| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Research Abstract |
For the construction of an overexpression system of the intracellular 1,2-α-mannosidase (EC3.2.1.113) gene (msdS) from Aspergillus saitoi(now designated A. phoenicis), the N-terminal sequence of the gene was replaced with that of the aspergillopepsin I (EC3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1,2-α-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 andT-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1(11.2 kbp) was constructed. The fused f-msdS gene has heen overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The recombinant enzyme has activity with methyl-2O-α-D-mannopyranosyl α-D-mannopyranoside at pH 5.0. The substate specificity of the enzyme was analyzed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man_<9-6>(GlcNAc)_2-PA. The enzyme hydr
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olysed Man_8GlcNAc_2-PA (type'M8A') fastest, and 'M6C'slowest, among the PA-sugar chains. Molecular mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superrose 12 respectively. The pl value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. Contents of the secondary structure (α-helix, β-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7 % respectively. The melting temperature, T_m of the enzyme was 71 ℃ by differential scanning calorimetry. Determination of 1 g-atom of Ca^<2+>/mol of enzyme was performed by atomic-absorption spectrophotometer.
Active site determination of the enzyme expressed in Aspergillus oryzae cells was performed by site-directed mutagenesis. Substitutions of Asp-269 to Asn and of the Glu-residues, Glu-273, Glu-411, Glu-414, Glu-474 and Glu-504, to Gln altered the drastic decrease of specific activities with Manα1-2Man-Ome and Man_9-GlcNAc_2-PA as substrates. From the present results, Asp-269, Glu-273, Glu-414 and Glu-474 are probably in the Ca^<2+>-binding ligands, whereas Glu-124 and Glu411 and are probably the catalytic residues for the 1,2-α-mannosidase. Less
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