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Multi-cellulase components produced by Sporotrichum cellulophilum

Research Project

Project/Area Number 63550725
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 発酵工学
Research InstitutionOsaka University

Principal Investigator

KINOSHITA Shinichi  Osaka Univ., ICBiotech., Assoc. Prof., 工学部, 助教授 (50029253)

Co-Investigator(Kenkyū-buntansha) 関 達治  大阪大学, 工学部, 助教授 (50029245)
Project Period (FY) 1988 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1989: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1988: ¥1,600,000 (Direct Cost: ¥1,600,000)
KeywordsSporotrichum cellulophilum / Cellulase / Monoclonal antibody / Cloning of cellulase gene / Multi-cellulase components / Relatedness of cellulases / DNA sequence of cellulase gene / Amino acid sequence of cellulase / セルラーゼ / セルラーゼの多様性 / モノクローナル抗体 / セルラーゼ間の類縁性 / セルラーゼの精製 / セルラーゼの修飾
Research Abstract

Cellulases CII1, CII2, CIII1, CIII2, CIII3, CIV were purified to homogeneity from the crude extract of S. cellulophilum, and sugar moieties of CIII2 and CIV were removed. Monoclonal antibodies against all the cellulases were tried to prepare, but only 4 clones were obtained. The reactivities of monoclonal antibodies against all the cellulases were analyzed by ELISA method. Cellulase CIV was most close to CIII2, more close to CIII1, close to CII1 and CIII3, and hardly related to CII2.
The amino terminus of cellulase CIV was blocked and CIV was hydrolyzed with lysyl endopeptidase. Two fractions were purified by HPLC on 5C_4.300 and amino acid sequences of 22 and 31 residues were determined. From those sequences, 8 DNA probes composing of 14 to 24 bases were synthesized. The RNA of S. cellulophilum was extracted, the cDNA library was prepared. By using probe T (20 mer), positive clones were screened from the library, those were recloned, and hybridized with probe (23 mer), but none of them was not.
In the above method the cDNA was not prepared with good yield. Then we extracted DNA from the cell, and after hydrolyzing it partially with SamIIIA1, the DNA library was prepared. It was screened with probe B.(23 mer), and 4.9 kbp fragment was obtained. After recloning to 400 bp AluI-AluII fragment, its DNA sequence was determined, but any part of the sequence did not correspond with the amino acid sequence. Next by using probe D (29 mer), 500 bp Smal-kpnI and 1050 EcoT22I-Xba1 fragments were isolated, those DNA sequences were determined, regions corresponding to amino acid sequence of 7 and 6 residues were found but the DNA sequences corresponding to other connecting amino acid sequence were not found near the regions. So cellulase gene is not cloned yet.

Report

(3 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • Research Products

    (3 results)

All Other

All Publications (3 results)

  • [Publications] 木下晋一: "モノクロ-ナル抗体によるSportrichum cellulophilumの数種のセルラ-ゼ成分の類縁性の検討" 日本発酵工学会講演要旨. 221-221 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Shinichi Kinoshita: "Analysis of relationship between cellulases produced by Sporotrichum cellulophilum by monoclonal antibodies" Abstr. Soc. Ferment. Technol.221 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 木下晋一: "モノクロ-ナル抗体によるSporotrichum cellulophilumの数種のセルラ-ゼ成分の類縁性の検討" 日本発酵工学会講演要旨. 221-221 (1989)

    • Related Report
      1989 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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