Characterization and mutagenesis of a novel Mycobacterium smegmatis-derived glutamate decarboxylase active at neutral...

γ-aminobutyric acid (GABA) has various physiological functions and is widely used in medicine, food, and other fields. Glutamate decarboxylase (GAD) is a key enzyme that catalyzes the decarboxylation of L-glutamate to synthesize GABA. However, the industrial application of microorganism-derived GAD is limited by its rapid loss of enzymatic activity with pH approaching neutrality. In this study, a novel glutamate decarboxylase, GAD_MSM, from Mycobacterium smegmatis was overexpressed and purified. On the basis of homologous modeling and substrate molecular docking, several GAD_MSM mutants were constructed, and their enzymatic properties were analyzed. The results showed that the optimal pH of wild-type GAD_MSM is 5.4; at pH 6.2, 22.8% enzymatic activity was retained. The T211I replacement in GAD and C-terminal deletion mutant GAD_MSMΔC showed relatively high catalytic activity in a pH range of 5.0-7.0. The V_max and K_m values of GAD_MSMΔC were 14.69 and 5.70, respectively, at pH 5.5, and 9.87 and 6.17, respectively, at pH 7.0. Compared with the wild-type GAD, GAD_MSMΔC maintained higher affinity and enzymatic activity of the substrate, maintaining 78.5% of the highest enzymatic activity even at pH 7.0, which is the highest reported activity retention for GAD under neutral pH condition. Therefore, GAD_MSMΔC can be used for the transformation of high-yielding strains and industrial production of GABA.