钝齿棒杆菌CD945丙酮酸羧化酶基因的克隆、序列分析及表达  

Cloning, Sequence Analysis and Expression of Pyruvate Carboxylase Gene in Corynebacterium crenatum CD945

作　　者：王绛[1] 刘阳剑[1] 王宇[1] 张英姿[1] 余志华[1] 丁久元[1] 

机构地区：[1]中国科学院微生物研究所微生物生物技术中心,北京100080

出　　处：《微生物学报》2003年第2期214-219,共6页Acta Microbiologica Sinica

基　　金：中国科学院重点项目的资助~~

摘　　要：以钝齿棒杆菌 (Corynebacteriumcrenatum)突变株CD945的基因组为模板 ,运用PCR方法 ,扩增出丙酮酸羧化酶的基因片段。核苷酸序列分析结果表明 ,该片段全长 365 7bp ,以GTG为起始密码子 ,编码一个ORF。该ORF的核酸序列与Corynebacteriumglutamicum ,Mycobacteri umsmegmatis以及Saccharomycescerevisiae的丙酮酸羧化酶结构基因相比 ,相似性分别为98 2 2 %、62 41 %和 49 61 %。由ORF推导出的氨基酸序列与上述属种的丙酮酸羧化酶相比 ,同源性分别是 99 30 %、64 65 %和 44 0 4 %。经证实对于酶的催化活性至关重要的一些保守区域 ,如ATP和生物素的结合位点等 ,在该氨基酸序列中都存在。将该基因片段转化钝齿棒杆菌 (C .crenatum)CD945 ,利用CTAB处理细胞和苹果酸脱氢酶偶联测定相结合的方法 ,进行酶活力的分析 ,结果表明 ,重组子与供体菌相比 ,丙酮酸羧化酶的活力提高Pyruvate carboxylase gene ( pyc ) from a mutant Corynebacterium crenatum CD945 was cloned and expressed in its parent strain. Analysis of the pyc sequence shows that only one ORF exists, which codes 1140 amino acids using GTG as the initiation codon and TAA as the termination codon. The similarities among this ORF and those from Corynebacterium glutamicum, Mycobacterium smegmatis and Saccharomyces cerevisiae are 98 22%,62 41%和52 93%, respectively. The amino acid sequence deduced from the ORF shows homologies of 99 30%, 64 65% and 46% to those from the organisms above, respectively. Several motifs believed essential to the enzyme activity are found in this sequence, such as ATP and biotin binding sites. The gene was transformed into C. crenatum CD945 In CTAB permeabilized cells, the enzyme activity was detected using the malate dehydrogenase coupling assay. Pyruvate carboxylase activity in the recombinant is about fivefold higher than that of the parent strain.

关 键 词：棒杆菌 丙酮酸羧化酶 氨基酸 序列分析 表达 基因 

分 类 号：Q786[生物学—分子生物学]