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Published on: January 18, 2014
Expression, characterization and mutagenesis of a novel glutamate decarboxylase from Bacillus megaterium
Qingdai Liu1, Haijiao Cheng1, Xiaoqian Ma1
1Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Ministry of Education, Tianjin, 300457, People's Republic of China.
Objectives:
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.
