(515av) Hydrogen Peroxide-Producing Nadh Oxidase (Nox-1) from Bacillus Cereus

NADH oxidases are useful biocatalysts for regenerating nicotinamide cofactors of many biological redox reactions. The sequence comparison-based approach was applied to develop a novel hydrogen peroxide-forming NADH oxidase (nox-1) from Bacillus cereus (B.cereus), which reduces oxygen to hydrogen peroxide. H₂O₂-forming NADH oxidase (nox-1) is also part of the alkyl hydroperoxide reductase system. The nox-1 gene (AhpF) was isolated from genomic B.cereus DNA by PCR and cloned into the expression vector. The His-tagged protein was overexpressed and purified by immobilized metal affinity chromatography. We studied the kinetics of the enzyme and found that nox-1 is turnover-limited as expected for enzymes with labile, redox-active thiols in the active site. The kinetics data on nox-1 from B. cereus will be compared with other known hydrogen peroxide-forming NADH oxidases, such as nox-1 from Lactococcus lactis.