酿酒酵母甲酸脱氢酶在大肠杆菌中的高效表达、纯化及其酶学性质

High Expression of Formate Dehydrogenase of Saccharomyces cerevisiae in E.coli and Purification and Enzymological Property of Expressed Product

目的克隆酿酒酵母甲酸脱氢酶(Formate dehydrogenase,FDH)基因,在大肠杆菌中表达、纯化重组蛋白,并进行酶学性质分析。方法以酿酒酵母基因组DNA为模板,采用温度梯度PCR法扩增fdh基因,重叠PCR技术校正fdh中的移码突变,构建重组表达质粒pET-28a-fdh,转化至大肠杆菌BL21(DE3)中,分别采用37℃IPTG诱导、15℃IPTG诱导和37℃乳糖自诱导表达,镍金属螯合亲和层析纯化重组FDH蛋白,并分析重组FDH的酶学性质。结果成功克隆了1 100 bp的fdh基因,碱基突变纠正了基因缺失和置换引起的编码错误;表达的重组FDH相对分子质量约为43 000,37℃乳糖诱导表达的重组FDH主要以可溶形式表达,表达量约占菌体总蛋白的30%;纯化的重组蛋白比活为7.69 U/mg;重组FDH的最适反应温度和pH分别为30℃和7.5,热稳定性较好,米氏常数分别为:KmNAD+=49μmol/L,Km甲酸=4.5 mmol/L。结论已在大肠杆菌中表达并纯化了重组FDH蛋白,为进一步研究酿酒酵母FDH,利用其构建NAD(P)H辅酶再生体系奠定了基础。

Objective To clone formate dehydrogenase（FDH） gene of Saccharomyces cerevisiae,express in E.coli,purify the expressed product and analyze its enzymological property.Methods The fdh gene was amplified by temperature gradient PCR using the genomic DNA of S.cerevisiae as a template,in which the shift mutation was corrected by overlap PCR.Recombinant plasmid pET-28a-fdh was constructed and transformed to E.coli BL21（DE3）,and induced with IPTG and 15 and 37℃ and with lactose at 37℃ respectively.The expressed recombinant FDH protein was purified by metal chelate chromatography with nickel ion and analyzed for enzymological property.Results The fdh gene at a length of 1 100 bp was successfully cloned,in which the coding error caused by gene deletion and substitution was corrected by base mutation.The relative molecular mass of expressed recombinant FDH was about 43 000.The recombinant FDH expressed in E.coli induced with lactose at 37℃ mainly existed in a soluble form,contained about 30% of total somatic protein,and reached a specific activity of 7.69 U/mg after purification.The optimal working temperature and pH value of recombinant FDH were 30℃ and 7.5 respectively.The recombinant FDH showed high thermal stability,of which KmNAD＋ and KMformate were 49 μmol/L and 4.5 mmol/L respectively.Conclusion Recombinant FDH was expressed in E.coli and purified,which laid a foundation of further study on FDH of S.cerevisiae and establishment of regeneration system of NAD（P）H coenzyme.