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X-ray diffraction study and reaction mechanism of bacterial alginate lyase

Research Project

Project/Area Number 07660111
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

MURATA Kousaku  Research Institute for Food Science Department of Food Design and Utilization : Professor, 食糧科学研究所, 教授 (90142299)

Co-Investigator(Kenkyū-buntansha) MIKAMI Bunzo  Research Institute for Food Science Department of Food Design and Utilization :, 食糧科学研究所, 助教授 (40135611)
Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥1,600,000 (Direct Cost: ¥1,600,000)
KeywordsBacterial infection / Pseudomonas aeruginosa / Alginate / X-ray diffraction / Alginate lyase
Research Abstract

Alginate is a highly viscous heteropolysaccharide composed of manuronate and gluronate. Alginate formation by Pseudomonas aeruginosa is a leading cause of bacterial infectious disease, which is also called biofilm infection. The effective therapeutic methods for the disease have not been developed. For the treatment of bacterial infectious disease, the removal of diofilm is expected as a useful and effective procedures. The present study aimed to apply bacterial enzyme : alginate lyase that depolymerizes alginate for the removal of biofilm and to analyze three-dimensional structure. Following results were obtained. (1) Preliminary X-ray diffraction study was performed and crystal constants were determined. However, the enzyme crystal did not accept heavy metal ions, although the reason for this resistance is unclear. (2) The reaction mechanism of the lyase was analyzed and reaction constants were obtained with an estimation of the active site structure. (3) The alginate lyase was chemically modified with polyyethylenglycol. The modified enzyme showed sufficient lyase activity with extremely low lebel of antigenic activity. The results, together with others, strongly suggested that the enzyme will be applied to the treatment of bacterial infectious patients. (4) To further decrease antigenic activity, the alginate lyase was fragmented and low molecular size enzyme was generated. (5) The bacterium producing alginate lyase had a pit on the cell surface. This is the first finding in the history of microbiology. (6) The fine structural analysis of the pit structure by electron microscopy and pit-deficient mutant indicated that the pit is responsible for the uptake of macromolecules with energy and information. Our paper summarized these results was awarded from Japan Society of Fermentation and Bioengineering in 1996.

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] 村田,幸作: "細菌の多糖リアーゼ:酵素学的、遺伝学的側面と医薬への応用" 日本応用酵素協会誌. (印刷中) (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tomohiro Hisano: "Pit structure on bacterial cell surface" Biochemical and Biophysical Research Communication. 220. 979-982 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Wataru Hashimoto: "Purification and characterization of microbial gellan lyase" Applied and Environmental Microbiology. 62(4). 1475-1477 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tomohiro Hisano: "Direct uptake of alginate molecule through a pit on bacterial cell surface : a novel mechanism to take up macromolecules." Journal of Fermentation and Bioengineering. 79(6). 538-544 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 村田幸作: "アルギン酸分解の新しい分子機構" バイオサイエンスとインダストリー. 53(5). 36-38 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 村田幸作: "アルギン酸分解の新しい分子機構" バイオサイエンスとインダストリー. 53(5). 36-38 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Bunzo Mikami, Tomohiro Hisano, Tetsuo Yamashita, Tomoko Inose, Masahiro Takagi, Kenji Sakaguchi, Akira Kimura, Tadayuki Imanaka and Kousaku Murata: "Crystalization and Preliminary X-Ray Diffraction Studies of Bacterial Al-ginate Lyase" Journal of Fermentation and Bioengineering. 77. 691-692 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kousaku Murata: "Bacterial Polysaccharide lyases : Enzymatic and genetic aspects and its application" Japanese Society for Applied enzymology. No.31. 28-39 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Wataru Hashimoto: "Purification and characterization of microbial gellan lyase." Applied and Environmental Microbiology. Volume 62 (4). 1475-1477 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tomohiro Hisano: "Direct uptake of alginate molecule through a pit on bacterial cell surface : a novel mechanism to take up macromolecules." Journal of Fermentation and Bioengineering. Volume 79 (6). 538-544 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tomohiro Hisano: "Pit structure on bacterial cell surface." Biochemical and Biophysical Research Communication. Volume 220. 979-982 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kousaku Murata: "Novel mechanism for alginate depolymerization" Bioscience & Industry. Volume 53 (5). 36-38 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 田村幸作: "細菌の多糖リアーゼ:酵素学的、遺伝学的側面と医薬への応用" 日本応用酵素協会誌. (印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Tomohiro Hisano: "Pit structure on bacterial cell surface." Biochemical and Biophysical Research Communication. 220. 979-982 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Wataru Hashimoto: "Purification and characterization of microbial gellan lyase." Applied and Environmental Microbiology. 62(4). 1475-1477 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Tomohiro Hisano: "Direct uptake of alginate molecule through a pit on bacterial cell surface : a novel mechanism to take up macromolecules." Journal of Farmentation and Bioengineering. 79(6). 538-544 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] 村田幸作: "アルギン酸分解の新しい分子機構。" バイオサイエンスとインダストリー. 53(5). 36-38 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] 村田幸作: "細菌多糖リアーゼのバイオフィルム感染症治療への応用" 平成8年度厚生省特定疾患調査研究班. (印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] 村田幸作: "アルギン酸分解の新しい分子機構" バイオサイエンスとインダストリー. 53. 36-38 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Mikami Bunzo: "Crystalization and Preliminary X-Ray Diffraction Studies of Bacterial Alginate Lyase" Journal of Fermentation and Bioengineering. 77. 691-692 (1994)

    • Related Report
      1995 Annual Research Report
  • [Publications] 村田幸作: "細菌によるアルギン酸分解の分子機構とバイオフイルム" Bulletin of the Research Institute for Food Science, Kyoto University. 58. 32-33 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Tomohiro Hisano: "Pit Structure on Bacterial Cell Surface" Biochemical and Biophysical Research Communications. (in press). (1996)

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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