通过体外定向进化提高谷氨酸脱羧酶的催化活性（英文）

摘要

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 Q51 H with high activity [55.4 mmol·L-1·min-1·(mg protein)-1, 120% higher than that of the wild type at p H 4.8] 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, Q51 P, and Q51 L, were identified. The kinetic parameters of the three mutants and Q51 H 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.