CN101952413A - 使用纤维素酶石洗织物的方法 - Google Patents
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Abstract
本发明涉及用包含纤维素酶的组合物,在防返染的染色纤维素织物的表面形成色密度局部改变的方法,所述纤维素酶包含SEQ ID NO:2的氨基酸序列或者与SEQ ID NO:2具有至少75%序列同一性的氨基酸序列。本申请还涉及通过使用新的内切葡聚糖酶用于将纤维素织物生物抛光的方法。
Description
技术领域
本发明涉及用组合物在染色纤维素织物的表面形成色彩密度局部变化的方法,所述组合物包含源自烟曲霉的新内切葡糖苷酶,其可促进磨蚀(abrasion)和控制返染(backstaining)。本申请还涉及通过使用新内切葡糖苷酶将纤维素织物生物抛光的方法。
技术背景
在由染色的纤维素织物生产服装(例如,从靛蓝染色的粗斜棉布制造蓝色牛仔裤)的过程中,经常处理粗斜棉布以提供“石洗的(stone-washed)”外观(粗斜棉布表面的颜色局部磨蚀)。人们深信内切葡聚糖酶对于粗斜棉布的性能依赖于酶的结构和化学性质。
在粗斜棉布的生化石磨中,靛蓝的磨蚀和返染是指定内切葡聚糖酶的两个关键的性能指标。用单一的内切葡聚糖酶提供粗斜棉布的高度磨蚀并将靛蓝返染控制在可接受的水平是很罕见的。内切葡聚糖酶-靛蓝、内切葡聚糖酶-纤维素和施用pH之间的相互作用也有助于粗斜棉布磨蚀和靛蓝返染。
WO 98/45395描述在低水洗涤过程中使用的洗涤剂组合物。WO 97/15660描述用于制备晶体内切葡聚糖酶的方法。WO 95/16782描述来自长枝木霉(Trichoderma longibrachiatum)的新截短型内切葡聚糖酶蛋白质,其可减少染料在织物上的重新沉积(返染),同时保持相当水平的磨蚀。
WO 91/17243和WO 95/09225(Novo Nordisk)描述使用分子量为43kD的表示为EGV的单组分内切葡聚糖酶的方法,所述内切葡聚糖酶源自特异腐质霉(Humicola insolens)菌株DSM 1800。WO 94/21801(Genencor)描述了源自长枝木霉的称为EGIII的单组分内切葡聚糖酶在“石洗”中的用途。WO 95/16782(Genencor International)提出了源自木霉属的其它单组分内切葡聚糖酶在“石洗”中的用途。
在已知的“石洗”方法中的常见问题是返染,即,通过磨蚀已经去除的染料重新沉积在织物或服装的部分上,从而使色密度的期望变化变平缓(even out)或使服装的任何浅色部分脱色。本领域需要在粗斜棉布磨蚀中具有良好性能的新内切葡聚糖酶。
发明概述
我们令人惊奇地发现,源自烟曲霉的内切葡聚糖酶在石洗中有非常良好的性能,同时降低返染。
因此,本发明的一个方面提供在染色纤维素织物的表面形成色密度局部变化的方法,其包括用包含内切葡聚糖酶的组合物接触所述织物,其中所述内切葡聚糖酶具有SEQ ID NO:1的氨基酸序列,或与SEQ ID NO:1具有至少70%序列同一性的氨基酸序列。
优选地,织物是靛蓝染色的粗斜棉布。
优选地,内切葡聚糖酶源自曲霉属菌株,优选烟曲霉。
本发明的另一方面提供在有色织物的石洗过程中减轻返染的方法,所述方法包括用包含内切葡聚糖酶的组合物接触织物,所述酶具有SEQ ID NO:1的氨基酸序列,或与SEQ ID NO:1具有至少70%序列同一性的氨基酸序列。
附图简述
图1显示pJZhAfum12A的表达质粒的构建概况。
图2描述烟曲霉CEL12A内切葡聚糖酶在不同温度在粗斜棉布磨蚀中的pH曲线。
图3描述里氏木霉(Trichoderma reesei)CEL12在pH 5.5和烟曲霉CEL 12A内切葡聚糖酶在pH 4.5的温度曲线。
图4描述从30℃至60℃,烟曲霉CEL12A内切葡聚糖酶、Super GX和Cellusoft L在生物抛光中的温度曲线。
图5描述烟曲霉CEL12A内切葡聚糖酶在55℃pH 5.0的剂量曲线。
发明详述
定义
在包含权利要求的本说明中,使用下述定义:
术语“内切葡聚糖酶活性”在本文中定义为内切-1,4-β-D-葡聚糖4-葡聚糖水解酶(endo-1,4-β-D-glucan 4-glucanohydrolase)(E.C.No.3.2.1.4),其催化纤维素、纤维素衍生物(例如羧甲基纤维素和羟乙基纤维素)、地衣淀粉(lichenin)中的1,4-β-D-糖苷键、混合的β-1,3葡聚糖例如谷类β-D-葡聚糖或木葡聚糖和含有纤维素成分的其它植物材料中的β-1,4键的内水解(endohydrolysis)。就本发明而言,根据Ghose,1987,Pure and Appl.Chem.59:257-268的方法,使用羧甲基纤维素(CMC)水解来测定内切葡聚糖酶活性。一个单位的内切葡聚糖酶活性定义为在50℃,pH 4.8每分钟生成1.0毫摩尔的还原糖。
在一个优选的方面,本发明的具有内切葡聚糖酶活性的多肽进一步对于选自下组的一种或多种底物具有酶活性:木聚糖、木葡聚糖、***木聚糖、1,4-β-D-甘露聚糖和半乳甘露聚糖。将底物(5mg每ml)与具有本发明的内切葡聚糖酶活性的多肽(5mg蛋白质每g底物)在pH 5.0(50mM乙酸钠)和50℃间歇搅拌温育24小时后,确定水解成还原糖的底物的百分比作为具有内切葡聚糖酶活性的多肽对于这些多糖底物的活性。通过对羟基苯甲酸酰阱(PHBAH)测定法确定水解混合物中的还原糖。
在一个更优选的方面,本发明的具有内切葡聚糖酶活性的多肽进一步对于木聚糖有酶活性。在另一个更优选的方面,本发明的具有内切葡聚糖酶活性的多肽进一步对于木葡聚糖有酶活性。在另一个更优选的方面,本发明的具有内切葡聚糖酶活性的多肽进一步对于***木聚糖有酶活性。在另一个更优选的方面,本发明的具有内切葡聚糖酶活性的多肽进一步对于1,4-β-D-甘露聚糖有酶活性。在另一个更优选的方面,本发明的具有内切葡聚糖酶活性的多肽进一步对于半乳甘露聚糖有酶活性。在另一个更优选的方面,本发明的具有内切葡聚糖酶活性的多肽进一步对于木聚糖、木葡聚糖、***糖基木聚糖、1,4-β-D-甘露聚糖和半乳甘露聚糖有酶活性。
本发明的多肽具有SEQ ID NO:2的成熟多肽的内切葡聚糖酶活性的至少20%,优选至少40%,更优选至少50%,更优选至少60%,更优选至少70%,更优选至少80%,更优选至少90%,更优选至少95%,并且甚至最优选至少100%。
根据Henrissat,B.等:Biochem.J.,(1991),280,p.309-16,和Henrissat,B.等:Biochem.J.,(1993),293,p.781-788所述分类***的氨基酸序列相似性,将内切葡聚糖酶分成多个家族。
本发明中使用的内切葡聚糖酶优选单组分,即,在本发明中使用的含水培养基除指明的组分之外不包含其它内切葡聚糖酶组分。单组分酶可以通过重组DNA技术经济地制备,即,它们可以通过克隆编码单组分的DNA序列,随后用DNA序列转化合适的宿主细胞,并在宿主中表达该组分而产生。
因此,可通过通常的方法分离编码有用的纤维素酶的DNA序列,所述方法涉及从本说明中稍后指出的一种微生物,在合适的载体中克隆DNA文库,用所述载体转化合适的酵母宿主细胞,在合适的条件下培养宿主细胞以表达由DNA文库中的克隆编码的任何目的酶,通过确定由这些克隆产生的酶的任何纤维素酶活性而筛选阳性克隆,和从这些克隆中分离编码酶的DNA。
通常的方法进一步公开于WO 94/14953(Novo Nordisk),其内容通过提述并入本文。
编码有用的内切葡聚糖酶的DNA序列可以,例如,通过筛选目的微生物的cDNA文库和选择表达合适的酶活性(即,内切葡聚糖酶活性)的克隆而分离。
编码同源酶的DNA序列,即类似的DNA序列,可获得自其它微生物。例如,DNA序列可通过相似地筛选另一真菌(如曲霉属菌种的菌株,特别是棘孢曲霉或黑曲霉的菌株;木霉属菌种的菌株,特别是里氏木霉、绿色木霉(T.viride)、长枝木霉、哈茨木霉(T.harzianum)或康宁木霉(T.koningji)的菌株,或新考玛脂霉属菌种(Neocallimastix sp.),瘤胃壶菌属菌种(Piromyces sp.),青霉属菌种(Penicillium sp.),伞菌属菌种(Agaricus sp.)或平革菌属菌种(Phanerochaete sp.)的菌株)的cDNA文库而得到。
可供选择地,编码有用的纤维素酶的DNA可以按照公知的方法,使用根据已知的DNA序列制备的合成寡核苷酸探针,便利地从来自合适来源(如上述任何生物体)的DNA分离。
然后可以将DNA序列***重组表达载体。这可以是任何载体,其可便利地进行重组DNA方法,并且载体的选择常常依赖于要导入的宿主细胞。因此,载体可以是自复制载体,即,作为染色体外实体存在的载体,其复制不依赖于染色体复制,例如,质粒。可供选择地,载体可以是如下的载体,其在导入宿主细胞时整合进宿主细胞基因组,并且与其导入的染色体一起复制。
在载体中,编码内切葡聚糖酶的DNA序列应与合适的启动子和终止子序列可操作地连接。启动子可以是在所选宿主细胞中表现出转录活性的任何DNA序列,并且可以源自编码与宿主细胞同源或异源的基因。用于分别连接编码内切葡聚糖酶的DNA序列、启动子和终止子,并且将它们***合适的载体中的方法为本领域技术人员所公知(例如,Sambrook等,Molecular Cloning.A Laboratory Manual,Cold Spring Harbor,NY,1989)。
用DNA序列转化的宿主细胞优选是真核细胞,特别是真菌细胞如酵母或丝状真菌细胞。特别地,细胞可以属于曲霉属或木霉属的菌种,最优选米曲霉或黑曲霉(Aspergillus niger)。可以将真菌细胞通过涉及原生质体形成、原生质体转化和细胞壁重建的方法以本身公知的方式转化。使用曲霉属作为宿主微生物在EP 238 023(Novo Nordisk AS)中描述,其内容通过提述并入本文。宿主细胞还可以是酵母细胞,例如,酵母属,特别是酿酒酵母(Saccharomyces cervisiae)或葡萄汁酵母(Saccharomyces uvarum)的菌株;裂殖酵母属菌种,如粟酒裂殖酵母(Schizosaccharomyces pombe)的菌株;汉逊酵母属菌种、毕赤酵母属菌种、西洋蓍霉属菌种,如解脂西洋蓍霉(Yarrowia lipolytica)的菌株;或克鲁维酵母属,如乳酸克鲁维酵母(Kluyveromyces lactic)的菌株。
在本发明中,术语“同源”或“同源序列”意欲表示与后文所示序列表中每一个所示的氨基酸序列相差一个或多个氨基酸残基的氨基酸序列。
同源序列可以是由这些列表中所示氨基酸序列的修饰而产生的,例如,涉及在氨基酸序列中一个或多个不同位点的一个或多个氨基酸残基的取代,在酶的任一端或两端或在氨基酸序列的一个或多个位点缺失一个或多个氨基酸残基,或者在氨基酸序列中一个或多个位点***一个或多个氨基酸残基。
然而,如对于技术人员显而易见的,氨基酸改变优选对性质是较不重要的(of a minor nature),即保守的氨基酸取代,其不显著影响蛋白质的折叠和/或活性;通常为1至大约30个氨基酸的小缺失;小的氨基或羧基末端延伸,例如氨基末端甲硫氨酸残基;多至大约20-25个残基的小接头肽;或通过改变净电荷或其它功能来促进纯化的小延伸,如多组氨酸序列(poly histidine tract)、抗原表位(antigenic epitope)或结合域(binding domain)。参见常用的Ford等,1991,Protein Expression and Purification 2:95-107。保守取代的实例是在以下组之内:碱性氨基酸组(精氨酸、赖氨酸和组氨酸)、酸性氨基酸组(谷氨酸和天冬氨酸)、极性氨基酸组(谷氨酰胺和天冬酰胺)、疏水性氨基酸组(亮氨酸、异亮氨酸和缬氨酸)、芳族氨基酸组(苯丙氨酸、色氨酸和酪氨酸)和小氨基酸组(甘氨酸、丙氨酸、丝氨酸、苏氨酸和甲硫氨酸)。
对于本领域技术人员还显而易见的是,可以在对分子的功能关键的区域外进行这种取代,并仍产生活性多肽。能够根据本领域已知的方法,例如定位诱变或丙氨酸分区诱变法(Cunningham和Wells,1989,Science 244:1081-1085)来鉴定由本发明的DNA构建体编码的多肽中的必需氨基酸(并且优选不进行取代)。在后一技术中,将单一丙氨酸突变引入到分子中的每个残基,并且测试所得突变分子的生物活性(即,内切葡聚糖酶活性)以鉴定对于所述分子的活性关键的氨基酸残基。
也能够通过分析如通过以下这些技术测定的晶体结构而确定底物-酶的相互作用部位:如核磁共振、晶体学或光亲和标记。参见,例如de Vos等,Science 255:306-312,1992;Smith等,J.Mol.Biol.224:899-904,1992;Wlodaver等,FEBS Lett.309:59-64,1992。
可以通过修饰编码酶的DNA序列,例如,按照公知的方法,通过定位或随机诱变或这些技术的组合而适当地进行氨基酸序列的修饰。可供选择地,同源序列可以是除分别对应于后文所示序列表中每个序列中所示氨基酸序列的内切葡聚糖酶之外的源自另一个来源的酶。因此,“同源物”可以,例如,表示由DNA编码的多肽,所述DNA在某些指定条件下(如在5x SSC中预浸,并在20%甲酰胺、5X Denhardt溶液、50mM磷酸钠,pH 6.8和50mg变性超声处理的小牛胸腺DNA的溶液中在400℃预杂交1小时,然后在补加100mMATP的相同溶液中在400℃杂交18小时)和与具有待研究氨基酸序列的内切葡聚糖酶的编码DNA相同的探针杂交。同源序列通常与后文所示序列表中每个序列所示的氨基酸序列分别显示至少50%,如至少60%、65%、70%、75%、80%、85%、90%或者甚至95%的同一性程度。
术语“同一性”在本文中定义为由参数“同一性”描述的两个氨基酸序列之间或两个核苷酸序列之间的相关性。
就本发明而言,两个氨基酸序列之间的同一性程度使用如EMBOSS软件包(EMBOSS:The European Molecular Biology Open Software Suite,Rice等,2000,Trends in Genetics 16:276-277)的Needle程序,优选3.0.0版或更高版本中所实施的Needleman-Wunsch算法(Needleman和Wunsch,1970,J.Mol.Biol.48:443-453)来测定。使用的可选参数为缺口开放罚分(gap open penalty)10,缺口延伸罚分(gap extension penalty)0.5和EBLOSUM62(BLOSUM62的EMBOSS版)取代矩阵。使用Needle标记为“最长同一性(longest identity)”的输出结果(使用-nobrief选项获得)作为同一性百分比,并计算如下:
(同样的残基×100)/(比对长度-比对中缺口的总数)
就本发明而言,两个核苷酸序列之间的同一性程度使用如EMBOSS软件包(EMBOSS:The European Molecular Biology Open Software Suite,Rice等,2000,见上文)的Needle程序,优选3.0.0版或更高版本中所实施的Needleman-Wunsch算法(Needleman和Wunsch,1970,J.Mol.Biol.48:443-453)来测定。使用的可选参数为缺口开放罚分10,缺口延伸罚分0.5和EDNAFULL(NCBI NUC4.4的EMBOSS版)取代矩阵。使用Needle标记为“最长同一性”的输出结果(使用-nobrief选项获得)作为同一性百分比,并计算如下:
(同样的脱氧核糖核苷酸×100)/(比对长度-比对中缺口的总数)
染色的纤维素织物
本发明的方法可以施用于任何类型的染色纤维素织物,其中理想地在表面形成色密度的局部变化。特别具有商业价值的实例是粗斜棉布,特别是靛蓝染色的用于蓝色牛仔裤等的粗斜棉布。
织物可以以未缝制织物或由这种织物制成的已缝制服装的形式处理。特别感兴趣的是对新的、洁净织物或服装施用本发明的方法。
工艺条件
本发明的方法可以在常规用于石洗的洗衣机(例如,洗涤脱水机(washer-extractor))中在常规条件下进行。
通常的条件是温度为25-60℃,并且织物∶液体比为1∶3-1∶20,15分钟-2小时。任选地,可以使用常规添加剂,例如,缓冲液、表面活性剂(阴离子和/或非离子,如PW100i)和/或聚合物(如PVP、聚丙烯酸酯和聚丙烯酰胺)。
实施例
实施例1-5说明了制备遗传工程改造的黑曲霉,使其能产生本发明的内切葡聚糖酶或根据本发明的需要产生特定的内切葡聚糖酶。
材料
作为缓冲液和底物使用的化学品是至少试剂等级的商业产品。
菌株
使用米曲霉Jal250菌株(WO 99/61651)表达烟曲霉cel12a基因。使用烟曲霉PaHa34作为编码糖基水解酶家族12内切葡聚糖酶的cel12a基因的来源。
培养基
马铃薯葡萄糖培养基由如下物质组成:每升39克马铃薯右旋糖(Difco)。
PDA平板由如下物质组成:每升39克马铃薯右旋糖琼脂。
MDU2BP培养基由如下物质组成:每升45g麦芽糖、1g MgSO4·7H2O、1g NaCl、2g K2SO4、12g KH2PO4、7g酵母提取物、2g脲和0.5ml AMG痕量金属溶液,pH至5.0。
AMG痕量金属溶液由如下物质组成:每升14.3g ZnSO4·7H2O、2.5g CuSO4·5H2O、0.5g NiCl2·6H2O、13.8g FeSO4·7H2O、8.5g MnSO4·H2O和3g柠檬酸。
YEG培养基由如下物质组成:每升5克酵母提取物和20克右旋糖。
实施例1:cel12a基因的克隆和米曲霉表达载体的构建
设计下述两个合成寡核苷酸引物,以从如WO 03/012071所述制备的基因组DNA中PCR扩增编码糖基水解酶家族12内切葡聚糖酶(CEL12A)的烟曲霉基因。使用InFusion克隆试剂盒(BD Biosciences,Palo Alto,CA,USA)将片段直接克隆入表达载体pAILo2,无需限制性消化和连接。
黑体字母代表编码序列。其余序列与pAILo2的***位点同源。
在PCR反应中使用上述引物各75皮摩尔,所述反应物在终体积50μl中包含120ng烟曲霉基因组DNA,1X Pfx扩增缓冲液(Invitrogen,Carlsbad,CA,USA),dATP、dTTP、dGTP和dCTP的10mM混合物1.5μl,1.9单位Platinum Pfx DNA聚合酶(Invitrogen,Carlsbad,CA,USA),和1μl 50mM MgSO4。扩增条件为94℃2分钟的1个循环;30个循环,每个循环为94℃15秒,55℃30秒,和68℃1.5分钟。
使用40mM Tris碱-20mM乙酸钠-1mM EDTA二钠(TAE)缓冲液,在1.0%琼脂糖凝胶上分离反应产物,其中从凝胶上切下0.9kb的产物条带,并+根据制造商的说明使用QIAQUICK凝胶提取试剂盒(QIAGEN Inc.,Valencia,CA,USA)纯化。
然后使用Infusion克隆试剂盒将片段克隆入pAILo2表达载体。用限制性内切酶NcoI和PacI(使用制造商指明的条件)消化载体。通过凝胶电泳和QIAQUICK凝胶纯化纯化片段。在反应中将基因片段和消化的载体融合在一起,得到表达质粒pJZhAfum12A(图1),其中cel12a基因的转录在NA2-tpi启动子的控制下。融合反应物(50μl)包含1X InFusion缓冲液(BD Biosciences,Palo Alto,CA,USA),1X BSA(BD Biosciences,Palo Alto,CA,USA),1μl Infusion酶(1∶10稀释)(BD Biosciences,Palo Alto,CA,USA),150ng用NcoI和PacI消化的pAILo2,和25ng烟曲霉cel12a纯化PCR产物。在室温温育反应物30分钟。使用1μl反应物转化大肠杆菌XL10 Solopac Gold细胞(Stratagene,La Jolla,CA,USA)。使用BIOROBOT9600(QIAGEN Inc.,Valencia,CA,USA)从一个转化体制备质粒DNA,并通过DNA测序确认同一性。
实施例2:编码CEL12A内切葡聚糖酶的烟曲霉基因组序列的表征
使用染料-终止子化学(Giesecke等,1992,J.Virol.Methods 38:47-60)用Perkin-Elmer Applied Biosystems Model 3700自动DNA测序仪(Perkin-Elmer/Applied Biosystems,Foster City,CA,USA)进行来自pJZhAfum12A的烟曲霉cel12a基因的DNA测序。审核核苷酸序列数据的质量,并在PHRED/PHRAP软件(University of Washington,Seattle,WA)的协助下将所有序列互相比较。
根据先前获得的全长cDNA序列构建烟曲霉序列的基因模型。核苷酸序列(SEQ ID NO:1)和推导的氨基酸序列(SEQ ID NO:2)如下所示。805bp的基因组片段(包括终止密码子)编码234个氨基酸的多肽,其中***54和46bp的2个内含子。所述基因的%G+C含量是56.2%,。使用SignalP软件程序(Nielsen等,1997,Protein Engineering 10:1-6)预测了16个残基的信号肽。预测的成熟蛋白质包含218个氨基酸,分子量为24.1kDa。
烟曲霉的cel12a基因的编码区域的核酸序列和翻译。内含子用斜体表示,并且预测的信号肽加下划线。
MKTFAILGAFFSSALAQTLCDQYATYSNGRYTVNNNLWGMSSGSGSQCT
YVDSISNSGVAWHTTWTWSGGDNQVKSYANS
QVSLTKKLVSQISSIPTTVQWSYDNTNTRADVAYDLFTAADPNHVTYSGD
YELMIWLARYGNVQPIGSQIASVNIGGHNW
ELWYGGSTQKTYSFVSATPITSFSGNVMDFWNYLTRNHGYPASSQYLINM
QFGTEPFTGGPATLTVSQWTASVN
烟曲霉的CEL12A前多肽的氨基酸序列。
QTLCDQYATYSNGRYTVNNNLWGMSSGSGSQCTYVDSISNSGVAWHTT
WTWSGGDNQVKSYANSQVSLTKKLVSQISSIP
TTVQWSYDNTNTRADVAYDLFTAADPNHVTYSGDYELMIWLARYGNVQ
PIGSQIASVNIGGHNWELWYGGSTQKTYSFVS
ATPITSFSGNVMDFWNYLTRNHGYPASSQYLINMQFGTEPFTGGPATLTVS
QWTASVN
烟曲霉的预测CEL12A成熟多肽的氨基酸序列。
使用如在EMBOSS的Needle程序中所实施的Needleman-Wunsch算法(Needleman和Wunsch,1970,见上文)及缺口开放罚分10、缺口延伸罚分0.5和EBLOSUM62矩阵来确定氨基酸序列的比较配对全局比对。比对显示,编码CEL12A成熟多肽的烟曲霉基因的推导氨基酸序列与来自烟曲霉(登录号Q4WGT4)、棘孢曲霉(Aspergillus aculeatus)(P22669)和川地曲霉(Aspergillus kawachii)(Q8NJY2)的糖基水解酶家族12蛋白的推导氨基酸序列分别具有99.5%、72.9%和70.2%的同一性(缺口除外)。
实施例3:烟曲霉cel12a内切葡聚糖酶基因在米曲霉JaL250中的表达
根据Christensen等,1988,Bio/Technology 6:1419-1422的方法制备米曲霉JaL250原生质体。使用5μg的pJZhAfum12A转化米曲霉JaL250。
用pJZhAfum12A转化米曲霉JaL250,得到约20个转化体。将八个转化体分离至单独的PDA平板。
用5ml 0.01%TWEEN 20洗涤八个转化体中五个的汇合PDA平板,并分别接种入125ml玻璃摇瓶中的25ml MDU2BP培养基中,并在34℃,250rpm温育。温育五天后,根据制造商的说明使用8-16%Tris-甘氨酸SDS-PAGE凝胶(Invitrogen,Carlsbad,CA,USA)分析每种培养物的0.5μl上清。培养物的SDS-PAGE谱显示,转化体之一(命名为米曲霉JaL250JZhAf12A)分泌约24kDa的主要条带。
如EP0489718所述,从米曲霉JaL250JZhAf12A产生用于进一步表征的蛋白。
实施例4:米曲霉基因组DNA提取
使用带挡板的摇瓶,在YEG培养基中在50ml培养体积中,使表达烟曲霉CEL12A蛋白的米曲霉菌株JaL250JZhAf12A在34℃,200rpm生长2天。生物质在液氮中冷冻并用研钵和杵研磨成粉末。将粉末悬浮于15ml 0.1M CAPS-NaOH pH 11.0,1mM EDTA,0.5%十二烷基硫酸锂中,并在60℃温育60分钟,定期通过倒转混合。加入等体积的中和苯酚,并在37℃温和振荡管1小时。加入5ml氯仿并剧烈搅拌1分钟。在1300xg离心10分钟后,用等体积的苯酚∶氯仿(1∶1)通过搅拌5分钟重新提取顶层水相。重复离心,并将水相移至2.5M乙酸铵中,并在-20℃保存20分钟。在4℃以17000xg离心20分钟后,通过加入0.7倍体积的异丙醇沉淀上清中的上清核酸。以17000xg离心10分钟后,倾析上清,用70%乙醇漂洗沉淀并风干。将沉淀溶于950μl去离子水中,然后加入50μl Promega细胞重悬溶液(Promega Corporation,Madison WI,USA),并在室温温育5分钟。加入乙酸铵至1.0M并通过加入2倍体积乙醇而沉淀核酸。以13000xg离心10分钟后,将沉淀溶于300μl1mM Tris-HCl,0.1mM EDTA,pH 8.0中,并在-20℃保存。
实施例5:从米曲霉表达菌株JaL250JZhAf12A的基因组DNA中PCR扩增烟曲霉cel12a基因组DNA
为了测序和克隆保藏物,从米曲霉菌株JaL250JZhAf12A基因组DNA扩增cel12a DNA。设计两个与pJZhAfum12A表达载体同源的合成寡核苷酸引物,以从米曲霉菌株JaL250JZhAf12A基因组DNA中PCR扩增cel12a基因。
正向引物:
5′ccacacttctcttccttcctc 3′(SEQ ID NO:5)
反向引物:
5′CCCCATCCTTTAACTATAGCG 3′(SEQ ID NO:6)
在PCR反应中使用上述引物各33皮摩尔,所述反应物在终体积30μl中包含约300ng米曲霉JaL250JZhAf12A基因组DNA(如实施例4所述),1X Pfx扩增缓冲液(Invitrogen,Carlsbad,CA,USA),dATP、dTTP、dGTP和dCTP的10mM混合物1.7μl,2.5单位Platinum Pfx DNA聚合酶(Invitrogen,Carlsbad,CA,USA),和1μl 50mM MgSO4。扩增在Stratagene Robocycler中进行,程序为96℃3分钟和72℃3分钟的1个循环(其间加入DNA聚合酶);和35个循环,每个循环为94℃50秒,56℃50秒,和68℃60秒钟;然后在68℃最终延伸7分钟。
使用TAE缓冲液,在1.2%琼脂糖凝胶上分离反应产物,其中从凝胶上切下0.9kb的产物条带,并根据制造商的说明使用QIAEXII凝胶提取试剂盒(QIAGEN Inc.,Valencia,CA,USA)纯化。
使用Zero Blunt TOPO PCR克隆试剂盒将片段克隆入pCR4Blunt-TOPO载体,并根据制造商的说明(Invitrogen,Carlsbad CA,USA)转化大肠杆菌TOP10细胞。使用QIAGEN BioRobot 9600从几个转化体制备质粒DNA。将来自一个转化体的质粒测序,并发现所述质粒包含cel12a DNA。将质粒命名为pPH53。
实施例6:在LOM中用烟曲霉CEL12A(如实施例3中所得)进行粗斜棉布磨蚀
在Launder-O-Meter(SDL-Atlas LP2)中确定烟曲霉CEL12A(后文称作AfCEL12A)在粗斜棉布磨蚀中的性质,包括温度曲线和pH曲线。使用的具有不同pH的缓冲液包括:pH 4.5(50mM乙酸盐缓冲液,2.93g NaAc·3H2O和1.71g HAc溶于1升去离子水),pH 5.5(50mM乙酸盐缓冲液,6.02gNaAc·3H2O和0.35gHAc溶于1升去离子水),pH 6.5(50mM磷酸盐缓冲液,5.64g Na2HPO4·12H2O和5.34g NaH2PO4·2H2O溶于1升去离子水),pH 7.5(50mM磷酸盐缓冲液,15.05g Na2HPO4·12H2O和1.25g NaH2PO4·2H2O溶于1升去离子水),pH 8.5(50mM磷酸盐缓冲液,17.61g Na2HPO4·12H2O和0.116g KH2PO4溶于1升去离子水)。酶处理时间为2小时,并且温度分别为25℃、30℃、40℃、50℃、60℃。将结果与里氏木霉CEL12(来自Genencor的Super GX)比较。
将原料粗斜棉布脱浆(desize)并剪成12.5cm高和23cm长。将粗斜棉布缝合,形成管状,高12.5cm和重约13.5g。将管在调节室(65%湿度,20℃)中放置24小时然后编号,通过分析天平称量并记录。在每个烧杯中放一个经过调节的管,蓝色一侧向内。对于每个烧杯,使用30个大螺母(M6M-SR-A4-80,耐酸),10个小螺母(M6M-SR-A4-80,耐酸),7个大的星形磁铁(直径17mm,产品编号3-CO-411117,Cowie,Schweiz via Bie & Berntsen)和3个小的星形磁铁(直径14mm,产品编号3-CO-11117,Cowie,Schweiz via Bie & Berntsen)提供机械辅助。然后根据酶溶液的计算结果加入缓冲液,总体积为200ml,其将产生约15∶1的液体比例。
在选择所需程序后启动LOM机器,并且在温度到达设定值后保持不变。之后,向分别包含不同pH缓冲液的烧杯加入54mg/L CEL12A,每个pH值有2个烧杯。对于每个pH,使用不含酶的两个烧杯作为空白对照。将包含54mg/L里氏木霉CEL12(纯化自配制的Super GX颗粒的蛋白质,后文称作Tr CEL12)的两个烧杯,和/或加入配制的Super GX颗粒以使Tr CEL12蛋白的浓度在pH 5.5达54mg/L的两个烧杯用于比较。每个烧杯配以衬有2个neoprin垫圈的盖子,并用例如金属夹持装置盖紧。将烧杯放入预热的LOM。使用金属架在4个鼓形位置中的每个在水平位置放置和固定6个烧杯。关闭LOM盖并继续进行洗涤程序。2小时后,去除所有烧杯并将粗斜棉布样品转移至失活溶液(2g/L碳酸钠),保持85℃10分钟。然后将试样在热水中漂洗2次,在冷水中漂洗2次。甩干粗斜棉布样品,并将样品在20℃,65%R.H调理24小时,然后评价。
通过用预先校准的DataColor SF450X测定反射比而确定粗斜棉布样品的磨蚀水平和返染水平。每个样品取四个读数。用样品蓝色一侧的指数CIE L*评价磨蚀水平,并用样品背面的指数CIE b*评价返染水平。
如图2所示,在所有测试温度,磨蚀水平(由ΔL*表示,ΔL*=用某种纤维素酶处理的试样的L*-在相同条件处理但不加纤维素酶的试样的L*)随缓冲液的pH值变化。这种pH曲线表明Af CEL12A在pH在4.5和5.5之间的缓冲液中性能非常好,说明它优选酸性缓冲液。
如图3所示,不同纤维素酶的磨蚀水平和返染水平(分别用ΔL*和Δb*代表,ΔL*的定义如上所述,Δb*=用某种纤维素酶处理的试样的b*-在相同的条件处理但不加纤维素酶的试样的b*)对温度作图。可以观察到,Af CEL12A能在从约25℃至60℃的宽温度范围内应用。为了获得相似的磨蚀水平,Af CEL12A引起的返染水平显著低于纯化自Super GX的Tr CEL12蛋白。
对于在25℃和60℃的试验,使用Tr CEL12(Super GX)的商业产品,并且对于其它温度,替代使用由配制的商业产品Super GX纯化的Tr CEL12。
实施例7:通过Af CEL12A进行生物抛光
将针织的双罗纹织物,100%棉(Knitted interlock fabric,100%cotton)或测试织物类型460(Test Fabric Style 460)剪成16.5cm*16.5cm,作为标准试样。将试样在调节室(65%湿度,20℃)中放置24小时,然后编号,通过分析天平称量并记录。在每个烧杯中放置两个经过调节的试样。对于每个烧杯,在每个烧杯(220g)中使用20个大钢珠提供机械辅助。然后根据酶溶液的计算结果加入缓冲液(50mM乙酸盐缓冲液,pH 5.0),总体积为100ml,其将产生约10∶1的液体比例。
在选择所需程序后启动LOM机器,并且在温度到达设定值后保持不变。对于温度为30℃、40℃、50℃、55℃和60℃的每个试验,两个烧杯包含30mg/LAf CEL12A,两个烧杯包含36mg/L Af CEL12A,使用分别包含36mg/L Super GX蛋白和143mg/L CELLUCLAST蛋白的两个烧杯作为基准(benchmark),使用不含酶的两个烧杯作为空白对照。将烧杯封口并放在LOM中,并在LOM中启动酶处理,进行1小时。结束时,移出所有烧杯并将试样转移至失活溶液(2g/L碳酸钠),保持85℃10分钟。然后将试样在热水中漂洗2次,在冷水中漂洗2次。甩干粗斜棉布样品,并将样品在20℃,65%R.H调理24小时,然后评价。
对于每个处理条件,有4个试样。将它们称重并记录以得到重量损失数据。将1个试样用于以Martindale起球测试仪进行起球记录测试(pilling notes test),且其余3个试样用于以燃烧强度测试仪进行燃烧强度测试以得到强度损失数据。
如图5所示,对于抗起球性能,18-44mg/L Af CEL12A与143mg/L Cellusoft L可比,表明在pH 5.0,55℃,Af CEL12A在生物抛光中是celluclast蛋白质的3-4倍强。
如表1所示,在相同的蛋白加载,从30℃至50℃,Af CEL12A和SuperGX能引起几乎相同的抗起球效果。而在强度损失方面,Af CEL12A略微优于Super GX。
表1.Af CEL12A和Super GX之间的生物抛光性能比较
Claims (17)
1.在染色的纤维素织物的表面形成色密度局部改变的方法,其包括用包含纤维素酶的组合物接触所述织物,其中所述纤维素酶具有SEQ ID NO:2的氨基酸序列或者与SEQ ID NO:2具有至少75%序列同一性的氨基酸序列。
2.权利要求1的方法,其中所述织物是靛蓝染色的粗斜棉布。
3.权利要求1或2的方法,其中所述纤维素酶是家族12的纤维素酶。
4.权利要求1-3中任一项的方法,其中所述纤维素酶源自曲霉属(Aspergillus)菌株,优选烟曲霉(Aspergillus fumigatus)。
5.用于在有色织物的石洗过程中减轻返染的方法,所述方法包括用包含纤维素酶的组合物接触织物,所述纤维素酶具有SEQ ID NO:2的氨基酸序列或与SEQ ID NO:2具有至少75%序列同一性的氨基酸序列。
6.权利要求5的方法,其中所述织物是靛蓝染色的粗斜棉布。
7.权利要求5或6的方法,其中所述纤维素酶是家族12的纤维素酶。
8.权利要求5-7中任一项的方法,其中所述纤维素酶源自曲霉属菌株,优选烟曲霉。
9.根据前述权利要求中任一项的方法,其中所述组合物进一步包含表面活性剂。
10.由权利要求1中所述的方法处理的织物。
11.由权利要求5中所述的方法处理的织物。
12.包含SEQ ID NO:2的氨基酸序列的新内切葡糖苷酶。
13.用于将含纤维素织物生物抛光的方法,所述方法包括用包含组合物的含水本体溶液接触所述织物,所述组合物包含纤维素酶,其中所述纤维素酶具有SEQ ID NO:2的氨基酸序列或与SEQ ID NO:2具有至少75%序列同一性的氨基酸序列。
14.如权利要求13所述的方法,其中所述含纤维素织物包含选自下组的纤维素纤维:棉、亚麻、苎麻、***、黄麻、人造丝、莱赛尔纤维,和前述任一种彼此或与非纤维素纤维的组合。
15.如权利要求5所述的方法,其中所述纤维素酶源自曲霉属菌株,优选烟曲霉。
16.如权利要求13-15中任一项所述的方法,其中所述生物抛光的特征在于选自下组的改进的性质:起球记录、手感,和外观。
17.如权利要求13-15中任一项所述的方法,其中所述本体溶液进一步包含选自下组的酶:蛋白酶、角质酶、淀粉酶、果胶消化酶和半纤维素酶。
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CN102876589A (zh) * | 2012-10-17 | 2013-01-16 | 贵州茅台酒厂(集团)习酒有限责任公司 | 高产纤维素酶活力的菌株及其筛选方法和使用方法 |
CN104947434A (zh) * | 2014-03-31 | 2015-09-30 | 河北科技大学 | 一种硫化染料染色织物处理用组合物、套组以及处理方法 |
CN111270394A (zh) * | 2019-02-18 | 2020-06-12 | 宜春希宇生物制品有限公司 | 一种抗菌面料及其制备方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
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WO1991017243A1 (en) | 1990-05-09 | 1991-11-14 | Novo Nordisk A/S | A cellulase preparation comprising an endoglucanase enzyme |
US5475101A (en) | 1990-10-05 | 1995-12-12 | Genencor International, Inc. | DNA sequence encoding endoglucanase III cellulase |
US5565006A (en) | 1993-01-20 | 1996-10-15 | Novo Nordisk A/S | Method for the treatment of dyed fabric |
US5861271A (en) | 1993-12-17 | 1999-01-19 | Fowler; Timothy | Cellulase enzymes and systems for their expressions |
US6190898B1 (en) | 1995-10-23 | 2001-02-20 | Genencor International, Inc. | Crystalline cellulase and method for producing same |
WO1998028410A1 (en) | 1996-12-20 | 1998-07-02 | Novo Nordisk A/S | A novel endoglucanase |
CN1240478A (zh) * | 1996-12-20 | 2000-01-05 | 诺沃挪第克公司 | 一种新的内切葡聚糖酶 |
WO1998045395A1 (en) | 1997-04-04 | 1998-10-15 | The Procter & Gamble Company | Low sudsing granular detergent composition containing optimally selected levels of a foam control agent and enzymes |
DE69836227T2 (de) | 1997-12-16 | 2007-08-23 | Genencor International, Inc., Palo Alto | Verfahren zur herstellung egiii-ähnlicher enzyme |
AU5685699A (en) | 1998-09-03 | 2000-03-27 | Genencor International, Inc. | Egiii-like cellulase compositions, dna encoding such egiii compositions and methods for obtaining same |
WO2002012464A2 (en) * | 2000-08-04 | 2002-02-14 | Genencor International, Inc. | Mutant trichoderma reesei egiii cellulases dna encoding such egiii compositions and methods for obtaining same |
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CN102876589B (zh) * | 2012-10-17 | 2014-07-16 | 贵州茅台酒厂(集团)习酒有限责任公司 | 高产纤维素酶活力的菌株及其筛选方法和使用方法 |
CN104947434A (zh) * | 2014-03-31 | 2015-09-30 | 河北科技大学 | 一种硫化染料染色织物处理用组合物、套组以及处理方法 |
CN111270394A (zh) * | 2019-02-18 | 2020-06-12 | 宜春希宇生物制品有限公司 | 一种抗菌面料及其制备方法 |
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