Studies on the role of proteases in the white-rot fungus Trametes versicolor: effect of PMSF and chloroquine on ligninolytic enzymes activity.

In the present study we investigated the possibility of proteinases, intracellular and extracellular, being involved in the regulation of ligninolytic activities in cultures of Trametes versicolor during the shift from primary growth (i.e. trophophase) to idiophase triggered by nitrogen or carbon starvation. These studies were performed using specific inhibitors added to the cultures of T. versicolor. Addition of PMSF (irreversible inhibitor of serine proteinases) or chloroquine (the lysosomotropic agent inhibiting intralysosomal degradation of proteins) revealed distinct differences in the activity of ligninolytic enzymes between nutrient-deprived and non-starved cultures. The addition of PMSF during the transfer of mycelia to the nutrient limited media significantly enhanced the activities of laccase (2-7-fold) and of unspecified peroxidases (2-4-fold). The activity of lignin peroxidase decreased with PMSF, both in tropho- and in idiophasic cultures. The enhanced activities of laccase and general peroxidases (horseradish peroxidase-like, HRP-like) were accompanied by markedly altered patterns of both intracellular and extracellular proteolytic activities revealed by electrophoretic analysis with polyacrylamide gels containing the copolymerized substrate (haemoglobin or gelatin, respectively). The experiments with chloroquine added to nutrient-deprived cultures showed that inhibition of vacuolar proteolysis resulted in lowered activities of laccase and peroxidase. Electrophoretic analysis revealed altered patterns of intracellular proteinases upon chloroquine addition to nutrient-starved cultures. Moreover, chloroquine was found to enhance the activity of proteases secreted in carbon-starved cultures. From the results it is concluded that both intracellular (including vacuolar) and extracellular proteases are involved in the regulation of laccase and peroxidase activity in cultures of T. versicolor under nutrient limitation.