海洋链霉菌酰基辅酶A硫酯酶基因的克隆及分析

Cloning and Analysis of Acyl-CoA Thioesterase II Gene from Marine Streptomyces

酰基辅酶A硫酯酶(ACOT)是催化脂酰辅酶A水解成自由脂肪酸和辅酶A的一类酶,而II型ACOT对底物具有较高的特异性,在脂肪酸合成途径中起着重要的作用,II型ACOT的缺乏或失调会导致机体脂肪酸代谢的紊乱,从而引起一系列疾病。从海洋链霉菌L1基因组DNA中克隆到786碱基的ACOT II基因,生物信息学分析表明其拟编码262个氨基酸,与Acyl-CoA thioesterase II(sequence ID:WP 069742521.1)相似性达到100%。BLAST比对发现,其属于热狗超家族成员,并包含一个II型硫酯酶催化反应的特有结构域。并构建了表达载体pET32a-ACOT II,0.5 mmol/L IPTG诱导目的基因表达,SDS-PAGE显示其分子量约为37.0 kD。通过对链霉菌中II型ACOT基因的研究,可为进一步深入研究II型硫酯酶的分子结构和生物学功能,进而指导药物研发提供参考。

Thioesterase is a widely-used thioester hydrolase,and acyl-coenzyme A thioesterase(ACOT)can hydrolyze various substrates containing coenzyme A.Tape II ACOT(ACOT II)has specificity to its substrates,the lack or imbalance of ACOT II will lead to the disorder of fatty acid metabolism and caused a serial malady accordingly.In this paper,a 786 base pairs ACOT II gene was cloned from Marine Streptomyces sp.L1 genomic DNA,and bioinformatics analysis showed that it coded a protein with 262 amino acids,and its similarity to acyl-CoA thioesterase II(SEQUENCE ID:WP 069742521.1)was 100%.BLAST comparison has found that it belonged to a member of hot dog superfamily and contains a unique domain of type II thioesterase-catalyzed reactions.And established an expression vector of pET32a-COT II,and gene expressed in E.coli BL21 induced by 0.5 mmol/L IPTG.SDS-PAGE showed that its molecular weight was about 37.0 kD.Through the study on type II ACOT gene,it could provide a reference for the further study on its molecular structure and biological function,and proceeded to guidance for the research and development of pharmaceutics.