大肠杆菌L-谷氨酸脱羧酶定点突变及其酶学性质初步研究

Study of site-directed mutagenesis and enzymatic properties of Escherichia coli L-glutamate decarboxylase

L-谷氨酸脱羧酶（L—glutamate decarboxylase，GAD）是生物法生产GABA的关键酶，但是野生型L-谷氨酸脱羧酶的最适pH过低，这限制了它的工业应用。本文对编码来源于大肠杆菌谷氨酸脱羧酶的gadB基因进行了定点突变，分别构建了表达野生型GadB和突变型GadBAHT的基因工程菌。在获得高表达的基础上，对两个酶的最适温度、最适pH、温度稳定性和pH稳定性进行了系统研究，证实了突变酶GadBAHT在保持了与野生型相当的催化活性的前提下，表现出更广泛的pH适应能力，在pH3．4～6．2的范围内，仍能保持50％以上的催化活性。突变酶更能适应工业生产的需要，降低生产成本，对于实际应用具有重要价值。

Gama-aminobutyric acid （GABA）is synthesized from L-glutamate using L-glutamate decarboxylase （GAD）.However,the optimum pH of wild-type L-glutamate decarboxylase is rather low,which limited its industrial application.In this report, sitedirected mutagenesis and over- expression of gadB gene in E. coli encoding L-glutamate decarboxylase was performed in E. coil host cells, and enzymatic characterization of recombinant enzymes were investigated in term of their temperature optimum, pH optimum, thermostability and pH stability. GadB △HT showed broader pH adaptability, at pH 3.4- 6.2, its relative catalytic activity maintain above 50% comparing to wild-type enzyme.Thus, the engineered Gad△HT overcomes the drawback of native enzyme by displaying broader pH tolerance,that could better meet the requirement of industrial bio-production of GABA.