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

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 [55.4mmol·L−1·min−1·(mg protein)−1, 120% higher than that of the wild type at pH4.8] 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 Q51H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well 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. A novel key amino acid residue site (Q51) affecting glutamate decarboxylase (GAD) activity and expression level remarkably was identified in this study. This site is distant from active sites according to the homology model of GAD but situated on a helix at the end of the N-terminal region (residues 1–59). One mutant Q51H with an activity of 1.2 times the wild type activity was obtained by directed evolution. The possible reason might be that the substitution will give rise to hydrogen bonding interactions with L47 and E55 and therefore alter the flexibility and position of the adjacent loop and improve the activity. [Display omitted]