2000 Fiscal Year Final Research Report Summary
Structure and function of yeast mannoprotein degrading enzymes
| Project/Area Number |
11660069
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Tohoku University |
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Principal Investigator |
NAKAJIMA Tasuku Tohoku University, Graduate School of Agricultural Science, Division of Life Science, Professor, 大学院・農学研究科, 教授 (20091720)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Mannoprotein / α-1, 2-mannosidase / Yeast cell wall / α-mannanase gene / Bacterial mannosidase / 1,6-α-マンナナーゼ |
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| Research Abstract |
To Clarify the structure of yeast mannoprotein degrading enzymes, at first, a gene (aman2) encoding 1, 2-α-D-mannosidase from Bacillus sp.M-90 was cloned. The putative aman2 was 5928 base pairs long and encoded a mature 1, 2-α-D-mannosidase protein of 1939 amino acids and a signal peptide of 37 amino acids. The deduced amino acid sequence exhibits no similarity to other 1, 2-α-D-mannosidases identified in various organisms. The recombinant protein was verified by immunoblotting with anti-1, 2-α-D-mannosidease antibodies and showed hydrolytic activity to 1, 2-α-D-mannobise and yeast mannan. Secondly, a gene (aman6) encoding endo-1, 6-α-mannanase, a yeast mannan backbone degrading enzyme from Bacilles circulans was cloned and the gene was expressed in Escherichis coli. The putative aman6 wa 1767 base pares long and encodes a mature 1, 6-α-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1, 6-α-D-mannanase from E.coli transformant showed 61-kDa protein, and N-terminal amino acid sequense and other general properties of the recombinant enzyme were identical to those of 1, 6-α-D-mannanase from Bacillus circulans TN-31.
A data base serch indicates both aman2 and aman6 were novel genes.
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[Publications] E.Ichishima, N.Taya, M.Ikeguchi, Y.Chiba, M.Nakamura, C.Kawabata, T.Inoue, K.Takahasi, T.Minetoki, K.Ozeki, C.Kumagai, K.Gomi, T.Yoshoda and T.Nakajima: "Molecular and enzymatic properties of recombinant 1, 2-a-mannosodase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells."Biochemical Journal. 339. 589-597 (1999)
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