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

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

DOI：10.3969/j.issn.1673-1689.2012.03.013

中文关键词: 谷氨酸脱羧酶 γ-氨基丁酸 克隆表达 亲和层析 细胞转化

英文关键词: glutamate decarboxylase gamma-Aminobutyric acid cloning and expression affinity chromatography cell conversion

基金项目:江苏省社会发展科技支撑项目(BE2010678;BE2010626)

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中文摘要:

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%。

英文摘要:

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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