Protease mediated processing of a Cu-induced laccase in Pleurotus ostreatus: a natural approach to improve protein stabilit

Laccase isoenzymes from Pleurotus ostreatus, a white rot basidiomycete fungus, have been extensively studied by our research group. More recently we have reported that the addition of CuSO4 to culture broth results in a large increase of the total laccase activity and in the production of a new isoenzyme: POXA1b. POXA1b secreted in the culture broth exhibits interesting properties with regard to pH stability. In fact the enzyme shows an increase of t1/2 from pH 3.0 to 10.0 and, surprisingly, displays a t1/2 value at pH 10.0 of about 100 days. Furthermore POXA1b is partly secreted, in fact analysis of proteins from cellular extract showed the presence of a larger amount of POXA1b in this extract than in the culture broth. The enzyme purified from cellular extract (POXA1b-I) shows some differences respect to the secreted enzyme (higher molecular mass, slightly different catalytic constants and lower pH stability). Extra-cellular POXA1b, named POXA1b-P, has been also purified from fungal culture in the presence of a serine protease inhibitor PMSF, in order to prevent protease action that may affect this isoenzyme. Comparison of properties of the three forms make evident significant similarity between POXA1b-P and POXA1b-I respect to POXA1b. A marked difference can be observed with regard to pH stability, as a fact POXA1b is the most stable form at alkaline pHs.