SCI和EI收录∣中国化工学会会刊

›› 2014, Vol. 22 ›› Issue (11/12): 1322-1327.DOI: 10.1016/j.cjche.2014.09.025

• 生物技术与生物工程 • 上一篇    下一篇

Enhancing the Activity of Glutamate Decarboxylase from Lactobacillus brevis by Directed Evolution

Ling Lin1, Sheng Hu2, Kai Yu1, Jun Huang3, Shanjing Yao1, Yinlin Lei2, Guixiang Hu2, Lehe Mei1,2   

  1. 1 Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;
    2 School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China;
    3 School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
  • 收稿日期:2013-06-04 修回日期:2013-10-29 出版日期:2014-12-28 发布日期:2014-12-24
  • 通讯作者: Lehe Mei
  • 基金资助:
    Supported by the National Natural Science Foundation of China (30970638, 21176220, and 31240054) and the National Natural Science Foundation of Zhejiang Province (LZ13B060002).

Enhancing the Activity of Glutamate Decarboxylase from Lactobacillus brevis by Directed Evolution

Ling Lin1, Sheng Hu2, Kai Yu1, Jun Huang3, Shanjing Yao1, Yinlin Lei2, Guixiang Hu2, Lehe Mei1,2   

  1. 1 Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;
    2 School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China;
    3 School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
  • Received:2013-06-04 Revised:2013-10-29 Online:2014-12-28 Published:2014-12-24
  • Supported by:
    Supported by the National Natural Science Foundation of China (30970638, 21176220, and 31240054) and the National Natural Science Foundation of Zhejiang Province (LZ13B060002).

摘要: Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ- aminobutyrate (GABA), which is in great demand in some foods and pharmaceuticals. In our previous study, gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51H with high activity [55.4 mmol·L-1·min-1·(mg protein)-1, 120% higher than that of the wild type at pH 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out, and three specific mutants, N-terminal truncated GAD, Q51P, and Q51L, were identified. The kinetic parameters of the three mutants and Q51Hwere characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity aswell as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure-function relationship of GAD.

关键词: Directed evolution, Recombinant DNA, Mutagenesis, Enzyme activity, Kinetic parameter, Bio-synthesis

Abstract: Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ- aminobutyrate (GABA), which is in great demand in some foods and pharmaceuticals. In our previous study, gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51H with high activity [55.4 mmol·L-1·min-1·(mg protein)-1, 120% higher than that of the wild type at pH 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out, and three specific mutants, N-terminal truncated GAD, Q51P, and Q51L, were identified. The kinetic parameters of the three mutants and Q51Hwere characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity aswell as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure-function relationship of GAD.

Key words: Directed evolution, Recombinant DNA, Mutagenesis, Enzyme activity, Kinetic parameter, Bio-synthesis