瘤胃菌中乳酸脱氢酶的异源表达与酶学性质研究

Heterologous Expression and Enzymatic Properties of Lactate Dehydrogenase in Rumen Bacteria

乳酸脱氢酶(ldh)在辅酶NAD^(+)/NADH的作用下能催化乳酸与丙酮酸的可逆转化。本研究从实验室保藏的瘤胃菌R1菌株中克隆出ldh基因并构建了pEGX 6p-1-ldh重组质粒,将其导入E.coli BL21(DE3)中实现了ldh异源表达。通过生物信息学分析,ldh呈疏水性,为胞外酶,不存在跨膜结构域,二级结构以α-螺旋和无规则卷曲为主,具有高度保守的功能位点和结构域。通过酶学性质分析,重组ldh最适反应温度为45℃,最适反应pH6.5,Na^(+)、Mn^(2+)和Co^(2+)对ldh酶活有促进作用。

Lactate dehydrogenase(ldh)can catalyze the reversible conversion of lactic acid and pyruvate under the action of coen-zyme NAD^(+)/NADH.The ldh gene was cloned from the rumen bacterium strain R1 preserved in the laboratory,and the pEGX 6p-1-ldh recombinant plasmid was constructed.The plasmid was introduced into E.coli BL21(DE3)to realize the heterologous expression of ldh.Through bioinformatic analysis,ldh is hydrophobic,and is an extracellular enzyme with no transmembrane domain.Its second-ary structure is dominated byα-helix and irregular curl,with highly conserved functional sites and domains.Through the analysis of its enzymatic properties,the optimum reaction temperature of the recombinant ldh was 45℃,the optimum reaction pH was 6.5,and Na^(+),Mn^(2+)and Co^(2+)had a promotion effect on the activity of ldh.