Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium

Abstract

Phanerochaete chrysosporium BKM-1767 secretes multiple lignin peroxidase isoenzymes when grown under nitrogen-limited conditions. Here we report the purification of these heme-containing peroxidases, and their physical and catalytic characterization. Ten hemeproteins, designated H1–H10, were separated by anion exchange HPLC. Six of them, H1, H2, H6, H7, H8, and H10, were lignin peroxidases, oxidizing veratryl alcohol in the presence of H 2O 2. The other four (three peaks were resolved) exhibited manganese-dependent peroxidase activity, oxidizing vanillylacetone in the presence of H 2O 2 and Mn +2. The lignin peroxidases have different isoelectric points, between p14.7 and 3.3, and molecular weights between 38 and 43 kDa, determined by SDS-PAGE. All are N- and probably O-glycosylated. Three organic substrates and H 2O 2 were used to compare their kinetic properties: the organic substrates were veratryl alcohol, 1,4-dimethoxybenzene, and the lignin model compound 1-(3,4-dimethoxyphenyl)-2-( o-methoxyphenoxy)-propane-1,3-diol. K M and TN values for each of these substrates varied significantly; e.g. for veratryl alcohol K M values were from 86 to 480 μ m and TN values were from 1.3 to 8.3 s -1. The ranking of the isoenzyme activities differed with the different substrates, suggesting differences in affinities or in active site accessibilities. The K M for H 2 O 2 varied between 13 and 77 μ m. Immunological blot analysis and partial proteolytic digestion patterns showed that the isoenzymes have a high degree of homology. The isoenzyme concentrations in extracellular culture fluid were found to vary relatively and absolutely with culture time. A nomenclature scheme for these 10 hemeproteins has been proposed. This scheme should simplify identification of these proteins in the literature as well as be adaptable to others found in Phanerochaete chrysosporium.