CN1108457A - 用于洗涤剂领域的新的脂酶变异体 - Google Patents

用于洗涤剂领域的新的脂酶变异体 Download PDF

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CN1108457A
CN1108457A CN94190243A CN94190243A CN1108457A CN 1108457 A CN1108457 A CN 1108457A CN 94190243 A CN94190243 A CN 94190243A CN 94190243 A CN94190243 A CN 94190243A CN 1108457 A CN1108457 A CN 1108457A
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J·M·范·德·兰
H·B·M·伦廷
L·J·S·M·马伦纳斯
M·M·J·考克斯
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Abstract

本发明公开了修饰的脂酶，其中，至少相当于野 生型类产碱假单胞菌脂酶21位上的甲硫氨酸被另 一种氨基酸取代，该脂酶表现出所需性质的改变，特 别是改善了洗涤性能。优选的实施方案中，所述甲硫 氨酸由亮氨酸、丝氨酸或丙氨酸取代。

Description

用于洗涤剂领域的新的脂酶变异体
本发明涉及修饰的脂酶及其在例如洗涤剂或清洗组合物中的用途。
脂酶是能够水解脂类的酶，它们被广泛用于如脂肪和油类加工、洗涤剂、诊断试剂等领域。
许多微生物生产细胞外脂酶(三酰基甘油酰基水解酶，E.C.3.1.1.3)。已在美国专利No.3,950,277中公开了适宜的脂酶。这些脂酶是从各种微生物如假单胞菌属、曲霉菌属、肺炎球菌属、葡萄球菌属、结核分枝杆菌、解脂芽生圆酵母、和核盘菌属中得到的。
在如EP463100(产碱假单胞菌)、EP0218272(类产碱假单胞菌)、EP0214761(洋葱假单胞菌)、EP0258068(Thermomyces)，EP206390(Pseccdomonas chromobacter、荧光假单胞杆菌、菌实假单胞菌、还原氮假单胞菌、划界假单胞菌和Chromobacter viscosum)中，给出了脂酶在洗涤剂组合物中用途的实例。
对于所需的应用领域，假单胞菌脂酶似乎具有良好的特征。因此，已经用假单胞菌属菌种来得到脂酶。为了在发酵中提高脂酶的产率，在同源和异源的宿主菌株中，已克隆并表达了几种脂酶基因。已经报道过的，从中克隆脂酶基因的假单胞杆菌属种的实例是洋葱假单胞菌(EP331376)、Pseudomonas glumae(EP464922)、类产碱假单胞菌(EP334462)、莓实假单胞菌(EP318775)。
假单胞杆菌属种的分类是以Palleroni等人(Int J.Syst.Bacteiol.23：333(1973))报道的，DNA-rRNA和DNA-DNA杂交研究为基础的。在Bergey′s Manual of Systematic Bacteriology(Vol.l，Seetion4，PP.160-161(1984)，Eds N.R.Kriegand J.G.Hoet，Williams and Wilkins，Baltimor/rondon)中，可见到更详细的综述。该综述同时报道的形态学数据和16s核糖体RNA同源性也支持该种分类法。
可以将具有良好应用特征的所述假单胞单菌菌株分成两个DNA同源组：类产碱假单胞菌、产碱假单胞菌、铜绿色假单胞杆菌(参见，如EP0334462)、施氏假单胞菌和门多萨假单胞菌非常相似，属于I类。例如铜绿色假单胞杆菌脂酶与类产碱假单胞菌脂酶有81％的同源性。Pseudomonas glumae和洋葱假单胞菌属于II类。表1列出了得自不同菌株的脂酶基因的同源性比较。从这种同源性比较中，可以看出，得自莓实假单胞菌的脂酶介于I类和II类之间。
来源于I类和II类的脂酶间有许多差异。发现属于I类假单胞菌的脂酶比属于II类的那些有更高的疏水性，因此是更亲脂的。这种特性对用于洗涤剂中是非常有益的。类产碱假单胞菌脂酶的疏水性使其容易粘附于表面。其它类或生物如Humicola Lanuginosa的脂酶常具有更高的亲水性。I类高疏水性脂酶仅需适当处理即可用于洗涤剂。
                                       表1
                          与其它假单胞菌脂酶的同源性
  No.     菌种     1     2     3     4     5
  1   类产碱假单胞菌     100     71     40     38     41
  2   铜绿假单胞菌     81     100     37     41     36
  3   洋葱假单胞菌     52     41     100     33     78
  4   莓实假单胞菌     56     51     46     100     34
  5   P.glumae     59     41     82     52     100
表的下部分是核酸序列的比较。上部分是氨基酸序列的比较。
可以用已提到的脂酶作为洗涤剂组合物中的组分。除脂酶外，根据其配方，洗涤剂组合物还可含有许多已知组分。
——洗涤剂粉末一般含有助洗剂(如沸石，磷酸盐)，表面活性剂(阴离子的，非离子的)聚合物(如，丙烯酰聚合物)，漂白剂前体(如硼酸盐)，漂白激活剂，结构剂(Structurant)(如硅酸盐)。调整pH的化合物(如，碱)。
——液体洗涤剂一般含有表面活性剂(如，阴离子的和非离子的)，漂白剂前体(如，硼酸盐)，漂白激活剂，调整pH的化合物(如，碱)。
也可将其它组分如酶(如，蛋白酶，淀粉酶)，有机酸，无机盐，纤维软化剂加到上述组合物中。
为了可在洗涤剂组合物中作为一种组分使用，除洗涤特性外，脂酶还应对所述组合物中的其它组分具有耐受性。
可以经若干途径，如经典的筛选方式或现代遗传和蛋白质工程技术，研制或发现用于洗涤剂组合物的酶。
对具有所需酶活性的生物或微生物的筛选可以通过如下完成，例如，从微生物或所述微生物的培养上清液中分离并纯化所述酶，确定其生化特性，检测这些生化特性是否符合特定用途的需要。如果不能从其天然的产生菌中得到所鉴定的酶，则可用重组DNA技术分离编码酶的基因，在另一种生物中表达该基因，分离并纯化所表达的酶，然后检测其是否适于所预期的用途。
另一种获得具有预期用途的新酶的方法是修饰现存的酶。特别是可经化学修饰方法(参见WO91/16423)达到该目的。通常，这些方法是非特异性的，也就是说：它们可修饰带一般侧链的所有可接触的残基，或它们依赖于所修饰的适当氨基酸的存在，而经常不能修饰难以达到的氨基酸，除非酶分子是未折叠的。
然而，通过诱变编码基因进行的酶修饰则没有上述的非特异性问题。可以用随机诱变或定点诱变完成诱变。
通过用化学诱变剂或放射诱变处理整个微生物而进行的随机诱变当然产生修饰过的酶。在这种情况下，必须要有极有效的选择方法以筛选这些特定、稀少的突变体。也可以作到提高经随机诱变分离突变体酶的可能性，即在克隆编码基因后，在体外或体内对其进行诱变，并在适当的宿主细胞内，通过再克隆突变基因表达所编码的酶。用于生产修饰酶的适当宿主有，例如，细菌(大肠杆菌、芽胞杆菌，假单胞菌)，酵母或真菌(曲霉菌属)。这种情况下，必须要有适宜的生物学选择方法，以选择所需的突变体酶。这些生物学选择方法不必直接选择酶，但最好适于工业化应用。例如，EP0407225描述了一种脂酶，它具有改善了的抗蛋白酶或氧化剂作用的稳定性。事实上，这些改进可以解决特定的问题，但不一定会产生适宜的，甚至更好的洗涤性能(参见，如EP0328229)。
EP0407225还公开了在Pseudomonas glumae脂酶序列中的突变。但该申请未说明：对于编码来源于其它微生物的脂酶的基因，应取代哪个残基。通常，对于一种特定酶的最佳突变选择并不是另一种同源酶自身最好的突变选择，而对于相关程度更低的酶当然更不是。
WO92/05249描述了带有不同疏水性或静电特性的脂类接触区的脂酶突变体，它可以产生改善的洗涤性能、稳定性、贮存稳定性以及特异性。但，该申请中描述的脂酶与假单胞杆菌属脂酶几乎没有同源性，并且实际上具有完全不同的特性，因此，不能用申请WO92/05249来确定哪个位置应被突变以得到具有所需特性的假单胞菌属脂酶。
本发明现提供了一种新的修饰过的脂酶，其中，至少相当于野生型、类产碱假单胞菌脂酶中21位的甲硫氨酸被另一种氨基酸取代，以得到所需的性质改变。在本发明优选的实施方案中，在使用条件下，这些脂酶变异体表现出改善的洗涤性能。优选的是其M21残基被亮氨酸、丝氨酸或丙氨酸取代的突变体。
图1：使用PCR的定点诱变
图2：将质粒pBRflank整合到染色体中
图3：从染色体中，重组缺失脂酶基因和质粒
图4：将突变体脂酶DNA导入染色体和质粒
图5：在3′侧区域重组。
在本说明书中，用术语“改善的洗涤性能”说明在应用条件下，即存在完整的洗涤剂组合物及已确立的条件下，洗涤结果改善。
在进行氨基酸序列排列比较时，铜绿色假单胞菌脂酶(Wohlfarth et al，(1992)，J.of General Microbiology 138(7)：1325-1335)16位的甲硫氨酸残基相当于类产碱假单胞菌酶在21位的甲硫氨酸。
优选地，野生型脂酶是由I类假单胞菌微生物生产的脂酶，所述野生型脂酶与类产碱假单胞菌脂酶优选地有70％或更高的同源性，同源性指：不同氨基酸序列(或蛋白质)排列比较时，相同的氨基酸数目。
我们第一次成功地得到突变体或修饰的酶，它们在工业生产条件下，表现出适当的甚至改善的洗涤性能。
使用能够使一个或多个氨基酸被任何其它所需氨基酸特异性取代的定点诱变，以构建并进一步选择具有改善了性能的酶。
具体地说，至少在相当于类产碱假单胞菌脂酶21位发生的特异性突变(其中原甲硫氨酸被另一种氨基酸取代)，在存在或不存在漂白剂的情况下，令人惊异地改善了洗涤性能，这说明，该位置对于洗涤性能的重要性，因此，在21位的甲硫氨酸取代，不仅会提高氧化稳定性，而且会改善洗涤性能。
以高度的氨基酸同源性为基础，类产碱假单胞菌脂酶与其它属于I类假单胞杆菌菌株的假单杆胞菌脂酶非常相近。这意味着这些脂酶的三维结构非常相似。因此，在假单胞菌菌株的属于I型的其它脂酶中的该结构位置进行修饰也有助于洗涤性能的改善。
适于取代所述甲硫氨酸的氨基酸实例包括Ala，Ser，Leu，Val，Phe，Asn和Asp。
在本发明优选的实施方案中，相当于野生型类产碱假单胞菌脂酶21位的原甲硫氨酸由亮氨酸取代，并表现出明显改善的性能，尽管在该突变体中，在疏水性或静电特性方面基本没有变化。
本发明也提供生产方法，包括在编码假单胞杆脂酶的DNA中，至少在相当于类产碱假单胞菌脂酶Met+21的位置上，进行突变，然后检测由所述突变产生的酶中，所需特性的变化。所述的特性变化包括：洗涤性能改善，特异性活性改变，pH活性改变，底物活性改变或氧化稳定性提高或所述特性的结合。
本发明的另一目的是提供一种选择方法，它能够选择洗涤性能提高了的突变体。在洗涤条件下确定脂酶性能的检测方法(SLM-试验)
为了本发明的目的，用所谓SLM-试验评估洗涤过程中的碱性脂酶突变体。SLM-试验使用的原理与由T.Hashimoto等人(Yukagaku 34(1985)，606-612)开展的方法的原理相同，但分析所需的时间大大减少。该方法包括：用纤维上固定的非乳化脂肪或油作为试验污渍，洗涤后提取小布块，分析提取物的脂肪及脂肪酸。根据所用的条件，洗涤过程中，形成的脂肪酸与残留的甘油三酯可以驻留在织物上，脂肪酸作为脂酶活性的效果。因此，在布块上剩余的产物数量看起来是洗涤过程中脂酶性能的良好尺度。
下面是如何优选地完成SLM试验的典型实例。用聚酯布块作为纤维，三油酸甘油酯或纯化的橄榄油(均是Sigma的产品，USA)作为底物。提取织物后，可接着对水解后的三油酸甘油酯进行色谱分析。
用于SLM试验目的的优选的洗涤过程如下：将80ml溶于正己烷(12.5％)的10mg橄榄油沾在聚酯布(3×3cm)上。室温下，将该布块空气干燥。将含10mLSTW(标准自来水：含2mM氯化钙和0.7mM氯化镁的蒸馏水)的洗涤液或溶于STW的洗涤剂放在带毛玻璃塞的Erlenmeyer烧瓶(50ml)中，然后于40℃保持在振荡水浴中。洗涤过程从将脂酶Ml(40ILU，见下文)和随后将污渍的布块加到Erlenmeyer烧瓶中开始，然后振荡40分钟。空白实验中，没有加入脂酶。洗涤后，用STW漂洗布块，随后于55℃干燥1小时，然后进行第二轮洗涤。在含5ml溶剂的玻璃试管中旋转提取干燥的布块，所述溶剂含有与底物及产物色谱分离所用的洗脱液相同的组成。
由HPLC测定所提取溶液中残留的甘油三酯量及形成的游离脂肪酸和1，2和1，3-二酰基甘油酯的量。设备及条件：泵：        LKB(2150型)检测：      折光监测仪(Jobin Yvon)注射***：  Perkin-Elmer Iss-101；10ul积分仪：    Spectra Physics，Chromjet柱：        CP Microspher-Si(Chrompack)，100×4.6mm洗脱液：    正己烷/异丙醇/甲酸：
        975∶25∶2.5(v/v)，1ml/分钟温度：      室温
在这些条件下，三油酸甘油酯、油酸，1.3和1.2-二酰基甘油酯的保留时间分别为1.2，1.6，2.4和3.4分钟。测量峰面积或峰高。在从布块提取后，它们是三甘油酯、游离脂肪酸和二酰基甘油酯的回收尺度。以从未洗涤的布块提取后，三甘油酯的回收率为100％。在上述条件下，以峰高度为基础，发现橄榄油、油酸、1.2和1.3-二酰甘油酯之间的折光指数反应比例为1.00，0.98，2.10和1.30.确定脂酶活性的检测方法
以橄榄油的水解为基础，确定按ILU表达的本发明脂酶及突变体的活性。在30℃，存在20mM氯化钠和10Mm氯化钙的情况下，在一含10％橄榄油的0.4mM Tris缓冲液(pH9)的恒pH槽中测量水解。将1 ILU定义为在检测条件下，每分钟释放1μmol脂肪酸所需的酶量。
提供下列实施例说明但不限制本发明。
                    实施例1
研制脂酶基因的诱变***
可以将目前的定点诱变法分成两类：
(1)缺口双链DNA由突变引物引导的错误植入(ill-in)会产生一个异源双链DNA，它含一条突变链。
(2)通过取代靶基因内的盒，可以导入适当突变的序列。然后将含所需突变的PCR片数换到表达盒内。
使用PCR的定点诱变
该方法来自Xiong，他在第三届假单胞杆菌会议(1991)上，对该方法作了综合描述。图1对该方法作了图示说明。
为了分离单链DNA，我们使用质粒pTMPv18A，在EP0334462中有描述。Vent-聚合酶(Biolabs)用于扩增反应。该聚合酶含3′校对(Proofreading)活性，因此减少了因PCR反应引起的错误掺入。
在下列缓冲液中完成PCR反应；所述缓冲液含：10mM KCl，10mM(NH4)2SO4，20mM Tris-HCl，pH8.8，2mM MgSO4，及0.1％Triton X-100，0.2mM dATP，0.2mM dCTP，0.2mM dGTP，0.2mMdTTP和1单位Vent聚合酶。
在步骤1中，将0.5μM引物A(含突变)，0.05μM引物B和1-10ng单链pTMPv18A加至50μl反应混合物中，并完成98℃ 2分钟，55℃ 2分钟；72℃ 2分钟的25个循环。
加入Klenow DNA聚合酶和连接酶，用含突变的片段作为引物补平单链DNA。这样产生一异源双链DNA，含一条突变的链。然后，用核酸外切酶处理混合物以除去单链DNA和引物A及B。然后沉淀DNA，并将其溶于PCR反应缓冲液。用含突变的新合成的DNA链作为步骤2中的模板。
步骤2中，将0.05μM引物C加到该混合物中。按上述完成第2次PCR。
然后用适当的限制酶消化PCR片数，然后将其亚克隆到整合的载体中。
                     实施例2
在活性位点附近导入突变：改变M21位
按上述导人突变。选XhoI和BclI作为适当的限制酶。所用的寡核苷酸列于表2：
                             表2
 Oligo 5′→3′ 引物号码     实变     位置
 CTGCGGGCTGGTGCTGGAGCTGCC 引物C      -   829-852R
 GGATGCAAGGATGGATCAGTGCCC 引物B      -   266-280
 CGAAGCCGAGNNNGCCGTGGGTC 引物A     M21X   426-450R
实施例3
类产碱假单胞菌M1(CBS473.85)脂酶基因的染色体失活
用***性整合质粒(它不能在类产碱假单胞菌中复制，但能在其它微生物中复制)使类产碱假单胞菌染色体中的脂酶基因失活。
将酯酶基因片段从质粒pTMPv18亚克隆到质粒pBR322(Bolivar等Gene 2(1977)95-113)上，该质粒能在大肠杆菌中复制，但不能在类产碱假单胞菌中复制。然后从该质粒中缺失一个内片段。所得质粒称为pBRflank。
用pBRflank转化类产碱假单胞菌M1(CBS473.85)。由于该质粒不能在假单胞菌中复制，所以只有经整合才能得到四环素抗性菌落。挑选数个四环素抗性(5mg/l)菌落。在这些菌株中，通过在5′或3′侧区域的单一重组，质粒pBRflank被整合到细菌染色体上(图2)。由于这些菌株仍含有能发挥功能的脂酶基因，因此，它们表现出脂酶阳性及四环素抗性表型。为了从染色体中缺失脂酶基因和该质粒，必须进行第二次重组(切割)。通过在没有抗生素的情况下，使菌株在BHI(脑心浸出物)培养基中生长数天，达到上述目的。
然后将细胞铺在含三丁酸甘油酯的琼脂培养基上。同样检测含脂酶阴性表型的菌落，看其是否不能在选择性琼脂盘中生长。将所得的脂酶阴性菌株称为Ps600。
在图2和3中，列出了该整合过程，然后是第二次重组的示意图。将突变的脂酶基因导入脂酶阴性菌落类产碱假单胞菌Ps600。
为了生产高产量的突变体酶，在质粒上的突变基因被整合到染色体中同时被导入相同的菌株质粒上。用与上述实施例中相似的方法，将突变脂酶基因整合到染色体中。
图4和5给出了该过程的示意图。生产突变体脂酶
按EP0334462所述，使菌株生长。然后从培养液中纯化脂酶蛋白质。EP0334462对该方法也作了描述。
                     实施例4
测定M21L突变体脂酶的特异性活性
用上述的活性检测法及对确定蛋白质含量的定量氨基酸分析，确定野生型和突变体的特异性活性。突变体的特异性活性为野生型酶的1.4倍：8000比6390ILU/mg蛋白质。
                    实施例5
在应用条件下(SLM试验)野生型脂酶M1(CBS47 3.85)和某些M21突变体的脂酶活性
按上文所述完成SLM试验。在下列条件下，于单和双循环洗涤试验中，检测野生型和突变体：——标准自来水(STW)——洗涤剂是Ariel UltraTM(2g/l)——脂酶量如所示Ariel UltraTM是Procter & Gamble的产品，可从市场上买到。该洗涤剂既不含蛋白酶也不含脂酶。
这些条件基本代表欧洲人的洗涤条件。
                      表3
有和没有脂酶M1及某些M21突变体的情况下，1和2个洗涤循环后，残留甘油三酯的百分比
                          表3
脂酶 浓度(μg/ml)     残留的甘油三酯(％)
    1个循环     2个循环
    M1M1M1M1M1M21LM21LM21LM21LM21LM21LM21AM21AM21AM21AM21AM21AM21SM21SM21SM21SM21SM21S     00.230.450.680.9100.160.310.470.631.250.250.500.751.001.502.000.250.500.751.001.502.00     8271686060845843393432605642352423696864615753     5845372920ndndndnd22281611720452933231713
nd＝未测定
从该表可以看出，所用的脂酶对织物有脂解特性。这些结果清楚地说明：当加入相同的重量时，在这些应用条件下，M21L和M21A突变体比野生型酶更有活性。M21S突变体的脂解特性可与野生型脂酶相比。
                     实施例6
根据SLM试验，在洗涤过程中，野生型脂酶(M1)与突变体M21L的性能。
在下列条件下，检测这些酶与粉末洗涤剂及漂白***的相容性：——标准硬度的水(0.75mM的钙和0.25mM的镁)——洗涤剂是TideTM(1g/l)——按标明的脂酶量——2个循环的洗涤试验
TideTM是Procter & Gamble的产品，可从市场上买到。该洗涤剂含蛋白酶但没有脂酶。
这些条件基本代表美国的洗涤条件。
从表4可清楚地看出：实施例中所用的脂酶对织物具有脂解特性，特别是在粉末洗涤剂中的洗涤性能。在含有和不含漂白剂的洗涤剂中，该突变体酶的洗涤性能优于野生型脂酶。
                                   表4
                       脂酶M1和突变体M21L的洗涤性能
脂酶     浓度.(μg/ml) 漂白剂*                   回收(％)
  TG**     DG     FFA     总量
    M1M1M1M1M1M1M1M1M1M1M21LM21LM21LM21LM21LM21LM21LM21LM21LM21LM21LM21LM21LM21LM21LM21L     00.230.450.680.9100.230.450.680.9100.080.160.310.470.630.941.2500.080.160.310.470.630.941.25     -----+++++--------++++++++     100896362509095989497102928143353329249878503933292314     038910000010458887605898786     029121600001015172120202305152022212323     100948083769095989499102979168646156539888736863575443
*漂白***(所用的浓度为0.3g/l)是过硼酸盐/NOBS(6.6∶1)；NOBS代表壬酰氧基苯磺酸酯**TG＝甘油三酯，DG＝甘油二酯，FFA＝游离脂肪酸
                      实施例7
根据SLM试验，在洗涤过程中野生型脂酶M1(CBS473.85)和M21S及M21A突变体的性能
在下列条件下，检测这些酶与粉末洗涤剂和漂白***的洗涤性能和相容性：——标准硬度的水(0.75mM的钙和0.25mM的镁)——洗涤剂是TideTM(1g/l)——按标明的脂酶量——2个循环的洗涤试验
TideTM是Procter & Gamble的产品，可从市场上买到。该洗涤剂含蛋白酶但没有脂酶。在本洗涤试验中所用的布块来自在洗涤中有不同行为的不同批次。
这些条件基本代表美国的洗涤条件。
从表5可清楚地表明：本实施例中所用的脂酶在含有和不含漂白***的洗涤剂中，对织物表现出脂解特性和其洗涤性能。
而M21S的洗涤性能可与野生型的相比，M21A的洗涤性能得到改善。
                                   表5
                 脂酶M1、突变体M21S和突变体M21A的洗涤性能
脂酶     浓度.(μg/ml) 漂白****                   回收(％)**
    TG     DG     FFA     总量
    M1M1M1M1M1M1M1M21SM21SM21SM21SM21SM21SM21SM21SM21SM21SM21AM21AM21AM21AM21AM21AM21AM21AM21AM21A     00.230.450.6800.230.6800.250.500.751.0000.250.500.751.0000.130.250.500.7500.130.250.500.75     ----+++-----+++++-----+++++     352416163420153525171713342317141335231516153416141110     000000000000000000000100000     0556021033540244406681104568     352921223422163528202217342521181735292124273420191718
*漂白***(所用的浓度为0.3g/l)是过硼酸盐/NOBS(6.6∶1)；NOBS代表壬酰氧基苯磺酸酯**TG＝甘油三酯，DG＝甘油二酯，FFA＝游离脂肪酸
                     实施例8
在不同温度的应用条件下，野生型M1脂酶和M21L突变体的脂酶活性
按上文所述完成SLM试验。在下列条件下的单一洗涤试验中检测野生型和突变体：——标准自来水(STW)——洗涤剂是Ariel UltraTM(2g/l)——按标明的脂酶剂量
Ariel UltraTM是Procter & Gamble的产品，可从市场上买到。它不含酶。
                              表6
    在有和无M21L突变体和野生型脂酶M1存在下，在不同温度下一
             个洗涤循环后，残留的甘油三脂的百分比
脂酶     浓度(μg/ml)   残留的TG*(％)   在T(℃)一个洗涤循环后
    40     50     60
    -M1M1M1M1M21LM21LM21LM21L     00.210.430.640.850.200.410.610.81     756055554647423636     856664565348352525     717168787265595653
*TG＝甘油三酯
本实施例清楚地说明：在检测的应用条件下，在所有的检测温度下，突变体的脂解能力均优于野生型。在这些应用条件下，发现脂酶M1的最佳温度(甘油三酯水解的最大值)40℃，突变体的值更高：50℃。
                    实施例9
配制的脂酶M1和某些M21突变体的存放稳定性
在-PEG成粒器(PEG-Prill)中配制脂酶M1和M21L，M21S及M21A突变体。然后在下列表7中标明的温度下，将这些颗粒存于密封的玻璃试管中。贮存8个星期后，在恒pH槽中，测定脂酶的残留活性。
正如从表中看到的，在37℃贮存8个星期后，所含的所有脂酶的残留活性均为其原活性的90％以上。
                     表7
     脂酶M1和各种M21突变体的贮存稳定性
      在℃贮存的残留活性4     25     27脂酶
  酯酶M1        98    95     95M21L突变体    97    102    102M21S突变体    97    94     92M21A变体      93    95     95
根据本发明的新脂酶变异体在洗涤方面，如洗衣，洗碗盘是特别有用的，但也可将其用于本领域已知的其它方面。优选的是以颗粒(如散颗粒)或液体的形式。通常与稳定剂一起用脂酶作为洗涤剂添加剂。Met+21的取代对洗涤剂的稳定性有积极效果，其程度取决于所用的特定氨基酸。在含和不含漂白***的情况下，Met+21突变体在溶液中的稳定性特别好。可以与其它酶，如淀粉酶、蛋白酶、纤维素酶和各种细胞壁降解酶一起使用。
本发明说明书中提到的所有公开文本(包括专利和专利申请)是与本发明有关的技术领域的专业人员可以理解的。所有公开文本均并入本文作为参考。
尽管为了清楚理解的目的，以说明及实施例的方式详细描述了本发明，但对本领域专业人员显而易见的是，在不背离本发明权利要求精神和范围内，也可以进行多种变化和修改。
                 序列目标
(1)一般信息
   (i)申请人
      (A)名称：GIST-BROCADES N.V.
      (B)街道：P.O.Box 1
      (C)城市：Delft
      (E)国家：Netherlands
      (F)邮政编码(ZIP)：NL-2600 MA
      (G)电话：31.15.791111
      (H)电话：31.15.793957
   (ii)发明题目：用于洗涤剂领域的新的脂酶变异体
   (iii)序列数目：3
   (iv)计算机阅读形式
       (A)介质类型Floppg盘
       (B)计算机：兼容
       (C)操作***：PC-DOS/MS-DOS
       (D)软件 PatentIn Release #1.0，Version #1.25(EPO)
   (vi)在先中请资料：
       (A)申请号： EP 93201212.3
       (B)申请日：27-APR-1993
(2)SEQ ID No.1的资料：
   (i)序列特征：
      (A)长度：23个碱基对
      (B)类型：核酸
      (C)链型：单链
      (D)拓扑结构：线性
   (ii)分子类型 ：DNA(合成)
   (iii)假设：无
   (vi)来源：
       (C)个体分离物：引物A
   (xi)序列描述 ：SEQ ID NO：1：CGAAGCCGAG NNNGCCGTGG GTC
(2)SEQ ID No.2的资料
   (i)序列特征：
       (A)长度：24个碱基对
       (B)类型：核酸
       (C)链型：单链
       (D)拓扑结构：线性
   (ii)分子类型：DNA(合成的)
   (iii)假设：无
   (vi)来源：
       (C)个体分离物 ：引物B
   (xi)序列描述：SEQ ID No：2：GGATGCAAGG ATGGATCAGT GCCC
(2)SEQ ID No.3的资料
   (i)序列特征：
      (A)长度：24个碱基对
      (B)类型：核酸
      (C)链型：单链
      (D)拓扑结构：线性
   (ii)分子类型：DNA(合成的)
   (iii)假设：无
   (vi)来源：
      (C)个体分离物：引物C
   (xi)序列描述 ：SEQ ID No：3：CTGCGGGCTG GTGCTGGAGC TGCC

Claims (13)

1、一种修饰过的脂酶，其中至少相当于野生型类产碱假单胞菌(Pseudomonas Pseudoalcaligenes)脂酶21位的甲硫氨酸被另一种氨基酸取代，所述脂酶表现出所需的特性变化。
2、根据权利要求1的修饰的脂酶，其中野生型脂酶来源于I型假单胞菌(Pseudomonas)微生物。
3、根据权利要求1或2的修饰的脂酶，其中野生型脂酶与类产碱假单胞菌脂酶有至少70％的同源性。
4、根据权利要求1-3之任一的修饰的脂酶，其中21位上的甲硫氨酸被Leu，Ser或Ala取代。
5、根据权利要求1-4之任一的修饰的脂酶，表现出改善的洗涤性能。
6、一种洗涤剂组合物，含有权利要求1-5之任一的修饰的脂酶。
7、权利要求1-5之任一的修饰的脂酶作为洗涤剂组分的用途。
8、一种重组DNA序列，其编码权利要求1-5之任一的修饰的脂酶。
9、一种重组DNA载体，其含权利要求8的重组DNA序列并可用于表达修饰的脂酶。
10、一种转化的宿主微生物，其含有权利要求8的重组DNA序列或权利要求9的重组DNA载体。
11、根据权利要求10的微生物，转化的宿主微生物是假单胞菌。
12、一种方法，包括在至少相当于类产碱假单胞菌脂酶Met+21的位置，在编码假单胞杆菌脂酶的DNA中进行突变，并检测在由所述突变产生的酶中，所需特性的变化。
13、根据权利要求12的方法，其中所述特性变化选自洗涤性能改善、特异性活性改变、pH活性改变、底物活性改变和氧化稳定性提高的一种或多种。
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WO2024126483A1 (en) 2022-12-14 2024-06-20 Novozymes A/S Improved lipase (gcl1) variants
EP4389864A1 (en) 2022-12-20 2024-06-26 Basf Se Cutinases
WO2024131880A2 (en) 2022-12-23 2024-06-27 Novozymes A/S Detergent composition comprising catalase and amylase

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DE69434242T2 (de) 2006-01-12
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FI116728B (fi) 2006-02-15
AU673078B2 (en) 1996-10-24
ATE287946T1 (de) 2005-02-15
EP0652946A1 (en) 1995-05-17
FI945998A0 (fi) 1994-12-21
KR950702240A (ko) 1995-06-19
JPH07508659A (ja) 1995-09-28
JP3618748B2 (ja) 2005-02-09
CA2138519A1 (en) 1994-11-10
EP0652946B1 (en) 2005-01-26
WO1994025578A1 (en) 1994-11-10
PL306812A1 (en) 1995-04-18
FI945998A (fi) 1995-02-10

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