Isoenzymes of manganese-dependent peroxidase and laccase produced by the lignin-degrading basidiomycete Ceriporiopsis subvermispora

摘要

The white-rot basidiomycete Ceriporiopsis subvermispora produces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pl values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pl 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II). mainly the MnPs with higher pl values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermispora when growing on wood chips of Pinus radiata. Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pl values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pl values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pl values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF. Immunoblotting studies showed that the molecular mass of MnP isoenzymes from liquid cultures was about 52.5 kDa, whereas the molecular masses of MnPs extracted from wood varied from 52.5 kDa to 62.5 kDa upon ageing of the cultures. The amino terminal sequences of seven MnP isoenzymes were determined. The consensus sequences of MnPs from liquid and solid cultures were clearly distinct, although both showed homology to MnPs from related white-rot fungi.