Abstract: The gene encoding glutamate decarboxylase (GAD), a key enzyme for the biosynthesis of γ-aminobutyric acid (GABA), from GABA-producing Lactobacillus plantarum BC114 was amplified by using PCR. Bioinformatics analysis of this gene was performed. Then, the target gene?was?subcloned?into the expression vector pGEM-T-gad and the recombinant plasmids were transformed into competent Escherichia coli BL21 (DE3) for heterogeneous expression. Real-time reverse transcription PCR (RT-PCR), polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC) were used to measure the mRNA and protein expression of GAD and GABA production, respectively. Results showed that the size of the GAD gene from L. plantarum BC114, encoding 469 amino acids, was 1 410 bp. The predicted secondary structure of the GAD protein contained 32.2% α-helix, 11.5% β-sheet and 56.3% random coil. Meanwhile, the three dimensional structure?was also predicted by using homologous modeling method. The recombinant E. coli harboring the gad gene was obtained successfully. The highest expression level of the gad gene was achieved after induction for 6 h, while the maximum protein expression level appeared 2 h later and the highest production of GABA of 2 387 mg/L was also obtained at this time.

Key words: Lactobacillus plantarum BC114, γ-aminobutyric acid, glutamate decarboxylase, cloning, expression, bioinformatics