摘要
甲酸脱氢酶(FDH)是氧化还原酶辅酶NAD+和NADH再生循环体系中的关键酶,具有重要的应用价值和产业化应用前景。通过采用重叠延伸PCR技术,人工合成编码FDH的基因fdh,构建原核表达载体pET30a-fdh,以E.coliBL21(DE3)为表达宿主,获得了能可溶性表达的甲酸脱氢酶的重组菌;经16℃IPTG诱导表达,重组酶的比活力为8.7 U/mgPro;为了提高FDH的稳定性,采用融合PCR方法对fdh基因进行了定点饱和突变,经筛选,获得了酶活未变但稳定性有所提高的突变酶Cys146AlaCys354Ala,其稳定性比野生酶提高了2.5倍。
Formate dehydrogenase（FDH）is a key enzyme in NAD＋-NADH coenzyme regeneration system,which has an important application value and industrial application prospect.In the paper,the gene（fdh）encoding format dehydrogenase（FDH）was artificially synthesized by overlapping PCR.Recombinant plasmid pET30a-fdh was constructed and transformed to E.coli BL21（DE3）.The recombinant FDH expressed in E.coli induced with IPTG at 16℃,mainly existed in a soluble form and reached a specific activity of 8.7 U/mgPro after purification.To improve the stability of FDH,the saturation mutagenesis of fdh was performed by using the overlapping PCR and the mutant enzyme Cys146 AlaCys354 Ala was screened.The activity of the two had no change,but the mutant gave rise to 2.5 fold enhancement of stability compared to that in its wild type.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第5期105-110,共6页
Biotechnology Bulletin
关键词
甲酸脱氢酶
基因合成
原核表达
饱和突变
Formate dehydrogenase Gene synthesis Prokaryotic expression Site-directed mutagenesis
作者简介
李天明，男，硕士，研究方向：分子定向进化与生物催化；E-mail：iamhm2000@hotmail．com
通讯作者：冯惠勇，女，教授，硕士生导师，研究方向：分子定向进化与生物催化；E-mail：fenghuiyong@163.com
