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

生物技术通报 ›› 2013, Vol. 0 ›› Issue (5): 105-110.

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

李天明¹, 朱灵桓², 刘天佳¹, 戴宝新¹, 冯惠勇¹

(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)

收稿日期:2012-11-27 修回日期:2013-05-24 出版日期:2013-05-24 发布日期:2013-05-24
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：iamltm2000@hotmail.com

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

Li Tianming¹, Zhu Linghuan², Liu Tianjia¹, Dai Baoxin¹, Feng Huiyong¹

(1. College of Biological Science and Engineering，Hebei University of Science and Technology，Shijiazhuang 050018；2. College of Biological Engineering，Dalian Polytecnic University，Dalian 116034)

Received:2012-11-27 Revised:2013-05-24 Published:2013-05-24 Online:2013-05-24
About author: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

摘要/Abstract

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.coli BL21(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。

关键词: 甲酸脱氢酶, 基因合成, 原核表达, 饱和突变

Abstract: 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 Cys146AlaCys354Ala 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.

Key words: Formate dehydrogenase, Gene synthesis, Prokaryotic expression, Site-directed mutagenesis

李天明, 朱灵桓, 刘天佳, 戴宝新, 冯惠勇. Pseudomonas sp. 101甲酸脱氢酶基因的合成、表达及定点饱和突变[J]. 生物技术通报, 2013, 0(5): 105-110.

Li Tianming, Zhu Linghuan, Liu Tianjia, Dai Baoxin, Feng Huiyong. Synthesis，Expression and Directed Evolution of the Formate Dehydrogenase Gene from Pseudomonas sp. 101 by Saturation Mutagenesis[J]. Biotechnology Bulletin, 2013, 0(5): 105-110.

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