饱和定点突变拓宽谷氨酸脱羧酶催化pH范围的研究

Expanding the Active p H Range of Lactobacillus brevis Glutamate Decarboxylase by Saturation Mutagenesis

谷氨酸脱羧酶(GAD)是生物法制备γ-氨基丁酸GABA以及一些含氮化合物的关键酶,其催化活力受p H值调控。研究GAD的pH值调控性质具有重要的理论和应用价值。研究通过氨基酸序列比对,发现了一个影响GAD 1407催化p H特性的关键位点:S307。通过对这个位点进行饱和突变,构建突变文库,从中筛选出催化pH范围拓宽的突变酶S307N,并对该突变酶的酶学性质进行了研究。为了解GAD 1407结构与功能之间的相互关系提供了一定的依据。

Glutamate decarboxylase （GAD） from LactobacilIus brevis is a promising candidate for biosynthesis of GABA and various other bulk chemicals derived from GABA. It exhibits an acidic pH optimum, and is regulated by pH sharply. Therefore, it is potential to investigate the characteristics of GAD and the corresponding structure-function relationships. 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 E.coli BL21（ED3）. Despite of its high activity towards L-MSG and good stability, a narrow optimal pH range （4.2-5.2） becomes an obstacle to its application. Here, we performed protein engineering to broaden the optimal pH range to facilitate its application in the bio-preparation of GABA. As expected, a mutant, S307N, with broader optimal pH range was created and exhibited 1-fold higher activity at pH 6.0 compared to the wild type. These results would be useful for researches on pH-dependent regulation of other GADs.