Hydrogen peroxide-producing NADH oxidase (nox-1) from Lactococcus lactis

We have successfully applied the sequence comparison-based approach to develop a novel hydrogen peroxide-forming NADH oxidase (nox-1) from Lactococcus lactis (L. lactis) that reduces oxygen to hydrogen peroxide. The nox-1 gene (AhpF) was isolated from genomic L. lactis DNA by PCR and cloned into the expression vector pET32. The His-tagged protein was overexpressed at 20 C after induction with 167lM IPTG, and purified by Co-IMAC. After purification, nox-1 was found to be an apo-protein, so we reconstituted the holo-flavoenzyme with FAD cofactor, finding a 1:1 stoichiometry of FAD and nox-1 subunit and a KM value of 54lM. The maximum specific activity of 15U/mg protein compares favorably to other nox-1 enzymes in the literature. While both products, NAD and H2O2, inhibit nox-1, the enzyme seems rather robust in presence of moderate H2O2 concentrations. Titration of H2O2 formed with Amplex Red demonstrates that only half of the electrons from NADH go to H2O2. 2004 Elsevier Ltd. All rights reserved.