Affiliation:
1. 0000 0000 9735 6249 grid.413109.e Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology Tianjin University of Science and Technology 300457 Tianjin People’s Republic of China
2. 0000 0000 9735 6249 grid.413109.e School of Computer Science and Information Engineering Tianjin University of Science and Technology 300457 Tianjin People’s Republic of China
Abstract
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 K m and V max values for the sole l-glutamate substrate were 5.26 and 3.45 μmol L−1 min−1, respectively.
Funder
National Basic Research Program (973 Program) of China
National Natural Science Foundation of China
National Natural Science Foundation of China (CN)
Youth Innovation Fund of Tianjin University
Publisher
Oxford University Press (OUP)
Subject
Applied Microbiology and Biotechnology,Biotechnology,Bioengineering
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