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 8.9mmoll^-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 2800nkatmg^-1, molecular mass 37kDa and pH optimum 5. The other properties were: respective half-lives (pH 4.5) of 575, 325 and 7min at 40, 50 and 60°C, K_m values 46.7; 9.5; 21.4μmoll^-1 and k_cat values 69.0; 64.2; 15.7s^-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.