Chin. J. Chem. Eng., 15(2) 157—161 (2007) Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk * HUANG Jun(黄俊) a,b , MEI Lehe(梅乐和) a,c, ** , SHENG Qing(盛清) d , YAO Shanjing(姚善泾) a and LIN Dongqiang(林东强) a a Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China b School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China c Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China d College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China Abstract 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% satura- tion (NH 4 ) 2 SO 4 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 K m 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. Keywords Lactobacillus brevis, glutamate decarboxylase, purification, anion-exchange chromatography, charac- terization 1 INTRODUCTION Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid is found in living cells of various organisms, from microorganisms to mammals, and acts as a major cerebral neurotransmitter in the central nervous system[1]. GABA has several physio- logical functions such as neurotransmission, induction of hypotensive effects, diuretic effects, treatment of epilepsy and tranquilizer effects[2,3]. Some recent studies show that GABA is also a strong secretagogue of insulin from the pancreas, and effectively prevents dia- betes[4,5]. Glutamate decarboxylase (GAD) is a unique pyridoxal enzyme, which catalyzes α-decarboxylation of L-glutamate or its salts to synthesize GABA[6]. It has been shown that many neurological disorders such as Huntington’s chorea and Parkinson’s disease are related to the alteration of the GAD level because the substrate (L-glutamate) acts as an excitant and the product (GABA) acts as an inhibitor[7]. During the last decade, GAD has received remarkable attention on account of its important physiological function and as a clinical diagnostic en- zyme for early detection of diabetes. The enzymic properties, molecular cloning, and expression of GAD in some bacteria such as Escher- chia coli[8], Neurospora crassa[9], Lactococcus lac- tis[10], and Lactobacillus brevis[11] have been re- ported. In this article, GAD from Lactobacillus brevis CGMCC 1306 has been purified, and its biochemical properties have been characterized. 2 MATERIALS AND METHODS 2.1 Materials GABA was purchased from Acros Organics (Geel, Belgium). Dansyl chloride was obtained from Sigma-Aldrich (St. Louis, MO, USA). 5′-Pyridoxal phosphate (5′-PLP) and β-mercaptoethanol were pro- vided by Sangon Inc (Shanghai, China). L-sodium glutamate (L-MSG) was purchased from China Medi- cine Shanghai Chemical Reagent Corp. (Shanghai, China). All chemicals were of analytical reagent grade. The chromatography system of the ÄKTA Ex- plorer 100, XK 16×20 column, Q sepharose FF, re- source Q, sephacryl S-200 media, and protein mo- lecular weight marker for sodium dodecyl sul- fate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from GE Healthcare (Uppsala, Swe- den). The electrophoresis unit was purchased from Bio-Rad Laboratories Ltd (USA). The French Press was provided by Thermo Electron Corp (USA). 2.2 Bacterial strain The strain used in the present study was isolated from fresh milk without pasteurization and then mutagenized by ultraviolet treatment and 60 Co radia- tion[12]. It was stored in the China General Microbi- ological Culture Collection Center (CGMCC) as Lac- tobacillus brevis CGMCC 1306. 2.3 Determination of protein concentration and GAD activity The protein concentration was determined ac- cording to the modified Braford method[13], with bo- vine serum albumin as a standard. GAD activity was measured by combining the enzyme extract and appropriate L-MSG, incubating the mixture, terminating the reaction by boiling, and Received 2006-03-02, accepted 2006-08-31. * Supported by the National Natural Science Foundation of China (No.30570411) and the Research Plan of Zhejiang Province, China. ** To whom correspondence should be addressed. E-mail: meilh@zju.edu.cn