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

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: POXA1 b. POXAlb secreted in the culture broth exhibits interesting properties with regard to pH stability. In fact the enzyme shows an increase of tl/2 from pH 3.0 to 10.0 and, surprisingly, displays a tl/2 value at pH 10.0 of about 100 days. Furthermore POXAlb is partly secreted, in fact analysis of proteins from cellular extract showed the presence of a larger amount of POXAlb in this extract than in the culture broth. The enzyme purified from cellular extract (POXAlb-I) shows some differences respect to the secreted enzyme (higher molecular mass, slightly different catalytic constants and lower pH stability). Extra-cellular POXAlb, named POXAlb-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 POXA1 b-P and POXA1 b-I respect to POXA1 b. A marked difference can be observed with regard to pH stability, as a fact POXAlb is the most stable form at alkaline pHs.