Isolation, purification, and study of some properties of the extracellular lignin peroxidases of the wood-destroying fungusPleurotus ostreatus

Abstract

Two bands with lignin peroxidase activity have been detected by isoelectric focusing in a total enzyme preparation obtained from a 15-day filtrate of the culture liquid of the fungusPleurotus ostreatus by fractionation with ammonium sulfate. Two homogeneous forms of the enzymes — LGP-I and LGP-II — have been obtained by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAF-Toyopearl 650 M gel, and rechromatography on Sephadex G-75, and also by electrophoresis in PAAG. The specific activities of the purified lignin peroxidases LGP-I and LGP-H amounted to 36.5 and 54.3 units/mg, their degrees of purification being 8.7 and 12.9, respectively. The molecular masses of LGP-I and LGP-II, determined by electrophoresis in PAAG in the presence of Na-DS and by gel filtration on TSK HW–65 gel were 42–44 and 61–63 kDa. The isoelectric points of LGP-I and LGP-II were 3.4 and 4.1, their pH optima 2.7 and 3.4, and the, temperatures of their optimum enzymatic action 28 and 34°C, respectively. The isoenzymes differed from one another substantially with respect to pH stability and resistance to heat. The values of KM determined from the rates of hydrolysis of the substrate by the enzymes in the presence of H2O2 at pH 3.7 were 0.09 mM for LGP-I and 0.07 mM for LGP-II. The values of KM with respect to veratryl alcohol, determined by the Lineweaver—Burk method, were 0.117 mM for LGP-II and 0.132 mM for LGP-II.