Agaricus bisporus secretes abundant laccase activity into the medium during mycelial growth. SDS-PAGE analysis of extracellular laccase protein, purified from compost extract, showed a predominant band of 65 kDa molecular mass, together with lesser amounts of smaller polypeptides. The main polypeptide was purified electrophoretically. Amino acid sequence analysis of the N-terminal region of the main polypeptide was used to specify the sequence of a 15-residue chemically synthesized peptide (N-terminal peptide). Rabbit antibodies were raised against pure laccase, electrophoretically purified main polypeptide and the synthetic N-terminal peptide. Electrophoretically purified main polypeptide antibody was further purified by affinity chromatography on laccase-CNBr-Sepharose. Western blot analysis showed that the antigenic behaviour of laccase in compost extract, culture filtrate from malt-extract culture, and the purified enzyme from both sources, differed. The patterns of bands revealed are most simply explained by generation of (proteolytically) partially cleaved enzyme molecules in the culture medium, possibly combined with differences in extent of glycosylation. [35S]Methionine incorporation and immunoprecipitation were used to follow laccase synthesis in cultures grown on malt extract. After short-term labelling, a single polypeptide of 68 kDa apparent molecular mass was immunoprecipitated from both mycelial extracts and the culture medium. When poly(A)-containing RNA from malt-extract-grown mycelium was translated in vitro in rabbit reticulocyte lysate, a single polypeptide of about 57 kDa molecular mass was immunoprecipitated, consistent with the previously measured carbohydrate content of 15% for the pure enzyme. After treatment with N-glycanase, the polypeptide showed an increase in mobility during SDS-PAGE consistent with a reduction in molecular mass of about 5 kDa, indicating about equal amounts of N- and O-linked carbohydrate. C-terminal labelling of pure laccase was attempted by transpeptidation with carboxypeptidase Y. Although some minor bands were labelled, the main polypeptide was not, indicating that the C-terminus of the enzyme may be blocked.
AnderP., ErikssonK. -E.1976; The importance of phenol oxidase activity in lignin degradation by the white rot fungus Sporotrichum pulverulentum.. Archives of Microbiology 109:1–8
AndrawisA., PeaseE. A., TienM.1990; Extracellular peroxidases of Phanerochaete chrysosporium:DNA cloning and expression.In Biotechnology in Pulp and Paper Manufacture.. Applications and Fundamental Investigations pp. 601–613KirkT. K., ChangH.-M. Edited by Boston: Butterworth-Heinemann;
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry 72:248–254
EsakaM., HattoriT., FujisawaK., SakajoS., AsahiT.1990; Molecular cloning and nucleotide sequence of full length cDNA for ascorbate oxidase from cultured pumpkin cells. European Journal of Biochemistry 191:537–541
GermanU. A., MullerG., HunzikerP. E., LerchK.1988; Characterisation of two allelic forms of Neurospora crassa laccase. Amino- and carboxyl-terminal processing of a precursor. Journal of Biological Chemistry 263:885–896
LeonardJ. C., DunhamS. M., ThurstonC. F.1981; Isolation of polysomes and poly(A)-containing RNA from Chlorella fusca var. vacuolata.. New Phytologist 87:39–51
SaloheimoM., Niku-PaavolaM.-L., KnowlesJ. K. C.1991; Isolation and structural analysis of the laccase gene from the lignindegrading fungus Phlebia radiata. Journal of General Microbiology 137:1537–1544
SmithJ. F., ClaydonN., LoveM. E., AllanM., WoodD. A.1989; Effect of substrate depth on extracellular endocellulase and laccase production of Agaricus bisporus. Mycological Research 93:292–296
TakahashiN., OrtelT. L., PutnamF. W.1984; Single chain structure of human ceruloplasmin: the complete amino acid sequence of the whole molecule. Proceedings of the National Academy of Sciences of the United States of America 81:390–394
TakahashiN., HottoT., IsiharoH., MoriM., TejimoS., BliguyR., AkazawaT., EudoS., ArataY.1986; Xylose-containing common structural unit in N-linked oligosaccharides of laccase from sycamore cells. Biochemistry 25:388–395
WesselD., FluggeU. I.1984; A method for quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry 138:141–143
WoodD. A., GoodenoughP. W.1977; Fruiting of Agaricus bisporus. Changes in extracellular enzyme activities during growth and fruiting. Archives of Microbiology 114:161–165