大肠杆菌UDP-葡萄糖脱氢酶基因的克隆、表达及酶活性测定

生物技术通报 ›› 2013, Vol. 0 ›› Issue (7): 136-143.

大肠杆菌UDP-葡萄糖脱氢酶基因的克隆、表达及酶活性测定

陈奕涵¹ 钱悦² 侯永泰² 周庆玮² 甘人宝² 管世敏¹ 荣绍丰¹

（1.上海应用技术学院香料香精技术与工程学院，上海 201418；2.上海昊海生物科技股份有限公司，上海 201613）

收稿日期:2012-12-24 修回日期:2013-07-19 出版日期:2013-07-19 发布日期:2013-09-02
作者简介:陈奕涵，男，硕士研究生，研究方向：医药产品的研究及工程菌的构建；E-mail：chenyhok1987@126.com
基金资助:
上海市科促会联盟项目（LM201123），上海市科委生物医药与农业领域重点项目（10431901900），上海市科委产学研医项目（11DZ1921405）

Gene Cloning，Expression and Enzyme Activity Assay of UDP-glucose Dehydrogenase in Escherichia coli

Chen Yihan¹ Qian Yue² Hou Yongtai² Zhou Qingwei² Gan Renbao² Guan Shimin² Rong Shaofeng¹

（1. School of Perfume and Aroma Technology，Shanghai Institute of Technology，Shanghai 201418；2. Shanghai Haohai Biological Technology Co. Ltd，Shanghai 201613）

Received:2012-12-24 Revised:2013-07-19 Published:2013-07-19 Online:2013-09-02

摘要/Abstract

摘要： UDP-葡萄糖脱氢酶（UDP-GlcDH）是透明质酸合成过程中关键的限速酶，可以将UDP-葡萄糖催化生成透明质酸必需的前体物质UDP-葡萄糖醛酸。分别采用大肠杆菌BL21（DE3）和YK537的基因组DNA为模板，通过PCR获得BL21（DE3）和YK537的UDP-GlcDH基因ugd，并采用分子生物学方法分别将两种菌株的ugd基因插入含有P _L启动子的pBLMVL2载体中，分别获得重组质粒pBLBugd和pBLYugd，再分别转化于大肠杆菌BL21（DE3）和YK537中，获得4株重组菌；采用升温诱导表达UDP-GlcDH，并测定不同重组菌株的UDP-GlcDH的活性，探索出不同温度诱导条件下UDP-GlcDH的表达量及活性差异。结果表明，采用双阶段升温诱导方式并添加适量酵母粉可以显著提高重组菌表达UDP-GlcDH的活性。

关键词: 透明质酸, UDP-葡萄糖脱氢酶, P_L启动子, 基因测序, 酶活

Abstract: UDP-glucose dehydrogenase, as the key rate-limiting enzyme in the hyaluronic acid synthesis process, can catalyze UDP-glucose to UDP-glucuronic acid which is essential for cell to produce hyaluronic acid. The ugd gene amplified from genomic DNA of Escherichia coli BL21（DE3）and YK537 by PCR were cloned into pBLMVL2 vector containing P _{L promoter. The plasmid pBLBugd and pBLYugd were constructed and transformed respectively into Escherichia coli BL21（DE3）and YK537 to get four recombinant strains. UDP-glucose dehydrogenase activity of different recombinant strains was measured by thermal induction. The differences of expression level and enzyme activity of UDP-glucose dehydrogenase were detected under different temperature induced conditions. These result demonstrate that adopting double stage of heating induction and adding some moderate amount of yeast extract to the cultivating medium can improve the expression level and enzyme activity of UDP - glucose dehydrogenase.}

Key words: Hyaluronic acid, UDP-glucose dehydrogenase, P_L promoter, Gene sequencing, Enzyme activity

陈奕涵,钱悦,侯永泰,周庆玮,甘人宝,管世敏,荣绍丰. 大肠杆菌UDP-葡萄糖脱氢酶基因的克隆、表达及酶活性测定[J]. 生物技术通报, 2013, 0(7): 136-143.

Chen Yihan,Qian Yue,Hou Yongtai,Zhou Qingwei,Gan Renbao,Guan Shimin,Rong Shaofeng. Gene Cloning，Expression and Enzyme Activity Assay of UDP-glucose Dehydrogenase in Escherichia coli[J]. Biotechnology Bulletin, 2013, 0(7): 136-143.

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