Isolation and characterization of novel pI 4.8 MnP isoenzyme from white-rot fungus Irpex lacteus

Growth of Irpex lacteus on polyurethane solid support led to secretion of various manganese-dependent peroxidase (MnP) isoenzymes with pI in the range of 3.8–6.7. The highest MnP activity was obtained at 8.9 mmol l−1 Mn2+ when predominantly a pI 4.8 isoenzyme was produced. Optimization of anion exchange chromatography (Mono Q) allowed the separation of MnP isoenzymes and collection of extensive sequence information by tandem LC–MS. The pI 4.8 isoenzyme was purified 109-fold (6% activity yield) by ion exchange chromatography, its specific activity of Mn2+ oxidation was 2800 nkat mg−1, molecular mass 37 kDa and pH optimum 5. The other properties were: respective half-lives (pH 4.5) of 575, 325 and 7 min at 40, 50 and 60 °C, Km values 46.7; 9.5; 21.4 μmol l−1 and kcat values 69.0; 64.2; 15.7 s−1 for Mn2+, H2O2 and DMP with Mn2+, respectively. The pI 4.8 isoenzyme was shown to be a true MnP affecting secondary substrates via the oxidation of Mn2+. Its biochemical properties and production by I. lacteus make the enzyme a candidate for biotechnological applications.