2001 Fiscal Year Final Research Report Summary
Phylogenetic analysis of inuin-degrading enzymes and efficient expression
| Project/Area Number |
12660297
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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 |
生物資源科学
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| Research Institution | MIYAZAKI UNIVERSITY |
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Principal Investigator |
OHTA Kazuyoshi Miyazaki University, Department of Biochemistry and Applied Biosciences, Associate Professor, 農学部, 助教授 (70112315)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Toyohiko Miyazaki University, Department of Biochemistry and Applied Biosciences, Professor, 農学部, 教授 (90040857)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Aspergillus niger / Penicillium sp. / Inulinase / Inulin / Inulooligosaccharide / Gene cloning / Immobilized enzyme / Bifidobacteria |
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| Research Abstract |
1. An endoinulinase gene of Penicillium sp. strain TN-88 was cloned and sequenced. An open reading frame of the gene was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein. The mature protein contained three Cys residues and ten potential N-linked glycosylation sites. The deduced amino acid sequence showed 72 and 85 % identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively. A neighbor-joining tree showed that fungal endoinulinases are closely related to bacterial levanases.
2. Endoinulinase was partially purified from the culture filtrate of a filamentous fungus A. niger mutant 817. The enzyme preparation was immobilized covalently onto a porous cellulose derivative, Amino-Cellulofine. A 5 % (w/v) solution (pH 5.0) of inulin was continuously hydrolyzed in a packed-bed column reactor containing the immobilized enzyme. The majority of the hydrolysis products were inulo-oligosaccharides with a degree of polymerization of 3 to 5. In vtro studies indicated that both the F_3 and F_4 was preferentially utilized in vitro by Bifidobacterium spp.
3. Intracellular exo- and endoinulinases were extracted from mycelia of A. niger strain 12 and purified by DEAE-Cellulofine A-500, Sephadex G-100, and Sephadex G-200 chromatographies. The purified exoinulinase P-II had specific activities of 6.6 U/mg toward inulin and 22 U/mg toward sucrose. The endoinulinase P-III showed 108 U/mg toward inulin, but no activity toward sucrose. M__-_rs of exoinulinase P-II and endoinulinase P-III were determined by gel filtration using Sephadex G-200 as 47 and 56 kDa, respectively. Optimal pH and temperature for enzyme activity were pH 5.0 and 55 ℃ for P-II, and pH 5.3 and 45 ℃, for P-III. Both the enzymes were activated by Mn^<2+>, and inactivated by Ag^+, Hg^<2+> or p__--chloromercuribenzoate. Inulinases P-II and P-III exhibited apparent K__-_m values of 5.8 and 0.80 mM, respectively.
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Research Products
(6 results)
