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

It has recently been reported that Pleurotus pulmonarius secretes a versati le peroxidase that oxidizes Mn2+, as well as different phenolic and nonphen olic aromatic compounds; this enzyme has also been detected in other Pleuro tus species and in Bjerkandera species. During culture production of the en zyme, the activity of the main peak was as high as 1,000 U/liter (measured on the basis of the Mn3+-tartrate formation) but this peak was very ephemer al due to enzyme instability (up to 80% of the activity was lost within 15 h), In culture filtrates inactivation was even faster; all peroxidase activ ity was lost within a few hours. Using different inhibitor compounds, we fo und that proteases were not responsible for the decrease in peroxidase acti vity. Peroxidase instability coincided with an increase in the H2O2 concent ration, which reached 200 mu M when filtrates were incubated for several ho urs. 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 sub strate of the enzyme aryl-alcohol oxidase, the main source of extracellular H2O2 in Pleurotus cultures, and addition of anisyl alcohol to filtrates co ntaining stable peroxidase activity resulted in rapid inactivation, A decre ase 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 3.2 5, which resulted in an increase in the level of inactivation by 10 mu M H2 O2 from 5 to 80% after 1 h, Moreover, stabilization of the enzyme was obser ved after addition of catalase, Mn2+, or some phenols or after dialysis of the culture filtrate. We concluded that extracellular H2O2 produced by the fungus during oxidation of aromatic metabolites is responsible for inactiva tion of the peroxidase and that the enzyme can protect itself in the presen ce of different reducing substrates.