Lactococcus lactis subsp.lactis IL1403谷氨酸脱羧酶的克隆表达、分离纯化及活性研究

doi:10.3969/j.issn.1673-1689.2012.03.013

首页 > 过刊浏览>2012年第31卷第3期 >302-306. DOI:10.3969/j.issn.1673-1689.2012.03.013

Lactococcus lactis subsp.lactis IL1403谷氨酸脱羧酶的克隆表达、分离纯化及活性研究
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                        10.3969/j.issn.1673-1689.2012.03.013
                    
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基金项目:江苏省社会发展科技支撑项目(BE2010678;BE2010626)

Cloning,Expression,Purification and Characterization of Glutamate Decarboxylase from Lactococcus lactis subsp.lactis IL1403

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谷氨酸脱羧酶(GAD)是功能因子γ-氨基丁酸(GABA)生物合成过程中的重要酶。为了得到一种有高效活性的GAD基因工程菌,克隆到一种GAD基因,来源于微生物Lactococcus lactis subsp.lactis IL1403。以pET-22b(+)为载体质粒,Escherichia coli BL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的重组GAD过量表达;经亲和层析纯化的重组蛋白质样品进行SDS-PAGE分析,在约54 000处出现显著的特征蛋白质条带;活性检测结果表明,该重组GAD的转化活性比野生菌株有明显提高,野生菌株经5h细胞转化,反应底物转化率为55.8%,而工程菌20min后转化率达到82.1%,30min后转化可达到98.3%。

Abstract:

Glutamate decarboxylase(GAD) is the main enzyme for biotransformation glutamate to gamma-Aminobutyric acid(GABA),a functional factor.In order to obtain a genetically engineered bacteria with high activity of GAD,a gene encoding glutamate decarboxylase from Lactococcus lactis subsp.lactis IL1403 was cloned and over-expressed in Escherichia coli BL21(DE3).The bacterium was induced by IPTG and analyzed by SDS-PAGE.The recombinant glutamate decarboxylase was purified to electrophoretic homogeneity by affinity chromatography.Approximately 54 kDa exogenous proteins were observed on the SDS-PAGE.The activity of recombinant GAD was also studied,the bioconversion rate reached 98.2% after 30 minutes,which was a significant improvement when compared with that of the wild type strain.

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张天萌,沐万孟,江波,张涛,倪靓霞. Lactococcus lactis subsp. lactis IL1403谷氨酸脱羧酶的克隆表达、分离纯化及活性研究[J].食品与生物技术学报,2012,31(3):302-306.

ZHANG Tian-meng, MU Wan-meng, JIANG Bo, ZHANG Tao, NI Liang-xia. Cloning, Expression, Purification and Characterization of Glutamate Decarboxylase from Lactococcus lactis subsp. lactis IL1403[J]. Journal of Food Science and Biotechnology,2012,31(3):302-306.

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在线发布日期: 2014-06-17
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