Mechanism of peroxidase inactivation in liquid cultures of the ligninolytic fungus Pleurotus pulmonarius

It has recently been reported that Pleurotus pulmonarius secretes a versatile peroxidase that oxidizes Mn(2+), as well as different phenolic and nonphenolic aromatic compounds; this enzyme has also been detected in other Pleurotus species and in Bjerkandera species. During culture production of the enzyme, the activity of the main peak was as high as 1,000 U/liter (measured on the basis of the Mn(3+)-tartrate formation) but this peak was very ephemeral due to enzyme instability (up to 80% of the activity was lost within 15 h). In culture filtrates inactivation was even faster; all peroxidase activity was lost within a few hours. Using different inhibitor compounds, we found that proteases were not responsible for the decrease in peroxidase activity. Peroxidase instability coincided with an increase in the H(2)O(2) concentration, which reached 200 micromolar when filtrates were incubated for several hours. It also coincided with the onset of biosynthesis of anisylic compounds and a decrease in the pH of the culture. Anisyl alcohol is the natural substrate of the enzyme aryl-alcohol oxidase, the main source of extracellular H(2)O(2) in Pleurotus cultures, and addition of anisyl alcohol to filtrates containing stable peroxidase activity resulted in rapid inactivation. A decrease in the culture pH could also dramatically affect the stability of the P. pulmonarius peroxidase, as shown by using pH values ranging from 6 to 3.25, which resulted in an increase in the level of inactivation by 10 micromolar H(2)O(2) from 5 to 80% after 1 h. Moreover, stabilization of the enzyme was observed after addition of catalase, Mn(2+), or some phenols or after dialysis of the culture filtrate. We concluded that extracellular H(2)O(2) produced by the fungus during oxidation of aromatic metabolites is responsible for inactivation of the peroxidase and that the enzyme can protect itself in the presence of different reducing substrates.