Purification and characterization of thermostable H₂O ₂-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°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.