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Domain Structure and Function of Gluoamylase

Research Project

Project/Area Number 05660089
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 InstitutionMIE UNIVERSITY

Principal Investigator

TANAKA Akiyoshi  Mie University, Faculty of Education, Associate Professor, 教育学部, 助教授 (10155111)

Co-Investigator(Kenkyū-buntansha) OKUMURA Katsuzumi  Mie University, Faculty of Bioresources, Associate Professor, 生物資源学部, 助教授 (30177183)
Project Period (FY) 1993 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
KeywordsGlucoamylase / Domain Structure / Subsite Structure / Calorimetry / Kinetics / Isozyme / Random Attack / Starch Binding Domain / DSC / 等温滴定熱量計
Research Abstract

Calorimetric and steady-state kinetic studies were done to investigate the structure and function of fungal glucoamylases.
(1) Thermal unfolding of two forms of Aspergillus niger glucoamylase was observed by adiabatic differential scanning calorimetry (DSC) at pH 7. The DSC traces of the larger form of the enzyme, G1, and the shorter form which lacks the C-terminal starch-binding domain of G1, G2, could be resolved by assuming two-state unfolding of five and four independent components, respectively. The thermal unfolding of the starch-binding domain was found to be reversible, but that of the catalytic domain was irreversible. The DSC observation of G1 and G2 in the presence of beta-cyclodextrin or 1-deoxynojirimycin showed that there was no domain interaction between the starch-binding domain and catalytic domain.
(2) Steady-state inhibitory kinetic studies of Aspergillus glucoamylase showed that gluconolactone and 1-deoxynojirimycin were non-competitive type and competitive type inihi … More bitor, respectively. These results indicate that gluconolactone binds to a site other than the active site of the enzyme forming nonproductive ternary complex of the enzyme, substrate, and gluconolactone, but 1-deoxynojirimycin binds to the active site (most probably subsite 2) of the enzyme without forming the ternary complex.
(3) The subsite structure of Rhizopus glucoamylase was identified for the hydrolytic reaction of isomaltooligosaccharides at 25゚C and at pH4.5. The arrangement of the subsite affinities A_i's (i is the subsite number) was evaluated as follows : A_1=-6.0, A_2=13.8, A_3=4.6, A_4=1.5, A_5=0.6, A_6=-0.5, and A_7=0.2 (in kj mol^<-1>) unit). The intrinsic rate constant for the hydrolytic reaction in the productive binding mode of substrates, k_<int>, was 10.2 s^<-1>. Larger values of K_m for isomaltooligosaccharides compared with those for maltooligosaccharides were attributed to smaller values A_1 and A_2 compared with those for maltooligosaccharides, and smaller values of k_0 were attributed t smaller values of A_1 and k_<int>.
(4) Hydrolytic reactions of maltooligosaccharides (degree of polymerization n=3-7) and isomaltooligosaccharides (n=3-7) by Rhizopus glucoamylase were observed by HPLC at pH 4.5,25゚C.It was confirmed that initial rates of the decrease of n-mer substrate and the increase of the products, glucose and (n-1)-mer oligosaccharide, are the same for all the substrates, indicating that the reaction mode of the enzyme is "random attack, " and no "multiple attack" or "single chain attack" is operating. Less

Report

(4 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • 1993 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Akiyoshi Tanaka: "Thermal Unfolding of Staphylococcal Nuclease and Several Mutant Forms Thereof Studied by Differential Scanning Calorimetry" Protein Science. 2. 567-576 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka: "Subsite Structure of Rhizopus Glucoamylase for the Hydrolytic Reaction of Isomaltooligosaccharides" Biosci.Biotech.Biochem.58. 1809-1813 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 田中晶善: "熱測定で見るタンパク質の高次構造" 化学と生物. 31. 439-446 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka: "Differential Scanning Calorimetric Studies on the Domain Structure of Aspergillus Glucoamylase" J.Biochem.117. 1024-1028 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka: "Random Attack as Hydrolytic Mode of Oligosaccharides by Rhizopus Glucoamylase" Biosci.Biotech.Biochem.59. 1372-1373 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 田中晶善: "タンパク質工学と熱測定" 熱測定. 22. 106-109 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka, John Flanagan, Julian M.Sturtevant: ""Thermal Unfolding of Staphylococcal Nuclease and Several Mutant Forms Thereof Studied by Differential Scanning Calorimetry"" Protein Science. 2. 567-576 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka: ""High-order Structure of Proteins Revealed by Calorimetry"" Kagaku to Seibutsu (Chemistry and Biology). 31. 439-446 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka, Shuichi Takeda: ""Subsite Structure of Rhizopus Glucoamylase for the Hydrolytic Reaction of Isomaltooligosaccharides"" Biosci.Biotech.Biochem.58. 1809-1813 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Masayuki Fukumura, Akiyoshi Tanaka, Kazuo Sakka, Kunio Oomiya: ""Process of Thermal Denaturation of Xylanase (XynB) from Clostridium stercorarium F-9"" Biosci.Biotech.Biochem.59. 47-50 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka, Harumi Fukada, Katsutada Takahashi: ""Differential Scanning Calorimetric Studies on the Domain Structure of Aspergillus Glucoamylase"" J.Biochem.117. 1024-1028 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka Shuichi Takeda: ""Random Attack as Hydrolytic Mode of Oligosaccharides by Rhizopus Glucoamylase"" Biosci.Biotech.Biochem.59. 1372-1373 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka: ""Protein Engineering and Calorimetry"" Netsu Sokutei (Calorimetry and Thermal Analysis). 22. 106-109 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka and Shuichi Takeda: ""Development of a Micro Scale Method for Determining Glucose Concentration and Its Application to Hydrolytic Reaction of Isomaltooligosaccharides"" Bulletin of the Faculty of Education Mie University (Natural Science). 46. 21-23 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Akiyoshi Tanaka: "Random Attack as Hydrolytic Mode of Oligosaccharides by Rhizopus Glucoamylase" Biosci. Biotech. Biochem. 59. 1372-1373 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 田中晶善: "タンパク質工学と熱測定" 熱測定. 22. 106-109 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Akiyosi TANAKA: "Subsite Structure of Rhizopus Glucoamylase for the Hydrolytic Reaction of Isomaltooligosaccharides" Biosci.Biotech.Biochem. 58. 1809-1813 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 田中晶善: "グルコース定量法の微量化と、イソマルトオリゴ糖水解反応の測定" 三重大学教育学部研究紀要. 46. 79-81 (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Akiyoshi TANAKA: "Differential Scanning Calorimetric Studies on the Domain Structure of Aspergillus Glucoamylase" J.Biochem.(印刷中). (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] 田中晶善: "熱測定で見るタンパク質の高次構造" 化学と生物. 7. 439-446 (1993)

    • Related Report
      1993 Annual Research Report

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Published: 1993-04-01   Modified: 2016-04-21  

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