Effect of Environmental Conditions on Extracellular Protease Activity in Lignolytic Cultures of Phanerochaete chrysosporium

Abstract

Two different types of extracellular protease activity were identified in the culture fluid of Phanerochaete chrysosporium wild-type BKM-F grown in submerged batch culture on N-limited media. The first activity, which appears to be inherent to the active growth phase, displayed a maximum on day 2 and decreased to a very low level on day 4. The second activity, which appeared at day 8 following the peak of ligninase activity, seems to be characteristic of late secondary metabolism and is stimulated by carbon starvation. Cultures started with half the amount of glucose of other cultures showed a remarkably earlier development of secondary activity. In contrast, the fed-batch addition of glucose started when ligninase activity was at a maximum (day 6) completely repressed secondary protease activity and enhanced ligninase production. The addition of exogenous veratryl alcohol increased the level of secondary protease activity, whereas the oxygen supply pattern significantly affected both the time course and the level of overall proteolytic activity. The addition of phenylmethylsulfonyl fluoride to growing cultures (0, 1, or 6 days) diminished overall protease activity, while it significantly enhanced ligninase activity. In all cases, the time courses of protease and ligninase activities were negatively correlated, indicating that protease activity promotes the decline of ligninase activity in batch culture.

Purification and characterization of a novel protease from culture filtrates of a Streptomyces sp.

“…They can also activate zymogenic enzymes by limited proteolysis and possibly release from bacterial cell walls enzymes that attack wood polymers [16]. Recent observations by Dosoretz et al [17] suggest the involvement of proteases in the decline of lignine peroxidase activity during secondary metabolism.…”

Comparison of two continuous fungal bioreactors for posttreatment of anaerobically pretreated weak black liquor from kraft pulp mills

“…Protease activity was measured with Azocasein (Sigma) as the substrate (Dosoretz et al, 1990). Samples were incubated with 30 mg of substrate in 50 mM sodium acetate (pH 4.5), with periodic shaking in a tube at 378C for 30 min.…”

Continuous production of manganese peroxidase byPhanerochaete chrysosporium immobilized on polyurethane foam in a pulsed packed-bed bioreactor

“…One unit of activity represents 1 mol of veratryl alcohol oxidized to veratryladehyde per minute. Protease activity was measured with azocoll as substrate (Dosoretz et al, 1990b) (Sigma Chemical Co). One unit was defined as the amount of enzyme that releases azodye, causing an absorbance change of 0.001 min −1 measured at 520 nm.…”

“…The main objective to be achieved in a continuous operation is the maintenance of a sustained secretion of MnP in steady-state conditions. A second important objective is to avoid extracellular protease secretion that is mainly responsible for destabilization of the produced enzymes (Dosoretz et al, 1990b). As reported for LIP, a residual glucose concentration minimizes extracellular proteases synthesis, thus avoiding enzyme inactivation (Dosoretz et al, 1990b;Dosoretz et al, 1990c).…”

“…A second important objective is to avoid extracellular protease secretion that is mainly responsible for destabilization of the produced enzymes (Dosoretz et al, 1990b). As reported for LIP, a residual glucose concentration minimizes extracellular proteases synthesis, thus avoiding enzyme inactivation (Dosoretz et al, 1990b;Dosoretz et al, 1990c). Another key factor related to the proper operation in a bioreactor is to avoid the excessive growth of filamentous fungi that would imply operational difficulties such as fouling of the fermenter probes and bed clogging.…”