Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Purification and Characterization of Glutamate Decarboxylase from Lactobacillus brevis IFO 12005
Yoshie UENOKiyoshi HAYAKAWASaori TAKAHASHIKohei ODA
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1997 Volume 61 Issue 7 Pages 1168-1171

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Abstract

Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobacillus brevis IFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60, 000 and 120, 000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH2-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30°C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effect was as follows: ammonium sulfate > sodium sulfate > magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with L-glutamic acid as asubstrate and the Km, kcat, and kcat/Km values were 9.3 mM, 6.5s-1, and 7×102 M-1s-1, respectively.

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