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J. Biochem, 1998, Vol. 123, No. 6 1156-1163
© 1998 Japanese Biochemical Society

research-article

Properties of Glutamate Racemase from Bacillus subtilis IFO 3336 Producing Poly-{gamma}-Glutamate1

Makoto Ashiuchi*, Kazuhiko Tani{dagger}, Kenji Soda{ddagger} and Haruo Misono*,{dagger},2

*Research Institute of Molecular Genetics, Kochi University Nankoku, Kochi 783-8502
{dagger}Department of Bioresources Science, Kochi University Nankoku, Kochi 783-8502
{ddagger}Faculty of Engineering, Kansai University Suita, Osaka 564-0073

2To whom correspondence should be addressed. E-mail: hmisono{at}cc.kochi-u.ac.jp

We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-{gamma}-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E, coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-{gamma}-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-{gamma}-glutamate production in B. subtilis IFO 3336.

1This work was partly supported by a Takano Agriculture Research Grant from The Takano Life Science Research Foundation.


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