CN1509330A - 具有纤维二糖酶活性的多肽和编码其的多核苷酸 - Google Patents
具有纤维二糖酶活性的多肽和编码其的多核苷酸 Download PDFInfo
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Abstract
本发明涉及具有纤维二糖酶活性的多肽和具有编码该多肽的核苷酸序列的多核苷酸。本发明还涉及核酸构建体、载体和包含该核酸构建体的宿主细胞以及制备和使用该多肽的方法。
Description
发明领域
本发明涉及具有纤维二糖酶(亦称β-葡糖苷酶)活性的多肽和具有编码本多肽的核苷酸序列的多核苷酸。本发明还涉及核酸构建体、载体和含本核酸构建体的宿主细胞,以及制备和使用本多肽的方法。
发明背景
纤维二糖酶是重要的降解生物量的酶。纤维二糖酶的这种用途是由植物材料生产乙醇过程中的一个关键步骤。考虑到环境因素,乙醇是一种吸引人的石油燃料代用品。纤维二糖酶的其它用途包括在果汁业中用于还原苦味化合物-如栎精。
本发明的一个目的是提供具有纤维二糖酶(亦称β-葡糖苷酶)活性的多肽和编码本多肽的核苷酸序列。
发明概述
本发明第一方面涉及具有纤维二糖酶活性的多肽,其选自:
(a)包含这样一个氨基酸序列的多肽,此氨基酸序列与SEQ ID NO:2
中1至842位氨基酸所示序列具有至少80%同一性或者与SEQ ID
NO:2中1至351位氨基酸所示序列具有至少90%同一性;
(b)包含这样一个氨基酸序列的多肽,此氨基酸序列与插入大肠杆菌(E.
coli)DSM 14240的质粒中的核苷酸序列的编码部分所编码的多肽具
有至少80%同一性;
(c)由能够在中度严紧条件下与多核苷酸探针杂交的核苷酸序列编码的
多肽,其中多核苷酸探针选自(i)SEQ ID NO:1中87至2612位核苷 酸所示序列的互补链,和(ii)SEQ ID NO:1中87至1139位核苷
酸所示序列的互补链;
(d)具有纤维二糖酶活性的(a)、(b)或(c)的片断。
本发明第二方面涉及多核苷酸,它具有能够编码本发明多肽的核苷酸序列。
本发明第三方面涉及核酸构建体,它包含能够编码本发明多肽的核苷酸序列,且该序列可操作地连接一个或多个指导此多肽在适当宿主中产生的调控序列。
本发明第四方面涉及包含本发明核酸构建体的重组表达载体。
本发明第五方面涉及包含本发明核酸构建体的重组宿主细胞。
本发明第六方面涉及用于产生本发明多肽的方法,其包括:
(a)培养野生型能够生产此多肽的菌株以产生此多肽;以及
(b)回收此多肽。
本发明第七方面涉及用于产生本发明多肽的方法,其包括:
(a)在有助于本多肽产生的条件下培养重组宿主细胞;以及
(b)回收本多肽。
本发明的其它方面可从下面的说明和所附的权利要求书中明显看出。
定义
进一步详细讨论本发明之前,首先定义下列术语和常规用语。
基本上纯的多肽:在本发明上下文中,术语“基本上纯的多肽”指的是多肽制备物中包含至多10%重量的其它天然相关的多肽物质(优选更低百分比——如至多8%重量,至多6%重量,至多5%重量,至多4%重量,至多3%重量,至多2%重量,至多1%重量和至多1/2%重量的其它多肽物质)。因此,优选的是基本上纯的多肽具有至少92%的纯度,即此多肽组成了制备物中总多肽物质重量的至少92%,优选更高的百分比纯度,如至少94%纯,至少95%纯,至少96%纯,至少97%纯,至少98%纯,至少99%纯,以及至多99.5%纯。本文公开的多肽优选是基本上纯的形式。尤其,优选的是本文公开的多肽是“实质上的纯形式”,即多肽制备物中实质上没有其它天然相关多肽物质。举例来说,这种纯度可通过熟知的重组法制备多肽来得到。在本文中,术语“基本上纯的多肽”与术语“分离的多肽”和“分离形式的多肽”同义。
纤维二糖酶活性:术语“纤维二糖酶活性”在本文中被定义为β-葡糖苷酶活性,如在酶分类EC3.2.1.21中的定义,它催化末端的非还原性β-D-葡萄糖残基水解释放出β-D-葡萄糖。对本发明来说,根据实施例部分中“纤维二糖酶试验”描述的步骤测定纤维二糖酶活性。一个单位的纤维二糖酶活性定义为在40℃,pH5下每分钟产生2μmol葡萄糖。
本发明多肽应优选具有由SEQ ID NO:2中1至842位氨基酸所示氨基酸序列组成的多肽的纤维二糖酶活性的至少20%。在特别优选的实施方案中,多肽应具有由SEQ ID NO:2中1至842位氨基酸所示氨基酸序列组成的多肽的纤维二糖酶活性的至少40%,如至少50%,优选至少60%,如至少70%,更优选至少80%,如至少90%,最优选至少95%,如大约或至少100%。
同一性:在本发明上下文中,两个氨基酸序列之间或两个核苷酸序列之间的同源性通过参量“同一性”来描述。
对本发明来说,两个氨基酸序列之间的同一性程度通过可用于蛋白质和DNA序列比对(align)的Needleman-Wunsch序列比对法进行确定。对于蛋白质比对,使用的默认评分矩阵是BLOSUM50,缺口(gap)中第一个残基的罚分是-12,而缺口中其它残基的罚分是-2。比对可利用FASTA程序包v20u6版(W.R.Pearson和D.J.Lipman(1988),“生物序列分析的改良工具”,PNAS 85:2444-2448;和W.R.Pearson(1990)“利用FASTP和FASTA进行快速灵敏的序列比较”,酶学方法(Methods in Enzymology),183:63-98)的比对软件来执行。
两个核苷酸序列之间的同一性程度可使用同一性矩阵为默认评分矩阵利用上述的相同计算法则和软件包进行确定。缺口中第一个残基的罚分是-16,而缺口中其它残基的罚分是-4。
片断:本文中,SEQ ID NO:2的“片断”是指从这个氨基酸序列的氨基和/或羰基末端缺失了一个或多个氨基酸的多肽。片断优选包含至少351个氨基酸残基,例如SEQ ID NO:2中的1至351位氨基酸。
等位变体:在本发明上下文中,术语“等位变体”表示占据同一染色体座位的一个基因的两种或多种可变形式中的任一种。等位变异可以由突变天然地引起,并可引起种群内多态性。基因突变可能是沉默突变(所编码的多肽没有变化)或者可能编码氨基酸序列发生改变的多肽。多肽等位变体是由基因等位变体所编码的多肽。
基本上纯的多核苷酸:本文使用的术语“基本上纯的多核苷酸”指的是一种多核苷酸制备物,其中此多核苷酸已经离开其天然遗传环境,从而它不含有其它外来或不想要的编码序列并且它存在的形式适于在遗传工程蛋白质生产系统内进行使用。因此,基本上纯的多核苷酸包含至多10%重量的其它天然相关多核苷酸物质(优选更低百分比,如至多8%重量,至多6%重量,至多5%重量,至多4%重量,至多3%重量,至多2%重量,至多1%重量和至多1/2%重量的其它多核苷酸物质)。然而,基本上纯的多核苷酸可以包括天然的5’和3’非翻译区,如启动子和终止子。优选的是基本上纯的多核苷酸具有至少92%的纯度,即此多核苷酸组成了制备物中总多核苷酸物质重量的至少92%,优选更高的百分比纯度如至少94%纯,至少95%纯,至少96%纯,至少97%纯,至少98%纯,至少99%纯,以及至多99.5%纯。本文所公开的多核苷酸优选为基本上纯的形式。尤其,优选的是本文所公开的多核苷酸是“实质上纯的形式”,即多核苷酸制备物中实质上没有其它天然相关的多核苷酸物质。在本文中,术语“基本上纯的多核苷酸”与术语“分离的核苷酸”和“分离形式的多核苷酸”同义。
修饰:在本发明上下文中术语“修饰”指的是对由SEQ ID NO:2中1至842位氨基酸所示氨基酸序列组成的多肽所进行的任何化学修饰,以及对编码此多肽的DNA所进行的遗传操作。修饰可以是在目的氨基酸内或其所在位置处进行的氨基酸侧链置换,以及氨基酸替代、缺失和/或插入。
人工变体:术语“人工变体”用于本文指的是具有纤维二糖酶活性的多肽,该多肽由表达修饰基因(对比SEQ ID NO:1而言)的生物体产生。修饰基因,即,在适当宿主中表达时产生所述变体的基因是经过人为干预对SEQ ID NO:1公开的核苷酸序列进行修饰得到的。
cDNA:术语“cDNA”用于本发明上下文意在覆盖这样的DNA分子,此DNA分子能够从成熟的、剪接的、真核细胞来源的mRNA分子的反转录制备得到。cDNA缺少通常在相应遗传组DNA中存在的内含子序列。最初的初级RNA转录物是mRNA的前体,它经过一系列加工事件才成为成熟剪接的mRNA。这些事件包括通过剪接过程去除内含子序列。cDNA由于衍生自mRNA因此缺少内含子序列。
核酸构建体:术语“核酸构建体”用于本文,既可以指单链的也可以指双链的核酸分子,它分离自天然基因或者它经修饰以非天然方式含有核酸片断。如果核酸构建体包含表达本发明编码序列所需的调控序列,则术语核酸构建体与术语“表达序列盒”同义。
调控序列:术语“调控序列”在本文被定义为包括所有的对表达本发明多肽必需或有利的组分。对编码多肽的核苷酸序列来说每个调控序列可以是天然的或外来的。这些调控序列包括,但不限于:前导序列、聚腺苷酸化序列、前肽序列、启动子、信号肽序列和转录终止子。最少地调控序列包括启动子及转录和翻译终止信号。为引入特异性限制位点以便于调控序列与编码多肽的核苷酸序列的编码区进行连接,可以提供带接头的调控序列。
可操作地连接:术语“可操作地连接”在本文定义为一种构型,在此构型中调控序列被置于相对于DNA序列的编码序列而言适当的位置,这样调控序列可指导多肽表达。
编码序列:本文使用的术语“编码序列”意在覆盖这样的核苷酸序列,该序列直接规定了其蛋白质产物的氨基酸序列。编码序列的边界一般由开放阅读框确定,通常开始于AGT起始密码子。编码序列一般包括DNA、cDNA、和重组核苷酸序列。
表达:在本发明上下文中,术语“表达”包括多肽合成中涉及的任何步骤,其包括但不限于:转录,转录后修饰,翻译,翻译后修饰及分泌。
表达载体:在本发明上下文中,术语“表达载体”包括这样的线状的或环状的DNA分子,它包含编码本发明多肽的片断,而该片段与为其转录提供条件的其它片断可操作地连接。
宿主细胞:用于本文的术语“宿主细胞”包括任一种易被核酸构建体转化的细胞。
术语“多核苷酸探针”、“杂交”及各种严紧条件在标题为“具有纤维二糖酶活性的多肽”一节中进行定义。
发明详述
具有纤维二糖酶活性的多肽
在第一实施方案中,本发明涉及具有纤维二糖酶活性的多肽,此多肽包含这样的氨基酸序列,并优选由这样的氨基酸序列组成,此氨基酸序列与SEQID NO:2中1至842位氨基酸(即成熟多肽)所示序列的同一性程度为至少65%,优选至少70%,如至少75%,更优选至少80%,如至少85%,甚至更优选至少90%,最优选至少95%,如至96%,至少97%,甚至最优选至少98%,如至99%(以下称“同源多肽”)。在有意义的一个实施方案中,此氨基酸序列有至多十个氨基酸(例如,十个氨基酸),尤其是至多五个氨基酸(例如,五个氨基酸),如至多四个氨基酸(例如,四个氨基酸),举例来说至多三个氨基酸(例如,三个氨基酸)与SEQ ID NO:2中1至842位氨基酸所示序列不同。在一个特别有意义的实施方案中,此氨基酸序列有至多两个氨基酸(例如,两个氨基酸),如一个氨基酸与SEQ ID NO:2中1至842位氨基酸所示序列不同。
在另一实施方案中多肽包含这样的氨基酸序列,并优选由这样的氨基酸序列组成,此氨基酸序列与SEQ ID NO:2中1至351位氨基酸(即催化核心)所示序列的同一性程度为至少65%,优选至少70%,如至少75%,更优选至少80%,如至少85%,甚至更优选至少90%,最优选至少95%,如至少96%,如至少97%,甚至最优选至少98%,如至99%。
利用上文(参见“定义”一节)所述方法,对由SEQ ID NO:2中1至842位氨基酸所示氨基酸序列组成的多肽与最新的现有技术进行序列比对,得到下列同一性百分数:
棘孢曲霉(Aspergillus aculeatus):79.6%
黑曲霉(Aspergillus niger):76.9%
川地曲霉(Aspergillus kawasachi):77.0%
对由SEQ ID NO:2中1至351位氨基酸(即催化核心)所示氨基酸序列组成的多肽与本领域最新现有技术进行序列比对,得到下列同一性百分数:
棘孢曲霉:86.6%
黑曲霉:81.9%
川地曲霉:80.3%
构巢曲霉(Aspergillus nidulans):25.1%
优选地是,本发明多肽包含SEQ ID NO:2所示的氨基酸序列;其等位变体;或者其具有纤维二糖酶活性的片断。在另一优选实施方案中,本发明多肽包含SEQ ID NO:2中1至842位氨基酸。在另一优选实施方案中,此多肽由SEQ ID NO:2中1至842位氨基酸组成。
本发明多肽可以是从天然源中鉴定分离出来的野生型纤维二糖酶。这种野生型多肽可通过本领域已知的标准技术进行特异性地筛选。此外,本发明多肽可利用DNA改组技术(如在J.E.Ness等,Nature Biotechnology 17,893-896(1999)一文中的描述)来制备。而且,本发明多肽可以是人工变体,它包含这样的氨基酸序列并优选由这样的氨基酸序列组成,此氨基酸序列同SEQ ID NO:2中1至842位氨基酸所示序列相比,有至少一个替代、缺失和/或插入的氨基酸。这种人工变体可利用本领域已知的标准技术,如通过对含有SEQ ID NO:2中1至842位氨基酸所示氨基酸序列的多肽进行定点/随机诱变来构建。在本发明一个实施方案中,氨基酸变化(在人工变体和野生型多肽中)是性质较小的,即,不会明显影响蛋白质折叠和/活性的保守性氨基酸替代;小缺失,一般有1至大约30个氨基酸;小的氨基或羧基末端延伸,如氨基末端的甲硫氨酸残基;不超过大约20-25个残基的小接头肽;或者可以通过改变净电荷或另一功能而利于纯化的小延伸,如聚组氨酸序列段、抗原表位或结合域。
保守性替代的实例为碱性氨基酸组(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),疏水氨基酸组(亮氨酸,异亮氨酸,缬氨酸和甲硫氨酸),芳香族氨基酸组(苯丙氨酸,色氨酸和酪氨酸),和小氨基酸组(甘氨酸,丙氨酸,丝氨酸和苏氨酸)内的替代。通常不改变特异性活性的氨基酸替代是本领域已知的并且在H.Neurath和R.L.Hill,1979《The Protein》,Academic Press,New York一书中进行过描述。最常发生的交换是Ala/Ser,Val/Ile,Asp/Glu,Thr/Ser,Ala/Gly,Ala/Thr,Ser/Asn,Ala/Val,Ser/Gly,Tyr/Phe,Ala/Pro,Lys/Arg,Asp/Asn,Leu/Ile,Leu/Val,Ala/Glu和Asp/Gly以及其反向交换。
在本发明一个有意义的实施方案中,氨基酸变化具有如此特性以至改变了多肽的理化性质。举例来说,可以实行氨基酸变化,以提高多肽的热稳定性,改变底物特异性,改变pH最适值等。
优选地,对比SEQ ID NO:2中1至842位氨基酸,这种替代、缺失和/或插入的数目至多为10,如至多为9,如至多为8,更优选至多为7,如至多为6,再如至多为5,最优选至多为4,如至多为3,再如至多为2,尤其至多为1。
本发明人已经从米曲霉(Aspergillus oryzae)中分离出编码具纤维二糖酶活性的多肽的基因并将此基因插入质粒pJaL621(参见实施例1)中,而该质粒被插入大肠杆菌中。根据国际承认用于专利程序保藏微生物的布达佩斯条约,于2001年4月19日在德意志微生物保藏中心(DSMZ-DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH,MascherorderWeg 1B,D-38124 Braunschweig Germany)保藏了携带此基因的大肠杆菌菌株,保藏号为DSM 14240。
这样,在第二实施方案中,本发明涉及包含如下氨基酸序列,优选由如下氨基酸序列组成的多肽,此氨基酸序列与插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的多肽具有至少65%的同一性。在本发明一个有意义的实施方案中,多肽包含如下氨基酸序列并优选由如下氨基酸序列组成,此氨基酸序列与插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的多肽具有至少70%,如至少75%,优选至少80%,如至少85%,更优选至少90%,最优选至少95%,如至少96%,如至少97%,甚至最优选至少98%,如至少99%的同一性(以下称“同源多肽”)。在一个有意义的实施方案中,此氨基酸序列有至多十个氨基酸(如十个氨基酸),尤其至多五个氨基酸(如五个氨基酸)如至多四个氨基酸(如四个氨基酸),至多三个氨基酸(如三个氨基酸)与插入在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的多肽不同。在特别有意义的实施方案中,此氨基酸序列有至多两个氨基酸(如两个氨基酸),如一个氨基酸与插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的多肽不同。
优选地,本发明多肽包含插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的多肽的氨基酸序列。在另一个优选实施方案中,本发明多肽由插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的多肽的氨基酸序列组成。
与上文所述相似,本发明多肽可以是人工变体,它包含这样的氨基酸序列并优选由这样的氨基酸序列组成,此氨基酸序列与插在大肠杆菌DSM14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的氨基酸序列相比,具有至少一个替代、缺失和/或插入的氨基酸。
在第三实施方案中,本发明涉及具有纤维二糖酶活性的多肽,它是由在极低严紧条件下,优选在低度严紧条件下,更优选在中度严紧条件下,更优选在中高度严紧条件下,甚至更优选在高度严紧条件下,以及最优选在极高严紧条件下,可与选自(i)SEQ ID NO:1中核苷酸87至2612位所示序列的互补链和(ii)SEQ ID NO:1中核苷酸87至1139位所示序列的互补链的多核苷酸探针进行杂交的核苷酸序列编码的(J.Sambrook,E.F.Fritsch,和T.Maniatus,1989,分子克隆实验室手册(Molecular Cloning,ALaboratory Manual),2d edition,Cold Spring Harbor,New York。)
按照本领域熟知的方法,SEQ ID NO:1所示的核苷酸序列或其亚序列,以及SEQ ID NO:2所示的氨基酸序列或其片断都可用于设计核苷酸探针以便从不同属或种的菌株中识别和克隆出能编码具纤维二糖酶活性的多肽的DNA。具体地,按照标准DNA印迹法利用这类探针与目的属或种的基因组或cDNA杂交,以便识别和分离出其中的相应基因。这类探针可能比整个序列短得多,但应该有至少15个,优选至少25个,更优选至少35个核苷酸长,如至少70个核苷酸长。然而,优选的是多核苷酸探针有至少100个核苷酸长。举例来说,多核苷酸探针可以是至少200个核苷酸长,至少300个核苷酸长,至少400个核苷酸长或者至少500个核苷酸长。甚至可利用更长的探针,如至少600个核苷酸长,至少700个核苷酸长,至少800个核苷酸长,或者至少900个核苷酸长的多核苷酸探针。DNA和RNA探针都可利用。探针一般都进行标记用于检测相应基因(例如,用32P、3H、35S、生物素或抗生物素蛋白进行标记)。
这样,可从制备自这些其它生物体的基因组DNA或cDNA文库中筛选出与上述探针杂交的并且编码具纤维二糖酶活性的多肽的DNA。来自这些其它生物体的基因组或其它DNA可通过琼脂糖或聚丙烯酰胺凝胶电泳,或其它分离技术进行分离。可以将来自文库的DNA或分离的DNA转移并固定在硝化纤维或其它适宜载体物质上。为鉴别出与SEQ ID NO:1所示序列同源的克隆或DNA,对载有固定化DNA的载体物质进行DNA印迹分析。
对于本发明,杂交指的是核苷酸序列杂交到标记的多核苷酸探针上,其中后者在极低到极高严紧条件下可与如SEQ ID NO:1所示的核苷酸序列杂交。在这些条件下与多核苷酸探针杂交的分子可利用X-射线胶片或通过任何其它本领域已知方法进行检测。只要在本发明上下文中使用术语“多核苷酸探针”,此术语应理解为是一种含有至少15个核苷酸的探针。
在一个有意义的实施方案中,多核苷酸探针是SEQ lD NO:1之87至1139位核苷酸所示序列的互补链。
在另一有意义的实施方案中,多核苷酸探针是能够编码SEQ ID NO:2多肽的核苷酸序列的互补链。在再一有意义的实施方案中,多核苷酸探针是如SEQ ID NO:1所示序列的互补链。在再一个有意义的实施方案中,多核苷酸探针是SEQ ID NO:1的成熟多肽编码区的互补链。在另一有意义的实施方案中,多核苷酸探针是包含在大肠杆菌DSM 14240中的质粒pJaL621中的核苷酸序列的互补链。再一个有意义的实施方案中,多核苷酸探针是包含在大肠杆菌DSM 14240的质粒pJaL621中的成熟多肽编码区的互补链。
对于至少100个核苷酸长的长探针来说,极低至极高严紧条件被规定为按照标准DNA印迹分析法,在42℃,在5XSSPE、1.0%SDS、5X Denhardt溶液、100μg/ml剪切变性的鲑鱼精子DNA中进行预杂交和杂交。优选地,至少100个核苷酸长的长探针不包括那些大于1000个核苷酸的探针。对于至少100个核苷酸长的长探针来说,载体物质最后利用2xSSC,0.1%SDS在42℃(极低严紧条件)洗涤三次每次洗涤15分钟;优选利用0.5x SSC,0.1%SDS在42℃(低度严紧条件)洗涤三次每次洗涤1 5分钟;更优选利用0.2xSSC,0.1%SDS在42℃(中度严紧条件)洗涤三次每次洗涤15分钟;甚至更优选利用0.2x SSC,0.1%SDS在55℃(中-高度严紧条件)洗涤三次每次洗涤15分钟;最优选利用0.1x SSC,0.1%SDS在60℃(高度严紧条件)洗涤三次每次洗涤15分钟;尤其优选利用0.1x SSC,0.1%SDS在68℃(极高严紧条件)洗涤三次每次洗涤15分钟。
尽管不是特别地优选,但也可考虑使用较短探针,可例举约15至99个核苷酸长,如约15至约70个核苷酸长的探针。对于这类短探针来说,严紧条件被规定为按照标准DNA印迹分析法,在低于计算出的Tm 5℃至10℃的温度下(利用Bolton和McCarthy(1962,Proceedings of the National Academyof Sciences USA,48:1390)的计算方法计算),在0.9M NaCl、0.09MTris-HCl pH7.6、6mM EDTA、0.5%NP-40、1XDenhardt溶液、1mM焦磷酸钠、1mM一代磷酸钠、0.1mM ATP及每ml 0.2mg的酵母RNA中进行预杂交、杂交及杂交后洗涤。
对于约15至99个核苷酸长的短探针,载体物质在低于计算出的Tm值5℃至10℃的温度下在6XSCC加0.1%SDS中洗涤一次洗15分钟,然后利用6XSCC洗涤两次每次洗15分钟。
具纤维二糖酶活性的多肽的来源
本发明多肽可从任何微生物属中获得。对于本发明来说,本处所用的术语“从......获得”应该指的是由核苷酸序列编码的多肽是通过这样的细胞产生的,其中此核苷酸序列天然存在于此细胞中或已经被插入到此细胞中。在优选实施方案中,多肽被分泌到细胞外。
本发明多肽可以是一种细菌多肽。例如,此多肽可以是革兰氏阳性细菌多肽,如芽孢杆菌属(Bacillus)多肽一-可例举有嗜碱芽孢杆菌(Bacillusalkalophilus)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、短芽孢杆菌(Bacillus brevis)、环状芽孢杆菌(Bacillus circulans)、凝结芽孢杆菌(Bacilluscoagulans)、灿烂芽孢杆菌(Bacillus lautus)、迟缓芽孢杆菌(Bacillus lentus)、地衣芽孢杆菌(Bacillus licheniformis)、巨大芽孢杆菌(Bacillus megaterium)、嗜热脂肪芽孢杆菌(Bacillus Stearothermophilus)、枯草芽孢杆菌(Bacillussubtilis)或苏云金芽孢杆菌(Bacillus thuringiensis)多肽;或链霉菌属(Streptomyces)多肽,可例举有青紫链霉菌(Streptomyces lividans)或鼠灰链霉菌(Streptomyces murinus)多肽;或革兰氏阴性细菌多肽,例如大肠杆菌或假单胞菌属(Pseudomonas)的多肽。
本发明多肽可以是一种真菌多肽,并且更优选酵母多肽如假丝酵母属(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Yarrowia多肽;或更优选丝状真菌多肽如枝顶孢属(Acremonium)、曲霉属、短梗霉属(Aureobasidium)、隐球酵母属(Cryptococcus)、线黑粉菌属(Filibasidium)、镰孢属(Fusarium)、腐质霉属(Humicola),Magnaporthe、毛霉属(Mucor)、毁丝霉属(Myceliophthora)、Noecallimastix、脉孢菌属(Neurospora)、拟青霉属(Paecilomyces)、青霉属(Penicillium)、Piromyces、裂褶菌属(Schizophyllum)、踝节菌属(Talaromyces)、热子囊菌属(Thermoascus)、梭孢壳属(Thielavia)、Tolypocladium或木霉属(Trichoderma)多肽。
在一个有意义的实施方案中,多肽是卡尔酵母(Saccharomycescalsbergensis)、酿酒酵母(Saccharomyces cerevisiae)、糖化酵母(Saccharomycesdiastaticus)、Saccharomyces douglasii、克鲁弗酵母(Saccharomyces kluyveri)、诺地酵母(Saccharomyces norbensis)或卵形酵母(Saccharomyces oviformis)多肽。
在另一个有意义的实施方案中,多肽是棘孢曲霉、泡盛曲霉(Aspergillusawamori)、臭曲霉(Aspergillus foretidus)、日本曲霉(Aspergillus Japonicus)、构巢曲霉、黑曲霉、米曲霉、杆孢状镰孢(Fusarium bactridioides)、Fusariumcerealis、Fusarium crookwellense、大刀镰孢(Fusarium culmorum)、禾谷镰孢(Fusarium graminearum)、禾赤镰孢(Fusarium graminum)、异孢镰孢(Fusarium heterosporum)、合欢木镰孢(Fusarium negundi)、尖镰孢(Fusariumoxysporum)、多枝镰孢(Fusarium reticulatum)、粉红镰孢(Fusarium roseum)、接骨木镰孢(Fusarium sambucinum)、肤色镰孢(Fusarium sarcochroum)、拟分枝孢镰孢(Fusarium sporotrichioides)、硫色镰孢(Fusarium sulphureum)、近念珠状镰孢(Fusarium torulosum)、Fusarium trichothecioides、有毒镰孢(Fusarium venenatum)、Humicola insolens、Humicola lanuginosa、米黑毛霉(Mucor miehei)、嗜热毁丝霉(Myceliophthora thermophila)、粗糙脉孢菌(Neurospora crassa)、产紫青霉(Penicillium purpurogenum)、Trichodermaharzianum、康宁木霉(Trichoderma koningii)、长枝木霉(Trichodermalongibrachiatum)、Trichoderma reesei或绿色木霉(Trichoderma viride)多肽。
在一个优选的实施方案中,多肽是米曲霉多肽,最优选大肠杆菌DSM14240多肽,举例来说,由SEQ ID NO:2中1至842位氨基酸序列组成的多肽。
对前面提及的菌种,应理解的是,不管已知的种名是什么,本发明既包括完全阶段和不完全阶段,还包括其它分类学上的等价物,如无性型。本领域技术人员能够很容易地识别出适当等价物的身份。
这些菌种的菌株可以容易地在许多培养物保藏机构为公众所得到,如美国典型培养物保藏中心(ATCC),德意志微生物保藏中心(DSM),真菌菌种保藏中心(CBS)和农业研究机构保藏中心(NRRL)。
此外,利用上述探针还可从其它来源,包括从自然界(如,土壤、堆肥、水域等)中分离出来的微生物中鉴别和获得这类多肽。从自然生境中分离微生物的技术是本领域熟知的。这样核苷酸序列可类似地通过筛选另一种微生物的基因组或cDNA文库获得。一旦探针检测出编码多肽的核苷酸序列,此序列就可用那些本领域普通技术人员已知的技术(实例参见Sambrook等,1989,同上)进行分离或克隆。
由本发明核苷酸序列编码的多肽还包括融合多肽或可断裂的融合多肽,其中另一多肽在本多肽或其片段的N-末端或C-末端与本多肽或其片段融合。可通过将编码另一个多肽的核苷酸序列(或其部分)与本发明的核苷酸序列(或其部分)融合而产生融合多肽。产生融合多肽的技术是本领域已知的,其中包括连接编码这些多肽的编码序列以使这些序列处于阅读框中,并使融合多肽的表达受同一启动子和终止子的调控。
多核苷酸和核苷酸序列
本发明还涉及具有编码本发明多肽的核苷酸序列的多核苷酸。尤其,本发明涉及这样的多核苷酸,其由编码本发明多肽的核苷酸序列组成。在优选的实施方案中,此核苷酸序列为SEQ ID NO:1中所示序列。在更优选的实施方案中,此核苷酸序列是SEQ ID NO:1的成熟多肽编码区。在另一更优选的实施方案中,此核苷酸序列是包含在大肠杆菌DSM 14240的质粒pJaL621中的成熟多肽编码区。本发明还包括含有这样的核苷酸序列并优选由这样的核苷酸序列组成的多核苷酸,此核苷酸序列能够编码由SEQ ID NO:2所示的氨基酸序列组成的多肽或其成熟多肽,但此核苷酸序列与SEQ ID NO:1因遗传密码简并性而不同。
本发明还涉及含有SEQ ID NO:1亚序列,并优选由此亚序列组成的多核苷酸,所述亚序列能够编码SEQ ID NO:2中具有纤维二糖酶活性的片断。SEQ ID NO:1的亚序列是SEQ ID NO:1所包含的核苷酸序列,但其在5’和/或3’末端有一个或多个核苷酸缺失。
本发明还涉及含有修饰核苷酸序列并优选由此序列组成的多核苷酸,所述修饰核苷酸序列在SEQ ID NO:1所示的成熟多肽编码序列中包含至少一个修饰,并且此修饰核苷酸序列编码的多肽由SEQ ID NO:2中的1至842位氨基酸组成。
用于分离或克隆编码多肽的核苷酸序列的技术是本领域已知的,其包括从基因组DNA进行的分离,从cDNA进行的制备,或者两者的组合。举例来说,通过利用熟知的聚合酶链反应(PCR)或抗体筛选表达文库检测出具有相同结构特征的克隆DNA片断,可以实现从基因组DNA克隆本发明核苷酸序列。实例参见Innis等,1990,PCR:方法和应用指南(PCR:A Guide toMethods and Application),Academic Press,New York。还可使用其它扩增方法如连接酶链反应(LCR),连接激活转录(LAT)及基于核苷酸序列的扩增(NASBA)。核苷酸序列可从曲霉属菌株,或者另外或相关生物体中克隆得到,因而它可以,举例来说,是此核苷酸序列的多肽编码区的等位基因变体或物种变体。
核苷酸序列可利用遗传工程的标准克隆方法使天然位点的核苷酸序列重定位在一个可使此核苷酸序列复制的不同位点而获得。克隆方法可能涉及切除和分离出所需要的含有编码多肽的核苷酸序列的片断,将片断插入载体分子,然后将重组载体导入宿主细胞中,在宿主细胞中复制出多个拷贝或克隆的核苷酸序列。核苷酸序列可来源于基因组,cDNA,RNA,半合成,合成或其任意组合。
本发明还涉及含有这样一个核苷酸序列并优选由此序列组成的多核苷酸,所述核苷酸序列与SEQ ID NO:1中87至2612位核苷酸所示序列具有至少65%的同一性。优选地,此核苷酸序列与SEQ ID NO:1中87至2612位核苷酸所示序列具有至少70%的同一性,例如至少80%的同一性,如至少90%的同一性,更优选至少95%的同一性,如至少96%的同一性,例如至少97%的同一性,甚至更优选至少98%的同一性,如至少99%的同一性。优选地,此核苷酸序列编码具有纤维二糖酶活性的多肽。两个核苷酸序列的同一性程度可如前文所述进行确定(参见标题”定义”一节)。优选地,此核苷酸序列包含SEQ ID NO:1中87至2612位核苷酸所示序列。在甚至更优选的实施方案中,此核苷酸序列由SEQ ID NO:1中87至2612位核苷酸组成。
在另一有意义的方面,本发明涉及包含这样一个核苷酸序列并优选由此序列组成的多核苷酸,所述核苷酸序列与插在大肠杆菌DSM 14240的质粒中的核苷酸序列的纤维二糖酶编码部分具有至少65%的同一性。在优选的实施方案中,与插在大肠杆菌DSM 14240的质粒中的核苷酸序列的纤维二糖酶编码部分具有的同一性是至少70%,举例来说至少80%,如至少90%,更优选至少95%,如至少96%,再例如至少97%,甚至更优选至少98%,如至少99%。优选地,此核苷酸序列包含插在大肠杆菌DSM 14240的质粒中的核苷酸序列的纤维二糖酶编码部分。在甚至更优选的实施方案中,此核苷酸序列由插在大肠杆菌DSM 14240的质粒中的核苷酸序列的纤维二糖酶编码部分组成。
对编码本发明多肽的核苷酸序列进行修饰可能是合成如下多肽所必需的,所述多肽包含与SEQ ID NO:2中1至842位氨基酸所示序列相比具有至少一个替代、缺失和/或插入的氨基酸序列。这些人工变体可以以某种工程化方式区别于从天然源分离的多肽,如在比活性、热稳定性、pH最适值等方面不同的变体。
对本领域技术人员来说,很明显这类修饰作用可在分子功能的关键区域之外进行且仍可得到活性多肽。对于本发明核苷酸序列编码的多肽的活性必需的氨基酸残基(因此优选不进行如替代这样的修饰),可按照本领域已知的方法——如定点诱变或丙氨酸扫描诱变进行鉴别(实例参见,Cunningham和Wells,1989,Science 244:1081-1085)。后一种技术中,向分子中每一个正电荷残基位置引入突变,然后检测所致的突变分子的纤维二糖酶活性以识别对分子活性起关键作用的氨基酸残基。底物-酶相互作用位点也可通过三维结构分析来确定,其中三维结构可通过核磁共振分析、晶体学或光亲和标记这类技术进行测定(实例参见de Vos等,1992,Science 255:306-312;Smith等,1992,Journal of Molecular Biology 224:899-904;Wlodaver等,1992,FEBS Letters 309:59-64)。
此外,编码本发明多肽的核苷酸序列可通过引入核苷酸替代来修饰,该替代不会导致此核苷酸序列编码的多肽具有另一氨基酸序列,但可使此核苷酸序列符合用来产酶的宿主生物体的密码子使用。
将突变引入核苷酸序列使一个核苷酸与另一个核苷酸交换可利用本领域任一已知方法通过定点诱变来完成。特别有用的是利用携带有目的插入物的超螺旋双链DNA和两个含有期望突变的合成引物的方法。寡聚核苷酸引物(每个引物各与载体的相反链互补)借助于Pfu DNA聚合酶在温度循环期间进行延伸。加入引物后产生出含有交错切口的突变质粒。温度循环之后,用特异针对甲基化和半甲基化DNA的DpnI处理产物,消化亲本DNA模板并筛选出含突变的合成DNA。还可以使用其它本领域已知的方法。对核苷酸替代的一般说明可参见Ford等,1991,蛋白质表达和纯化(Protein Expressionand Purification)2:95-107。
本发明还涉及具有这样一个核苷酸序列并优选由此序列组成的多核苷酸,所述核苷酸序列编码具有纤维二糖酶活性的多肽,并且可以在极低严紧条件下,优选在低度严紧条件下,更优选在中度严紧条件下,更优选在中一高度严紧条件下,甚至更优选在高度严紧条件下,最优选在极高严紧条件下与选自:(i)SEQ ID NO:1中核苷酸87至2612位所示序列的互补链,(ii)SEQ ID NO:1中核苷酸87至1139位所示序列的互补链的多核苷酸探针进行杂交。
应该知道,关于核苷酸序列杂交的细节与详情与标题为“具有纤维二糖酶活性的多肽”一节中论述的杂交方面相同或相似。
核酸构建体
本发明还涉及包含可操作地与一个或多个调控序列连接的本发明核苷酸序列的核酸构建体,所述调控序列能够指导此编码序列在适当宿主细胞中在与调控序列相适应的条件下进行表达。
可利用多种方法处理编码本发明多肽的核苷酸序列以备多肽表达。根据表达载体的不同,核苷酸序列在插入载体之前所进行的操作可能是需要的或必需的。利用重组DNA法修饰核苷酸序列的技术是本领域所熟知的。
调控序列可以是适当的启动子序列,即可被宿主细胞识别用于表达本发明核苷酸序列的核苷酸序列。启动子序列包含可调节多肽表达的转录调控序列。启动子可以是任意的能够在所挑选的宿主细胞中表现出转录活性的核苷酸序列,包括突变启动子、截短启动子、和杂合启动子,并且可从与宿主细胞同源或异源的编码细胞外或细胞内多肽的基因中获得。
尤其在细菌宿主细胞中,可指导本发明核酸构建体转录的适用启动子的实例为来自大肠杆菌lac操纵子、天蓝色链霉菌(Streptomyces coelicolor)琼脂糖酶基因(dagA)、枯草芽孢杆菌果聚糖蔗糖酶基因(sacB)、地衣芽孢杆菌α-淀粉酶基因(amyL)、嗜热脂肪芽孢杆菌生麦芽糖淀粉酶基因(amyM)、解淀粉芽孢杆菌α-淀粉酶基因(amyQ)、地衣芽孢杆菌青霉素酶基因(penP)、枯草芽孢杆菌xylA和xylB基因及原核β-内酰胺酶基因(Villa-Kamaroff等,1978,Proceedings of the National Academy of Science USA,75:3727-3731)的启动子,以及tac启动子(DeBoer等,1983,Proceedings ofthe National Academy of Science USA,80:21-25)。对其它启动子的说明参见Scientmc American,1980,242:74-94中的“来自重组细菌的有用蛋白质”;和Sambrook等,1989,同上引文。
在丝状真菌宿主细胞中,可指导本发明核酸构建体转录的适用启动子的实例是来自米曲霉TAKA淀粉酶基因、米黑根毛霉(Rhizomucor miehei)的天冬氨酸蛋白酶基因、黑曲霉中性α-淀粉酶基因、黑曲霉酸稳定α-淀粉酶基因、黑曲霉或泡盛曲霉的葡糖淀粉酶(glaA)基因、米黑根毛霉脂肪酶基因、米曲霉碱性蛋白酶基因、米曲霉磷酸丙糖异构酶基因、构巢曲霉乙酰胺酶基因、和尖镰孢胰蛋白酶样蛋白酶(WO 96/00787)基因的启动子,以及NA2-tpi启动子(来自黑曲霉中性α-淀粉酶和米曲霉磷酸丙糖异构酶基因的启动子杂合体),和它们的突变、截短及杂合启动子。
在酵母宿主中,有用的启动子来自酿酒酵母烯醇化酶(ENO-1)基因、酿酒酵母半乳糖激酶(GAL1)基因、酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)基因和酿酒酵母3-磷酸甘油酸激酶基因。Romanos等,1992,酵母(Yeast)8:423-488对其它适用于酵母宿主细胞的启动子进行了说明。
调控序列还可以是适当的转录终止序列,即可被宿主细胞识别进而终止转录的序列。终止序列可操作地与编码多肽的核苷酸序列的3’末端连接。任何在所选宿主细胞中起作用的终止子都可用于本发明。
对于丝状真菌宿主细胞,优选的终止子来自米曲霉TAKA淀粉酶基因、黑曲霉葡糖淀粉酶基因、构巢曲霉邻氨基苯甲酸合酶基因、黑曲霉α-葡糖苷酶基因和尖镰孢胰蛋白酶样蛋白酶基因。
对于酵母宿主细胞,优选的终止子来自酿酒酵母烯醇化酶基因、酿酒酵母细胞色素C(CYC1)基因和酿酒酵母甘油醛-3-磷酸脱氢酶基因。Romanos等,1992,同上引文中对其它适用于酵母宿主细胞的终止子进行了说明。
调控序列还可以是适当的前导序列,即对宿主细胞的翻译来说很重要的mRNA非翻译区。前导序列可操作地与编码多肽的核苷酸序列的5’末端连接。任何在所选宿主细胞中起作用的前导序列都可用于本发明。
适用于酵母宿主细胞的前导序列可以获自酿酒酵母烯醇化酶基因(ENO-1)、酿酒酵母3-磷酸甘油酸激酶基因、酿酒酵母α-因子基因和酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)基因。
调控序列还可以是聚腺苷酸化序列,即可操作性地与核苷酸序列的3’末端连接的序列,在转录中,此序列可被宿主细胞识别作为将聚腺苷残基添加到转录的mRNA上的信号。任何在所选宿主细胞中起作用的聚腺苷酸化序列都可用于本发明。
对于丝状真菌宿主细胞优选聚腺苷酸化序列获自米曲霉TAKA淀粉酶基因、黑曲霉葡糖淀粉酶基因、构巢曲霉邻氨基苯甲酸合酶基因、尖镰孢胰蛋白酶样蛋白酶基因和黑曲霉α-葡糖苷酶基因。
Guo和Sherman,1995,分子细胞生物学(Molecular Cellular Biology)15:5983-5990对适用于酵母宿主细胞的聚腺苷酸化序列进行了说明。
调控序列还可以是信号肽编码区,它编码的氨基酸序列与多肽的氨基末端连接并且指导此编码多肽进入细胞分泌途径。核苷酸序列的编码序列的5’末端可能固有地包含一个信号肽编码区,此信号肽编码区在翻译阅读框中与编码分泌多肽的编码区片断天然连接。或者,编码区的5’末端可以包含一个对编码序列而言外源的信号肽编码区。如果编码序列天然不含有信号肽编码区,则可能需要外源信号肽编码区。或者,外源信号肽编码区可以简单地替代天然信号肽编码区以增强多肽的分泌。然而,任何可以指导表达的多肽进入所选宿主细胞的分泌途径的信号肽编码区均可用于本发明。
信号肽编码区可以是编码SEQ ID NO:2中-19至-1位氨基酸的SEQID NO:1中30至86位核苷酸。
对于细菌宿主细胞有效的信号肽编码区有获自芽孢杆菌NCIB 11837生麦芽糖淀粉酶基因、嗜热脂肪芽孢杆菌α-淀粉酶基因、地衣芽孢杆菌枯草杆菌蛋白酶基因、地衣芽孢杆菌β-内酰胺酶基因、嗜热脂肪芽孢杆菌中性蛋白酶(nprT,nprS,nprM)基因和枯草芽孢杆菌prsA基因的信号肽编码区。Simonen和Palva,1993在Microbiological Reviews 57:109-137中对其它信号肽进行了说明。
对于丝状真菌宿主细胞有效的信号肽编码区有来自米曲霉TAKA淀粉酶基因、黑曲霉中性淀粉酶基因、黑曲霉葡糖淀粉酶基因、米黑根毛霉天冬氨酸蛋白酶基因、Humicola insolens纤维素酶基因和Humicola lanuginosa脂肪酶基因的信号肽编码区。
对于酵母宿主细胞适用的信号肽来自酿酒酵母α-因子基因和酿酒酵母转化酶基因。Romanos等,1992,同上引文中对其它适用的信号肽编码区进行了说明。
调控序列还可以是编码位于多肽氨基末端的氨基酸序列的前肽编码区。所得多肽被称作前酶或前多肽(或者有时称作酶原)。前多肽一般是无活性的,并可通过催化或自我催化从前多肽中切除前肽而转变成成熟活性多肽。前肽编码区可来自枯草芽孢杆菌碱性蛋白酶(aprE)基因、枯草芽孢杆菌中性蛋白酶(nprT)基因、酿酒酵母α-因子基因、米黑根毛霉天冬氨酸蛋白酶基因和嗜热毁丝霉漆酶基因(WO 95/33836)。
当信号肽和前肽区同时存在于多肽的氨基末端时,前肽区与多肽的氨基末端连接,信号肽区与前肽的氨基末端连接。
还可能需要加上调节序列以使多肽的表达可以相对于宿主细胞的生长进行调节。可例举的调节系统有那些能够响应化学物理刺激(包括存在调节化合物)而引起基因表达开启或关闭的调节系统。原核生物系统中的调节系统包括lac,tac和trp操纵子系统。在酵母中,可使用ADH2系统或GAL1系统。在丝状真菌中,TAKAα-淀粉酶启动子、黑曲霉葡糖淀粉酶启动子和米曲霉葡糖淀粉酶启动子可用作调节序列。可例举的调节序列的其它实例是那些允许基因扩增的序列。在真核生物系统中,此类序列包括有氨甲蝶呤存在时进行扩增的二氢叶酸还原酶基因,以及有重金属存在时进行扩增的金属硫蛋白基因。在这些情况下,编码多肽的核苷酸序列与调节序列可操作地连接。
表达载体
本发明还涉及含有本发明核酸构建体的重组表达载体。可以将上文所述的各种核苷酸和调控序列连接起来产生重组表达载体,此重组表达载体可包括一个或多个方便的限制酶切位点使得编码多肽的核苷酸序列可以在这些位点进行插入或替代。或者,本发明核苷酸序列的表达可以通过将本核苷酸序列或含有本序列的核酸构建体插入适当表达载体中来进行。构建表达载体时,将编码序列置于载体中以使编码序列可操作地与适当的表达调控序列连接。
重组表达载体可以是任何便于接受重组DNA操作并能引起核苷酸序列表达的载体(如,质粒或病毒)。载体的选择一般取决于载体与引入此载体的宿主细胞之间的相容性。此载体可以是线状的或闭合环状的质粒。
载体可以是自主复制的载体,即载体以染色体外实体形式而存在,其复制独立于染色体的复制,举例来说,质粒、染色体外因子、微型染色体或人工染色体。
载体可以包含任何用于确保自主复制的手段。或者,载体可以是这样一个载体,它被引入宿主细胞之后,将整合进入基因组并与被整合的染色体一起复制。此外,可利用单一载体或质粒或者共同含有待引入宿主细胞基因组的全部DNA的两个或多个载体或质粒或者转座子。
本发明载体优选包含一个或多个选择标记以允许容易地挑选出转化细胞。选择标记是一种基因,其产物提供抵抗杀生物剂或病毒的抗性、抗重金属抗性,对营养缺陷体的原养等。
可例举的细菌选择标记有枯草芽孢杆菌或地衣芽孢杆菌的dal基因,或提供抗生素抗性如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的标记。适用于酵母宿主细胞的标记有ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。适用于丝状真菌宿主细胞的选择标记包括,但不限于:amdS(乙酰胺酶)、argB(鸟氨酸氨基甲酰基转移酶)、bar(膦丝菌素乙酰转移酶),hygB(潮霉素磷酸转移酶)、niaD(硝酸还原酶)、pyrG(乳清酸核苷-5’-磷酸脱羧酶)、sC(硫酸腺苷酸转移酶)、trpC(邻氨基苯甲酸合酶)及其等价物。
优选用于曲霉属细胞的是构巢曲霉或米曲霉的amdS和pyrG基因及吸水链霉菌(Streptomyces hygroscopicus)的bar基因。
本发明载体优选包含允许载体稳定整合到宿主细胞的基因组中或者允许载体在细胞中不依赖于基因组自主复制的元件。
对整合到宿主细胞基因组中来说,载体可依靠编码多肽的核苷酸序列或任何其它载体元件以通过同源或非同源重组使载体稳定整合到基因组中。或者,载体可包含附加的核苷酸序列用来指导通过同源重组整合进入宿主细胞的基因组。此附加核苷酸序列能使载体整合到宿主细胞基因组中染色体的精确位置上。为提高整合在精确位置的可能性,整合元件应优选包含足够数目的与相应靶序列高度同源的核苷酸,如100至1,500个碱基对,优选400至1,500个碱基对,最优选800至1,500个碱基对,以提高同源重组的几率。整合元件可以是任何与宿主细胞基因组中的靶序列同源的序列。此外,整合元件可以是非编码或编码核苷酸序列。另一方面,载体可通过非同源重组整合到宿主细胞的基因组中。
对自主复制来说,载体还可包含复制起点以使载体在所研究的宿主细胞中自主复制。可例举的细菌复制起点的实例有允许在大肠杆菌中复制的质粒pBR322、pUC19、PACYC177和pACYC184的复制起点,和允许在芽孢杆菌中复制的质粒pUB110、pE194、pTA1060和pAMβ1的复制起点。可例举的用于酵母宿主细胞中的复制起点的实例是2μ复制起点、ARS1、ARS4、ARS1和CEN3的组合,以及ARS4和CEN6的组合。复制起点可具有使其可在宿主细胞中以温度敏感方式起作用的突变(实例参见,Ehrlich,1978,Proceedings of the National Academy of Science USA,75:1433)。
可将一个以上拷贝的本发明核苷酸序列插入到宿主细胞中以提高基因产物的产量。增加核苷酸序列的拷贝数可通过将至少一个附加拷贝的序列整合进宿主细胞基因组中来得到,或者通过同时包括核苷酸序列与可扩增的选择标记基因、并在适当的选择试剂存在时培养此细胞以筛选出含有扩增拷贝的选择标记基因及由此含有额外拷贝的核苷酸序列的细胞而得到。
用于连接上文所述的元件以构建本发明重组表达载体的方法是本领域技术人员熟知的(实例参见Sambrook等,1989,同上引文)。
宿主细胞
本发明还涉及重组的含有本发明核酸构建体的宿主细胞,它们可有利地用于多肽的重组生产。如前所述,可将含本发明核苷酸序列的载体引入宿主细胞以使载体作为染色体的整合体或作为自主复制的染色体外载体而存在。
宿主细胞可以是单细胞微生物,如原核生物、或者是多细胞微生物,如真核生物。
可适用的单细胞是细菌细胞如革兰氏阳性细菌,包括,但不限于:芽孢杆菌属细胞,例如嗜碱芽孢杆菌、解淀粉芽孢杆菌、短芽孢杆菌、环状芽孢杆菌、Bacillus clausii、凝结芽孢杆菌、灿烂芽孢杆菌、迟缓芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌、嗜热脂肪芽孢杆菌、枯草芽孢杆菌和苏云金芽孢杆菌;或链霉菌属细胞,例如青紫链霉菌或鼠灰链霉菌;或者革兰氏阴性细菌如大肠杆菌和假单胞菌属种。在一个优选实施方案中,细菌宿主细胞是迟缓芽孢杆菌、地衣芽孢杆菌、嗜热脂肪芽孢杆菌或枯草芽孢杆菌细胞。在另一个优选实施方案中,芽孢杆菌属细胞是嗜碱芽孢杆菌。
载体导入细菌宿主细胞可以例如通过原生质体转化(实例参见Chang和Cohen,1979,Molecular General Genetics 168:111-115)、利用感受态细胞(实例参见Young和Spizizn,1961,Journal of Bacteriology 81:823-829,或Dubnau和Davidoff-Abelson,1971,Journal of Molecular Biology 56:209-221)、电穿孔(实例参见Shigekawa和Dower,1988,Biotechniques 6:742-751)或接合(实例参见Koehler和Thorne,1987,Journal ofBacteriology 169:5771-5278)实现。
宿主细胞可以是真核生物如哺乳动物,昆虫,植物或真菌细胞。
在一个优选实施方案中,宿主细胞是真菌细胞。本文所用的“真菌”包括子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、壶菌门(Chytridiomycota)、和接合菌门(Zygomycota)(如Hawsksworth等在Ainsworth and Bisby’Dictionary of The Fungi,8th edition,1995,CABinternational,University Press,Cambridge,UK中的定义)以及卵菌门(Oomycota)(如在Hawsksworth等,1995,上引书171页中的引用)以及所有有丝分裂孢子真菌(Hawsksworth等,1995,上引书)。
在一个更优选的实施方案中,真菌宿主细胞是酵母细胞,本文所用的“酵母”包括产子囊酵母(内孢霉目(Endomycetales))、产担子孢酵母,以及属于半知菌类(芽孢纲(Blastomycetes))的酵母。由于酵母的分类将来还会变化,对于本发明来说,酵母应按照“酵母生物学及活性”(Skinner,F.A.,Passmore,S.M.,和Davenport,R.R.,eds,Soc.App.Bacteriol.Symposium Series No.9,1980)”中的描述进行定义。
在一个甚至更优选的实施方案中,酵母宿主细胞是假丝酵母属、汉逊酵母属(Hansenula)、克鲁维酵母属、毕赤酵母属、酵母属、裂殖酵母属或Yarrowia细胞。
在一个最优选的实施方案中,酵母宿主细胞是卡尔酵母、酿酒酵母、糖化酵母、Saccharomyces douglasii、克鲁弗酵母、诺第酵母或或卵形酵母细胞。在另一最优选的实施方案中,酵母宿主细胞是乳酸克鲁维酵母(Kluyveromyces lactis)细胞。在另一最优选的实施方案中,酵母宿主细胞是Yarrowia lipolytica细胞。
在另一个更优选的实施方案中,真菌宿主细胞是丝状真菌细胞。“丝状真菌”包括所有真菌门(Eumycota)和卵菌门亚门中的丝状真菌(如Hawksworth等,1995,上引文中定义)。丝状真菌的特征是菌丝体壁由几丁质,纤维素,葡聚糖,脱乙酰壳多糖、甘露聚糖及其它复合多糖组成。营养生长表现为菌丝延长,碳代谢是专性需氧的,相反,酵母如酿酒酵母的营养生长表现为单细胞菌体的出芽而碳代谢可以是发酵的。
在甚至更优选的实施方案中,丝状真菌宿主细胞是,但不限于枝顶孢属、曲霉属、镰孢属、腐质霉属、毛霉属、毁丝霉属、脉孢菌属、青霉属、梭孢壳属、Tolypocladium或木霉属种的细胞。
在最优选的实施方案中,丝状真菌宿主细胞是泡盛曲霉、臭曲霉、日本曲霉、构巢曲霉、黑曲霉、或米曲霉细胞。在另一最优选的实施方案中,丝状真菌宿主细胞是杆孢状镰孢、Fusarium cerealis、Fusarium crookwllense、大刀镰孢、禾谷镰孢、禾赤镰孢、异孢镰孢、合欢木镰孢、尖镰孢、多枝镰孢、粉红镰孢、接骨木镰孢、肤色镰孢、拟分枝孢镰孢、硫色镰孢、近念珠状镰孢、Fusarium trichothecioides或有毒镰孢细胞。在甚至最优选的实施方案中,丝状真菌亲本细胞是有毒镰孢(Nirenberg新种)细胞。在另一最优选的实施方案中,丝状真菌宿主细胞是Humicola insolens、Humicolalanuginosa、米黑毛霉、嗜热毁丝霉、粗糙脉孢菌、产紫青霉、Thielaviaterrestris、Trichoderma harzianum、康宁木霉、长枝木霉、Trichoderma reseei或绿色木霉细胞。
真菌细胞可以以本身已知的方式通过涉及原生质形成、原生质转化和细胞壁重建的过程进行转化。在EP238 023和Yelton等,1984,PNAS 81:1470-1474中描述了适合曲霉属宿主细胞转化的方法。Malardier等,1989,基因(Gene)78:147-156和WO 96/00787中描述了适合转化镰孢属种的方法。酵母可利用Becher和Guarente在Abelson,J.N.和Simon,M.I.编著的酶学方法(Methods in Enzymotgy)一书中的酵母遗传学和分子生物学指南,194卷,182-187页,Academic Press,Inc.,New York;Ito等,1983,细菌学杂志153:163;和Hinnen等,1978,PNAS 75:1920中描述的方法进行转化。
生产方法
本发明还涉及用于生产本发明多肽的方法,其包括(a)培养菌株,该菌株的野生型能够产生本多肽;以及(b)回收本多肽。优选地,所述菌株为曲霉属菌株,更优选米曲霉菌株。
本发明还涉及生产本发明多肽的方法,其包括(a)在有益于本多肽生产的条件下培养宿主细胞;以及(b)回收本多肽。
在本发明的生产方法中,利用本领域已知的方法在适于生产本多肽的营养培养基中对细胞进行培养。例如,细胞培养可在实验室或工业发酵罐中在适当的培养基中并在可使本多肽表达和/或分离的条件下,通过摇瓶培养、小型或大型发酵(包括连续、分批、补料分批或固态发酵)来进行。利用本领域已知的方法,培养在含有碳源、氮源和无机盐的适当营养培养基中进行。适用的培养基可从供应商购买或者根据已发表的(如,在美国典型培养物保藏中心的目录中)培养基组成制备。如果多肽分泌到营养培养基中,可直接从培养基回收多肽。如多肽不进行分泌,则可从细胞裂解物回收多肽。
多肽可利用本领域已知的特异针对本多肽的方法进行检测。这些检测方法包括特异性抗体的利用、酶产物的形成或酶底物的消失。例如,酶测定试验可用来确定本文所述的多肽的活性。
所得多肽可通过本领域已知的方法回收。例如,可以利用常规方法,包括,但不限于:离心、过滤、抽提、喷雾干燥、蒸发或沉淀从营养培养基中回收多肽。
本发明多肽可通过本领域已知的许多方法纯化,这些方法包括,但不限于:层析法(如离子交换层析、亲和层析、疏水层析,层析聚焦和分子筛排阻层析),电泳法(如制备性电点聚焦电泳),差别溶解性法(如硫酸铵沉淀),SDS-PAGES,或提取法(实例参见“蛋白质纯化”(Protein Purification),J.-C.Janson和Lars Ryden,editors,VCH Publishers,New York,1989)。
从生物量生产乙醇
乙醇可通过生物量的酶降解并将释放出的多糖转化成乙醇来生产。这种乙醇通常称为生物乙醇或生物燃料。它可在混合物(燃料替代物)中用作燃料添加剂或增量剂,浓度从不到1%直到100%。在一些国家如巴西,乙醇在很大程度上正在取代汽油。
生物量的初生细胞壁中占优势的多糖是纤维素,含量第二的是半纤维素,第三是果胶。次生细胞壁,即细胞停止生长之后产生的细胞壁,也含有多糖并且通过聚合的木质素与半纤维素的共价交联得到强化。纤维素是脱水纤维二糖的同聚物,即线性β-(1-4)-D-葡聚糖,而半纤维素包括许多种化合物,如带有多种取代基的复杂分支结构的木聚糖、木葡聚糖、阿拉伯木聚糖和甘露聚糖。虽然纤维素一般来说是多形的,但在植物组织中发现的纤维素主要是以不溶性的平行葡聚糖链的晶体矩阵的形式存在。半纤维素通常与纤维素以及另外的半纤维素氢键结合,这有助于稳定细胞壁的矩阵结构。
有三大类别的纤维素酶被用于裂解生物量:
●“内切-1,4-β-葡聚糖酶”或“1,4-β-D-葡聚糖-4-葡
聚糖水解酶”(EC 3.2.1.4),它随机地作用于可溶的和不溶的1,4
-β-葡聚糖底物。
●“外切-1,4-β-D-葡聚糖酶”,既包括1,4-β-D-葡聚糖葡
糖水解酶(EC 3.2.1.74),它可从1,4-β-D-葡聚糖中释放出D
-葡萄糖并缓慢水解D-纤维二糖;又包括1,4-β-D-葡聚糖纤
维二糖水解酶(EC 3.2.1.91),又称为纤维二糖水解酶I,它可从1,
4-β-D-葡聚糖中释放出D-纤维二糖。
●“β-D-葡糖苷酶”或β-D-葡糖苷葡糖水解酶(EC 3.2.1.21),
它可从纤维二糖和可溶的纤维糊精以及一批配糖物中释放出D-葡
萄糖单元。
这三类酶在复杂的相互影响中协同作用,导致有效的解晶作用并水解生物量中的天然纤维素,产生还原糖,进而可以通过发酵将这些糖转化成乙醇。
因此本发明还涉及从生物量生产乙醇的方法,其包括使生物量接触本发明多肽;并涉及本发明多肽在生产乙醇中的用途。
植物
本发明还涉及经编码本发明具纤维二糖酶活性的多肽的核苷酸序列转化以致可以表达和生产可回收量的本多肽的转基因植物、植株部分或植物细胞。本多肽可从植物或植株部分中回收。或者,将含有重组多肽的植物或植株部分就此用于改善食物或饲料的质量,如改良营养价值、适口性和流变学性质或破坏抗营养因子。
转基因的植物可以是双子叶的(dicot)或单子叶的(monocot)。可例举的单子叶植物有牧草如草地早熟禾(蓝草,早熟禾属(Poa));饲料草如羊茅属(Festuca)、黑麦草属(Lolium);温带牧草,如剪股颖属(Agrostis);和谷类如小麦、燕麦、黑麦、大麦、稻、高梁和玉米(corn)。
可例举的双子叶植物有烟草、马铃薯、甜菜、豆类如羽扇豆、豌豆、蚕豆、大豆;以及十字花科植物(Brassicaceae family)如花椰菜、菜籽油菜,和亲缘很近的模式生物体鼠耳芥(Arabidopsis thaliana)。
可例举的植株部分有茎、愈合组织、叶、根、果实、种子和块茎。特殊的植物组织,如叶绿体、质外体、线粒体、液泡、过氧物酶体和细胞质也被认为是植株部分。此外,任何植物细胞,不论什么组织来源,也被认为是植株部分。
这些植物、植株部分和植物细胞的子代也包括在本发明的范围之内。
可按照本领域已知的方法构建能够表达本发明多肽的转基因植物或植物细胞。简而言之,转基因植物或植物细胞的构建可以通过将一个或多个编码本发明多肽的表达构建体并入植物宿主基因组中并繁殖所致的修饰植物或植物细胞得到转基因的植物或植物细胞来进行。
有利地,此表达构建体是这样一个核酸构建体,它含有编码本发明多肽的核苷酸序列,其中此核苷酸序列与在选定的植物或植株部分中表达此核苷酸序列所需的适当调节序列可操作地连接。而且,表达构建体可包含用于识别整合了此表达构建体的宿主细胞的选择标记,和将此构建体引入所研究的植物所需的DNA序列(后者取决于使用的DNA引入方法)。
对调控序列,如启动子和终止序列和任选地信号或转运序列的选择,举例来说要根据需要多肽在何时、何处和如何进行表达来确定。例如,编码本发明多肽的基因表达可以是组成型的或可诱导的;或者可以是发育、阶段特异性的或组织特异性的;基因产物可以靶向特异性的组织或植株部分如种子或叶。例如,Tague等,1988,植物生理学(Plant Physiology)86:503对调控序列进行了说明。
对于组成型表达来说,可使用35S-CaMV启动子(Franck等,1980,细胞(Cell)21:285-294)。器官特异性启动子可以是例如,来自贮藏库组织如种子、马铃薯块茎和果实的启动子(Edwards&Coruzzi,1990,遗传学综述年鉴(Ann.Rev.Genet.)24:275-303),或者是来自代谢库组织如分生组织的启动子(Ito等,1994,植物分子生物学(Plant Mol.Biol.)24:863-878),种子特异性启动子如水稻的谷蛋白、谷醇溶蛋白、球蛋白或清蛋白启动子(Wu等,1998,植物和细胞生理学(Plant and Cell Physiology)39:885-889),豆球蛋白B4的蚕豆(Vicia faba)启动子和蚕豆中未知的种子蛋白基因的启动子(Conrad等,1998,植物生理学杂志(Journal of PlantPhysiology)152:708-711),种子油体蛋白的启动子(Chen等,1998,植物和细胞生理学39:935-941),欧洲油菜(Brassica napus)的贮藏蛋白napA启动子,或者如在WO 91/14772中描述的本领域已知的任何其它种子特异性启动子。此外,启动子可以是叶特异性启动子如水稻或番茄的rbcs启动子(Kyozuka等,1993,植物生理学(Plant Physiology)102:991-1000),小球藻病毒腺嘌呤甲基转移酶基因启动子(Mitra和Higgins,1994,植物分子生物学(Plant Molecular Biology)26:85-93),或水稻aldP基因启动子(Kagaya等,1995,分子和普通遗传学(Molecular and General Genetics)248:668-674),或者伤口诱导的启动子如马铃薯pin2启动子(Xu等,1993,植物分子生物学22:573-588)。
还可利用启动子增强子元件在植物中获得酶的更高表达。举例来说,启动子增强子元件可以是位于启动子和编码本发明多肽的核苷酸序列之间的内含子。如Xu等(1993,同上引文)公开了利用水稻肌动蛋白1基因的第一个内含子增强表达。
选择标记基因和表达构建体的任何其它部分可从本领域可利用的那些中挑选。
利用本领域已知的常用技术可将核酸构建体并入植物基因组,这些技术包括土壤杆菌(Agrobacterium)介导的转化、病毒介导的转化、微注射、粒子轰击、生物轰击转化和电穿孔(Gasser等,1990,Science244:1293;Potrykus,1990,Bio/Technology 8:535;Shimamoto等,1989,Nature 338:274)。
目前,根癌土壤杆菌(Agrobacterium tumefaciens)介导的基因转移是所选的用于产生转基因双子叶植物的方法(综述参见Hooykas和Schilperoort,1992,植物分子生物学19:15-38)。然而此方法也可以转化单子叶植物,虽然对于单子叶植物一般优选其它的转化方法。目前所选的产生转基因单子叶植物的方法是粒子轰击胚性愈伤组织或正在发育的胚胎(用转化DNA包被的金或钨微粒)(Christou,1992,植物杂志(Plant Journal)2:275-281;Shimamoto,1994,Current Opinion Biotechnology,5:158-162;Vasil等,1992,Bio/Technology 10:667-674)。如Omirulleh等,1993,植物分子生物学21:415-428所述,转化单子叶植物的一个替代方法是基于原生质体转化来进行。
按照本领域熟知的方法,在转化之后,挑选出掺入表达构建体的转化体并使其再生成完整植株。
本发明还涉及用于生产本发明多肽的方法,其包括(a)在有利于本多肽产生的条件下,对含有编码本发明具纤维二糖酶活性的多肽的核苷酸序列的转基因植物或植物细胞进行培养;并且(b)回收本多肽。
组合物
在另一方面,本发明涉及包含本发明多肽的组合物。
本组合物可包含作为主要酶组分的本发明多肽,如单组分组合物。或者,本组合物可包含多种酶活性,如氨肽酶、淀粉酶、糖酶、羧肽酶、过氧化氢酶、纤维素酶、几丁质酶、角质酶(cutinase)、环糊精糖基转移酶、脱氧核糖核酸酶、酯酶、α-半乳糖苷酶、β-半乳糖苷酶、葡糖淀粉酶、α-葡糖苷酶、β-葡糖苷酶、卤代过氧化物酶(haloperoxidase)、转化酶、漆酶、脂肪酶、甘露糖苷酶、氧化酶、果胶分解酶、肽谷氨酰胺酶(peptidoglutaminase)、过氧化物酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶或木聚糖酶。
本组合物可按照本领域已知的方法进行制备并且可以以液体或干组合物形式存在。举例来说,本多肽组合物可以是颗粒或微粒形式。包含在本组合物中的多肽可按照本领域已知的方法进行稳定。
下文给出本发明多肽组合物的优选用途实例。本发明多肽组合物的剂量和其它使用条件可根据本领域已知的方法进行确定。
去污剂组合物
本发明的纤维二糖酶可被添加到去污剂组合物中并因此成为其组分。
例如,可以将本发明的去污剂组合物配制成手洗或机洗洗衣去污剂组合物,包括适用于预处理污染织物的洗衣添加剂组合物和漂洗时添加的织物柔顺组合物,或配制成用于普通家庭硬表面清洁处理的去污剂组合物,或适用于手洗或机洗碗碟操作的组合物。
在一个特定方面,本发明提供了含本发明纤维二糖酶的去污剂添加剂。此去污剂添加剂以及去污剂组合物中可包含一种或多种其它的酶如蛋白酶、脂肪酶、角质酶、淀粉酶、糖酶、纤维素酶、果胶酶、甘露聚糖酶、阿拉伯聚糖酶、半乳聚糖酶、木聚糖酶,氧化酶,如漆酶和/或过氧化物酶。
一般来说,所选择的酶的性质应该与所选去污剂相兼容,(即最适pH,与其它酶的或非酶的组分的相容性等),并且酶应该以有效量存在。
蛋白酶:适宜的蛋白酶包括动物,植物或微生物来源的蛋白酶,优选微生物来源的蛋白酶。还包括化学修饰过的或蛋白质工程化的突变体。蛋白酶可以是丝氨酸蛋白酶或金属蛋白酶,尤其是碱性微生物蛋白酶或胰蛋白酶样蛋白酶。碱性蛋白酶的实例有枯草杆菌蛋白酶,尤其来源于芽孢杆菌的那些,如枯草杆菌蛋白酶Novo,枯草杆菌蛋白酶Carlsberg,枯草杆菌蛋白酶309,枯草杆菌蛋白酶147和枯草杆菌蛋白酶168(说明参见WO89/06279)。胰蛋白酶样蛋白酶的实例有胰蛋白酶(如猪或牛源的)和镰孢霉蛋白酶(对其描述参见WO89/06270和WO94/25583)。
有用的蛋白酶的实例是在WO92/19729,WO98/20115,WO98/20116和WO98/34946中描述的变体,尤其是在一个或多个如下位点发生替代的变体:27,36,57,76,87,97,101,104,120,123,167,170,194,206,218,222,224,235和274。
优选的商业化的蛋白酶包括ALCALASETM,SAVINASETM,PRIMASETM,EVerlaseTM,ESPERASETM和KANNASETM(Novo Zymes A/S),MAXATASETM,MAXACALTM,MAXAPEMTM,PROPERASETM,PURAFECTTM,PURAFECT OXPTM,FN2TM,和FN3TM(GenencorInternational Inc.)。
脂肪酶:适宜的脂肪酶包括细菌和真菌来源的脂肪酶。还包括化学修饰过的或蛋白质工程化的突变体。有用的脂肪酶的实例包括腐质霉属(同义词Thermomyces),(如EP258 068和EP305 216中描述的)H.lanuginosa(T.lanuginosus)或(如WO96/13580中描述的)H.insolens的脂肪酶;假单胞菌属,如产碱假单胞菌(P.alcaligenes)或类产碱假单胞菌(P.pseudoalcaligenes)(EP218 272),洋葱假单胞菌(P.cepacia)(EP331 376),施氏假单胞菌(P.stutzeri)(GB1,372,034),荧光假单胞菌(P.fluorescens),假单胞菌属菌株SD 705(WO95/06720和WO96/27002),P.wisconsinensis(WO96/12012)的脂肪酶;芽孢杆菌属,如枯草芽孢杆菌(Dartois等(1993),Biochemica et Biophysica Acta,1131,253-360),嗜热脂肪芽孢杆菌(JP 64/744992)或短小芽孢杆菌(B.pumillus)(WO91/16422)的脂肪酶。
其它实例有例如在WO92/05249,WO94/01541,EP407 225,EP260105,WO95/35381,WO96/00292,WO95/30744,WO94/25578,WO95/14783,WO95/22615,WO97/04079和WO97/07202中描述的脂肪酶变体。
优选的商业化的脂肪酶包括LIPOLASETM和LIPOLASE ULTRATM(Novo Zymes A/S)。
淀粉酶:适宜的淀粉酶(α和/或β)包括细菌和真菌来源的淀粉酶。还包括化学修饰过的或蛋白质工程化的突变体。淀粉酶包括,举例来说,芽孢杆菌属如地衣芽孢杆菌的特定菌株的α-淀粉酶,详细说明参见GB1,296,839。
有用的淀粉酶的实例有在WO94/02597,WO94/18314,WO96/23873和WO97/43424中描述的变体,特别是在一个或多个如下位点发生替代的变体:15,23,105,106,124,128,133,154,156,181,188,190,197,202,208,209,243,264,304,305,391,408和444。
商业化的淀粉酶有DURAMYLTM,TERMAMYLTM,FUNGAMYLTM和BANTM(Novo Zymes A/S),RAPIDASETM和PURASTARTM(来自Genencor International Inc.)。
纤维素酶:适宜的纤维素酶包括细菌和真菌来源的纤维素酶。还包括化学修饰过的或蛋白质工程化的突变体。适宜的纤维素酶包括芽孢杆菌属,假单胞菌属,腐质霉属,镰孢属,梭孢壳属,枝顶孢属的纤维素酶,例如在US4,435,307,US5,648,263,US5,691,178,US5,776,757和WO89/09259中公开的由Humicola insolens,嗜热毁丝霉和尖镰孢产生的真菌纤维素酶。
尤其适宜的纤维素酶是有益于颜色维护的碱性或中性纤维素酶。这样的纤维素酶的实例有EP0 495 257,EP0 531 372,WO96/11262,WO96/29397,WO98/08940中描述的纤维素酶。其它实例有如在WO94/07998,EP0 531315,US5,457,046,US5,686,593,US5,763,254,WO95/24471,WO98/12307和PCT/DK98/00299中描述的纤维素酶变体。
商业化的纤维素酶包括CELLUZYMETM,和CAREZYMETM(NovoZymes A/S),CLAZINASETM,和PURADAX HATM(Genencor InternationalInc.),和KAC-500(B)TM(Kao Corporation)。
过氧化物酶/氧化酶:适宜的过氧化物酶/氧化酶包括植物,细菌或真菌来源的过氧化物酶/氧化酶,还包括化学修饰过的或蛋白质工程化的突变体。有用的过氧化物酶的实例包括鬼伞属(Coprinus)如灰盖鬼伞(C.cinereus)的过氧化物酶,及其变体,如在WO93/24618,WO95/10602,和WO98/15257中描述的那些。
可以通过添加含一种或多种酶的各单独添加剂,或通过添加包含所有这些酶的组合添加剂,使去污剂组合物中包含这些去污剂酶。本发明的去污剂添加剂,即单独添加剂或组合添加剂可以以如粒状、液体、浆液等形式配制。优选的去污剂添加剂制剂是颗粒剂,尤其是无尘颗粒;液体,尤其是稳定液体;或浆液。
无尘颗粒可如US4,106,991和4,661,452中的公开进行生产并可任选地利用本领域已知的方法加包衣。蜡质包衣材料的实例有平均摩尔重量1000-20000的聚(环氧乙烷)产物(聚乙二醇,PEG);具有16-50个环氧乙烷单位的乙氧基化壬基酚;具有15-80个环氧乙烷单位的乙氧基化脂肪醇,其中醇含12-20个碳原子;脂肪醇;脂肪酸;以及脂肪酸的单-和二-和三甘油酯。GB1483591中给出了适于通过流化床技术来使用的成膜包衣材料的实例。举例说来,液体酶制备物可按照成熟方法通过添加多元醇如丙二醇、糖或糖醇、乳酸或硼酸来稳定化。受保护的酶可按照EP238,216中公开的方法制备。
本发明的去污剂组合物可以采取任一种方便形式,如条状、片状、粉末、颗粒、糊或液体。液体去污剂可以是水性的,一般含高达70%的水和0-30%的有机溶剂,或者是非水性的。
去污剂组合物可以包含一种或多种表面活性剂,其可以是非离子的(包括半极性的)和/或阴离子的和/或阳离子的和/或两性离子的表面活性剂。表面活性剂的含量按重量计一般是0.1%到60%。
当去污剂中包括阴离子表面活性剂时,去污剂通常包含大约1%到大约40%的阴离子表面活性剂,如直链烷基苯磺酸盐,α-烯属磺酸盐,烷基硫酸盐(脂肪醇硫酸盐),脂肪醇乙氧基硫酸盐,仲链烷磺酸盐,α-磺基脂肪酸甲酯,烷基-或链烯基丁二酸或皂。
当去污剂中包含非离子表面活性剂时,去污剂通常含有约0.2%到约40%的非离子表面活性剂,例如乙氧基化醇、乙氧基化壬基酚、烷基多苷、烷基二甲基胺氧化物、乙氧基化脂肪酸单乙醇酰胺、脂肪酸单乙醇酰胺、多羟基烷基脂肪酸酰胺或葡糖胺的N-酰基N-烷基衍生物(“葡糖酰胺”)。
去污剂可以包含0-65%的去污剂助洗剂或络合剂,例如沸石、二磷酸盐、三磷酸盐、膦酸盐、碳酸盐、柠檬酸盐、次氮基三乙酸、乙二胺四乙酸、二亚乙基三胺五乙酸、烷基-或链烯基琥珀酸、可溶性硅酸盐或层叠式硅酸盐(例如购自Hoechst的SKS-6)。
去污剂可以包含一种或多种聚合物。实例为羧甲基纤维素、聚(乙烯吡咯烷酮)、聚(乙二醇)、聚(乙烯醇)、聚(乙烯基吡啶-N-氧化物)、聚(乙烯基咪唑)、聚羧酸酯如聚丙烯酸酯、马来酸/丙烯酸共聚物和甲基丙烯酸月桂醇酯/丙烯酸共聚物。
去污剂可包含漂白系统,该系统可包含H202源如过硼酸盐或过碳酸盐,并可以同形成过酸的漂白活化剂如四乙酰基乙二胺或壬酰氧基苯磺酸盐进行组合。或者,漂白系统可包含如酰胺,二酰亚胺,或砜类型的过氧酸。
本发明的去污剂组合物中的酶可以通过诸如以下的常规稳定剂稳定:多元醇如丙二醇或丙三醇、糖或糖醇、乳酸、硼酸或硼酸衍生物如芳香硼酸酯或苯基硼酸衍生物如4-甲酰基苯基硼酸,且所述的组合物也可以按WO92/19709和WO92/19708中所述配制。
本去污剂还可包含其它常规去污剂成分,例如织物调理剂(包括黏土)、增泡剂、泡沫抑制剂、抗腐蚀剂、污物悬浮剂、抗污物再沉淀剂、染料、杀菌剂、荧光增白剂、水溶助长剂、晦暗抑制剂或香料。
目前认为在去污剂组合物中任何酶,尤其是本发明的纤维二糖酶都可以以相当于每升洗涤液中具有0.01-100mg酶蛋白质,优选每升洗涤液0.05-5mg酶蛋白质,尤其是每升洗涤液中具有0.1-1mg酶蛋白质的量加入。
此外,本发明的纤维二糖酶可以添加到如WO97/07202所公开的去污剂制剂中,该文献特此引入作为参考。
DNA重组(改组)
如SEQ ID NO:1所示的核苷酸序列可用于DNA重组(或改组)过程。
如此得到的新多核苷酸序列可编码具有如下改良性质的新纤维二糖酶活性多肽:如改良的稳定性(贮藏稳定性,热稳定性)、改良的比活性、改良的pH最适值,和/或改良的对特定化合物的耐受性。
两个或多个同源输入多核苷酸(起始点多核苷酸)之间的改组包括使多核苷酸片段化并重组这些片断,获得与输入多核苷酸相比有许多核苷酸片断发生交换的输出多核苷酸(即已经过一个改组循环的多核苷酸)。
DNA重组或改组可以是从两个或多个起始基因产生出一个嵌合基因文库的(部分的)随机过程。可利用许多已知的方式来实施这样的改组或DNA重组。
此方法可涉及亲本DNA的随机片断化,然后通过PCR重新组装成新的全长基因,如在US5605798,US5811238,US5830721,US6117679中的说明。体外基因重组可按照如US6159687,WO98/41623,US6159688,US5965408,US6153510中的说明进行。此重组过程可在活细胞体内进行,如WO97/07205和WO98/28416中的说明。
如Kikuchi等(2000a,Gene 236:159-167)的说明,亲本DNA可通过DNA’se I处理或限制性内切酶消化而片段化。两个亲本的改组可通过改组两亲本的单链亲本DNA来完成,如Kikuchi等(2000b,Gene 243:133-137)的说明。
一个具体的改组方法是按照Crameri等,1998,Nature,391:288-291和Ness等,Nature Biotechnology 17:892-896中说明的方法操作。另一方式是如US6159687中:实例1和2所述的方法。
本发明通过下列实施例进一步说明,这些实施例不应视为限制本发明的范围。
实施例
用作缓冲剂和底物的化学制品是至少试剂纯的上市产品。
纤维二糖酶试验
PNP葡萄糖加终止试剂
底物溶液:5mM PNP β-D-葡萄糖(Sigma N-7006)底物置于0.1M醋酸钠缓冲液中,pH4.0;
终止试剂:0.1M碳酸钠,pH11.5。
50μl纤维二糖酶溶液与1ml底物溶液混合并在40℃下孵育20分钟。反应通过加入5ml终止试剂而终止。测量404nm的吸光度。
纤维二糖的降解(CBU分析)
纤维二糖酶水解纤维二糖中的β-1,4键,释放出两个葡萄糖分子。释放的葡萄糖量可利用适当的葡萄糖分析方法,如己糖激酶法测定(如Bergmeyer等:D-葡萄糖,利用己糖激酶和葡萄糖-6-磷酸脱氢酶的测定,在Bergmeyer HU,Gawehn K.编《(Methods of Enzymatic Analysis》NewYork:Academic Press;1974:196-201中;或Kunst,A.,Draeger,B.&Ziegenhorn,J.(1984)《Methods of Enzymatic Analysis》(Bergmeyer,HU,ed),第3版,Vol.6,pp.163-172,VCH,Weinheim,W.Germany-Deer-field),其可购自Roche(#127183)。一个纤维二糖酶单位(CBU)是以纤维二糖为底物,在40℃,pH5下每分钟释放出2μmol葡萄糖的酶量。
将纤维二糖酶样品在0.1M醋酸缓冲液,pH5.0中稀释到大约0.05-0.25CBU/ml。0.5ml此溶液与2.5ml纤维二糖底物溶液(0.2g D-(+)纤维二糖,Sigma C-7252,于0.1M醋酸缓冲液中,pH5.0)混合并以40℃加热15分钟。反应通过加入300μl 1N高氯酸终止并测量释放的葡萄糖。
米曲霉纤维二糖酶的比活性是每mg蛋白质150CBU,黑曲霉纤维二糖酶的比活性是每mg蛋白质10CBU。
材料
菌株
BECh2:本菌株的构建参见WO00/39322。
JaL406:本菌株的构建参见实施例2。
质粒
pYES2.0:购自Invitrogen Corporation,San Diego,CA,USA;
pJaL621:本质粒的构建参见实施例1。
pJaL660:本质粒的构建参见实施例2。
pMT2188:本质粒的构建参见实施例2。
pCaHj527:本质粒的构建参见WO 00/70064。
实施例1
米曲霉纤维二糖酶(β-葡糖苷酶)的克隆
米曲霉菌株IFO4177的定向cDNA文库的构建
以尿素和酵母提取物为氮源使米曲霉菌株IFO4177以10升规模在20升实验室发酵罐中生长,分批培养基中以右旋糖为碳源,而补料中以nutriose(麦芽糖浆)为碳源。发酵罐中生长培养基的组成是(g/l):右旋糖27.5,酵母提取物5.0,MgSO4-7H2O 2.0,K2SO4 3.0,KH2PO4 2.0,柠檬酸4.0,尿素5.0及微量元素0.5ml/l。真菌菌丝体在30℃下生长68.3小时后进行收获,立即在液氮中冷冻并在-80℃贮藏。
总RNA的提取
总RNA利用硫氰酸胍提取,再利用超离心通过5.7M CsCl垫(Chirgwin等,1979 Biochemistry 18:5294-5299)来制取,该方法具有如下改良。在液氮中用研钵和研杵将冷冻菌丝体磨成细粉末,然后在预冷的咖啡豆磨具中磨碎,立即悬浮于5倍体积的RNA提取缓冲液中(4M GuSCN,0.5%十二烷基肌氨酸钠,25mM柠檬酸钠,pH7.0,0.1M β-巯基乙醇)。在室温下搅拌混合物30分钟,离心(20分钟,10000rpm,Beckman)沉淀细胞碎片。收集上清液,按照每12.0ml CsCl垫取用26.5ml上清液,小心地将上清液铺在5.7M CsCl垫层(5.7M CsCl,10mMEDTA,pH7.5,0.1%DEPC;使用前高压灭菌)上分层,然后离心得到总RNA(Beckman,SW28转头,25000rpm,室温,24小时)。离心后小心除去上清液,切下含总RNA沉淀的试管底部并以70%EtOH漂洗。将总RNA沉淀转移到Eppendorf管,悬浮于500ml TE,pH7.6中(如果有难度,可在65℃下间断地加热5分钟),用酚抽提并在-20℃下用乙醇进行12小时的沉淀(2.5体积EtOH,0.1体积3M NaAc,pH5.2)。RNA经离心收集,在70%的EtOH中洗涤后重新悬浮于最小体积的DEPC-DIW中。RNA浓度通过测量OD260/280来确定。
poly(A)+RNA的分离
poly(A)+RNA通过寡聚(dT)-纤维素亲和层析分离(Aviv&Leder,1972,Proc.Natl.Acad.Sci.U.S.A.69:1408-1412)。通常,将0.2g寡聚(dT)-纤维素(Boehringer Mannheim)在10ml的1X柱上样缓冲液(20mM Tris-Cl,pH7.6,0.5M NaCl,1mM EDTA,0.1%SDS)中预溶胀,然后加到DEPC-处理的加盖塑料柱上(Poly Prep ChromatographyColumn,Bio Rad),并以20ml的1x上样缓冲液平衡。总RNA(1-2mg)在65℃下加热8分钟,在冰上淬火5分钟,并将1体积2x柱上样缓冲液加入到RNA样品中之后,上柱。收集洗脱物并重新加样2-3次,而且每次加样前都如上述加热样品并在冰上淬火。寡聚(dT)柱用10体积的1x上样缓冲液洗涤,再用3体积的中间盐缓冲液(20mM Tris-Cl,pH7.6,0.1M NaCl,1mM EDTA,0.1%SDS)洗涤,之后用预热到+65℃的3体积洗脱缓冲液(10mM Tris-Cl,pH7.6,1mM EDTA,0.05%SDS)洗脱poly(A)+RNA,最后收集500μl的洗脱级分。读取收集的每一级分的OD260值,将含mRNA的级分合并并在-20℃用乙醇沉淀12小时。离心收集poly(A)+RNA,重新悬浮于DEPC-DIW并在-80℃以5-10μg等分试样贮藏。
cDNA的合成
利用F.S.Hagen(私人交流)研发的发夹改良法,通过RNA酶H法(Gulber&Hoffman 1983 Gene 25:263-269,Sambrook等,1989分子克隆:实验室手册.Cold Spring Harbor Lab.,Cold Spring Harbor NY)从5μg米曲霉A1560的poly(A)+RNA合成出双链cDNA。将poly(A)+RNA(5μl DEPC-处理过的水中5μg)置于预先硅化的无RNA酶的Eppendorph管中,在70℃下加热8分钟,冰上淬火,并与含1mM dATP,dGTP和dTTP以及0.5mM 5-甲基-dCTP(Pharmacia)、40单位的人胎盘核糖核酸酶抑制剂(Rnasin,Promega)、4.82μg寡聚(dT)18-NotI引物(Pharmacia)和1000单位的SuperScriptII RNAse H逆转录酶(Bethesda ResearchLaboratories)的逆转录酶缓冲液(50mM Tris-Cl,pH8.3,75mM KCl,3mM MgCl2,10mM DTT,Bethesda Research Laboratories)混合,终体积为50μl。反应混合物在45℃下孵育1小时合成cDNA第一链。合成之后,按照厂商说明书对mRNA:cDNA杂合体混合物通过MicroSpin S-400 HR(Pharmacia)离心柱进行凝胶过滤。
凝胶过滤之后,杂合体在含200μM各种dNTP、60单位大肠杆菌DNA聚合酶I(Pharmacia)、5.25单位RNase H(Promega)和15单位大肠杆菌DNA连接酶(Boehringer Mannheim)的250μl第二链缓冲液(20mMTris-Cl,pH7.4,90mM KCl,4.6mM MgCl2,10mM(NH4)2SO4,0.16mM βNAD+)中稀释。反应试管在16℃下孵育2小时,25℃下再孵育15分钟进行第二链cDNA的合成。反应通过加入终浓度为20mM的EDTA终止,再进行苯酚和氯仿提取。
双链(ds)cDNA通过加入2体积的96%EtOH,0.2体积的10M NH4Ac,在-20℃下放置12小时进行乙醇沉淀,经离心回收,在70%EtOH中洗涤,干燥(Speed Vac),再重新悬浮于30μl的含25单位绿豆核酸酶(Pharmacia)的绿豆核酸酶缓冲液中(30mM NaAc,pH4.6,300mM NaCl,1mM ZnSO4,0.35mM DTT,2%甘油)。单链发夹DNA的修剪通过下列步骤进行:使反应物在30℃下孵育30分钟,然后加入70μl 10mM Tris-Cl,pH7.5、1mMEDTA,苯酚提取,以2体积96%EtOH和0.1体积3M NaAc,pH5.2置于冰上30分钟进行乙醇沉淀。
ds cDNA经离心(20000rpm,30分钟)回收,并与30μl的T4 DNA聚合酶缓冲液(20mM Tris-醋酸,pH7.9,10mM MgAc,50mM Kac,1mMDTT)(其中包含0.5mM各种dNTP和5单位T4 DNA聚合酶)中的T4 DNA聚合酶进行反应,反应混合物在+16℃下孵育1小时,产生出平端。反应通过加入终浓度为20mM的EDTA而终止,然后进行苯酚和氯仿提取,加入2体积96%EtOH和0.1体积3M NaAc,pH5.2置于-20℃下12小时进行乙醇沉淀。
补平反应之后,cDNA经离心(同上)回收,在70%EtOH中洗涤,DNA沉淀在SpeedVac中干燥。将cDNA沉淀重新悬浮于含2μg EcoRI衔接物(0.2μg/μl,Pharmacia)和20单位T4连接酶(Promega)的25μl连接缓冲液(30mM Tris-Cl,pH7.8,10mM MgCl2,10mM DTT,0.5mM ATP)中,反应混合物在+16℃下孵育12小时。反应通过65℃加热20分钟,然后在冰上放置5分钟而终止。带衔接物的cDNA通过加入20μl高压灭菌水,5μl的10xNotI限制性酶缓冲液(New England Biolabs)和50单位的NotI(NewEngland Biolabs),然后+37℃孵育3小时,而实现NotI限制性酶消化。反应通过65℃加热样品15分钟而终止。利用琼脂糖凝胶电泳(在1×TBE(于高压灭菌水中)中于0.8%SeaPlaque GTG低溶点琼脂糖凝胶上)对cDNAs进行大小分级,分离出未连接的衔接物和小cDNA。凝胶电泳在15V运行12小时,通过在0.7kb位置处切下琼脂糖凝胶的下部对cDNA进行大小选择。然后将1.5%琼脂糖凝胶倾倒在含cDNA的凝胶的前面,反向进行凝胶电泳浓缩ds cDNA,直到其在凝胶上显示为一个压缩带。从凝胶上切下含cDNA的凝胶块并利用GFX凝胶带纯化试剂盒(Amersham-Pharmacia)按照如下步骤从凝胶中提取cDNA。将修剪好的凝胶切块置于2ml Biopure Eppendorf管中称重,然后以每10mg凝胶切块加入10ml的Capture Buffer,凝胶切块的溶解通过60℃孵育10分钟直到琼脂糖完全溶解来实现,样品经瞬时离心而位于管底部。将熔融样品转移到收集管中放置的GFX离心柱上,25℃孵育1分钟,然后在台式离心机中全速旋转30秒。除去流出液,以500μl的洗涤缓冲液洗涤柱子,然后再全速离心30秒。
丢掉收集管,将柱子放置在1.5ml的Eppendorf管中,再将50μl TE,pH7.5加到柱子中心,25℃孵育1分钟,最后以最大速度离心1分钟洗脱cDNA。将洗脱得到的cDNA贮藏在-20℃以待文库的构建。
制备EcoRI/NotI切割的载体用于文库构建
按厂商操作说明书利用Qiagen Tip-100对用于制备含EcoRI-NotI插入片断的pYES 2.0 cDNA克隆的质粒DNA进行纯化。10μg纯化的质粒DNA在加有6μl用于EcoRI的10xNE缓冲液(New England Biolabs)、40单位NotI(New England Biolabs)、和20单位EcoRI(New England Biolabs)的总体积60μl混合液中37℃孵育6小时,而实现EcoRI和NotI的完全消化。反应通过65℃加热样品20分钟而终止。消化的质粒DNA用苯酚-氯仿提取一次,然后用氯仿提取一次,再通过加入2体积96%EtOH和0.1体积3MNaAc,pH5.2在-20℃放置12小时进行乙醇沉淀。将沉淀DNA重新悬浮于25μl 1xTE,pH7.5中,加载到1xTBE(在高压灭菌水中)中的0.8%SeaKem琼脂糖凝胶上,在60V跑凝胶3小时。从凝胶上切下消化的载体,利用GFX凝胶带纯化试剂盒(Amersham-Pharmacia Biotech)按照使用说明从凝胶上提取DNA。用OD260/280测量DNA浓度之后,将洗脱得到的载体贮藏在-20℃以待文库构建。
米曲霉IF04177 cDNA文库的构建
为建立cDNA文库的最适连接条件,在含7μl ds cDNA(相当于cDNA样品的总体积的大约1/10),2单位T4连接酶(Promega)和分别25ng、50ng和75ng的EcoRI-NotI切割的pYES2.0载体(Invitrogen)的10μl连接缓冲液(30mM Tris-Cl,pH7.8,10mM MgC12,10mM DTT,0.5mM ATP)中进行了四个连接试验。载体背景对照连接反应中含有75ng的EcoRI-NotI切割的pYES2.0载体而不含cDNA。连接反应通过16℃孵育12小时,65℃加热20分钟来进行,然后向每个试管中加入10μl的高压灭菌水。1μl的连接混合物经电穿孔(200W,2.5kV,25mF)进入40μl电感受态的大肠杆菌DH10B细胞(Bethesda Research Laboratories)中。向每个转化混合物中加入1ml SOC之后,使细胞在+37℃生长1小时,从每个电穿孔反应物中取50μl和5μl铺在LB+氨苄青霉素平板(100mg/ml)上并在37℃生长12小时。利用最适条件,在大肠杆菌中建立了含2.5×107个独立菌落形成单位的米曲霉(A.oryzae)A1560 cDNA文库,载体背景为大约1%。cDNA文库以多个单独库(25000c.f.u./库)的形式在20%甘油中,-80℃进行贮藏。
利用Qiaprep系统从各单菌落中分离出质粒DNA并按照厂商使用说明在ABI 3700毛细管测序仪上进行序列测定。对比这些序列与SWISSPROT数据库,鉴定到命名为pJaL621的cDNA克隆能够编码纤维二糖酶。对2771bp(碱基对)的cDNA克隆(SEQ ID NO:1)测序揭示一个编码计算分子量为93,437 Da的861个氨基酸的多肽(SEQ ID NO:2)的开放阅读框。
实施例2
在米曲霉中表达米曲霉纤维二糖酶
米曲霉纤维二糖酶表达质粒的构建
曲霉属表达质粒pCaHj527(公开于WO00/70064)由一个表达序列盒组成,此表达序列盒基于黑曲霉中性淀粉酶II启动子与构巢曲霉丙糖磷酸异构酶非翻译前导序列(Pna2/tpi)及黑曲霉淀粉糖苷酶终止子(Tamg)的融合。质粒中还存在使得菌株能以乙酰胺为唯一氮源进行生长的来自构巢曲霉的曲霉属选择标记amdS和能使pyrF缺陷型埃希氏大肠杆菌(Escherichiacoli)菌株DB6507(ATCC 35673)生长的酿酒酵母URA3标记。
利用酿酒酵母(S.cerevisiae)URA3基因为选择标记转化大肠杆菌DB6507的步骤如下:
利用Mandel和Higa方法(Mandel,M.和A.Higa(1970)J.Mol.Biol.45,154)使大肠杆菌DB6507处于感受态。从补充有1g/l酪蛋白氨基酸、500μg/l硫胺素和10mg/l卡那霉素的固体M9培养基(Sambrook等(1989)分子克隆,实验室手册,2 edition,Cold Spring Harbor Laboratory Press)上挑选出转化体。
pCaHj527的修饰步骤如下:
-利用简单PCR法使存在于pCaHj527上的Pna2/tpi启动子发生定点突变;
-利用突变引物141223(SEQ ID NO:5)使核苷酸第134-144位从SEQID NO:3转变为SEQ ID NO:4;
-利用突变引物141222(SEQ ID NO:8)使核苷酸第423-436位从SEQID NO:6转变为SEQ ID NO:7。
所得质粒命名为pMT2188。
将纤维二糖酶基因克隆到pMT2188的步骤如下:
利用下列两个寡聚核苷酸引物:B2902E12(SEQ ID NO:9)和B2902F01(SEQ ID NO:10)通过PCR扩增米曲霉纤维二糖酶编码区。为便于克隆,将限制性酶切位点插入每个引物的5’末端,引物B2902E12含一个BglII位点,引物B2902F01含一个XhoI位点。
米曲霉cDNA克隆pJaL621被用作PCR反应的模版。反应在含2.5单位Taq聚合酶,100ng pJaL621,250nM的各种dNTP,以及如上所述两种引物各10pmol的100μl体积反应缓冲液(50mM KCl,10mM Tris-Cl pH8.0,1.5mM MgCl2)中进行。
扩增在Perkin-Elmer Cetus DNA Termal 480中进行,由94℃3分钟一个循环,之后25个循环每个循环为94℃1分钟、55℃30秒和72℃1分钟组成。PCR反应产生一个2615bp长的DNA片断。此片断用BglII和XhoI消化,凝胶电泳分离,纯化,然后克隆到BamHI和XhoI消化的pMT2188中,得到一个质粒,命名为pJaL660。这样,通过质粒pJaL660的构建产生了米曲霉纤维二糖酶cDNA基因的真菌表达质粒。
在米曲霉中表达米曲霉纤维二糖酶
菌株BECh2(公开于WO 00/30322)利用pJaL600进行转化,方法如Christensen等;Biotechnology 1988,6,1419-1422一文所述。米曲霉的菌丝体一般在丰富营养肉汤中生长。菌丝体经过滤从肉汤中分离出来。将酶制品Novozyme(Novozyme A/S)添加到渗压稳定化缓冲液(如1.2M MgSO4,以磷酸钠缓冲到5.0)中的菌丝体中。此悬浮液在37℃搅拌孵育60分钟。通过微孔布(mira-cloth)过滤原生质体以除去菌丝体碎片。收获原生质体并用STC(1.2M山梨糖醇,10mM CaCl2,10mM Tris-ClpH7.5)洗涤两次。最后将原生质体重新悬浮于200-1000μlSTC中。
为进行转化,将5μg DNA添加到100μl原生质体悬浮液中,然后再加入200μl PEG溶液(60%PEG4000,10mM CaC12,10mM Tris-Cl pH7.5)并且混合物在室温下孵育20分钟。收获原生质体并以1.2M山梨糖醇洗涤两次。最后将原生质体重新悬浮于200μl 1.2M山梨糖醇中,在选择性平板上(基本培养基+10g/L细菌用琼脂(Bacto-Agar)(Difco))铺平板,37℃培养。在37℃下生长3-4天之后,稳定转化体表现为旺盛生长和形成孢子的菌落。对转化体进行两次孢子分离。
使转化体在含100mlYPM培养基(2g/l酵母提取物,2g/l胨,和2%麦芽糖)的摇瓶中30℃下生长4天。按照厂商使用说明在SDS-PAGE凝胶(NovexNuPAGE10%Bis-Tris凝胶)上对上清液(20μl)进行分析。第四号转化体被命名为JaL406。
实施例3
米曲霉纤维二糖酶的生产
利用标准底物使转化的米曲霉JAL406在发酵罐中生长,培养5天后收获发酵液。使整个发酵液通过Whatman公司的玻璃纤维滤器,先经过“D”滤器然后经过“F”滤器,最后使澄清酶溶液经过Millipore公司的孔径22μm的无菌滤器。除去菌丝体。利用Filtron交叉流动膜(截留值10kDa)对澄清酶溶液浓缩。为获得纯酶,使浓缩液经过0.1M醋酸钠pH6缓冲液平衡的21 Sephacryl 200柱。纯酶在SDS-PAGE中的分子量为91kDa。在DSC中pH6.0时的熔解温度是67℃。比较来说,纯化的黑曲霉纤维二糖酶的熔解温度是62℃。米曲霉纤维二糖酶的摩尔消光系数是169540。
如果对硝基苯基β-D-葡萄糖为底物,在pH4.0和40℃下黑曲霉纤维二糖酶的催化活性比米曲霉纤维二糖酶的催化活性低三倍。在pH3.0至7.0之间黑曲霉和米曲霉的纤维二糖酶都显示出大于50%的催化活性。
当将强启动子和终止子基因转化回米曲霉中时,克隆的米曲霉纤维二糖酶获得高得多的产量。
生物材料的保藏
下列生物材料按照布达佩斯条约的条款保藏在德意志微生物保藏中心(DSMZ-Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH,Mascherorder Weg 1B,D-38124 Braunschweig Germany),授予如下保藏号:
保藏 保藏号 保藏日期
NN049573 DSM 14240 2001-04-19
0-10-1-1 | 表格-PCT/RO/134(EASY)与保藏的微生物或其它生物材料有关的声明(PCT细则第13条之2)制备时利用 | PCT-EASY版本2.92(01.01.2002更新) |
0-2 | 国际申请号 | |
0-3 | 申请人或代理人的参考卷号 | 10128-WO |
11-11-2 | 下面列出与说明书中提及的保藏的微生物或其它生物材料有关的声明:页行 | 819-23 |
1-31-3-11-3-21-3-31-3-4 | 保藏鉴定保藏机构名称保藏机构地址保藏日期保藏号 | DSMZ-德意志微生物保藏中心Mascheroder weg 1b,D-38124 Braunschweig,德国2001年4月19日(19.04.2001)DSMZ 14240 |
1-4 | 其它声明 | 无 |
1-5 | 声明适用的指定国 | 所有指定国 |
1-6 | 声明的单独提供这些声明之后将提交给国际局 | 无 |
序列表
<110>诺和酶股份有限公司(Novozymes A/s)
<120>具有纤维二糖酶活性的多肽和编码其的多核苷酸
<130>10128.000-DK
<160>10
<170>patentIn version 3.0
<210>1
<211>2771
<212>DNA
<213>米曲霉(Aspergillus oryzae)
<220>
<221>CDS
<222>(30)..(2612)
<220>
<221>mat_peptide
<222>(87)..()
<400>1
ctgttctgct ggttacctgc cacgttatc atg aag ctt ggt tgg atc gag gtg 53
Met Lys Leu Gly Trp Ile Glu Val
-15
gcc gca ttg gcg gct gcc tca gta gtc agt gcc aag gat gat ctc gcg 101
Ala Ala Leu Ala Ala Ala Ser Val Val Ser Ala Lys Asp Asp Leu Ala
-10 -5 -1 1 5
tac tcc cct cct ttc tac cct tcc cca tgg gca gat ggt cag ggt gaa 149
Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp Gly Gln Gly Glu
10 15 20
tgg gcg gaa gta tac aaa cgc gct gta gac ata gtt tcc cag atg acg 197
Trp Ala Glu Val Tyr Lys Arg Ala Val Asp Ile Val Ser Gln Met Thr
25 30 35
ttg aca gag aaa gtc aac tta acg act gga aca gga tgg caa cta gag 245
Leu Thr Glu Lys Val Asn Leu Thr Thr Gly Thr Gly Trp Gln Leu Glu
40 45 50
agg tgt gtt gga caa act ggc agt gtt ccc aga ctc aac atc ccc agc 293
Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu Asn Ile Pro Ser
55 60 65
ttg tgt ttg cag gat agt cct ctt ggt att cgt ttc tcg gac tac aat 341
Leu Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg Phe Ser Asp Tyr Asn
70 75 80 85
tca gct ttc cct gcg ggt gtt aat gtc gct gcc acc tgg gac aag acg 389
Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala Thr Trp Asp Lys Thr
90 95 100
ctc gcc tac ctt cgt ggt cag gca atg ggt gag gag ttc agt gat aag 437
Leu Ala Tyr Leu Arg Gly aln Ala Met Gly Glu Glu Phe Ser Asp Lys
105 110 115
ggt att gac gtt cag ctg ggt cct gct gct ggc cct ctc ggt gct cat 485
Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly Pro Leu Gly Ala His
120 125 130
ccg gat ggc ggt aga aac tgg gaa ggt ttc tca cca gat cca gcc ctc 533
Pro Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro Asp Pro Ala Leu
135 140 145
acc ggt gta ctt ttt gcg gag acg att aag ggt att caa gat gct ggt 581
Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile Gln Asp Ala Gly
150 155 160 165
gtc att gcg aca gct aag cat tat atc atg aac gaa caa gag cat ttc 629
Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn Glu Gln Glu His Phe
170 175 180
cgc caa caa ccc gag gct gcg ggt tac gga ttc aac gta agc gac agt 677
Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn Val Ser Asp Ser
185 190 195
ttg agt tcc aac gtt gat gac aag act atg cat gaa ttg tac ctc tgg 725
Leu Ser Ser Asn Val Asp Asp Lys Thr Met His Glu Leu Tyr Leu Trp
200 205 210
ccc ttc gcg gat gca gta cgc gct gga gtc ggt gct gtc atg tgc tct 773
Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala Val Met Cys Ser
215 220 225
tac aac caa atc aac aac agc tac ggt tgc gag aat agc gaa act ctg 821
Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu Asn Ser Glu Thr Leu
230 235 240 245
aac aag ctt ttg aag gcg gag ctt ggt ttc caa ggc ttc gtc atg agt 869
Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly Phe Val Met Ser
250 255 260
gat tgg acc gct cat cac agc ggc gta ggc gct gct tta gca ggt ctg 917
Asp Trp Thr Ala His His Ser Gly Val Gly Ala Ala Leu Ala Gly Leu
265 270 275
gat atg tcg atg ccc ggt gat gtt acc ttc gat agt ggt acg tct ttc 965
Asp Met Ser Met Pro Gly Asp Val Thr Phe Asp Ser Gly Thr Ser Phe
280 285 290
tgg ggt gca aac ttg acg gtc ggt gtc ctt aac ggt aca atc ccc caa 1013
Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn Gly Thr Ile Pro Gln
295 300 305
tgg cgt gtt gat gac atg gct gtc cgt atc atg gcc gct tat tac aag 1061
Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala Ala Tyr Tyr Lys
310 315 320 325
gtt ggc cgc gac acc aaa tac acc cct ccc aac ttc agc tcg tgg acc 1109
Val Gly Arg Asp Thr Lys Tyr Thr Pro Pro Asn Phe Ser Ser Trp Thr
330 335 340
agg gac gaa tat ggt ttc gcg cat aac cat gtt tcg gaa ggt gct tac 1157
Arg Asp Glu Tyr Gly phe Ala His Asn His Val Ser Glu Gly Ala Tyr
345 350 355
gag agg gtc aac gaa ttc gtg gac gtg caa cgc gat cat gcc gac cta 1205
Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg Asp His Ala Asp Leu
360 365 370
atc cgt cgc atc ggc gcg cag agc act gtt ctg ctg aag aac aag ggt 1253
Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu Leu Lys Asn Lys Gly
375 380 385
gcc ttg ccc ttg agc cgc aag gaa aag ctg gtc gcc ctt ctg gga gag 1301
Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val Ala Leu Leu Gly Glu
390 395 400 405
gat gcg ggt tcc aac tcg tgg ggc gct aac ggc tgt gat gac cgt ggt 1349
Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn Gly Cys Asp Asp Arg Gly
410 415 420
tgc gat aac ggt acc ctt gcc atg gcc tgg ggt agc ggt act gcg aat 1397
Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser Gly Thr Ala Asn
425 430 435
ttc cca tac ctc gtg aca cca gag cag gcg att cag aac gaa gtt ctt 1445
Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln Asn Glu Val Leu
440 445 450
cag ggc cgt ggt aat gtc ttc gcc gtg acc gac agt tgg gcg ctc gac 1493
Gln Gly Arg Gly Asn Val Phe Ala Val Thr Asp Ser Trp Ala Leu Asp
455 460 465
aag atc gct gcg gct gcc cgc cag gcc agc gta tct ctc gtg ttc gtc 1541
Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val Ser Leu Val Phe Val
470 475 480 485
aac tcc gac tca gga gaa agc tat ctt agt gtg gat gga aat gag ggc 1589
Asn Ser Asp Ser Gly Glu Ser Tyr Leu Ser Val Asp Gly Asn Glu Gly
490 495 500
gat cgt aac aac atc act ctg tgg aag aac ggc gac aat gtg gtc aag 1637
Asp Arg Asn Asn Ile Thr Leu Trp Lys Asn Gly Asp Asn Val Val Lys
505 510 515
acc gca gcg aat aac tgt aac aac acc gtg gtc atc atc cac tcc gtc 1685
Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val Ile Ile His Ser Val
520 525 530
gga cca gtt ttg atc gat gaa tgg tat gac cac ccc aat gtc act ggt 1733
Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp His Pro Asn Val Thr Gly
535 540 545
att ctc tgg gct ggt ctg cca ggc cag gag tct ggt aac tcc atc gcc 1781
Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn Ser Ile Ala
550 555 560 565
gat gtg ctg tac ggt cgt gtc aac cct ggc gcc aag tct cct ttc act 1829
Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys Ser Pro Phe Thr
570 575 580
tgg ggc aag acc cgg gag tcg tat ggt tct ccc ttg gtc aag gat gcc 1877
Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ser Pro Leu Val Lys Asp Ala
585 590 595
aac aat ggc aac gga gcg ccc cag tct gat ttc acc cag ggt gtt ttc 1925
Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe Thr Gln Gly Val Phe
600 605 610
atc gat tac cgc cat ttc gat aag ttc aat gag acc cct atc tac gag 1973
Ile Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr Pro Ile Tyr Glu
615 620 625
ttt ggc tac ggc ttg agc tac acc acc ttc gag ctc tcc gac ctc cat 2021
Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Leu Ser Asp Leu His
630 635 640 645
gtt cag ccc ctg aac gcg tcc cga tac act ccc acc agt ggc atg act 2069
Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro Thr Ser Gly Met Thr
650 655 660
gaa gct gca aag aac ttt ggt gaa att ggc gat gcg tcg gag tac gtg 2117
Glu Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp Ala Ser Glu Tyr Val
665 670 675
tat ccg gag ggg ctg gaa agg atc cat gag ttt atc tat ccc tgg atc 2165
Tyr Pro Glu Gly Leu Glu Arg Ile His Glu Phe Ile Tyr Pro Trp Ile
680 685 690
aac tct acc gac ctg aag gca tcg tct gac gat tct aac tac ggc tgg 2213
Asn Ser Thr Asp Leu Lys Ala Ser Ser Asp Asp Ser Asn Tyr Gly Trp
695 700 705
gaa gac tcc aag tat att ccc gaa ggc gcc acg gat ggg tct gcc cag 2261
Glu Asp Ser Lys Tyr Ile Pro Glu Gly Ala Thr Asp Gly Ser Ala Gln
710 715 720 725
ccc cgt ttg ccc gct agt ggt ggt gcc gga gga aac ccc ggt ctg tac 2309
Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn Pro Gly Leu Tyr
730 735 740
gag gat ctt ttc cgc gtc tct gtg aag gtc aag aac acg ggc aat gtc 2357
Glu Asp Leu Phe Arg Val Ser Val Lys Val Lys Asn Thr Gly Asn Val
745 750 755
gcc ggt gat gaa gtt cct cag ctg tac gtt tcc cta ggc ggc ccg aat 2405
Ala Gly Asp Glu Val Pro Gln Leu Tyr Val Ser Leu Gly Gly Pro Asn
760 765 770
gag ccc aag gtg gta ctg cgc aag ttt gag cgt att cac ttg gcc cct 2453
Glu Pro Lys Val Val Leu Arg Lys Phe Glu Arg Ile His Leu Ala Pro
775 780 785
tcg cag gag gcc gtg tgg aca acg acc ctt acc cgt cgt gac ctt gca 2501
Ser Gln Glu Ala Val Trp Thr Thr Thr Leu Thr Arg Arg Asp Leu Ala
790 795 800 805
aac tgg gac gtt tcg gct cag gac tgg acc gtc act cct tac ccc aag 2549
Asn Trp Asp Val Ser Ala Gln Asp Trp Thr Val Thr Pro Tyr Pro Lys
810 815 820
acg atc tac gtt gga aac tcc tca cgg aaa ctg ccg ctc cag gcc tcg 2597
Thr Ile Tyr Val Gly Asn Ser Ser Arg Lys Leu Pro Leu Gln Ala Ser
825 830 835
ctg cct aag gcc cag taaggggcaa gtcctgattg tacagagcat ttcgagattt 2652
Leu Pro Lys Ala Gln
840
atgatgtaca tgtttatgaa tgacctaggg tagggtaata cttagtaggg ttagttctaa 2712
ttcttggagt caagtattga ctcactgggc cgataaaaaa aaaaaaaaaa aaaaaaaaa 2771
<210>2
<211>861
<212>PRT
<213>米曲霉
<400>2
Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val
-15 -10 -5
Val Ser Ala Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser
-1 1 5 10
Pro Trp Ala Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala
15 20 25
Val Asp Ile Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr
30 35 40 45
Thr Gly Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser
50 55 60
Val Pro Arg Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu
65 70 75
Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn
80 85 90
Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala
95 100 105
Met Gly Glu Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro
110 115 120 125
Ala Ala Gly Pro Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu
130 135 140
Gly Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr
145 150 155
Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr
160 165 170
Ile Met Asn Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly
175 180 185
Tyr Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys
190 195 200 205
Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala
210 215 220
Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr
225 230 235
Gly Cys Glu Asn Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu
240 245 250
Gly Phe Gln Gly Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly
255 260 265
Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val
270 275 280 285
Thr Phe Asp Ser Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly
290 295 300
Val Leu Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val
305 310 315
Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr
320 325 330
Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His
335 340 345
Asn His Val Ser Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp
350 355 360 365
Val Gln Arg Asp His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser
370 375 380
Thr Val Leu Leu Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu
385 390 395
Lys Leu Val Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly
400 405 410
Ala Asn Gly Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met
415 420 425
Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu
430 435 440 445
Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala
450 455 460
Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln
465 470 475
Ala Ser Val Ser Leu Val Phe Val Asn Ser Asp Ser Gly Glu Ser Tyr
480 485 490
Leu Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp
495 500 505
Lys Asn Gly Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn
510 515 520 525
Thr Val Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp
530 535 540
Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly
545 550 555
Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn
560 565 570
Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr
575 580 585
Gly Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln
590 595 600 605
Ser Asp Phe Thr Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys
610 615 620
phe Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr
625 630 635
Thr Phe Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg
640 645 650
Tyr Thr Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu
655 660 665
Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile
670 675 680 685
His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser
690 695 700
Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu
705 710 715
Gly Ala Thr Asp Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly
720 725 730
Ala Gly Gly Asn Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val
735 740 745
Lys Val Lys Asn Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu
750 755 760 765
Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys
770 775 780
Phe Glu Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr
785 790 795
Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp
800 805 810
Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser
815 820 825
Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln
830 835 840
<210>3
<211>11
<212>DNA
<213>合成构建体
<400>3
gtactaaaac c 11
<210>4
<211>11
<212>DNA
<213>合成构建体
<400>4
ccgttaaatt t 11
<210>5
<211>45
<212>DNA
<213>合成构建体
<400>5
ggatgctgtt gactccggaa atttaacggt ttggtcttgc atccc 45
<210>6
<211>14
<212>DNA
<213>合成构建体
<400>6
atgcaattta aact 14
<210>7
<211>14
<212>DNA
<213>合成构建体
<400>7
cggcaattta acgg 14
<210>8
<211>44
<212>DNA
<213>合成构建体
<400>8
ggtattgtcc tgcagacggc aatttaacgg cttctgcgaa tcgc 44
<210>9
<211>27
<212>DNA
<213>合成构建体
<400>9
agatctacca tgaagcttgg ttggatc 27
<210>10
<211>24
<212>DNA
<213>合成构建体
<400>10
ctcgagttac tgggccttag gcag 24
Claims (31)
1、具纤维二糖酶活性的多肽,其选自:
(a)包含这样的氨基酸序列的多肽,此氨基酸序列与SEQ ID NO:2中1至842位氨基酸所示序列具有至少80%同一性或者与SEQ ID NO:2中1至351位氨基酸所示序列具有至少90%同一性;
(b)包含这样的氨基酸序列的多肽,此氨基酸序列与插在大肠杆菌DSM 14240的质粒中的核苷酸序列的纤维二糖酶编码部分所编码的多肽具有至少80%同一性;
(c)由能够在中度严紧条件下与多核苷酸探针杂交的核苷酸序列编码的多肽,其中多核苷酸探针选自
(i)SEQ ID NO:1中87至2612位核苷酸的互补链,和(ii)SEQ IDNO:1中87至1139位核苷酸的互补链;
(d)具有纤维二糖酶活性的(a)、(b)或(c)的片断。
2、如权利要求1所述的多肽,其包含与SEQ ID NO:2中1至842位氨基酸所示序列具有至少85%同一性,优选与SEQ ID NO:2中1至842位氨基酸所示序列具有至少90%同一性、至少95%同一性或至少99%同一性的氨基酸序列。
3、如权利要求2所述的多肽,其包含SEQ ID NO:2中1至842位氨基酸。
4、如权利要求1-3任一项所述的多肽,其由SEQ ID NO:2中1至842位氨基酸组成。
5、如权利要求1-3任一项所述的多肽,其中所述多肽是含有这样的氨基酸序列的人工变体,此氨基酸序列与SEQ ID NO:2中1至842位氨基酸所示序列相比具有至少一个氨基酸替代、缺失和/或插入。
6、如权利要求1所述的多肽,其包含这样的氨基酸序列,该序列与插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的多肽具有至少85%同一性,优选与插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的多肽具有至少90%同一性、至少95%同一性或至少99%同一性。
7、如权利要求6所述的多肽,其包含插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的氨基酸序列。
8、如权利要求6或7所述的多肽,其由插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的氨基酸序列组成。
9、如权利要求6或7所述的多肽,其中所述多肽是含有这样的氨基酸序列的人工变体,此氨基酸序列与插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分所编码的氨基酸序列相比有至少一个替代、缺失和/或插入的氨基酸。
10、如权利要求1所述的多肽,其是由在中度严紧条件下,优选在高度严紧条件下可与多核苷酸探针进行杂交的核苷酸序列编码的,所述多核苷酸探针选自:
(i)SEQ ID NO:1中核苷酸87至2612位的互补链,和
(ii)SEQ ID NO:1中核苷酸87至1139位的互补链。
11、如权利要求10所述的多肽,其是由在中度严紧条件下,优选在高度严紧条件下可与多核苷酸探针进行杂交的核苷酸序列编码的,所述多核苷酸探针为SEQ ID NO:1中核苷酸87至2612位的互补链。
12、具有编码如权利要求1-11中任一项所述的多肽的核苷酸序列的多核苷酸。
13、含有如权利要求12所述的核苷酸序列的核酸构建体,其中所述核苷酸序列可操作地与一个或多个能够指导多肽在适宜宿主中产生的调控序列连接。
14、含有如权利要求13所述的核酸构建体的重组表达载体。
15、含有如权利要求13所述的核酸构建体的重组宿主细胞。
16、生产如权利要求1-11任一项所述的多肽的方法,该方法包括:
(a)培养野生型能够产生所述多肽的菌株以产生所述多肽;以及
(b)回收此多肽。
17、生产如权利要求1-11任一项所述的多肽的方法,该方法包括:
(a)在有利于所述多肽产生的条件下培养如权利要求16所述的重组宿主细胞:以及
(b)回收此多肽。
18、多核苷酸,其具有与SEQ ID NO:1中核苷酸87至2612位所示序列至少85%相同的核苷酸序列。
19、多核苷酸,其具有与插在大肠杆菌DSM 14240的质粒内的核苷酸序列的纤维二糖酶编码部分至少85%相同的核苷酸序列。
20、含有以下核苷酸序列的多核苷酸,此核苷酸序列编码具纤维二糖酶活性的多肽,并且在中度严紧条件下能与选自(i)SEQ ID NO:1中87至2612位核苷酸的互补链、和(ii)SEQ ID NO:1中87至1139位核苷酸的互补链的多核苷酸探针杂交。
21、含有修饰的核苷酸序列的多核苷酸,其中所述修饰的核苷酸序列在SEQ ID NO:1的成熟多肽编码区中包含至少一个修饰,并且所述修饰的核苷酸序列编码由SEQ ID NO:2中1至842位氨基酸组成的多肽。
22、含有如权利要求1-11任一项所述的多肽和表面活性剂的组合物。
23、用于从生物量生产乙醇的方法,其包括使生物量与如权利要求1-11任一项所述的多肽接触。
24、如权利要求1-11任一项所述的多肽在乙醇生产中的用途。
25、DNA改组方法,其包括利用如权利要求12和18-21任一项所述的多核苷酸。
26、DNA改组方法,其包括利用SEQ ID NO:1的87至1139位核苷酸。
27、编码具纤维二糖酶活性的多肽的多核苷酸,其可利用如权利要求25或26所述的方法获得。
28、由如权利要求27所述的多核苷酸编码的具纤维二糖酶活性的多肽。
29、如权利要求12和18-21任一项所述的多核苷酸在DNA改组中的用途。
30、SEQ ID NO:1中87至1139位核苷酸在DNA改组中的用途。
31、转基因植物、植物部分或植物细胞,其已经被编码如权利要求1-11任一项所述的具纤维二糖酶活性的多肽的核苷酸序列转化。
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- 2006-09-28 US US11/529,169 patent/US20070118930A1/en not_active Abandoned
Cited By (1)
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CN108728428A (zh) * | 2004-01-30 | 2018-11-02 | 诺维信股份有限公司 | 具有分解纤维增强活性的多肽及其编码多核苷酸 |
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AU2002316785A1 (en) | 2002-12-03 |
JP2004527261A (ja) | 2004-09-09 |
US20060075519A1 (en) | 2006-04-06 |
WO2002095014A3 (en) | 2003-12-24 |
WO2002095014A9 (en) | 2004-05-21 |
EP1395653A2 (en) | 2004-03-10 |
US20070118930A1 (en) | 2007-05-24 |
WO2002095014A2 (en) | 2002-11-28 |
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