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Molecular Analyses of Starch-debranching Enzymes involved in Amylopectin Biosynthesis

Research Project

Project/Area Number 15580115
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Food science
Research InstitutionFukuyama University

Principal Investigator

IWAMOTO Hiroyuki  Fukuyama Univ., Dept.Life Sci.and Biotech., Assoc.Professor, 生命工学部, 助教授 (90213321)

Co-Investigator(Kenkyū-buntansha) HIROSE Junzo  Fukuyama Univ., Dept.Life Sci.and Biotech., Professor, 生命工学部, 教授 (70080215)
Project Period (FY) 2003 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000)
KeywordsAmylopectin / Biosynthesis / Starch debranching enzymes / Enzyme kinetics / Structural Biology / X-ray crystallography / Rice / Potato / プルラナーゼ / イソアミラーゼ / X線結晶解析 / 大腸菌での発現系構築 / 変異酵素 / 枝切り酵素
Research Abstract

Starch debranching enzymes hydrolyze α-1,6-glucosidic linkage of amylopectin, glycogen, pullulan and other glucan containing branching points. These enzymes are usually classified to two categories, isoamylase and pullulanase. In plants, isoamylase is coded in sugary gene and considered to be involved in amylopectin biosynthesis, whereas the role of pullulanase is not known. In this study, we purified and characterized two plant pullulanases, rice and potato, and have solved the crystal structures of Klebsiella pullulanase.
1.Rice endosperm pullulanase : Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L.cv.Fujihikari). The optimum pH of the enzyme was 5.5 to 7.5 and the optimum temperature was around 50C. Kinetic parameters, such as Km and kcat, for pullulan were very close to those of Klebsiella pullulanase, but the inhibition experiments using various maltooligosaccharides indicates some differences in the … More active sites of two enzymes.
2.Potato tuber pullulanase : The enzyme was purified from potato tuber using various chromatography columns. Molecular weight of this enzyme was 100 kDa by SDS-PAGE and 320 kDa by gel filtration, which indicates that potato pullulanase has trimeric or tetrameric structure. The optimum pH of the enzyme was 5.5 to 6.5 and the optimum temperature was around 40C. Michaelis constant of this enzyme was almost the same as rice pullulanase, while the kcat was less than one tenth of rice enzyme.
3.Klebsiella pullulanase : In collaboration with Mikami et al., Kyoto University, we have refined the crystal structures of bacterial pullulanase derive from Klebsiella pneulnoniae and its complexes with various maltooligosaccharides at around 1.7-1.9 A resolution by using a synchrotron radiation source at SPring8. This enzyme is composed of 5 domains (N1,N2,N3,A, and C). The N1 and N2 domains are characteristic of Klebsiella pullulanase, while N3,A, and C domains have only weak similarities with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain classified to CBM41 in CAZy database. Less

Report

(4 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • Research Products

    (2 results)

All 2006

All Journal Article (2 results)

  • [Journal Article] Crystal Structure of Pullulanase : Evidence for Parallel Binding of 0ligosaccharides in the Active Site2006

    • Author(s)
      Mikami, B.
    • Journal Title

      Journal of Molecular Biology 359

      Pages: 690-707

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Crystal Structure of Pullulanase : Evidence for Parallel Binding of Oligosaccharidesin the Active Site2006

    • Author(s)
      Mikami, B.
    • Journal Title

      J.Mol.Biol. 359

      Pages: 690-707

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary

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Published: 2003-04-01   Modified: 2016-04-21  

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