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1995 Fiscal Year Final Research Report Summary

Protein engineering of beta-amylase

Research Project

Project/Area Number 06660104
Research Category

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

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

Principal Investigator

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

Project Period (FY) 1994 – 1995
Keywordsbeta-amylase / protein engineering / x-ray crystal analysis
Research Abstract

For the study of protein engineering of enzymes, X-ray crystallographic analysis and the over-production of the mutated enzymes are inevitable techniques. The author has determined the SH-modified soybean beta-amylases as a model of the mutated enzyme and also developed the over-production system of soybean beta-amylase in Escherichia coli.
1. X-ray crystallography of the SH-modified soybean beta-amylase
Two sulfhydryl groups (Cys95 and Cys343) in soybean beta-amylase exist near the active site of the enzyme. The role of SH (S-S) groups of b-amylase from soybean and Bacillus cereus have been investigated by chemical modification. The three dimensional structures of the complexes of Cys95 and Cys343 blocked soybean b-amylase with maltose or glucose have been determined at around 2.0 A resolution. Two maltose molecules could bind to subsite 1-4 as is the case of the native enzyme complex except for the position of the flexible loop (residues from 96 to 103). In the modified enzyme, the loop position could not be determined owing to high temperature factors, suggesting that the modified side chain of Cys95 inhibit the loop movement from open to closed conformation. The X-ray crystal analysis of Cys343-blocked enzyme/maltose complex showed the normal maltose binding except for the main chain positions of Thr342 and Cys343. It seems that the altered Thr342 residue affect the catalysis of the enzyme by the break of hydrogen bond with catalytic water.
2. Over-production system of soybean beta-amylase
Soybean beta-amylase cDNA has been efficiently expressed in Escherichia coli.with pkk233-2 vector. The purified enzyme from the extract of Escherichia coli.showed the same enzymatic properties as the enzyme from soybean seeds except for the N-terminal blocked residue. The qurified enzyme has been crystallized in the same way as the seed enzyme. The X-ray crystallographic study of the expressed enzyme is now in progress.

  • Research Products

    (13 results)

All Other

All Publications (13 results)

  • [Publications] B. Mikami: "Two sulfhydryl groups near the active site of soybean β-amylase." Biosci Biotech. Biochem.58. 126-132 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K. Nomura: "The role of SH and S-S groups in Bacilluls cereus β-amylase." J. Biochem.118. 1124-1130 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 三上文三: "β-アミラーゼの構造と機能" 日本農芸化学会誌. 69. 315-323 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 三上文三: "β-アミラーゼの活性中心" 日本農芸化学会誌. 69. 1046-1049 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] B. Mikami, M. Degano, EJ. Hehre, and J. C. Sacchettini: "Crystal structure of soybean β-amylase reacted with β-maltose and maltal: Active site components and thier apparent roles in catalysis." Biochemistry. 33. 7779-7787 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] B. Mikami: "Structure of β-amylase. in Enzyme chemistry and molecular biology of amylase and related enzymes" Ed. by the amylase research society of Japan, CRC Press Inc, Tokyo, 9 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 三上文三: "β-アミラーゼ タンパク質化学 第4巻-III" 廣川書店(予定), 3 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] B.Mikami, K.Nomura, and Y.Morita: "Two sulfhydryl groups near the active site of soybean beta-amylase" Biosci.Biotech.Biochem. 58. 126-132 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] B.Mikami, M.Degano, E.J.Hehre, and J.C.Sacchettini: "Crystal structure of soybean beta-amylase reacted with beta-maltose and maltal : Active site components and their apparent roles in catalysis" Biochemistry. 33. 7779-7787 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Nomura, I.Yoneda, T.Nammori, R.Shinke, Y.Morita, and B.Mikami: "The role of SH and S-S groups in Bacillus cereus beta-amylase" J.Biochem. 118. 1124-1130 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] B.Mikami: "Studies on structure and functions of beta-amylase by X-ray crystallography" Nippon Nogeikagaku Kaishi. 69. 315-323 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] B.Mikami: "Active site of beta-amylase" Nippon Nogeikagaku Kaishi. 69. 1046-1049 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] B.Mikami, E.J.Hehre, J.C.Sacchettini and Y.Morita: "Structure of beta-amylase. in Enzyme chemistry and molecular biology of amylase and related enzymes" Ed.by the amylase research society of Japan, CRC Press Inc, Tokyo. (1994)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1997-03-04  

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