Journal
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
Volume 44, Issue 6, Pages 817-824Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s10295-017-1906-3
Keywords
Glutamate decarboxylase; gamma-Aminobutyric acid; Enterococcus raffinosus; Purification; Characterization
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Funding
- National Basic Research Program (973 Program) of China [2013CB734004]
- Natural Science Foundation of China [31370075, 31471725, 61603273]
- Youth Innovation Fund of Tianjin University of Science and Technology of China [2014CXLG28]
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Glutamate decarboxylase (GAD) is the sole enzyme that synthesizes gamma-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 A degrees C, while the K (m) and V (max) values for the sole l-glutamate substrate were 5.26 and 3.45 mu mol L-1 min(-1), respectively.
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