| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
A lignin-degrading basidiomycete, Pleurotus ostreatus produces manganese-dependent peroxidase (MnP) which is considered to be one of ligninolytic enzymes. I have investigated on this enzyme enzymologically and genetically, and obtained the following results.
1.P.ostreatus produced much higher amount of MnP in the medium containg Mn^<2+> than in the medium without added Mn^<2+>, indicating that MnP is induced by Mn^<2+>.
2.MnP has been purified to homogeneity and characterized.
3.A cDNA and genomic DNA encoding MnP were cloned and characterized. The sequnce analyzes of MnP and its cDNA revealed that a mature form of MnP consists of 332 amino acid residues preceeded by a signal peptide of 29 amino acid residues. Consistent with the peroxidase mechanism of MnP,the proximal histidine, the distal histidine, and the distal arginine are all conserved and regions flanking these residues display homology with other peroxidases. A single potential N-glycosylation site with the general sequence Asn-X-Ser/Thr is present in the deduced MnP sequence. Comparison of the sequences of cDNA and genomic DNA revealed that the protein encoding sequence of the MnP gene is interrupted by 15 introns which conform to the universal G-T/A-G splicing rule observed for the 3' and 5' intron boudaries. The putative eukaryotic regulatory sequences, "TATA" and "CAAT", are present in the 5' non-coding region of the MnP gene.
4.To express the MnP-encoding cDNA in the fungus, Aspergillus oryzae a MnP expression plasmid (pMAR5-MnP) was constructed, and it was introduced into the A.oryzae strain. The MnP cDNA was expressed in A.oryzae under the alpha-amylase-encoding amybeta promoter. The MnP mRNA was produced in A.oryzae transformants. No active MnP protein, however, could be detected.
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