title: Expression, characterization and mutagenesis of a novel glutamate decarboxylase from Bacillus megaterium.
abstract: OBJECTIVE
  To search for a novel glutamate decarboxylase (GAD) with an optimum pH towards near-neutrality in order to improve production of gamma-aminobutyric acid (GABA) in recombinant hosts.
  
  
  RESULTS
  A novel glutamate decarboxylase, BmGAD, from Bacillus megaterium was overexpressed and purified. BmGAD was approximately 53 kDa by SDS-PAGE analysis. Its optimum activity was at pH 5 and 50 °C. BmGAD had a specific activity of 59 ± 5.2 U mg(-1) at pH 6, which is the highest value reported so far. The apparent Km and Vmax values of BmGAD were 8 ± 0.5 mM and 150 ± 4.7 U mg(-1), respectively. Through site-directed mutagenesis, two BmGAD mutants (E294R and H467A) showed higher Vmax values than that of wild-type, with the values of 210 ± 6.9 and 180 ± 4.1 U mg(-1) at pH 5 and 50 °C, respectively.
  
  
  CONCLUSIONS
  The unusual high activity of BmGAD at pH 6 makes it an attractive GABA-producing candidate in industrial application.
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Ministry of Education, Tianjin, 300457, People's Republic of China.
authors.forename: Qingdai
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Ministry of Education, Tianjin, 300457, People's Republic of China.
authors.forename: Haijiao
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Ministry of Education, Tianjin, 300457, People's Republic of China.
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Ma
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China.
authors.forename: Ning
authors.initials: N
authors.lastname: Xu
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. liu_jun@tib.cas.cn.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. liu_jun@tib.cas.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase
keywords: Bacillus megaterium; Gamma-aminobutyric acid (GABA); Glutamate decarboxylase (GAD); Mutagenesis