Purification and properties of nitroalkane oxidase from Fusarium oxysporum

Abstract

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium, was purified to homogeneity from an extract of F. oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(NO2)CH2CH3 + O2 + H2O .fwdarw. OHCCH2CH3 + HNO2 + H2O2. In addition to 1-nitropropane, 3-nitro-2-pentanol, 2-nitropropane and nitrocyclohexane are good substrates; the enzyme is designated nitroalkane oxidase (EC class 1.7.3). The enzyme has a MW of approximately 185,000 and consists of 4 subunits identical in MW (47,000). FAD was required for the enzyme activity and could be replaced in part by riboflavine 5''-phosphate. Maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate and N-ethylmaleimide. The Km''s are: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM and FAD, 1.33 .mu.M.