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, intrac ellular and extracellular, being involved in the regulation of ligninolytic activities in cultures of Trametes versicolor during the shift from primar y growth (i.e, trophophase) to idiophase triggered by nitrogen or carbon st arvation. These studies were performed using specific inhibitors added to t he cultures of T. versicolor. Addition of PMSF (irreversible inhibitor of s erine proteinases) or chloroquine (the lysosomotropic agent inhibiting intr alysosomal degradation of proteins) revealed distinct differences: in:the a ctivity of ligninolytic enzymes between nutrient-deprived and non-starved c ultures. The addition of PMSF during the transfer of mycelia to the nutrien t limited media significantly enhanced the activities of laccase (2-7-fold) and of unspecified peroxidases (2-4-fold). The activity of lignin peroxida se decreased with PMSF, both in tropho- and in idiophasic cultures. The enh anced activities of laccase and general per oxidases (horseradish peroxidas e-like, HRP-like) were accompanied by markedly altered patterns of both int racellular and extracellular proteolytic activities revealed by electrophor etic analysis with polyacrylamide gels containing the copolymerized substra te (haemoglobin or gelatin, respectively). The experiments with chloroquine added to nutrient-deprived cultures showed that inhibition of vacuolar pro teolysis resulted in lowered activities of laccase and peroxidase. Electrop horetic analysis revealed altered patterns of intracellular proteinases upo n chloroquine addition to nutrient-starved cultures. Moreover, chloroquine was found to enhance the activity of proteases secreted in carbon-starved c ultures. From the results it is concluded that both intracellular (includin g vacuolar) and extracellular proteases are involved in the regulation of l accase and peroxidase activity-in cultures of T. versicolor under nutrient limitation.