Multi-cellulase components produced by Sporotrichum cellulophilum

Research Project

Project/Area Number	63550725
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	発酵工学
Research Institution	Osaka 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)
Keywords	Sporotrichum 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.