A physiological, biochemical and molecular biological study of laccases in the edible straw mushroom, Volvariella volvacea.

摘要

Volvariella volvacea, produced multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as carbon source, and on 'composts' representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis was associated with the onset of secondary growth, and was positively regulated by copper (up to 200 muM CuSO4) and by various aromatic compounds. In solid-state systems, only low levels of laccase were detectable during the vegetative growth phase but enzyme activity increased sharply at the onset of fruiting and during sporophore development.;A major laccase protein (Lac1) secreted by V. volvacea was purified using ammonium sulphate precipitation combined with ion-absorption and gel filtration chromatography. The purified laccase had a molecular weight of 58 kDa and an isoelectric point of 3.7. Lac1 oxidized ABTS, syringaldazine, 2,6-dimethoxyphenol (2,6-DMP), guaiacol and catechol, but not ferulic acid, tyrosine and beta-(3,4-dihydroxyphenyl)alanine (DOPA). The pH optimum for Lac1 activity was dependent upon the test substrate and bell-shaped pH activity profiles with optima of 3, 4.6 and 5.6 were obtained for ABTS, 2,6-dimethoxyphenol and syringaldazine, respectively. Lac1 was most active at 45°C and stable up to 40°C. The Km values determined for ABTS, 2,6-DMP and syringaldazine were 0.03 mM, 0.057 mM and 0.01 mM, respectively. Lac1 activity was inhibited 100% by the putative laccase inhibitors dithiothreitol, L-cysteine, sodium azide and thioglycollic acid. Concentrated Lac1 solutions lacked the typical blue colour and the spectral maxima near 600nm that characterize all the blue oxidases. The N-terminal amino acid sequence revealed a very low homology with other fungal laccases.;Degenerate primers designed on the basis of consensus sequences of enzyme copper-binding regions and on the N-terminal amino acid sequence of Lac1 were used to generate cDNA and genomic fragments encoding laccase genes. A total of six different laccase genomic DNAs and four cDNA fragments were amplified. Eight different laccase homologues were obtained by analysis of these deduced amino acid sequences after removal of introns in genomic DNA sequences.;Lac1 and Lac4 were chosen for transcriptional analysis because of their high mRNA abundances. RT-PCR analysis revealed that the expression of lac1 but not lac4 was regulated at the transcription level by copper and various aromatic compounds. (Abstract shortened by UMI.)