Purification and characterization of the conidial laccase of Aspergillus nidulans.
Conidial laccase of Aspergillus nidulans was purified by standard protein purification methods. Although the purified material showed a cluster of several protein bands on a nondenaturing gel, each of these protein bands had laccase activity. All bands of activity, however, were absent in a strain carrying a mutation in the structural gene for laccase. Concentrated solutions (greater than 1 mg/ml) were bright blue, suggesting that, like other laccases, this enzyme contains copper. The enzyme contained asparagine-linked carbohydrate (12% by weight) which could be removed by digestion with endo-beta-N-acetylglucosaminidase H. The molecular weight of native enzyme as determined by gel filtration was 110,000, but the largest component in a sodium dodecyl sulfate gel was 80,000. Several smaller components (55,000 and 36,000 molecular weight) were also visible. We present evidence which suggests that the smaller components are in vivo cleavage products tightly associated with enzymatically active molecules. Comparison of the laccase from a white-spore (wA) and a green-spore (wA+) strain showed, surprisingly, that the enzymes differed in electrophoretic pattern, in vitro heat stability, and in vivo metabolic stability. The difference was manifested for enzymes isolated from cultures after conidial pigmentation of the wA+ strain had occurred. If examined earlier, before pigmentation, the enzymes were indistinguishable. Since wA strains lack the precursor of the wild-type green pigment, i.e., the laccase substrate, we suggest that the transformation of the enzyme of the wA strain is due to its failure to interact with its normal substrate.
D001233:Aspergillus nidulans / Q000201:enzymology*; D004586:Electrophoresis; D006358:Hot Temperature; D042845:Laccase; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D010088:Oxidoreductases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D013172:Spores, Fungal / Q000201:enzymology
D046911:Macromolecular Substances; D010088:Oxidoreductases; D042845:Laccase