Gene cloning and biochemical characterization of a catalase from Gluconobacter oxydans(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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Gluconobacter oxydans has a large number of membrane-bound dehydrogenases linked to the respiratory chain that catalyze incomplete oxidation of a wide range of organic compounds by oxidative fermentation. Because the respiratory chain is a primary site of reactive oxygen species (ROS) production, the bacterium is expected to have a high capacity to detoxify nascent ROS. In the present study, a gene that encodes a catalase of G. oxydans, which might act as a potential scavenger of H_2O_2, was cloned, and the expression product (termed rGoxCat) was characterized biochemically. rGoxCat is a heme b-containing tetrameric protein (molecular mass, 320kDa) consisting of identical subunits. The recombinant enzyme displayed a strong catalase activity with a k_<cat> of 6.28×10^4s^<-1> and a K_m for H_2O_2 of 61mM; however, rGoxCat exhibited no peroxidase activity. These results, along with the phylogenetic position of the enzyme, provide conclusive evidence that rGoxCat is a monofunctional, large-subunit catalase. The enzyme was most stable in the pH range of 4-9, and greater than 60% of the original activity was retained after treatment at pH 3.0 and 40℃ for 1h. Moreover, the enzyme exhibited excellent thermostability for a catalase from a mesophilic organism, retaining full activity after incubation for 30min at 70℃. The observed catalytic properties of rGoxCat, as well as its stability in a slightly acidic environment, are consistent with its role in the elimination of nascent H_2O_2 in a bacterium that produces a large amount of organic acid via oxidative fermentation.
著者
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Nakayama Toru
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Hosoya Miho
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Nakayama Toru
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Takahashi Seiji
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Takahashi Seiji
Department Of Applied Chemistry Faculty Of Engineering Kyushu Institute Of Technology
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Yamaguchi Haruhiko
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Sugiyama Keigo
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Yamaguchi Haruhiko
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Sugiyama Keigo
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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NAKAYAMA Toru
Department of Applied Microbial Technology, The Kumamoto Institute of Technology:(Present office)Institute for Fundamental Research, Suntory Ltd.
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