A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase (GAD) activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps: 30%—90% saturation (NH4)2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel fil-tration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The purified GAD had a half-life of 50min at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22. 5′-pyridoxal phosphate (PLP) had little effect on the regulation of its activity.
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