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 γ-aminobutyrate (GABA). Due to the physiological functions of GABA, such as the regulation of neurological disorders, and the induction of hypotensive, diuretic effects, development of foods and pharmaceuticals containing highly concentrated GABA is in demand. In our previous study, gad, the gene coding one glutamate decarboxylase (GAD) from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was accomplished. In order to improve its activity, error-prone PCR, followed by a screening, was conducted. A mutant Q51H with much higher activity towards L-MSG (55.4 mM/(min×mg_Protein), 120% higher than that of the wild type at pH4.8) was screened 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. Kinetic parameters of the three mutants and Q51H were characterized and revealed that aspartic acid at site 88 and N-terminal domain were essential to the activity as well as correct folding of GAD. This study not only improved the activity of GAD and enlarged its optimal pH range, but also shed new light on the structure-function relationship of GAD.