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J. Biochem, 2003, Vol. 134, No. 6 805-812
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Recombinant Expression, Biochemical Characterization and Stabilization through Proteolysis of an L-Glutamate Oxidase from Streptomyces sp. X-119-6

Jiro Arima1, Takashi Tamura1, Hitoshi Kusakabe2, Makoto Ashiuchi3, Toshiharu Yagi3, Hidehiko Tanaka1 and Kenji Inagaki*,1

1 Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530; 2 Research Laboratories, Yamasa Shoyu Co., Ltd., Choshi, Chiba 288-0056; and 3 Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502

L-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure {alpha}2ß2{gamma}2. The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of {alpha}2ß2{gamma}2 and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119–-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.

* To whom correspondence should be addressed. Tel: +81-86-251-8299, Fax: +81-86-251-8299, E-mail: kinagaki{at}cc.okayama-u.ac.jp


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