L-谷氨酸氧化酶的克隆表达、纯化及酶学性质研究

中国生物工程杂志

2013, Vol. 33

Issue (6): 38-44

研究报告

L-谷氨酸氧化酶的克隆表达、纯化及酶学性质研究

卢婵, 郑璞, 孙志浩

江南大学生物工程学院工业生物技术教育部重点实验室无锡 214122

Cloning, Expression, Purification and Characterization of L-glutamate Oxidase

LU Chan, ZHENG Pu, SUN Zhi-hao

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China

全文: PDF(792 KB) HTML

摘要：

为提高微生物产L-谷氨酸氧化酶水平,将Streptomyces sp.X-119-6的L-谷氨酸氧化酶基因(LGOX)与表达载体pET28a连接,导入E.coli BL21(DE3),实现LGOX的高效表达。采用HisTrap^TMFF亲和层析柱对重组L-谷氨酸氧化酶进行纯化, 并对纯酶的酶学性质进行了研究。结果表明:在IPTG终浓度为0.4 mmol/L下,30℃诱导6 h,可以获得比酶活为1.1 U/mg的粗酶液;重组酶的最适反应温度和pH分别为37℃和5.0;K_m值为2.12 mmol/L,V_max为1.06μmol/min·mg, 对L-谷氨酸具有专一性;具有良好的应用前景。

关键词： L-谷氨酸氧化酶; 克隆; 表达; 酶学性质

Abstract:

In order to improve microbial production of L-glutamate oxidase, the L-glutamate oxidase gene (LGOX) from Streptomyces sp.X-119-6 was successfully expressed by connected to the expression vector pET28a and then transformed into E.coli BL21(DE3). The HisTrap^TMFF affinity chromatography column was used for the purification of LGOX. Enzymatic properties of the recombinant LGOX were also investigated. Results showed that the recombinant LGOX activity could reach 1.1 U/mg at the IPTG concentration of 0.4 mmol/L at 30℃ for induction 6 h. The optimum reaction temperature and pH were 37℃ and 5.0, respectively. The K_m was 2.12 mmol/L and V_max was 1.06μmol/min·mg. The LGOX had high substrate specificity of L-glutamic acid with application prospect.

Key words: L-glutamate oxidase Cloning Expressing Enzymatic properties

收稿日期: 2013-03-04 出版日期: 2013-06-25

ZTFLH:

Q786

基金资助:

国家"863"计划资助项目(2006AA020301-012)

通讯作者: 郑璞 E-mail: zhengpu@jiangnan.edu.cn

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引用本文:

卢婵, 郑璞, 孙志浩. L-谷氨酸氧化酶的克隆表达、纯化及酶学性质研究[J]. 中国生物工程杂志, 2013, 33(6): 38-44.

LU Chan, ZHENG Pu, SUN Zhi-hao. Cloning, Expression, Purification and Characterization of L-glutamate Oxidase. China Biotechnology, 2013, 33(6): 38-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I6/38

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