Enhancing The Activity Of Glutamate Decarboxylase By Directed Evolution And Semi-rational Evolution

Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of glutamate into y-aminobutyrate (GABA) and is useful for the biosynthesis of GABA. In our previous study, gad gene from Lactobacillus brevis CGMCC1306was cloned and soluble expression of this gad gene was obtained. Unfortunately, its activity is not as high as that of the E.coli. what’s more, a narrow optimal pH range (4.2～5.2) becomes an obstacle of its application. Here, we performed protein engineering to modify this enzyme to facilitate its utilization in the bio-preparation of GABA.Despite there have been many rational and semi-rational protein engineering methods to modify enzymes, error-prone PCR, especially when there is no available crystal structure of the objective enzyme, also provides a useful first approach for gaining insights into the functionally significant regions of protein. To improve the decarboxylase activity of this enzyme, error-prone PCR, followed by a screening was conducted. A mutant Q51H, with much higher activity towards L-MSG, was screened from the library. In order to investigate the potential role of site51in the regulation of enzymatic activity, site-directed saturation mutagenesis at this site was carried out. Kinetic analysis of Q51H and the characteristics of the N-terminal truncated GAD, Q51P and Q51L, provide evidence for the important role of N-terminal region in the regulation of activity and correct folding of this enzyme. And also, the resulting mutant, Q51H, would be useful in a bioreactor for continuous production of GABA.We also applied a semi-rational directed evolution method. First, we constructed a3-D model of GAD1407through homologous modeling using3fz7F, a GAD subunite from E.coli, as the template. According to the sequence and structure alignment of arabidopsis GAD1(the optimum catalytic pH5.5-7.0) and the GAD1407, we selected some key amino acid sites that may be important in regulating its activity. Based on the hypothesis, we designed a semi-rational means to engineer GAD1407by site-directed mutagenesis. As expected, a mutant with broader optimal pH range was created, and exhibited2-fold higher activity at pH6.0compared to the wild type. What’s more, it was proved that aspartic acid at site88and N-terminal domain were essential to the activity or correct folding of GAD.This study not only improved the activity of GAD1407and enlarged its optimal pH range, but also shed new light on the structure-function relationships of GAD.