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A new laccase isoenzyme (POXA1b, where POX is phenol oxidase), produced by Pleurotus ostreatus in cultures supplemented with copper sulphate, has been purified and fully characterized. The main characteristics of this protein (molecular mass in native and denaturing conditions, pI and catalytic properties) are almost identical to the previously studied laccase POXA1w. However, POXA1b contains four copper atoms per molecule instead of one copper, two zinc and one iron atom per molecule of POXA1w. Furthermore, POXA1b shows an unusually high stability at alkaline pH. The gene and cDNA coding for POXA1b have been cloned and sequenced. The gene coding sequence contains 1599 bp, interrupted by 15 introns. Comparison of the structure of the poxa1b gene with the two previously studied P. ostreatus laccase genes (pox1 and poxc) suggests that these genes belong to two different subfamilies. The amino acid sequence of POXA1b deduced from the cDNA sequence has been almost completely verified by means of matrix-assisted laser desorption ionization MS. It has been demonstrated that three out of six putative glycosylation sites are post-translationally modified and the structure of the bound glycosidic moieties has been determined, whereas two other putative glycosylation sites are unmodified.

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K m value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca 2؉ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.White rot basidiomycetes have received extensive attention because of their lignin-degrading activity. The biochemistry of lignin degradation is a complex process involving a series of enzymatic and nonenzymatic reactions. Extracellular enzymes which catalyze oxidative reactions are lignin peroxidases, laccases, manganese peroxidases, and hydrogen peroxide-producing enzymes (3, 13). Even though their catalyzed reactions have been studied in detail, their in vivo coordination and, possibly, synergistic action are not clearly understood.Pleurotus ostreatus is a white rot basidiomycete which belongs to the subclass of ligninolytic microorganisms that produce laccases, manganese peroxidases, and veratryl alcohol oxidases but no lignin peroxidase. Among these enzymes, laccases have been the most widely studied and characterized (11,12,17). In a recent study (16), it has been demonstrated that a laccase isoenzyme (POXA1b; phenol oxidase A1b) is specifically degraded in the early phase of fungal growth by proteases present in P. ostreatus culture broth; hence, the disappearance of POXA1b seems to be correlated with the appearance of extracellular protease activity. A similar relationship was observed for lignin peroxidases in Phanerochaete chrysosporium, and, in this case, the extracellular proteases caused an almost complete disappearance of lignin peroxidase activity due to degradation of all lignin peroxidase isoenzymes (8). Furthermore, a recent report (21) suggests that both intracellular and extracellular proteases are involved in the regulation of ligninolytic activities in cultures of Trametes versicolor under nutrient limitation. In contrast, it has been reported (2) that proteases are not responsible for the decrease in peroxidase activity in Pleurotus pulmonarius ...

A new laccase isoenzyme (POXA1b, where POX is phenol oxidase), produced by Pleurotus ostreatus in cultures supplemented with copper sulphate, has been purified and fully characterized. The main characteristics of this protein (molecular mass in native and denaturing conditions, pI and catalytic properties) are almost identical to the previously studied laccase POXA1w. However, POXA1b contains four copper atoms per molecule instead of one copper, two zinc and one iron atom per molecule of POXA1w. Furthermore, POXA1b shows an unusually high stability at alkaline pH. The gene and cDNA coding for POXA1b have been cloned and sequenced. The gene coding sequence contains 1599 bp, interrupted by 15 introns. Comparison of the structure of the poxa1b gene with the two previously studied P. ostreatus laccase genes (pox1 and poxc) suggests that these genes belong to two different subfamilies. The amino acid sequence of POXA1b deduced from the cDNA sequence has been almost completely verified by means of matrix-assisted laser desorption ionization MS. It has been demonstrated that three out of six putative glycosylation sites are post-translationally modified and the structure of the bound glycosidic moieties has been determined, whereas two other putative glycosylation sites are unmodified.