tailieunhanh - Báo cáo khóa học: Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea

We have isolated a laccase (lac1) from culture fluid ofVol-variella volvacea, grown in a defined medium containing 150lMCuSO4 , by ion-exchange and gel filtration chroma-tography. Lac1 has a molecular mass of 58 kDa as deter-mined by SDS/PAGE and an isoelectric point of . Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. . | Eur. J. Biochem. 271 318-328 2004 FEBS 2003 doi Biochemical and molecular characterization of a laccase from the edible straw mushroom Volvariella volvacea Shicheng Chen Wei Ge and John A. Buswell Department of Biology and the Centre for International Services to Mushroom Biotechnology The Chinese University of Hong Kong Shatin New Territories Hong Kong SAR China We have isolated a laccase lac1 from culture fluid of Vol-variella volvacea grown in a defined medium containing 150 M CuSO4 by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS PAGE and an isoelectric point of . Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lacl contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lacl is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcrip tion in fungal mycelia grown on rice-straw revealed that apart from during the early stages of substrate colonization lacl was expressed at every stage of the mushroom developmental cycle defined in this study although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lacl increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages primordium formation pinhead stage of fruit body morphogenesis. Gene expression then declined to w 20-30 of peak levels throughout the subsequent stages of sporophore development. Keywords Volvariella volvacea laccase edible mushroom gene expression. .