• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Studies on heat tolerance and substrate-recognition mechanism of cellulase

Research Project

Project/Area Number 04454076
Research Category

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

Allocation TypeSingle-year Grants
Research Field 応用微生物学・発酵学
Research InstitutionMie University

Principal Investigator

OHMIYA Kunio  Mie Univ., Fac.Bioresources, Professor, 生物資源学部, 教授 (60023488)

Co-Investigator(Kenkyū-buntansha) TANAKA Akiyoshi  Mie Univ., Fac.Education, Assoc.Professor, 生物資源学部, 助教授 (10155111)
SAKKA Kazuo  Mie Univ., Fac.Bioresources, Assoc.Professor, 生物資源学部, 助教授 (20154031)
SHIMADA Kyo  Mie Univ., Fac.Bioresources, Professor, 生物資源学部, 教授 (20024549)
Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1992: ¥5,200,000 (Direct Cost: ¥5,200,000)
Keywordscellulase / xylanase / Clostridium thermocellum / gene / heat tolerance / Clostridium stercorarium / lignocellulose / plant fiber / clostridium thermocellum / clostridium stercorarium / クローニング
Research Abstract

The present research aimed at getting some properties of heat tolerance and substrate recognition of cellulases from Clostridium thermocellum and Clostridium stercorarium F-9 by gene analysis and protein characterization. The results obtained here were as follows ; AC.stercorarium gene consisting of 1101 bp, i.c., 367 amino acids with no cysteine was encoded a cellulase XynB, which belongs to cellulase family F.In the amino acid sequence of the protein, 8 conserved regions were found outo by homology analysis. The glutamic acid in the 6th conserved region, ITELD, was estimated to be the active center of the enzyme. Glutamic and aspartic acids in the other conserved regions were evaluated to contribute to the recognition of substrates. Purified XynB revealed cellulase activity in addition to the xylanase activity. This enzyme had maximum activity at pH6.1, 80C and recovered about 60% of initial activity at 60C, even after heating at 100C for 10 min at pH 6.1. This activity recovery was negligible at pH5.8 due to the formation of protein aggregates. Solubilization of the aggregates with 8 M urea showed a partial activity recovery, suggested that activity loss might be caused by the irreversible denaturation in addition to the reversible denaturation. Differential scanning calorimetry (DSC) analysis showed that this protein had a power to rewind the peptide for forming a native protein structure. For studying on the substrate-recognition mechanism, a substrate analog, 1-deoxynogirimicine which binds to active center of the enzyme hydrolyzing glucoside bond, was added to XynB but it was not inhibited. This indicate that the substrate recognition mechanism is different from amylase. The C.thermocellum cellulase(CelC) gene was modified by Sodium nitrate to form random mutation. The mutated gene was encoded the cellulase, in which two amino acids, 107Glu and 209Lys, were replaced to 107Gly and 209 Glu, respectively. This mutation lowered temperature and pH optima. Adding

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] T.Miyagi et al: "Expression of a cellulase gene from Ruminococcus albus in Bacillus subtilis by electroporation" Anim.Sci.Technol.65 (in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] S.Karita et al: "Cloning and sequencing of a novel endo-1,4-β-glucanase gene from Ruminococcus albus" J.Ferment.Technol.76. 433-444 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.Fujino et al: "Nucleotide sequence of cel B gene encoding endo-1,4_1-β-glueanase-2 and ORF1 forming aputative cellulase gene cluster of Clostridiumjosui." J.Ferment.Technol.76. 243-250 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] K.Sakka et al: "Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanase A" Biosci.Biotech.Biochem.52. 273-277 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] K.Sakka et al: "Nucleotide sequence of the Clostridium stercorarium xynA gene encoding a bifunctional protein with β-O-xylosidase and α-L-alabinosidase" Biosci.Biotech.Biochim.52. 268-272 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 大宮邦雄ら: "ルーメン細菌の遺伝子工学とその応用 I.セルロース分解酵素と遺伝子の特性 II遺伝子組換えによるセルロース分解ルーメン細菌の育種" 獣医畜産新報. 46. 687-695,767-773 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] K.Shimada et al.: "Recombinant Microbes for Industrial and Agricultural Application" Y.Murooka and T.Imanaka, 550 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] K.Sakka et al.: "嫌気微生物学" 上木勝司・永井史郎, 323 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Miyagi, T., K.Kaneichi, S.Karita, K.Sakka, K.Shimada and K.Ohmiya: "Expression of a cellulase gene from Ruminococcus albus in Bacillus subtilis by electroporation" Anim.Sci.Technol.(in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Karita, S., K.Morioka, T.Kajino, K.Sakka, K.Shimada and K.Ohmiya: "Cloning and sequencing of a novel endo-1,4-beta-glucanase gene from Ruminococcus albus." J.Ferment.Technol.76(6). 433-444 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Fujino, T., S.Karita and K.Ohmiya: "Nucleotide sequence of celB gene encoding endo-1,4-beta-glucanase-2 and ORF1 forming a putative cellulase gene cluster of Clostridium josui." J.Ferment.Bioeng.76(4). 243-250 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Sakka. K., K.Yoshikawa, Y.Kojima, S.Karita, K.Ohmiya and K.Shimada: "Nucleotide sequence of the Clostridium stercorarium xylA gene encoding a bifunctional protein with b-D-xylosidase and a-L-arabinofuranosidase activities, and properties of the translated product." Biosci.Biotech.Biochem.57(2). 268-272 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Sakka, K., Y,Kojima, T.Kondo, S.Karita, K.Ohmiya and K.Shimada: "Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanase A : Identification of catalytic and cellulose binding domains." Biosci.Biotech.Biochem.57(2). 273-277 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Ohmiya, K., K.karita, K.Sakka and K.Shimada: "Genetic engineering of rumen bacteria and their use. I.Properties ofcellulolytic enzymes and their genes." J.Vet.Med.46(8). 687-695 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Ohmiya, K., K.Karita, K.Sakka and K.Shimada: "Genetic engineering of rumen bacteria and their use II.Genetic breeding of cellulolytic rumen bacteria." J.BVet.Med.46(9). 767-773 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Shimada, K., S.Karita, K.Sakka and K.Ohmiya, (ed)Y.Murooka and T.Imanaka: Recombinant microbes for industrial and agricultural application. Marcel Dekker, Inc., 395-429 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] K.Sakka, S.Karita, K.Ohmiya and K.Shimada, (ed) K.Ueki and S.Nagai: Anaerobic Microbes.Yokendo Ltd., 239-263 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.Miyagi: "Expression of a cellecease gene from Ruminococcus alfus in Bacillus subtilis by electroporation" Anim.Sci.Technol. 65(in press). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] S.Karita: "Cloning and sequencing of a novel endo-1,4-β-glucanase gene from Ruminococcus alfus" J.Ferment.Technol.76. 493-444 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] T.Fujino: "Nucleotide sequence of cel B gene encoding endo-1,4-β-glucanase-2 and ORF1 forming a putative cellulase gene cluster of clostridium josui" J.Ferment.Technol.76. 243-250 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Sakka: "Nucleotide sequence of the clostridium stercorarium xynA gene encoding xylanase A" Biosci Biotech.Biochem. 52. 273-277 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Sakka: "Nucleotide sequence of the clostridium stercorarium xylA gene encoding a bifunctional protein with β-D-xylasidase and α-L-albinosidase" Biosci.Biotech.Biochem.52. 268-272 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Shimada: "Reco★binant Microbes for Industrial and Agricultural Applications" Y.Murooka and T.Imanaka, 550 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 大宮 邦雄: "植物繊維分解ルーメン細菌における遺伝子工学" 日本畜産学会報. 63. 520-536 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Kazuo Sakka: "Nucleotide sequence of the clostridium stercorarium xyn A gene encoding xylanase A:Identifieation of catalytic and cellulose binding domains." Biosci. Biotech.Biochem.57. 273-277 (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Kazuo Sakka: "Nucleoticle sequence of the clostridium stercorarium xyl A gene encoding a bifunctional proteims with B-O-Xylosidase and α-L-arabinofuranse activities,and propertics of the translated product" Biosci. Biotech.Biochem.57. 268-272 (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Masayuki Fukumura ed.by Kuwahara and shimada: "Biotechnology in pulp and paper industry" Uni Publishers, 545 (1992)

    • Related Report
      1992 Annual Research Report

URL: 

Published: 1992-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi