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Protein engineering of beta-amylase

Research Project

Project/Area Number 08660109
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

MIKAMI Bunzo  KYOTO UNIVERSITY,Research Institute for Food Science Associate Professor, 食糧科学研究所, 助教授 (40135611)

Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1997: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥1,600,000 (Direct Cost: ¥1,600,000)
Keywordsbeta-amylase / protein engineering / X-ray crystal analysis / thermostability
Research Abstract

In order to elucidate the relationship between structure and function of beta-amylases and to perform the structure based protein engineering of beta-amylase, the three-dimensional structures of the enzymes from soybean, barley and Bacillus cereus has been determined by X-ray crystallography. beta-Amylases from soybean and B.cereus has been produced in E.coli for mutation experiments.
1. X-ray crystal analysis of Bacillus cereus beta-amylase. Bacillus cereus beta-amylase expressed in E.coli.has been purified and crystallized. The structure has been determined by X-ray crystallography at 2.1* resolution by multiple isomorphous replacement method. The enzyme has a core and a C-terminal starch binding domain instead of the C-terminal loop found in plant beta-amylases. The structure of the enzyme complex with maltose clealy showed that maltose binds to three different sites, the active site in core, C-terminal starch binding domain and a maltose binding site in core. These results explain t … More he ability of raw starch digestion found in only bacterial beta-amylase.
2. X-ray crystal analysis of a mutant barley beta-amylase. The structure of a seven-fold mutant barley beta-amylase with increased thermostability has been determined by X-ray crystallography at 2.5* resolution. The comparison of the structure with that of soybean enzyme elucidated the individual mechanisms of the increased thermostabilty at four mutated amino acid residues.
3. Mutation and X-ray crystal analysis of soybean beta-amylase. Mutants of soybean beta-amylase of which two catalytic residues(E 178 and E 380)were mutated to glutamine were produced and crystallized. The complex of E 380 Q with maltose clearly showed that maltose bind to subsites 1-2 and 4-5 in contrast to subsites 1-2 and 3-4 in the case of the wild type enzyme. This strongly suggested the activation mechanism of a catalytic water by E 380 in the catalytic step. The complex of the wild type enzyme with beta-cyclodextrin showed the importance of subsite 4 for the multiple attack mechanism of the enzyme. Less

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] 三上 文三、新田 康則: "タンパク質化学4ヒドロラーゼ 3巻" 廣川書店, 3 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 三上 文三: "丸善食品総合辞典" 丸善株式会社出版事業部, 1 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] B.Mikami: "Protein engineering of beta-amylase(in Japanese)" Bul.Res.Inst.Food.Sci.61. 62-64 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] B.Mikami: "Structure of beta-amylase(in Japanese)" Kagaku to Seibutsu. 35. 199 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] B.Mikami and Y.Nitta: beta-amylase in Tanpakushitu Kagaku(in Japanese). Hirokawa Publishing Co., (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] "タンパク質科学4ヒドロラーゼ3巻" 3 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] 三上文三: "丸善食品総合辞典" 丸善株式会社出版事業部, 1 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] 三上 文三: "β-アミラーゼの構造を見る" 化学と生物. 35巻3号. 199-199 (1997)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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