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Genetic analysis of inuin-degrading enzymes and their application to oligosaccharide production

Research Project

Project/Area Number 10660312
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 生物資源科学
Research InstitutionMiyazaki University

Principal Investigator

OHTA Kazuyoshi  Miyazaki University, Department of Biological Resource Sciences, Associate Professor, 農学部, 助教授 (70112315)

Co-Investigator(Kenkyū-buntansha) NAKAMURA Toyohiko  Miyazaki University, Department of Biological Resource Sciences, Professor, 農学部, 教授 (90040857)
Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
KeywordsAspergillus niger / Penicillium sp. / Inulinase / Inulin / Inulooligosaccharide / Gene cloning / Immobilized enzyme / Aspergillus niger / penicillium sp.
Research Abstract

1. Aspergillus niger 12 contained two copies of endoinulinase genes (inuA and inuB) in the genome. Only the inuB gene was transcribed constitutively. Four distinct 5'-ends of the transcripts were observed at positions -80 (A), -72 (G), -69 (A), and -65 (A) from the start codon. The inuB mRNAs were polyadenylated at various sites between 94 and 297 bp downstream of the stop codon. We have determined the nucleotide sequences of the 1201- and 1017-bp 5'-noncoding regions of the inuA and inuB genes, respectively. The inuB promoter region included a putative TATA box at -116 (TATATA).
2. An A. niger endoinulinase has been immobilized covalently onto bromoacetyl-cellulose (BAC), cellulose-carbonate (CC) and CNBr-activated Sepharose 4B (CAS). All three immobilized enzymes increased the resistance to inactivations by p-chloromercuribenzoate or FeィイD13+ィエD1. The marked rise from 45 to 60℃ in the optimal temperature for inulin hydrolysis was observed in the CAS-immobilized enzyme. The pH stability decreased on the acidic side and increased slightly to alkaline side in the BAC-immobilized endoinulinase, and shifted to acidic side in the CC-immobilized enzyme. The KィイD2mィエD2 value decreased in the BAC-immobilized endoinulinase and increased in the CAS-immobilized enzyme.
3. An endoinulinase gene from Penicillium sp. Strain TN-88 was cloned and sequenced. The open reading frame (ORF) of 1,545 bp was not interrupted by introns, and it encoded 25 amino acid signal peptide and 490 amino acid mature protein. The mature enzyme contained three Cys residues and ten potential N-linked glycosylation sites. The deduced amino acid sequence showed 72 and 85% identities to those of A. niger and Penicillium purpurogenum endoinulinases, respectively. A neighbor-joining tree showed that fungal endoinulinases form a distinct group from other β-fructofuranosidases. It is postulated that the fungal endoinulinase genes are of bacterial origin.

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (9 results)

All Other

All Publications (9 results)

  • [Publications] Kazuyoshi Ohta: "Molecular cloning and sequence analysis of two endoinulinase genes from Aspergillus niger"Bioscience, Biotechnology, and Biochemistry. 62(9). 1731-1738 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] hidetoshi Akimoto: "Transcriptional analysis of two endoinulinase genes inuA and inuB in Aspergillus niger and nucleotide sequences of their promoter regions"Journal of Bioscience and Biengineering. 88(6). 599-604 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Norihiro Zaita: "Preparation and enzymatic properties of Aspergillus niger endoinulinase immobilized onto various polysaccharide supports"Food Science and Technology Research. 6(1). 34-39 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Kazuyoshi Ohta et al.: "Molecular cloning and sequence analysis of two endoinulinase genes from Aspergillus niger"Bioscience, Biotechnology, and Biochemistry. 62(9). 1731-1738 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hidetoshi Akimoto et al.: "Transcriptional analysis of two endoinulinase genes inuA and inuB in Aspergillus niger and nucleotide sequences of their promoter regions"Journal of Bioscience and Bioengineering. 8(6). 599-604 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Norihiro Zaita el al.: "Preparation and enzymatic properties of Aspergillus niger endoinulinase immobilized onto various polysaccharide supports"Food Science and Technology Research. 6(1). 34-39 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hidetoshi Akimoto: "Transriptional analysis of two endoinulinase genes inuA and inuB in Aspergillus niger and nucletide sequnces of their promoter regions"Journal of Biscience and Bioengineering. 88(6). 599-604 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Norihiri Zaita: "Preparation and enzymatic properties of Aspergillus niger endinulinase immobillzed onto various polysaccharide supports"Food Science and Tecnology Rsearch. 6(1). 1-6 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kazuyoshi OHTA: "Molecular Cloning and Sequence Analysis of Two Endoinulinase Genes from Aspergillus niger" Bioscience, Biotechnology, and Biochemistry. 62(9). 1731-1738 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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