...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Annals of Microbiology >Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain Lactobacillus brevis CGMCC 1306
【24h】

Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain Lactobacillus brevis CGMCC 1306

机译：产GABA的短乳杆菌CGMCC 1306的谷氨酸脱羧酶基因的克隆，测序和表达

获取原文
获取原文并翻译 | 示例
   

获取外文期刊封面封底 >>

       
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

In previous work, our research group isolated and bred a γ-aminobutyric acid (GABA)-producing microorganism, Lactobacillus brevis 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 gad gene from L. brevis 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 gad gene with a hexa-His tag was inserted into pET-28a(+) and expressed in Escherichia coli 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 α-decarboxylation of L-sodium glutamate into GABA, which confirmed that the protein was derived from a gad gene. GAD activity was the highest at pH 4.8 and 48°C. Using Lineweaver–Burk plots, the K m and V max 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 gad 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.
机译：在先前的工作中，我们的研究小组从新鲜的未经巴氏灭菌的牛奶中分离并繁殖了产生γ-氨基丁酸（GABA）的微生物，即短乳杆菌CGMCC 1306，并发现该菌株具有较高的谷氨酸脱羧酶（GAD）活性。然而，该菌株的细胞在深层发酵过程中生长较差，最高干细胞重量仅为2.78 g / L。为了提高GABA生物合成的效率并在高细胞密度发酵中实现GAD的产生，使用简并PCR克隆了来自短乳杆菌CGMCC 1306的gad基因。核苷酸测序分析表明，克隆的基因包含1407 bp，编码468个氨基酸的蛋白质。将带有hexa-His标签的gad基因插入pET-28a（+），并在大肠杆菌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，与推导蛋白的预测大小（53.47 kDa）相符。高效液相色谱分析表明，纯化的重组蛋白具有催化L-谷氨酸钠α-脱羧成GABA的作用，证实了该蛋白来源于gad基因。 GAD活性在pH 4.8和48°C时最高。使用Lineweaver–Burk图，重组酶的K m 和V max 分别为10.26 mM和8.86 U / mg。其活性不依赖于吡ido醛5'-磷酸的添加。这些结果强烈表明，克隆gad基因不仅对研究人员感兴趣的是通过蛋白质工程策略影响GAD的产生，而且可能通过高细胞密度发酵增加GAD的产生并提高GABA生物合成的效率。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号