Purification and characterization of thermostable H2O2-forming NADH oxidase from 2-phenylethanol-assimilating Brevibacterium sp. KU1309.
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 degrees 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.
Hirano
Jun-ichiro
J
Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama, Japan.
Miyamoto
Kenji
K
Ohta
Hiromichi
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001951:Brevibacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010626:Phenylethyl Alcohol / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D002784:Cholesterol; D006861:Hydrogen Peroxide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D010626:Phenylethyl Alcohol