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1995 Fiscal Year Final Research Report Summary

Improvement of milk-clotting enzyme, Mucor rennin, by protein engineering and 「genetic engineering.

Research Project

Project/Area Number 06556014
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 応用微生物学・応用生物化学
Research InstitutionThe University of Tokyo

Principal Investigator

HORINOUCHI Sueharu  University of Tokyo, Biotechnology Faculty of Agricalture Deptment of , Professor, 農学部, 教授 (80143410)

Co-Investigator(Kenkyū-buntansha) NISHIYAMA Makoto  University of Tokyo, Biotechnology Center, Assoc.Prof., 生物生産工学研究センター, 助教授 (00208240)
Project Period (FY) 1994 – 1995
KeywordsMilk-clotting enzyme / Mucor rennin / Protein engineering / Host-vector system / Mucor pusillus
Research Abstract

(1) Mutagenesis of a fungus Rhizomucor pusillus, a producer of an aspartic proteinase named Rhizomucor pepsin (MPP), was performed to obtain the mutated enzymes with decreased thermostability, which is desirable for practical use of the enzyme as a milk coagulant for cheese manufacturing. Two different mutant strains, Ala94Thr and Gly169Asp which produced the mutant enzyme with distinctly reduced thermostability was isolated.
(2) We obtained the mutant enzyme, Tyr75Asn/Trp190Phe, with decreased thermostability and increased substrate specificity by sitedirected mutagenesis of the MPP subsite. We also obtained the mutant enzymes, Thr218Ser and Asn303Ala, to have different optimum pH compare with that of wild MPP.
(3) For development of a homologous transformation system for the zygomycete fungus, R.pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R.pusillus IFO 4578. The leuA gene was introduced into protoplasts of a leuA-mutant of R.pusillus that was obtained by UV mutagenesis. Using this system, we succeed to overexpress the mpp gene.

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] M.Yamashita: "Mutation of a fungal aspartic proteinase, Mucor pusillus rennin, to decrease thermostability for use as a milk coagulant." J. Biotechnol.32. 17-28 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y. N. Park: "Role of tyrosine 75 on the flap in catalytic function of Mucor pusillus pepsin analyzed by site-directed mutagenesis." Protein Engineering. 7. 1152-1152 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 朴栄男: "微生物由来の凝乳酵素の蛋白工学的改良" 乳業技術. 45. 16-26 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T. Beppu: "Tyrosine 75 on the flap contributes to enhance catalytic efficiency of a fungal aspartic proteinase, Mucor pusillus pepsin." Advances in Experimental Medicine and Biology. 374. 501-509 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y. N. Park: "Site-directed mutagenesis revealed role of subsite residues of Mucor pusillus pepsin in catalytic function." Advances in Experimental Medicine and Biology. 374. 559-563 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y. N. Park: "Role of S_3 subsite residues in the catalytic function of a fungal aspartic proteinase, Rhizomucor pusillus pepsin." Protein Engineering. 8. 961-961 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Wada: "Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination" J. Appl. Environ. Microbiol.(印刷中).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Yamashita, M.Nishiyama, S.Horinouchi, and T.Beppu: "Mutation of a fungal aspartic proteinase, Mucor pusillus rennin, to decrease thermostability for use as a milk coagulant" J.Biotechnol. 32. 17-28 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.N.Park, K.Takeuchi, M.Nishiyama, S.Horinouchi, and T.Beppu: "Role of tyrosine 75 on the flap in catalytic function of Mucor pusillus pepsin analyzed by site-directed mutagenesis" Protein Engineering. 7. 1152-1152 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.N.Park, S.Horinouchi: "Protein engineering of milk-clotting enzymes, chymosin and Rhizomucor pepsin" Bulletin of Japan Dairy Technical Association. 45. 16-26 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Beppu, Y.N.Park, J.Aikawa, M.Nishiyama, and S.Horinouchi: "Tyrosine 75 on the flap contributes to enhance catalytic efficiency of a fungal aspartic proteinase, Mucor pusillus pepsin" Advances in Experimental Medicine and Biology. 374. 501-509 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.N.Park, M.Nishiyama, S.Horinouchi, and T.Beppu: "Sitedirected mutagenesis revealed role of subsite residues of Mucor pusillus pepsin in catalytic function." Advances in Experimental Medicine and Biology. 374. 559-563 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.N.Park, M.Sugiyama, M.Nishiyama, S.Horinouchi, and T.Beppu: "Role of S_3 subsite residues in the catalytic function of a fungal aspartic proteinase, Rhizomucor pusillus pepsin" Protein Engineering. 8. 961-961 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M.Wada, T.Beppu, and S.Horinouchi18GB07 : Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination: J.Appl.Environ.Microbiol. (in press).

    • 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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