Pseudom onas sp.101甲酸脱氢酶基因的合成、表达及定点饱和突变  

Synthesis , Expression and Directed Evolution of the Formate Dehydrogenase Gene from Pseudomonas sp. 101 by Saturation Mutagenesis

作　　者：李天明[1] 朱灵桓[2] 刘天佳[1] 戴宝新[1] 冯惠勇[1] 

机构地区：[1]河北科技大学生物科学与工程学院,石家庄050018 [2]大连工业大学生物工程学院,大连116034

出　　处：《生物技术通报》2013年第5期105-110,共6页Biotechnology Bulletin

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.

关 键 词：甲酸脱氢酶 基因合成 原核表达 饱和突变 

分 类 号：Q78[生物学—分子生物学]