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Protein engineering of the cellulase catalyzing transglycosylation and condensation of lactose unit.

Research Project

Project/Area Number 16580081
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied biochemistry
Research InstitutionIchinoseki National College of Technology

Principal Investigator

TOTANI Kazuhide  Ichinoseki National College of Technology, Chemical Engineering, Associate Professor, 物質化学工学科, 助教授 (40369913)

Co-Investigator(Kenkyū-buntansha) WATANABE Takashi  Ichinoseki National College of Technology, Chemical Engineering, Associate Professor, 物質化学工学科, 助教授 (90300516)
MURATA Takeomi  Shizuoka University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (30273171)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥2,600,000 (Direct Cost: ¥2,600,000)
Keywordscellulase / lactose / condensation / transglycosylation / N-アセチルラクトサミン / エンドグルカナーゼ / Trichoderma reesei
Research Abstract

In a crude enzyme preparation of Trichoderma reesei, we found activities not only to transfer lactose (Lac) and N-acetyllactosamine (LacNAc) disaccharides to hydroxyl groups of various aglycons (R-OH) but also to catalyze the condensation reaction between those disaccharides and the aglycons. We have been suggesting the condensation enzyme are particular endo-B-1,4-glucanases, however, they have not been identified yet. In the study, we tried to purify the lactose condensation activity by serial column chromatographic methods to improve the condensation activity by protein engineering such as the site-directed mutagenesis.
The crude enzyme was purified by two kinds of anion exchange chromatography (HiTrap DEAE FF and UNO-Q1) after 70% ammonium sulfate saturated precipitation. The purified enzyme was homogeneous in SDS-PAGE and showed Lac condensing and disaccharide-releasing activities. The enzyme had Mr 54,000 and pI4.8-5.1, which are close to those of EGI. Several Lac condensing and d … More isaccharide-releasing activity were observed after chromato-focusing in Mono P column. The enzyme showed strong CMC'ase but no Avicel'ase activity, which is characteristic of endo-B-1,4-glucanase. Recombinant enzymes of EGI and EGII expressed in different host cells, gifted from Dr.Okada in Nagaoka University of Technology, were also tested for various activities. The rEGI expressed in A. oryzae showed both activity condensing Lac and releasing Lac and LacNAc while rEGII expressed in S. pombe showed none of them. In result, we concluded one of the disaccharides-condensing enzymes from T resei as EGI. The cDNA of EGI from T reesei was prepared by RT-PCR for protein engineering of EGI.
Dr.Yoshida et al. in Hirosaki University also found Lac condensation activity in culture broth of Aspergillus oryzae TB1 which is a mutant strain with highly expression of Ce1B, an endo-type cellulase. The homology of amino acid sequence is approximately 50% between EGI of T reesei and Ce1B of A. oryzae, both of which are endo-B-1,4-glucanases in Glycoside Hydrolase Family 7. We also tried to compare the dissacharides-releasing, transglycosylating, and condensing activities of CelB to these of T reesei EGI and rEGI. Less

Report

(3 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • Research Products

    (8 results)

All 2007 2005 2004

All Journal Article (8 results)

  • [Journal Article] メカブ水不溶性画分を可溶化する微生物および酵素のスクリーニング2007

    • Author(s)
      戸谷一英
    • Journal Title

      三陸総合研究 第29号

      Pages: 78-80

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] 酵素による水産バイオマス資源の有効利用と新糖質マテリアルの創出2005

    • Author(s)
      戸谷一英
    • Journal Title

      三陸総合研究 第27号

      Pages: 75-78

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary
  • [Journal Article] Design and Facile Synthesis of Neoglycolipids as Lactosylceramide Mimetics and Their Transformation into Glycoliposomes.2005

    • Author(s)
      Harada Y, Murata T, Totani K, Kajimoto T, Masum S-M, Tamba Y, Yamazaki M, Usui T
    • Journal Title

      Biosci. Biotechnol. Biochem. 69

      Pages: 166-178

    • NAID

      130000030568

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Design and Facile Synthesis of Neoglycolipids as Lactosylceramide Mimetics and Their Transformation into Glycolinosomes.2005

    • Author(s)
      Harada Y, Murata T, Totani K, Kajimoto T, Masum S-M, Tamba Y, Yamazaki M, Usui T
    • Journal Title

      Biosci. Biotechnol. Biochem. 69

      Pages: 166-178

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Design and Facile Synthesis of Neoglycolipids as Lactosylceramide Mimetics and Their Transformation into Glycoliposomes.2005

    • Author(s)
      Harada, Yoichiro
    • Journal Title

      Biosci.Biotechnol.Biochem. 69・1

      Pages: 166-178

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-β-galactosidase-catalyzed hydrolytic and transglycosylation reaction2005

    • Author(s)
      Takeomi Murata
    • Journal Title

      Biochim.Biophys.Acta 1722

      Pages: 60-68

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Transglycosylation and Condensation of Disaccharides Units Using Endo-type Glycosidases.2004

    • Author(s)
      Totani K, Yasutake N, Murata T, Usui T
    • Journal Title

      Trends. Glycosi. Glycotech. 16

      Pages: 383-392

    • NAID

      10014076271

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Trransglycosylation and Condensation of Disaccharides Units Using Endo-type Glycosidases.2004

    • Author(s)
      Totani, Kazuhide
    • Journal Title

      Trends.Glycosi.Glycotech. 16・92

      Pages: 383-392

    • Related Report
      2004 Annual Research Report

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Published: 2004-04-01   Modified: 2016-04-21  

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