| Project/Area Number |
06453164
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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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 | HOKKAIDO UNIVERSITY |
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Principal Investigator |
CHIBA Seiya Hokkaido Univ., Fac.of Agr., Pro., 農学部, 教授 (30001449)
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1996: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1995: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1994: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Keywords | alpha-Glucosidase / alpha-galactofuranosyl residue / alpha-Secondary kinetic isotope effect / D-Glucal hydration / High mannose type sugar chain / 全一次構造 / ジフルフィド結合 / 第二次アイソトープ効果 / Aspergillus niger / ジスルフィド結合 / 化学修飾 |
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| Research Abstract |
An crystalline alpha-glucosidase from Asperillus niger (ANGase), a so-called "maltase"-type enzyme, was composed of two different subunits, p1 (227 amino acids) and P2 (719 amino acids), which were not linked via S-S bridge. Their primary structures were determined by Edman degradation and genetic methods. The genomic nucleotide sequence revealed theat the P1 and P2 polypeptides were encoded by a single reading frame in this order. Between two subunits 14 amino acids were removed, suggesting an unique post-translational modification with limited proteolysis. The Asp (224) of P2 was identified to be one of catalytic amino acids. Seven N-linked high-mannose type oligosaccharides were isolated, and their structures were determined. Four sugar chains contained one galactofuranosyl residue bound via alpha- (1*2) -linkage to Man (A). Three of them are novel types of N-linked oligosaccharides. The O-linked sugars also were isolated from ANGase. Structural analysis of two compounds indicated that mannose and alpha-mannosyl- (1*2) -mannose bound directly to Ser/Thr of ANGase.
Based on the above findings, A.niger alpha-glucosidase was compared in the primary structures with alpha-glucosidases from other origins. alpha-Glucosidases were elucidated to be classified into the two families on the primary structures. In adittion, the mode of substrate recognition and the model of reaction mechanism in the carbohydrate-hydrolases were discussed and proposed on the analysis for the hydration reaction of D-glucal and the alpha-secondary kinetic isotope effects by alpha-glucosidase and glucoamylase.
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