Extraction and determination of enzymes produced by Ceriporiopsis subvermispora during biopulping of Pinus taeda wood chips

Ceriporiopsis subvermispora is a white-rot basidiomycete that degrades lignin selectively and often is used in biopulping. Development of appropriate experimental procedures for recovery and determination of enzymes produced by C. subvermispora during biopulping is necessary to evaluate the role of each enzyme in this process. In this work, enzymes produced during biopulping were extracted and determined by using a series of substrates. Manganese-dependent peroxidases (MnP) predominated over laccases, which were of minor significance. The lack of laccases was further confirmed by assaying several substrates such as syrindaldazine, o-dianisidine and ABTS. A basal oxidation of syringaldazine and phenol red in the absence of exogenous Mn²⁺ led to the assumption that the extracts contained some Mn-independent peroxidase or enough Mn²⁺ to initiate MnP catalyzed reactions. The presence of Mn²⁺ was evidenced since phenol red was not oxidized by a 16-h-dialysed extract. Addition of 111 μM of Mn²⁺ to the reaction medium restored the oxidative activity. Lignin peroxidase activity was not detected in the extracts based on the Azure B assay. Cellulase activities were very low while xylanases predominated. The xylanolytic complex was deficient in beta-xylosidase. Successive extractions were performed for a quantitative recovery of the enzymes adsorbed on wood chips. The first 4-h extraction recovered an average of 50% of the total enzymatic activities.