CN101960008B - 具有脂肪酶活性的多肽和编码该多肽的多核苷酸 - Google Patents
具有脂肪酶活性的多肽和编码该多肽的多核苷酸 Download PDFInfo
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Abstract
本发明提供通过将突变引入在亲本脂肪酶中鉴定的一个或多个区域而获取的多肽。令人惊奇的发现本发明的多肽针对短链脂肪酸具有低比活性，导致与本领域已知的脂肪酶相比减少的气味产生和增加的BR。
Description
发明领域
本发明涉及相对气味产生具有改进的洗涤效果的脂肪酶变体，及制备它们的方法。其特别涉及细毛嗜热霉(Thermomyceslanuginosus)脂肪酶的变体。
背景技术
脂肪酶是有用的，例如，作为洗涤剂酶用于从衣物和其它纺织品去除脂质或脂肪污渍，及作为用于面包和其它烘焙产品的生面团的添加剂。因此，源自细毛嗜热霉(Thermomyceslanuginosus)(同物异名疏棉状腐质霉(Humicolalanuginosa)，EP258068和EP305216)的脂肪酶以商品名(NovozymesA/S的产品)出售用于洗涤剂用途。WO0060063描述了细毛嗜热霉脂肪酶的变体，其在洗涤剂溶液中具有特别好的初次洗涤性能(first-washperformance)。WO9704079、WO9707202和WO0032758也公开了细毛嗜热霉脂肪酶的变体。
在一些应用中，感兴趣的是将产生气味的短链脂肪酸的形成降至最低。因此，已知的是含有脂肪酶的洗衣洗涤剂有时可能留下残留的气味沾在受到牛奶污染的衣物上(EP430315)。WO02062973公开了脂肪酶变体，其中通过附接(attaching)C末端延伸减少了气味的产生。最近公开的WO07087508公开了脂肪酶变体，其中通过在亲本脂肪酶中鉴定的一个或多个区域中引入突变而减少了气味产生。WO07087503描述了具有脂肪酶活性的多肽，且其在该说明书中给定的测试条件下进一步具有至少0.8的RP和至少1.1的BR。
发明内容
在第一方面，本发明涉及具有脂肪酶活性的第一多肽，其中所述多肽是具有下列中的至少一项的多肽：(a)相对于280nm处的吸光度(A280)的脂肪酶活性(LU)少于500LU/A280，其中一单位的LU(1LU)定义为能够在30℃，pH7每分钟释放1μmol的丁酸的的酶量，且所述多肽的吸光度在280nm测定；(b)风险性能气味(Riskperformanceodor，R)低于0.5，其中R计算为自多肽洗涤样本释放的丁酸量和自参照多肽洗涤样本释放的丁酸量的比例，计算比例之前两个值均相对于自非多肽洗涤样本释放的丁酸量进行了校正；或者(c)效益风险因子(BenefitRiskfactor，BR)至少为1.8，其中BR定义为平均洗涤性能(averagewashperformance，RPavg)除以风险性能气味(R)。
在第二方面，本发明涉及具有脂肪酶活性的第二多肽，其包含在下述位置的氨基酸的改变：T231R+N233R+I255A+P256K及下列中的至少一种：(a)S58A+V60S+A150G+L227G；或(b)E210V/G，所述位置对应于SEQIDNO：2。
在进一步的方面，本发明涉及编码具有脂肪酶活性的多肽的分离的多核苷酸、包含该多核苷酸的核酸构建体、包含所述核酸构建体的重组表达载体以及包含所述核酸构建体或所述重组表达载体的转化的宿主细胞。
在进一步的方面，本发明涉及制备所述多肽的方法，包括下述步骤：(a)在有益于所述多肽产生的条件下培养包含所述核酸构建体或重组表达载体的转化宿主细胞，所述核酸构建体或重组表达载体包含所述多肽；以及(b)回收该多肽。
在进一步的方面，本发明涉及包含所述多肽的配制物。
在进一步的方面，本发明涉及通过使用所述多肽在脂质水解过程中减少产生气味的短链脂肪酸形成的方法。
附图简述
图1显示脂肪酶的比对。
序列列表
SEQIDNO：1显示编码来自细毛嗜热霉的脂肪酶的DNA序列。
SEQIDNO：2显示来自细毛嗜热霉的脂肪酶的氨基酸序列。
SEQIDNO：3显示来自反射犁头霉(Absidiareflexa)的脂肪酶的氨基酸序列。
SEQIDNO：4显示来自伞枝犁头霉(Absidiacorymbifera)的脂肪酶的氨基酸序列。
SEQIDNO：5显示来自曼赫根毛霉(Rhizmucormiehei)的脂肪酶的氨基酸序列。
SEQIDNO：6显示来自米根霉(Rhizopusoryzae)的脂肪酶的氨基酸序列。
SEQIDNO：7显示来自黑曲霉(Aspergillusniger)的脂肪酶的氨基酸序列。
SEQIDNO：8显示来自塔宾曲霉(Aspergillustubigensis)的脂肪酶的氨基酸序列。
SEQIDNO：9显示来自尖镰孢(Fusariumoxysporum)的脂肪酶的氨基酸序列。
SEQIDNO：10显示来自异孢镰孢(Fusariumheterosporum)的脂肪酶的氨基酸序列。
SEQIDNO：11显示来自米曲霉(Aspergillusoryzea)的脂肪酶的氨基酸序列。
SEQIDNO：12显示来自沙门柏干酪青霉(Peniciliumcamembertii)的脂肪酶的氨基酸序列。
SEQIDNO：13显示来自臭曲霉(Aspergillusfoetidus)的脂肪酶的氨基酸序列。
SEQIDNO：14显示来自黑曲霉的脂肪酶的氨基酸序列。
SEQIDNO：15显示来自米曲霉的脂肪酶的氨基酸序列。
SEQIDNO：16显示来自Landerinapenisapora的脂肪酶的氨基酸序列。
发明详述
使用脂肪酶去除脂质和脂肪污渍(fattystain)在本领域是已知的，其中导致游离短链脂质(如丁酸)的释放的脂肪酶活性与令人不快的(undesirable)气味相关。底物三丁酸甘油酯的水解导致丁酸的释放。令人惊讶地发现本发明的多肽在中性pH针对三丁酸甘油酯具有低比活性(作为LU/A280测定)，参见实施例2和表3。
效益风险因子(BR)通过相对(洗涤)性能(效益，RP)除以风险性能气味(风险，R)来计算。所述洗涤性能可通过自动机械应力试验(automatedmechanicalstressassay，AMSA)来测定，参见实施例3，而气味产生可直接通过气相层析来测定，参见实施例4和表3。气味减少影响BR，且可导致BR增加。另外还发现本发明的多肽与本领域已知的脂肪酶相比较具有减少的气味产生和增加的BR，参见实施例5和表3。
脂肪酶活性(LU)：如本文所使用的术语“脂肪酶活性”指催化在二酰基甘油和羧酸的形成下三酰基甘油的水解的羧酸酯水解酶活性。就本发明而言，脂肪酶活性通过下述过程来确定：脂肪酶的底物通过使用阿拉伯胶作为乳化剂对三丁酸甘油酯(甘油三丁酸)进行乳化来制备。接着在30℃，pH7或9在pH恒定的滴定实验中水解三丁酸甘油酯。一个单位的脂肪酶活性(1LU)定义为能够在30℃，pH7每分钟释放1μmol丁酸的酶量。
风险性能气味(R)：如本文所使用的术语“风险性能气味”指自多肽洗涤样本释放的丁酸量和自参照多肽洗涤样本释放的丁酸量之间的比例，之前两个值均相对于自非多肽洗涤样本释放的丁酸量进行了校正。
相对性能(RP)：如本文所使用的术语“相对性能”指与参照多肽的洗涤性能相比较的所述多肽的洗涤性能。就本发明而言，相对性能根据在实施例3中所描述的过程来确定。
参照多肽：如本文所使用的术语“参照多肽”、“参照酶”或“参照脂肪酶”指具有取代T231R+N233R的SEQIDNO：2的成熟部分。
效益风险因子(BR)：如本文所使用的术语“效益风险因子”指与气味产生风险(R)相比较的平均相对性能(RPavg)，且具有下列公式：BR＝RPavg/R。
氨基酸修饰的命名法
为了易于参考，在描述本发明的脂肪酶变体时，采用了以下命名法：
原始氨基酸：位置：取代氨基酸
根据这种命名法，例如，在位置195中用谷氨酸取代甘氨酸表示为G195E。在相同的位置中缺失甘氨酸表示为G195*，而插入额外的氨基酸残基例如赖氨酸表示为G195GK。与其它脂肪酶相比，当特定脂肪酶含有“缺失”并且在该位置进行插入的情况，就在位置36插入天冬氨酸而言，将这种情况表示为*36D。
用加号分隔多个突变，即：R170Y+G195E，分别表示在位置170用酪氨酸取代精氨酸，和在位置195用谷氨酸取代甘氨酸。
当应用所述排列方法时，X231表示在亲本多肽中对应于位置231的氨基酸。X231R表示用R置换所述氨基酸。就SEQIDNO：2而言，X是T，因此X231R表示用R取代位置231的T。当某个位置(例如231)中的氨基酸可以由选自一组氨基酸的另一个氨基酸取代的情况，例如由R和P和Y组成的组，将这种情况表示为X231R/P/Y。
在所有的情况下，采用公认的IUPAC单字母或三字母的氨基酸缩写。
同一性：如本文所使用的术语“同一性”指两个氨基酸序列之间或两个核苷酸序列之间的相关性，由参数“同一性”来描述。
就本发明而言，通过使用来自EMBOSS软件包(http://emboss.org)版本2.8.0的Needle程序来测定两个氨基酸序列之间的比对。Needle程序执行总体比对算法(globalalignmentalgorithm)，其在Needleman，S.B.andWunsch，C.D.(1970)J.Mol.Biol.48，443-453中描述。使用的取代矩阵是BLOSUM62，缺口开启罚分(gapopeningpenalty)是10，并且缺口延伸罚分(gapextensionpenalty)是0.5。
本发明的氨基酸序列(“发明序列”；例如SEQIDNO：2的氨基酸1至269)和不同氨基酸序列(“外源序列”)之间的同一性程度如下计算：用两个序列比对中的精确匹配数除以“发明序列”的长度或“外源序列”的长度中最短的一个。将结果表示为百分比同一性。
当“发明序列”和“外源序列”在重叠的相同位置中具有相同的氨基酸残基时，发生精确匹配。序列的长度是序列中氨基酸残基的数目(例如SEQIDNO：2的长度是269)。
可以使用上述方法计算同一性和同源性，并用于比对。在本发明的上下文中，同源性和比对如下所述计算。
同源性和比对
就本发明而言，可以通过本领域已知的计算机程序的手段合适地测定同源性的程度，所述程序例如GCG程序包中提供的GAP(ProgramManualfortheWisconsinPackage，Version8，August1994，GeneticsComputerGroup，575ScienceDrive，Madison，Wisconsin，USA53711)(Needleman，S.B.andWunsch，C.D.，(1970)，JournalofMolecularBiology，48，443-45)，按照用于多肽序列比较的以下设定来使用GAP：GAP产生罚分(GAPcreationpenalty)为3.0，和GAP延伸罚分(GAPextensionpenalty)为0.1。
在本发明中，通过图1所示的比对定义反射犁头霉、伞枝犁头霉(Absidiacorymbefera)、曼赫根毛霉、德氏根霉、黑曲霉、塔宾曲霉、尖镰孢、异孢镰孢、米曲霉、沙门柏干酪青霉、臭曲霉、黑曲霉、细毛嗜热霉(同物异名：疏棉状腐质霉)和Landerinapenisapora的脂肪酶序列中相应(或同源)的位置。
为了发现所述比对中未显示的脂肪酶序列中的同源位置，将感兴趣的序列与图1中显示的序列进行比对。通过使用GAP比对将新序列与GAP程序发现的最同源的序列进行比对，将所述新序列排入图1中的现有比对。GAP在GCG程序包(ProgramManualfortheWisconsinPackage，Version8，August1994，GeneticsComputerGroup，575ScienceDrive，Madison，Wisconsin，USA53711)(Needleman，S.B.andWunsch，C.D.，(1970)，JournalofMolecularBiology，48，443-45)中提供。使用以下设置用于多肽序列比较：GAP产生罚分为3.0，且GAP延伸罚分为0.1。
具有脂肪酶活性多肽的来源
可以使用任何合适的多肽。在一些实施方案中，所述多肽可以是真菌多肽。
所述多肽可为源自下列属的酵母多肽：如，念珠菌属(Candida)、克鲁维酵母属(Kluyveromyces)、毕赤酵母属(Pichia)、酵母属(Saccharomyces)、裂殖酵母属(Schizosaccharomyces)或西洋蓍霉属(Yarrowia)多肽；或更优选是源自下列属的丝状真菌多肽：如，枝顶孢霉属(Acremonium)、曲霉属(Aspergillus)、短梗霉属(Aureobasidium)、隐球菌属(Cryptococcus)、Filobasidium、镰孢属(Fusarium)、腐质霉属(Humicola)、梨孢菌属(Magnaporthe)、毛霉属(Mucor)、毁丝霉属(Myceliophthora)、新考玛脂霉属(Neocallimastix)、脉孢菌属(Neurospora)、拟青霉属(Paecilomyces)、青霉属(Penicillium)、瘤胃壶菌属(Piromyces)、裂褶菌属(Schizophyllum)、踝节菌属(Talaromyces)、嗜热子囊菌属(Thermoascus)、梭孢壳属(Thielavia)、弯颈霉属(Tolypocladium)、嗜热霉属(Thermomyces)或木霉属(Trichoderma)。
另外，所述多肽可为具有脂肪酶活性的卡尔酵母(Saccharomycescarlsbergensis)、酿酒酵母(Saccharomycescerevisiae)、糖化酵母(Saccharomycesdiastaticus)、道格拉氏酵母(Saccharomycesdouglasii)、克鲁弗酵母(Saccharomyceskluyveri)、诺地酵母(Saccharomycesnorbensis)或卵形酵母(Saccharomycesoviformis)多肽。
或者，所述多肽是棘孢曲霉(Aspergillusaculeatus)、泡盛曲霉(Aspergillusawamori)、烟曲霉(Aspergillusfumigatus)、臭曲霉、日本曲霉(Aspergillusjaponicus)、构巢曲霉(Aspergillusnidulans)、黑曲霉、米曲霉、塔宾曲霉、杆孢状镰孢(Fusariumbactridioides)、禾谷镰孢(Fusariumcerealis)、库威镰孢(Fusariumcrookwellense)、大刀镰孢(Fusariumculmorum)、禾本科镰孢(Fusariumgraminearum)、禾赤镰孢(Fusariumgraminum)、异孢镰孢、合欢木镰孢(Fusariumnegundi)、尖镰孢、多枝镰孢(Fusariumreticulatum)、粉红镰孢(Fusariumroseum)、接骨木镰孢(Fusariumsambucinum)、肤色镰孢(Fusariumsarcochroum)、拟分枝孢镰孢(Fusariumsporotrichioides)、硫色镰孢(Fusariumsulphureum)、圆镰孢(Fusariumtorulosum)、拟丝孢镰孢(Fusariumtrichothecioides)、镶片镰孢(Fusariumvenenatum)、特异腐质霉(Humicolainsolens)、细毛嗜热霉(同物异名：疏棉状腐质霉)、米赫毛霉(Mucormiehei)、嗜热毁丝霉(Myceliophthorathermophila)、粗糙脉孢菌(Neurosporacrassa)、产紫青霉(Penicilliumpurpurogenum)、哈茨木霉(Trichodermaharzianum)、康宁木霉(Trichodermakoningii)、长枝木霉(Trichodermalongibrachiatum)、里氏木霉(Trichodermareesei)或绿色木霉(Trichodermaviride)多肽。
在一些实施方案中，本发明涉及是嗜热霉属脂肪酶的多肽。
在一些实施方案中，本发明涉及是细毛嗜热霉脂肪酶的多肽。
在一些实施方案中，本发明涉及下述多肽，其中所述多肽与SEQIDNO：2至少50％，至少60％，至少70％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或100％相同。
鉴定具有脂肪酶活性的多肽中的改变
下面提及的位置是SEQIDNO：2中氨基酸残基的位置。将在“同源性和比对”段落所描述的过程用于在不同脂肪酶中寻找氨基酸残基的对应或同源位置。
在一些实施方案中，本发明涉及具有脂肪酶活性的第一多肽，其中所述多肽是具有下列中的至少一项的多肽：(a)相对于280nm处的吸光度(A280)的脂肪酶活性(LU)少于500，少于450，少于400，少于350，少于300，少于250，少于200，少于150，少于150或少于50LU/A280，其中一单位的LU(1LU)定义为能够在30℃，pH7每分钟释放1μmol的丁酸的酶量，且所述多肽的吸光度在280nm测定；(b)风险性能气味(Riskperformanceodor，R)低于0.5，低于0.4，低于0.3，低于0.2，低于0.1或低于0.05，其中将R计算为自多肽洗涤样本释放的丁酸量和自参照多肽洗涤样本释放的丁酸量之间的比例，计算比例之前两个值均相对于自非多肽洗涤样本释放的丁酸量进行了校正；或(c)效益风险因子(BenefitRiskfactor，BR)为至少1.8，至少1.9，至少2.0，至少2.5，至少3.0，至少4.0，至少5.0或至少6.0，其中BR定义为平均洗涤性能(averagewashperformance，RPavg)除以风险性能气味(R)。
在一些实施方案中，本发明涉及第一多肽，其中所述多肽包含在下述位置的氨基酸的改变：T231R+N233R+I255A+P256K和下列中的至少一项：(a)S58A+V60S+A150G+L227G；或(b)E210V/G；所述位置对应于SEQIDNO：2。
在一些实施方案中，本发明涉及进一步包含在位置I86V或T143S的氨基酸改变中的至少一种的第一多肽。
在一些实施方案中，本发明涉及第一多肽，其中所述多肽包含至少一个选自下列的进一步改变：在对应于SEQIDNO：2的位置E1、D27、N33、S83、G91、N94、K98、E99、D102、D111、G163、I202、E210、S216、L259或L269的位置取代、缺失或添加至少一个氨基酸。
在一些实施方案中，本发明涉及第一多肽，其中所述至少一个改变选自下组：SEQIDNO：2的E1N/*、D27N、N33Q、S83T、G91N、N94R、K98I、E99K、D102A、D111N、G163K、I202L、E210A、S216P、L259F或L269APIA。
在一些实施方案中，本发明涉及第二多肽，其包含在下列位置的氨基酸的改变：T231R+N233R+I255A+P256K和下列中的至少一种：(a)S58A+V60S+A150G+L227G；或(b)E210V/G；所述位置对应于SEQIDNO：2。
在一些实施方案中，本发明涉及进一步包含在位置I86V或T143S的氨基酸改变中的至少一种的第二多肽。
在一些实施方案中，本发明涉及第二多肽，其中所述多肽包含至少一个选自下列的进一步改变：在对应于SEQIDNO：2的位置E1、D27、N33、S83、G91、N94、K98、E99、D102、D111、G163、I202、E210、S216、L259或L269位置的位置取代、缺失或添加至少一个氨基酸。
在一些实施方案中，本发明涉及第二多肽，其中所述至少一个改变选自下组：SEQIDNO：2的E1N/*、D27N、N33Q、S83T、G91N、N94R、K98I、E99K、D102A、D111N、G163K、I202L、E210A、S216P、L259F或L269APIA。
在一些实施方案中，本发明涉及第一多肽，其中所述多肽包含选自下组的改变：(a)T231R+N233R+L269APIA；(b)S58T+V60K+A150G+T231R+N233I+D234G；(c)S58T+V60K+I86V+D102A+A150G+L227G+T231R+N233R+P256K；(d)S58N+V60S+I86P+T231R+N233R+P256S；(e)S58N+V60S+I86S+L227G+T231R+N233R+P256S；以及(f)S58N+V60S+I86T+L227G+T231R+N233R+P256L。
在一些实施方案中，本发明涉及第一或第二多肽，其中所述多肽包含选自下组的改变：(a)S58A+V60S+S83T+A150G+L227G+T231R+N233R+I255A+P256K；(b)S58A+V60S+I86V+A150G+L227G+T231R+N233R+I255A+P256K；(c)S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(d)S58A+V60S+I86V+T143S+A150G+G163K+S216P+L227G+T231R+N233R+I255A+P256K；(e)E1*+S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(f)S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(g)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K+L259F；(h)S58A+V60S+I86V+K98I+E99K+D102A+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(i)N33Q+S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(j)E1*+S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(k)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+S216P+L227G+T231R+N233R+I255A+P256K；(l)D27N+S58A+V60S+I86V+G91N+N94R+D111N+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(m)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+E210A+S216P+L227G+T231R+N233R+I255A+P256K；(n)A150G+E210V+T231R+N233R+I255A+P256K；(o)I202L+E210G+T231R+N233R+I255A+P256K；(p)E1N+A18K+V60K+I86V+A150G+E210A+L227G+T231R+N233R+P256K；(q)E1L+D27K+V60K+I86V+A150G+S219P+L227G+T231R+N233R+P256K；(r)E1N+S58A+V60S+S83T+A150G+L227G+T231R+N233R+I255A+P256K；(s)E1N+S58T+V60K+I86V+D102A+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(t)E1N+S58A+V60S+I86V+K98I+E99K+D102A+T143S+A150G+S216P+L227G+T231R+N233R+I255A+P256K；和(u)S58A+V60S+S83T+A150A+L227G+T231R+N233R+I255A+P256K。
表1：在所述多肽中可能包含的改变
在一些实施方案中，本发明涉及第一多肽，其中所述多肽包含选自下组的改变：(a)T231R+N233R+L269APIA；(b)S58T+V60K+A150G+T231R+N233I+D234G；(c)S58T+V60K+I86V+D102A+A150G+L227G+T231R+N233R+P256K；(d)S58N+V60S+I86P+T231R+N233R+P256S；(e)S58N+V60S+I86S+L227G+T231R+N233R+P256S；和(f)S58N+V60S+I86T+L227G+T231R+N233R+P256L。
在一些实施方案中，本发明涉及第一或第二多肽，其中所述多肽包含选自下组的改变：(a)S58A+V60S+S83T+A150G+L227G+T231R+N233R+I255A+P256K；(b)S58A+V60S+I86V+A150G+L227G+T231R+N233R+I255A+P256K；(c)S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(d)S58A+V60S+I86V+T143S+A150G+G163K+S216P+L227G+T231R+N233R+I255A+P256K；(e)E1*+S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(f)S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(g)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K+L259F；(h)S58A+V60S+I86V+K98I+E99K+D102A+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(i)N33Q+S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(j)E1*+S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(k)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+S216P+L227G+T231R+N233R+I255A+P256K；(l)D27N+S58A+V60S+I86V+G91N+N94R+D111N+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(m)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+E210A+S216P+L227G+T231R+N233R+I255A+P256K；(n)A150G+E210V+T231R+N233R+I255A+P256K；(o)I202L+E210G+T231R+N233R+I255A+P256K；(p)E1N+A18K+V60K+I86V+A150G+E210A+L227G+T231R+N233R+P256K；(q)E1L+D27K+V60K+I86V+A150G+S219P+L227G+T231R+N233R+P256K；(r)E1N+S58A+V60S+S83T+A150G+L227G+T231R+N233R+I255A+P256K；(s)E1N+S58T+V60K+I86V+D102A+T143S+A150G+L227G+T231R+N233R+I255A+P256K；(t)E1N+S58A+V60S+I86V+K98I+E99K+D102A+T143S+A150G+S216P+L227G+T231R+N233R+I255A+P256K；和(u)S58A+V60S+S83T+A150A+L227G+T231R+N233R+I255A+P256K。
多肽的多核苷酸、表达载体、宿主细胞、产生
在一些实施方案中，本发明涉及编码所述多肽的分离的多核苷酸。这些多核苷酸在非常低严紧性条件下，优选低严紧性条件下，更优选中严紧性条件下，更优选中-高严紧性条件下，甚至更优选高严紧性条件下，并且最优选非常高严紧性条件下，可以与以下杂交：(i)SEQIDNO：1的核苷酸178至660，(ii)SEQIDNO：1的核苷酸178至660中包含的cDNA序列，(iii)(i)或(ii)的亚序列，或(iv)(i)、(ii)或(iii)的互补链(J.Sambrook，E.F.Fritsch，andT.Maniatus，1989，MolecularCloning，ALaboratoryManual，2dedition，ColdSpringHarbor，NewYork)。SEQIDNO：1的亚序列含有至少100个连续的核苷酸或优选至少200个连续的核苷酸。此外，所述亚序列可编码具有脂肪酶活性的多肽片段。
对于长度至少100个核苷酸的长探针，将非常低至非常高的严紧条件定义为：于42℃在5×SSPE、0.3％SDS、200μg/ml剪切和变性的鲑精DNA，以及对于非常低和低严紧性25％的甲酰胺，对于中和中-高严紧性35％的甲酰胺，或者对于高和非常高严紧性50％的甲酰胺中，根据标准Southern印迹方法最佳进行12至24小时的预杂交和杂交。
对于长度至少为100个核苷酸的长探针，使用2×SSC、0.2％SDS，优选至少在45℃(非常低严紧性)，更优选至少50℃(低严紧性)，更优选至少55℃(中等严紧性)，更优选至少60℃(中-高严紧性)，甚至更优选至少65℃(高严紧性)，和最优选至少70℃(非常高严紧性)将载体材料最终清洗三次，每次15分钟。
在一些实施方案中，本发明涉及包含所述多核苷酸的核酸构建体，所述多核苷酸可操作连接于至少一个在表达宿主中指导所述多肽产生的调控序列。
在一些实施方案中，本发明涉及包含所述核酸构建体的重组表达载体。
在一些实施方案中，本发明涉及包含所述核酸构建体或所述重组表达载体的转化的宿主细胞。
编码所述多核苷酸的分离的多核苷酸、包含所述多核苷酸的核酸构建体、包含所述核酸构建体的重组表达载体以及包含所述核酸构建体或所述重组表达载体的转化的宿主细胞均可通过本领域已知的方法获取。
在一些实施方案中，本发明涉及制备多肽的方法，包括下述步骤：(a)在有益于所述多肽生产的条件下培养所述转化的宿主细胞，所述宿主细胞包含所述核酸构建体或包含所述核酸构建体的重组表达载体；和(b)回收该多肽。本方法可根据本领域已知的原则加以实施。
用途
本发明的酶可用于，包括供去除脂肪物质用的工业用途。
在一些实施方案中，本发明涉及包含所述多肽的配制物。在进一步的实施方案中，本发明涉及配制物，其中所述配制物可为固体或液体配制物。所述多肽可用于固体以及液体配制物两者。
在一些实施方案中，本发明涉及通过使用所述多肽在脂质水解过程中减少产生气味的短链脂肪酸形成的方法。
实施例
本发明进一步通过下述实施例进行描述，其不应理解为对本发明范围的限制。
用作缓冲剂和底物的化学品是至少试剂等级的商业产品。
实施例1-脂肪酶变体的产生
使用本领域的标准方法构建含有编码所述多肽的基因的质粒，并且将所述质粒转化至合适的宿主细胞中。
使用34℃的恒定培养基温度和1.2升的起始体积，按照补料分批发酵来进行发酵。将培养基的初始pH设为6.5。一旦pH增加至7.0，则通过添加10％H3PO4来保持该值。通过改变搅拌速率和使用1.0升空气每升培养基每分钟的固定通气速率来控制培养基中的溶解氧水平。在整个补料分批阶段期间，将补料添加速率保持在恒定水平。
分批培养基含有麦芽糖糖浆作为碳源，尿素和酵母提取物作为氮源，以及痕量金属和盐的混合物。在补料分批阶段期间连续添加的补料含有麦芽糖糖浆作为碳源，而添加酵母提取物和尿素以确保氮的充足供应。
可以通过使用本领域已知的标准方法来进行所述多肽的纯化，例如通过过滤发酵上清，继之以疏水层析和离子交换层析，例如如EP0851913EP，实施例3中所述。
实施例2-相对于280nm处吸光度的脂肪酶活性单位(LU)(LU/A280)
脂肪酶活性(LU)如上面在脂肪酶活性部分所述来确定。测定所述脂肪酶在280nm的吸光度(A280)。多肽的比活性可表达为LU/A280的比例。
相对LU/A280由所述多肽的LU/A280除以参照酶的LU/A280来计算。在本发明的背景下，所述参照酶是具有取代T231R+N223R的SEQIDNO：2的脂肪酶。
实施例3-由自动机械应力试验(AMSA)获取的数据计算相对性能(RP)
使用自动机械应力试验(AutomaticMechanicalStressAssay；AMSA)测试本发明的多肽。使用AMSA测试，能够检查大量小体积酶洗涤剂溶液的洗涤性能。AMSA平板具有用于测试溶液的多个槽(slot)，和将待洗涤的纺织品样本相对于所有槽的开口(slotopening)压紧的盖。在洗涤时间中，剧烈摇动平板、测试溶液、纺织品和盖以使测试溶液与纺织品接触并且施以机械应力。进一步的描述参见WO02/42740，特别是第23-24页的段落“特殊方法实施方式”。含有洗涤剂测试溶液的容器由金属平板中的圆柱形孔洞(直径6mm，深10mm)组成。沾污的织物(测试材料)放置在金属平板顶部，并将沾污的织物用作盖子密封在所述容器上。另一个金属平板放置在沾污织物的顶部以避免从各个容器中的任何溢出。以2mm振幅和30Hz的频率将两块金属平板连同沾污织物一起上下振动。
表2：AMSA的实验条件
通过将5g姜黄(SantaMaria，Denmark)与100g奶油(38％脂肪，Arla，Denmark)和100克咖啡稀奶油(coffeecream)(9％脂肪Arla，Denmark)在50℃混合来分别制备奶油-姜黄样本和咖啡稀奶油姜黄样本。将所述混合物在该温度保持大约20分钟，并且过滤(50℃)以去除任何未溶解的颗粒。将混合物冷却至20℃，并且将编织的棉布样本EMPA221浸入所述奶油-姜黄混合物，之后允许其在室温干燥过夜，并且冷冻直到使用。奶油-姜黄样本的制备在WO06125437中公开。
将多肽的性能作为用所述特定多肽洗涤的纺织品样品的颜色亮度来测量。也可以将亮度表示为当用白光照射时，从纺织品样品反射的光的强度。当纺织品被沾污时，反射光的强度与清洁纺织品的反射光强度相比较低。因此，能够使用反射光的强度来测量多肽变体的洗涤性能。
使用专业平板扫描仪(PFUDL2400pro)进行颜色测量，使用该扫描仪来捕捉经洗涤纺织品样品的图像。使用200dpi的分辨率和24比特(bit)的输出色深度来进行扫描。为了获得精确的结果，经常性地使用Kodak反射IT8指标(KodakreflectiveIT8target)校准扫描仪。
为了从扫描的图像提取光强度的值，使用特殊设计的软件应用程序(NovozymesColorVectorAnalyzer)。该程序从所述图像恢复24比特像素值，并且将它们转化为红、绿和蓝的值(RGB)。通过将RGB值作为矢量加在一起，再取所得矢量的长度来计算强度值(Int)：
根据下式计算多肽的洗涤性能(P)：
P＝Int(v)-Int(r)，
其中Int(v)是用酶洗涤的纺织品表面的光强度值，且Int(r)是不用酶洗涤的纺织品表面的光强度值。
根据以下定义，给出相对性能评分作为AMSA洗涤的结果：
相对性能评分(RP)概括了测试多肽相对于参照多肽的性能(P)：
RP＝P(测试多肽)/P(参照多肽)。
RPavg表示在0.5mgep/1处进行的测定中与参照多肽比较的平均相对性能。
如果多肽表现优于参照，则认为多肽呈现出改进的洗涤性能。在本发明的上下文中，参照酶是具有取代T231R+N233R的SEQIDNO：2的脂肪酶。
实施例4-由固相微抽提气相层析(SolidPhaseMicroExtractionGasChromatograph)测定计算风险因子(R)
使用以下方法通过固相微抽提气相层析(SolidPhaseMicroExtractionGasChromatography)(SPME-GC)测量来自经脂肪酶洗涤的样本的丁酸释放。将在含有0.5mg/L脂肪酶的表2中的指定溶液中洗涤的四个纺织品片(直径5mm)转移至气相层析(GC)小瓶。将样品在30℃下温育24h，并随后加热至140℃30分钟，并在20℃-25℃下储藏至少4小时直至进行分析。在装备有Stabilwax-DAw/Integra-Guard柱(30m，0.32mmID和0.25微米df)和CarboxenPDMSSPME纤维(85微米)的Varian3800GC上进行分析。对每个GC小瓶的采样在50℃下用SPME纤维在纺织品片之上的顶部空间(headspace)中进行8分钟，且继而将所采样的化合物注射到柱上(注射器温度＝250℃)。柱流速＝2ml氦/分钟。柱加热炉温度梯度：0分钟＝50℃，2分钟＝50℃，6分45秒＝240℃，11分45秒＝240℃。使用火焰离子化检测器(FlameIonizationDector，FID)进行检测，并使用可靠的标准鉴别出丁酸的保留时间。
多肽的风险性能气味(R)指自多肽洗涤样本释放的丁酸量(峰面积)和自参照多肽洗涤样本释放的丁酸量(峰面积)之间的比例，之前两个值均相对于自非多肽洗涤样本(空白)释放的丁酸量(峰面积)进行了校正。所述参照多肽是具有取代T231R+N223R的SEQIDNO：2的多肽。所述多肽的风险性能气味(R)根据下式计算：
气味＝测得的自纺织物表面释放的丁酸(峰面积)
α测试酶＝气味测试酶-气味空白
α参照酶＝气味参照酶-气味空白
R＝α测试酶/α参照酶
如果R因子小于1，则认为多肽与参照相比表现出减少的气味。
实施例5-效益风险因子(BR)
效益风险因子描述的是与减少的气味风险相比的洗涤性能，因此将其定义为：
BR＝RPavg/R
如果BR因子高于1，则认为变体显示出改进的洗涤性能和减少的气味。
表3：一些本发明多肽的比活性(LU/A280)、风险性能气味(R)和效益风险因子(BR)
Claims (10)
1.一种具有脂肪酶活性的多肽，所述多肽是具有下列中的至少一项的多肽：
(a)相对于280nm处的吸光度(A280)的脂肪酶活性(LU)少于500LU/A280，其中一单位的LU(1LU)定义为能够在30℃，pH7每分钟释放1μmol的丁酸的酶量，且所述多肽的吸光度在280nm处测定；
(b)风险性能气味(R)低于0.5，其中R计算为自多肽洗涤样本释放的丁酸量和自参照多肽洗涤样本释放的丁酸量的比例，计算比例之前两个值均相对于自非多肽洗涤样本释放的丁酸量进行了校正；或
(c)效益风险因子(BR)至少为1.8，其中BR定义为平均洗涤性能(RPavg)除以风险性能气味(R)，
所述多肽是在如SEQIDNO：2所示的氨基酸序列基础上包括以下突变位点的组合：T231R+N233R+I255A+P256K和下列中的至少一种：
(a)S58A+V60S+A150G+L227G；或
(b)E210V/G；
其中所述多肽还包含至少一个选自下组的改变：SEQIDNO：2的E1N/*、D27N、N33Q、S83T、G91N、N94R、K98I、E99K、D102A、D111N、G163K、E210A、S216P或L259F。
2.权利要求1的多肽，其中所述多肽包含选自下组的改变：
(a)S58A+V60S+I86V+A150G+L227G+T231R+N233R+I255A+P256K；
(b)S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；
(c)S58A+V60S+I86V+T143S+A150G+G163K+S216P+L227G+T231R+N233R+I255A+P256K；
(d)E1*+S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；
(e)S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K；
(f)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K+L259F；
(g)S58A+V60S+I86V+K98I+E99K+D102A+T143S+A150G+L227G+T231R+N233R+I255A+P256K；
(h)N33Q+S58A+V60S+I86V+T143S+A150G+L227G+T231R+N233R+I255A+P256K；
(i)E1*+S58A+V60S+I86V+K98I+E99K+T143S+A150G+L227G+T231R+N233R+I255A+P256K；
(j)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+S216P+L227G+T231R+N233R+I255A+P256K；
(k)D27N+S58A+V60S+I86V+G91N+N94R+D111N+T143S+A150G+L227G+T231R+N233R+I255A+P256K；
(1)E1N+S58A+V60S+I86V+K98I+E99K+T143S+A150G+E210A+S216P+L227G+T231R+N233R+I255A+P256K；
(m)A150G+E210V+T231R+N233R+I255A+P256K；
(n)I202L+E210G+T231R+N233R+I255A+P256K；
(o)E1N+S58A+V60S+S83T+A150G+L227G+T231R+N233R+I255A+P256K；
(p)E1N+S58T+V60K+I86V+D102A+T143S+A150G+L227G+T231R+N233R+I255A+P256K；
(q)E1N+S58A+V60S+I86V+K98I+E99K+D102A+T143S+A150G+S216P+L227G+T231R+N233R+I255A+P256K。
3.一种编码权利要求1-2任一所述的多肽的分离的多核苷酸。
4.一种包含权利要求3的多核苷酸的核酸构建体，所述多核苷酸可操作连接于至少一个在表达宿主中指导所述多肽产生的调控序列。
5.一种包含权利要求4的核酸构建体的重组表达载体。
6.一种包含权利要求4的核酸构建体或权利要求5的重组表达载体的转化的宿主细胞。
7.一种制备权利要求1-2任一所述的多肽的方法，包括下述步骤：
(a)在有益于所述多肽产生的条件下培养转化的宿主细胞，所述宿主细胞包含含有所述多肽的重组表达载体或核酸构建体；和
(b)回收该多肽。
8.一种包含权利要求1-2任一所述的多肽的配制物。
9.权利要求8的配制物，其中所述配制物可为固体或液体配制物。
10.一种通过使用权利要求1-2任一所述的多肽在脂质水解过程中减少产生气味的短链脂肪酸形成的方法。
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| EP08152163 | 2008-02-29 | ||
| EP08152163.5 | 2008-02-29 | ||
| PCT/EP2009/052246 WO2009109500A1 (en) | 2008-02-29 | 2009-02-26 | Polypeptides having lipase activity and polynucleotides encoding same |
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| ATE125865T1 (de) | 1987-08-28 | 1995-08-15 | Novo Nordisk As | Rekombinante humicola-lipase und verfahren zur herstellung von rekombinanten humicola-lipasen. |
| GB8921995D0 (en) | 1989-09-29 | 1989-11-15 | Unilever Plc | Perfumed laundry detergents |
| CN1193346A (zh) | 1995-07-14 | 1998-09-16 | 诺沃挪第克公司 | 一种具有脂解活性的修饰酶 |
| ATE267248T1 (de) | 1995-08-11 | 2004-06-15 | Novozymes As | Neuartige lipolytische enzyme |
| US7312062B2 (en) * | 1998-11-27 | 2007-12-25 | Novozymes A/S | Lipolytic enzyme variants |
| NZ511340A (en) | 1998-11-27 | 2003-07-25 | Novozymes As | Lipolytic enzyme variants |
| US6939702B1 (en) | 1999-03-31 | 2005-09-06 | Novozymes A/S | Lipase variant |
| WO2002042740A1 (en) | 2000-11-27 | 2002-05-30 | Novozymes A/S | Automated mechanical stress assay for screening cleaning ingredients |
| DE60231700D1 (de) * | 2001-01-10 | 2009-05-07 | Novozymes As | Variante eines lipolytischen enzyms |
| CN1491278A (zh) | 2001-02-07 | 2004-04-21 | ŵά�Ź�˾ | 脂酶变体 |
| DK1888764T3 (da) | 2005-05-27 | 2014-10-27 | Novozymes As | Stofprøve til afprøvning af et enzyms vaskeevne |
| ES2629332T3 (es) | 2006-01-23 | 2017-08-08 | Novozymes A/S | Variantes de lipasa |
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2009
- 2009-02-26 CN CN200980106985.3A patent/CN101960008B/zh active Active
- 2009-02-26 JP JP2010548100A patent/JP5650543B2/ja not_active Expired - Fee Related
- 2009-02-26 ES ES09716837.1T patent/ES2603979T3/es active Active
- 2009-02-26 CA CA2715829A patent/CA2715829C/en not_active Expired - Fee Related
- 2009-02-26 AR ARP090100662A patent/AR070490A1/es unknown
- 2009-02-26 MX MX2010009072A patent/MX2010009072A/es active IP Right Grant
- 2009-02-26 WO PCT/EP2009/052246 patent/WO2009109500A1/en active Application Filing
- 2009-02-26 US US12/393,283 patent/US7919298B2/en active Active
- 2009-02-26 EP EP09716837.1A patent/EP2250259B1/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1484693A (zh) * | 2001-01-10 | 2004-03-24 | 诺维信公司 | 脂解酶变体 |
| CN1675355A (zh) * | 2002-08-19 | 2005-09-28 | 帝斯曼知识产权资产管理有限公司 | 新颖的脂肪酶及其用途 |
| WO2007087503A1 (en) * | 2006-01-23 | 2007-08-02 | Novozymes A/S | Polypeptides having lipase activity and polynucleotides encoding same |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2603979T3 (es) | 2017-03-02 |
| CA2715829A1 (en) | 2009-09-11 |
| EP2250259A1 (en) | 2010-11-17 |
| WO2009109500A1 (en) | 2009-09-11 |
| EP2250259B1 (en) | 2016-08-31 |
| MX2010009072A (es) | 2010-09-24 |
| US20090221033A1 (en) | 2009-09-03 |
| WO2009109500A8 (en) | 2010-10-14 |
| JP2011512808A (ja) | 2011-04-28 |
| US7919298B2 (en) | 2011-04-05 |
| JP5650543B2 (ja) | 2015-01-07 |
| CA2715829C (en) | 2017-05-23 |
| AR070490A1 (es) | 2010-04-07 |
| CN101960008A (zh) | 2011-01-26 |
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