title: Purification and characterization of glutamate decarboxylase from Enterococcus raffinosus TCCC11660.
abstract: Glutamate decarboxylase (GAD) is the sole enzyme that synthesizes γ-aminobutyric acid through the irreversible decarboxylation of L-glutamate. In this study, the purification and characterization of an unreported GAD from a novel strain of Enterococcus raffinosus TCCC11660 were investigated. The native GAD from E. raffinosus TCCC11660 was purified 32.3-fold with a recovery rate of 8.3%, using ultrafiltration and ammonium sulfate precipitation, followed by ion-exchange and size-exclusion chromatography. The apparent molecular weight of purified GAD, as determined by SDS-PAGE and size-exclusion chromatography was 55 and 110 kDa, respectively, suggesting that GAD exists as a dimer of identical subunits in solution. In the best sodium citrate buffer, metal ions of Mo6+and Mg2+had positive effects, while Cu2+, Fe2+, Zn2+and Co2+showed significant adverse effects on enzyme activity. The optimum pH and temperature of GAD were determined to be 4.6 and 45 °C, while the Kmand Vmaxvalues for the sole L-glutamate substrate were 5.26 and 3.45 μmol L-1min-1, respectively.
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Chuanyou
authors.initials: C
authors.lastname: Chang
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Shenxi
authors.initials: S
authors.lastname: Ma
authors.affiliation: School of Computer Science and Information Engineering, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Depei
authors.initials: D
authors.lastname: Wang
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China. gaoqiang@tust.edu.cn.
authors.forename: Qiang
authors.identifier: 0000-0003-4075-7115
authors.initials: Q
authors.lastname: Gao
mesh_terms: D016983:Enterococcus / Q000201:enzymology*; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals; D008970:Molecular Weight; D013696:Temperature
chemical_list: D008670:Metals; D018698:Glutamic Acid; D005968:Glutamate Decarboxylase
keywords: Characterization; Enterococcus raffinosus; Glutamate decarboxylase; Purification; γ-Aminobutyric acid