PROPERTIES OF LACCASE ISOENZYMES PRODUCED BY THE BASIDIOMYCETE CERIPORIOPSIS-SUBVERMISPORA

Laccase is one of the ligninolytic enzymes found in liquid cultures of the fungus Ceriporiopsis subvermispora in defined medium. As an appro ach to a clarification of the role of laccases during the attack on li gnin by the fungus, the enzyme has been characterized further. The lev els of this phenol oxidase increase 2-fold in the presence of p-anisid ine and are severely affected when addition of either Mn(II) or Cu(II) ions to the medium is omitted. Isoelectrofocusing allowed the resolut ion of two laccase isoenzymes, with pIs of 3.65 and 3.59. In rich medi um, laccase activity is 10-fold higher than in salt medium, and it is not affected by the external addition of p-anisidine or Ran(II). Four isoenzymes were detected in these cultures, with pIs between 3.76 and 3.60. In a wheat bran medium, four isoenzymes with pIs in the range 3. 63-3.46, plus a fifth isoenzyme of high pI (4.82), were also identifie d. The absorption spectrum of a pool containing the four isoenzymes fr om rich medium shows a maximum at 600 nn, typical of laccase possessin g a type I copper atom. The molecular mass of the isoenzyme with pI 3. 60 is 79 kDa, as determined by SDS/PAGE. Upon treatment with endoglyco sidase F, the molecular mass of this isoform decreases to 63 kDa, indi cating a high degree of glycosylation. Substrate specificity studies c onducted with the four isoenzymes from rich medium and a combination o f isoenzymes from salt medium showed marked differences among them. Th e amino-terminal sequences (24 residues) of three isoenzymes isolated from rich medium were determined. Two of them are identical, whereas t he third one differs from these in three amino acid residues. The cons ensus sequence reveals clear homology with laccases from other microor ganisms.