红冬孢酵母苹果酸酶基因RKME1的克隆与表达

摘要

苹果酸酶(malic enzyme,ME)普遍存在于各种生物体中,在二价阳离子(Mg2+或者Mn2+)的存在下,它可以催化L-苹果酸进行氧化脱羧反应,产生丙酮酸、CO2和NADPH前期分析结果预测红冬孢酵母YM25235菌株具有2个苹果酸酶同功酶基因RKME1和RKME2,而且RKME1基因在15℃低温条件下mRNA转录水平显著提高 为了验证RKME1的结构与功能,以红冬孢酵母YM25235 cDNA为模板,PCR扩增得到大小为1 623 bp的开放阅读框,共编码540个氨基酸序列分析结果显示该序列含有苹果酸酶保守的4个结构域(Ⅰ～Ⅳ),同源建模结果显示该序列的三级结构和蛔虫中的苹果酸酶晶体结构有较高相似性且高度保守 进一步将RKME1插入到载体pET32a(+)中构建重组表达质粒pET32a-RKME1,然后导入大肠杆菌BL21中进行表达 经IPTG诱导,获得了相对分子质量约为70 kD的蛋白质条带将该蛋白质进行镍柱亲和层析纯化后,酶活分析的结果表明,重组表达的RKME1蛋白可催化苹果酸脱氢生成丙酮酸,同时生成NADPH,酶活为160 U/mg以上.上述结果表明,RKME1是一个新的苹果酸酶基因,这为深入研究苹果酸酶与红冬孢酵母低温条件下生长适应性之间的关系奠定了基础.%Malic enzyme (ME) is a widely distributed enzyme involved in different metabolic pathways in prokarvotic and eukaryotic microorganisms.It catalyzes the oxidative decarboxylation of L-malate to yield pyruvate,CO2 and NADPH in the presence of a bivalent cation (Mg2+ or Mn2+).The results of the previous analysis showed that the YM25235 strain had the expression of two ME iso-enzyme genes RKME1 and RKME2,and the mRNA transcription level of RKME1 gene was significantly improved at 15 ℃.Thus,the sequence of RKME1,which encoded a total of 540 amino acids,with an open reading frame of 1 623 bp,was obtained by PCR with YM25235 cDNA as template.Sequence analvsis showed that there are four conserved domains of malic enzyme (Ⅰ～Ⅳ) in the sequence,and homology modeling showed the tertiary structure of the sequence had high similarity with that of the malate enzyme in Ascaris suum.In order to verify its function,RKME1 was further inserted into vector pET32a(+) to construct a recombinant expression plasmid pET32a-RKME1,which was then introduced into E.coli BL21 for expression.A new protein band with relative molecular mass of about 70 kD was obtained by IPTG induction.The result of enzymatic activity analysis after purification by nickel affinity chromatography showed that recombinant expression of RKME1 protein could catalyze the dehydrogenation of malic acid to pyruvate and produce NADPH,and the cnzyme activity was more than 160 U/mg.The above results indicate that RKME1 is a novel malic enzyme gene and could lay a foundation for further study of the relationship between malic acid enzyme and growth adaptability of Rhodosporidium kratochvilovae at low temperature.