Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain CGMCC 1306
Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain CGMCC 1306
复制标题
产 GABA 菌株短乳杆菌 CGMCC 1306 的谷氨酸脱羧酶基因的克隆、测序和表达
DOI：
10.1007/s13213-011-0307-5
复制
发表时间：
2012-06-01
影响因子：
3
通讯作者：
Yu, Kai
中科院分区：
文献类型：
--
作者：
Fan, Enyu;Huang, Jun;Yu, Kai
在前期工作中，本课题组从鲜牛乳中分离并培育了一株产γ-氨基丁酸（GABA）的微生物，命名为CGMCC 1306，并发现该菌株具有较高的谷氨酸脱羧酶（GAD）活性。但在深层发酵过程中，菌体生长缓慢，最高干重仅为2.78g/L。为了提高GABA的生物合成效率，实现高密度发酵生产GAD，利用简并PCR技术从CGMCC1306中克隆了GAD基因。核苷酸序列分析表明，该基因全长1407 bp，编码468个氨基酸。将带有hexa-His标签的基因插入到pET-28a（+）中，并在BL21中表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，重组蛋白产物的分子量约为53 kDa，这对应于推导的蛋白质（53.47 kDa）的预测大小。高效液相色谱分析表明，纯化的重组蛋白能够催化α-脱羧谷氨酸钠成GABA，这证实了该蛋白是来自一个基因。GAD活性在pH4.8和48A ℃时最高。用Lineweaver-Burk作图法测得重组酶的活力为10.26mM和8.86U/mg。其活性不依赖于5 '-磷酸吡哆醛的添加。这些结果强烈表明，该基因的克隆不仅是感兴趣的研究人员在影响GAD生产的蛋白质工程策略，但它可能会增加GAD生产的高细胞密度发酵和提高GABA生物合成的效率。
In previous work, our research group isolated and bred a gamma-aminobutyric acid (GABA)-producing microorganism, CGMCC 1306, from fresh unpasteurized milk and found that the strain possessed high glutamate decarboxylase (GAD) activity. However, the cells of the strain grew poorly during submerged fermentation, with the highest dry cell weight only 2.78 g/L. To enhance the efficiency of GABA biosynthesis and achieve GAD production in high cell-density fermentations, the gene from CGMCC 1306 was cloned using degenerate PCR. Nucleotide sequencing analysis showed that the cloned gene comprised 1407 bp and encoded a 468-amino acid protein. The gene with a hexa-His tag was inserted into pET-28a(+) and expressed in BL21. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the recombinant protein product had a molecular weight of approximately 53 kDa, which corresponded to the predicted size of the deduced protein (53.47 kDa). High-performance liquid chromatography analysis showed that the purified recombinant protein was capable of catalyzing alpha-decarboxylation of -sodium glutamate into GABA, which confirmed that the protein was derived from a gene. GAD activity was the highest at pH 4.8 and 48A degrees C. Using Lineweaver-Burk plots, the and of the recombinant enzyme were 10.26 mM and 8.86 U/mg, respectively. Its activity was not dependent on the addition of pyridoxal 5'-phosphate. These results strongly suggest that cloning of the gene is not only of interest to researchers in terms of affecting GAD production by protein engineering strategies, but that it may increase GAD production by high cell-density fermentation and enhance the efficiency of GABA biosynthesis.