Purification and characterization of glutamate decarboxylase of Lactobacillus brevis CGMCC   1306 isolated from fresh milk

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Purification and characterization of glutamate decarboxylase of Lactobacillus brevis CGMCC 1306 isolated from fresh milk

HUANGJun^a,b;MEILehe^a,c;SHENGQing^d;YAOShanjing^a; LINDongqiang^a

^aDepartment 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

Received:1900-01-01 Revised:1900-01-01 Online:2007-04-28 Published:2007-04-28
Contact: HUANG Jun

牛奶中分离的乳酸菌CGMCC1306中谷氨酸脱羧酶的分离纯化及酶学性质研究

黄俊^a,b; 梅乐和^a,c; 盛清^d; 姚善泾^a; 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

通讯作者: 黄俊

Abstract

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% saturation (NH4)2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel filtration, 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.

Key words: Lactobacillus brevis, glutamate decarboxylase, purification, anion-exchange chromatography, characterization

摘要： 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 filtration, 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.

关键词: Lactobacillus brevis;glutamate decarboxylase;purification;anion-exchange chromatography;characterization

HUANGJun,MEILehe,SHENGQing,YAOShanjing, LINDongqiang. Purification and characterization of glutamate decarboxylase of Lactobacillus brevis CGMCC 1306 isolated from fresh milk[J]. .

黄俊, 梅乐和, 盛清, 姚善泾, 林东强. 牛奶中分离的乳酸菌CGMCC1306中谷氨酸脱羧酶的分离纯化及酶学性质研究[J]. CIESC Journal.

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