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Purification and characterization of thermostable H2O2-forming NADH oxidase from 2-phenylethanol-assimilating Brevibacterium sp. KU1309

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Abstract

A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70°C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or l-phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.

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Acknowledgments

This work was partially supported by the Ministry of Education, Culture, Sports, Science and Technology, Grant-in-Aid for the 21st century COE Program entitled “Understanding and Control of Life’s Function via Systems Biology”, and Global COE program entitled “Center of Human Metabolomic Systems Biology” Keio University. The analysis of N-terminal amino acid sequence of NOX was assisted by Prof. Midori MATHUMOTO (Keio University), Associate Prof. Hiroyuki KAWAHARA (Hokkaido University), Mr. Tomohiro HIROSE (Hokkaido University), and Mr. Akira MIYAO (Hokkaido University), to whom we would like to express our thanks.

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  1. Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama, 223-8522, Japan

    Jun-ichiro Hirano, Kenji Miyamoto & Hiromichi Ohta

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  1. Jun-ichiro Hirano
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  2. Kenji Miyamoto
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  3. Hiromichi Ohta
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Correspondence to Kenji Miyamoto.

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Hirano, Ji., Miyamoto, K. & Ohta, H. Purification and characterization of thermostable H2O2-forming NADH oxidase from 2-phenylethanol-assimilating Brevibacterium sp. KU1309. Appl Microbiol Biotechnol 80, 71–78 (2008). https://doi.org/10.1007/s00253-008-1535-x

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  • DOI: https://doi.org/10.1007/s00253-008-1535-x

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