺等。
本发明的洗涤剂组合物，如前所述，含有表面活性剂、脂肪酶、碱性剂及无机电解质作为必要的组成成分，但是，按需要，也可含有两性表面活性剂；如过碳酸氢钠、过硼酸氢钠等的漂白剂；色素；助洗剂；如聚乙二醇、聚乙烯醇、聚乙烯基吡咯烷酮、羧甲基纤维素等的再污染防止剂；防结块剂、抗氧化剂；蛋白酶等其它的酶。
可用任何方法将脂肪酶或蛋白酶之类的其它的酶掺用于本发明的洗涤剂组合物中。但是，如以微粉末状掺用时，则在操作洗涤剂时散发出粉尘，对洗涤剂的使用者及洗涤剂生产的操作者来说，有安全和卫生上的问题，不甚理想。最好是，以溶液状态，或预先赋与一定的形状，以控制粉尘的散发。所述赋形方法可采用通常使用的圆形造粒、挤出造粒、流动造粒、离心流动造粒及其它的方法。但是，用于本发明中的洗涤剂组合物的脂肪酶或蛋白酶之类的其它酶的形状并不限于由上述方法所赋于的形状。
下面，举实施例说明本发明，但是，本发明并不限于下述的实施例。另外，在下述的说明中，如无特别说明，则％皆指重量％。
实施例1
脂肪酶产生菌(SD705菌株)的培养
将大豆粉2％、磷酸氢二铵0.1％、橄榄油1％、磷酸氢二钾0.5％、硫酸镁七水合物0.1％及碳酸钠0.3％浓度的液体培养基2ml装入18mm口径的试验管中，在121℃作高压蒸汽灭菌20分钟后，接种假单胞菌属(Pseudomonas)SD705菌株1白金环，在35℃，于130rpm下培养24小时。培养后，离心分离去除菌体，得到脂肪酶液。所得的脂肪酶液的脂肪酶活性为5U/ml。
实施例2
脂肪酶产生菌(SD705菌株)的培养及脂肪酶的取得
将大豆粉2％、磷酸氢二铵0.1％、磷酸氢二钾0.5％、硫酸镁七水合物0.1％、碳酸钠0.3％及Tween85的1.0％浓度的液体培养基2立升装入5立升的培养槽中，在121℃作高压蒸汽灭菌20分钟后，接种假单胞菌属(Pseudomonas)SD705菌株，在35℃，于1000rpm下通气搅拌培养24小时。培养后，离心分离去除菌体，得到脂肪酶液。所得的脂肪酶液的脂肪酶活性为20U/ml。
从如此所得的脂肪酶液中，由硫酸铵沉淀法得到20-40％饱和部分的沉淀。再以通常的方法脱盐后，冷冻干燥得脂肪酶的原粉。
实施例3
脂肪酶的精制
将实施例2中所得的脂肪酶原粉溶解于10％的硫酸铵溶液中，以Butyl-Toyopear1650M(商品名，东ソ株式会社)作疏水色谱，得到活性成分。将该活性成分用含有0.3mM的氯化钙的10mM的三羟甲基氨基甲烷盐酸缓冲液(pH8)作透析后，使其吸附于由该缓冲液平衡过的离子交换色谱树脂(DEAE-CellulofineA-800，商品名，生化学株式会社)上，得到以氯化钠浓度梯度溶出的活性成分。脱盐后，冷冻干燥，得到精制的酶。
上述冷冻干燥制品经SDS聚丙烯酰胺凝胶电泳法确认为单一
制品。
实施例4
本发明的脂肪酶和其它的脂肪酶在洗涤剂溶液中的活性比较
使用实施例2中所得的脂肪酶原粉，作洗涤剂溶液中的活性比较，比较美国专利第5069810号上所载的Pseudomonas alcaligenesSD2菌株(ATCC 53877)所产生的脂肪酶SD2，和欧洲专利第218272号上所载的Pseudomo n as Pseudoalcaligenes CBS 467.85菌株、CBS468.85菌株、CBS 471.85菌株、CBS 473.85菌株所产生的脂肪酶在洗涤剂溶液中的活性测定结果。Pseudomo n as alcaligenesSD2菌株的酶为在硫酸铵0.5％、磷酸氢二钾0.05％、硫酸镁七水合物0.025％、胰化蛋白胨2.0％、聚氧乙烯(20)十六烷基醚1.0mM的培养基中、30℃下培养16小时而得，而上述四种Pseudomo n asPseudoalcaligenes菌株的酶为分别将其菌株在脱脂牛奶10％、pH7的培养基中，在20℃下培养48小时，使用其经离心分离后所得的培养液上清液。
以三油酸甘油酯乳液为基质，将下述的标准使用浓度的各种市售洗涤剂(4种)和代表性的洗涤剂用的碱性蛋白酶API-21(特公昭60-55118号)以0.3nkat/ml的浓度添加其中，调节反应pH为10，反应时间为30分钟，其余如同前述的效力测定法，进行测量。这里使用的市售洗涤剂有，Attack(商品名，花王株式会社制，直链烷基苯磺酸钠，烷基硫酸酯钠及含有聚氧乙烯烷基醚的阴离子系表面活性剂的洗涤剂)；Ultra Ariel(商品名，P&G远东株式会社制；直链烷基苯磺酸钠，烷基硫酸酯钠、烷酰基羟基苯磺酸钠及含有聚氧乙烯烷基醚的阴离子系表面活性剂的洗涤剂)；Ultra Tide(商品名，P&G株式会社制，阴离子型表面活性剂)FreshStart(商品名，Colgate-Palmolive株式会社制，非离子型表面活性剂)。作为标准的使用浓度，分别添加最终浓度为，Attack 833ppm,Ultra Ariel 1000ppm,Ultra Tide 1000ppm,FreshStart 624ppm。
设不添加市售洗涤剂及API-21时的活性为100，则上述洗涤剂的相对活性示于表2。
表2洗涤剂溶液中的相对活性(单位％)
                            洗涤剂酶产生菌株 洗涤剂 Attack Ultra Ariel Ultra Tide Fresh StartSD705      100    78     84          57         98ATCC 53877 100    31     29          33         80CBS 467.85 100    17     18          29         102CBS 468.85 100    54     50          55         62CBS 471.85 100    20     16          35         85CBS 473.85 100    50     49          40         95
从上表可以明白，本发明的脂肪酶比起其它的脂肪酶来，可在洗涤剂的溶液中发挥更高的活性。
即，在比较用的其它脂肪酶中，虽然，有时，有的洗涤剂也可显示与本发明的脂肪酶同等水准的相对活性，但是，如果洗涤剂的种类发生变化，则其相对活性就显著降低(来自CBS 467.85菌株及CBS468.85菌株的酶)。而相比之下，本发明的脂肪酶的特征是，其所显示的高的相对活性不随洗涤剂的种类而变化。
实施例5
洗涤剂组合物及洗涤效果评价
在市售洗涤剂的Attack中，以2400u/g的比率掺用实施例2中所得的脂肪酶原粉，按本发明配制成含脂肪酶的洗涤剂组合物。
按如下所述地进行洗涤效果的评价。即，使脱脂棉布(15cm×15cm)浸透溶于苯中的三油酸甘油酯70mg，在室温下干燥过夜，用作污染布。洗净装置使用Terg-O-Tometer。在加有氯化钙的最终浓度为50ppm的蒸馏水1立升中，分别以标准使用浓度溶入前述掺和了脂肪酶的洗涤剂组合物及市售洗涤剂Attack。在前者的场合，液中的脂肪酶浓度为2单位/ml。再有，添加蛋白酶API-21(特公昭60-55448)至0.3nkat/ml。在每1立升的洗涤液中加入上述六块三油酸甘油酯污染布，用30C的洗涤温度、120rpm的转速洗涤30分钟。洗净后用前述含钙的蒸馏水1立升洗，共进行二次，各洗3分钟，然后，室温下干燥。未洗净及洗净后的污垢布上的三油酸甘油酯的量由用正己烷提取后的前述TLC-FID法测定，由下述的计算式求得洗净效率。
Figure C9419322000181
其结果示于表3。
                  表3洗涤评价
            添加有本发明的脂肪酶     不添加脂肪酶洗净效率(％)    78.4                      68.5
如表3所示，在添加有本发明的脂肪酶时，其对脂质污垢的洗净效率比起不添加脂肪酶的要高。
实施例6
洗涤剂组合物及洗涤效果评价
分别配制成下述三种脂肪酶/蛋白酶的洗涤剂组合物：在市售的洗涤剂All(lever兄弟公司制，商品名)中，以2400u/g的比率掺用实施例2中所得的脂肪酶原粉而成的掺以脂肪酶的洗涤剂组合物；在All中以270nkat/g的比率掺用添加蛋白酶API-21(特公昭60-55448)配制而得的掺以蛋白酶的洗涤剂组合物；以及在All中以2400u/g的比率掺用实施例2中所得的脂肪酶原粉和以270nkat/g的比率掺用添加蛋白酶API-21配制而得的掺有脂肪酶/蛋白酶的洗涤剂组合物。
按如下所述地进行洗涤效果的评价。将市售的污染布EMPA112用作污染布。洗净装置使用Terg-O-Tometer。在添加氯化钙至最终浓度为50ppm的蒸馏水1立升中，分别按标准使用浓度(最终浓度1100ppm)溶入前述的洗涤剂组合物及市售洗涤剂All。在添加了脂肪酶的场合，液中的脂肪酶浓度为2.6单位/ml。在添加了蛋白酶的洗涤剂组合物的场合，液中的蛋白酶浓度为0.3nkat/ml。在每1立升的洗涤液中加入上述六块三油酸甘油酯污染布，用30℃的洗涤温度、120rpm的转速洗涤30分钟。洗净后用前述含钙的蒸馏水1立升洗，共进行二次，各洗3分钟，然后，室温下干燥。测得洗净后的污染布的反射率(460nm)。酶对洗净的添加效果以经掺酶的洗涤剂组合物的洗净后的反射率和经不掺酶的洗涤剂组合物的洗净后的反射率之差表示。其结果示于表4。
                    表4洗涤评价洗涤剂组合物                    添加酶的效果掺用脂肪酶                       8.2掺用蛋白酶                       9.8掺用脂肪酶/蛋白酶               15.6
如表4所示，在添加有本发明的脂肪酶时，不论是否添加有蛋白酶，其洗净效果皆比不添加脂肪酶的要高。
                        发明效果
本发明的脂肪酶在各种市售的洗涤剂溶液中，又在与表面活性剂、蛋白酶等的洗涤剂成分共存时，具有较高的稳定性和活性，可以在洗涤条件下有效地去除油脂污垢，可期望配合洗涤剂增加洗净力。
又，本发明的假单胞菌属(Pseudomonassp.)SD705菌株及在菌学上来说与其同等的菌株及其变异菌株，可有效地用于产生本发明的脂肪酶。
再有，本发明的优点在于，使用所述菌株的制造具有上述性质的脂肪酶的方法可有效地产生所述的脂肪酶。
又，通过本发明，将脂肪酶掺用于洗涤剂中，可以提供一种具有优异的洗净性能的洗涤剂组合物。
又，通过本发明，将脂肪酶及其它的酶掺用于洗涤剂中，可以提供一种具有优异的洗净性能的洗涤剂组合物。

Claims (8)

1．一种脂肪酶，其特征在于，所述脂肪酶具有下述性质：
(1)作用pH及最佳pH
以三油酸甘油酯乳液为基质，在pH范围为3.5-12内所测定的其作用pH为3.5-12，最佳pH为10-12；
(2)作用温度及最佳温度
以三油酸甘油脂乳液为基质，在温度范围为30-80℃内，所测定的作用温度为30-80℃，最佳温度为55-65℃；
(3)分子量
以SDS-聚丙烯酰胺凝胶电泳法所得到的分子量为31000±2000；
(4)等电点
由等电点聚丙烯酰胺凝胶电泳法所测得的等电点为5.2±0.5。
2．如权利要求1所述的脂肪酶，其特征在于，所述脂肪酶系从属于假单胞菌属的细菌的培养物得到。
3．如权利要求1所述的脂肪酶，其特征在于，所述脂肪酶从假单胞菌属SD705菌株的培养物得到。
4．一种细菌，其特征在于，所述细菌属于产生权利要求1所述的脂肪酶的假单胞菌属。
5．一种假单胞菌属SD705菌株(FERM BP-4772)，其特征在于，所述菌株为产生权利要求1所述的脂肪酶的假单胞菌属。
6．一种脂肪酶的制造方法，其特征在于，培养如权利要求4或权利要求5所述的细菌，从培养液中回收权利要求1所述的脂肪酶。
7．如权利要求1所述的脂肪酶的用途，其特征在于，所述脂肪酶用于洗涤剂组合物。
8．如权利要求7所述的脂肪酶的用途，其特征在于，在所述洗涤剂组合物还含有其它的酶。
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CN1129954A (zh) 1996-08-28
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US5827718A (en) 1998-10-27
EP0721981A1 (en) 1996-07-17
EP0721981A4 (en) 2002-07-31
FI960949A (fi) 1996-02-29
JPH0767636A (ja) 1995-03-14
AU7508794A (en) 1995-03-22
JP2859520B2 (ja) 1999-02-17

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