Overproduction of lignin peroxidase by Phanerochaete chrysosporium (BKM-F-1767) under nonlimiting nutrient conditions

The ligninolytic enzymes synthesized by Phanerochaete chrysosporium BKM-F-1767 immobilized on polyurethane foam were characterized under limiting, sufficient, and excess nutrient conditions. The fungus was grown in a nonimmersed liquid culture system under conditions close to those occurring in nature, with nitrogen concentrations ranging from 2.4 to 60 mM. This nonimmersed liquid culture system consisted of fungal mycelium immobilized on porous pieces of polyurethane foam saturated with liquid medium and highly exposed to gaseous oxygen. Lignin peroxidase (LIP) activity decreased to almost undetectable levels as the initial NH4+ levels were increased over the range from 2.4 to 14 mM and then increased with additional increases in initial NH4+ concentration. At 45 mM NH4+, LIP was overproduced, reaching levels of 800 U/liter. In addition, almost simultaneous secretion of LIP and secretion of manganese-dependent lignin peroxidase were observed on the third day of incubation. Manganese-dependent lignin peroxidase activity was maximal under nitrogen limitation conditions (2.4 mM NH4+) and then decreased to 40 to 50% of the maximal level in the presence of sufficient or excess initial NH4+ concentrations. Overproduction of LIP in the presence of a sufficient nitrogen level (24 mM NH4+) and excess nitrogen levels (45 to 60 mM NH4+) seemed to occur as a response to carbon starvation after rapid glucose depletion. The NH4+ in the extracellular fluid reappeared as soon as glucose was depleted, and an almost complete loss of CO2 was observed, suggesting that an alternative energy source was generated by self-proteolysis of cell proteins. The peak level of CO2 concentration in the cultures increased with increasing NH4+ concentrations, reaching an almost asymptotic value at 24 mM NH4+ and paralleling glucose consumption. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anion-exchange high-performance liquid chromatography analyses of heme protein composition in the extracellular fluid revealed that LIP synthesis in the presence of sufficient and even excess nitrogen concentrations was characterized not only by increasing activity but also by isoenzyme distribution. The R2 heme protein was the predominant LIP isoenzyme (around 70% of the total heme content) under nonlimiting conditions, in contrast to the substantial levels of all of to ligninolytic isoenzymes under N-limiting conditions.