| Project/Area Number |
06556014
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
HORINOUCHI Sueharu University of Tokyo, Biotechnology Faculty of Agricalture Deptment of , Professor, 農学部, 教授 (80143410)
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| 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)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥14,300,000 (Direct Cost: ¥14,300,000)
Fiscal Year 1995: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 1994: ¥10,000,000 (Direct Cost: ¥10,000,000)
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| Keywords | Milk-clotting enzyme / Mucor rennin / Protein engineering / Host-vector system / Mucor pusillus / 蛋白質工学 / 部位特異的変異法 / Rhigomucor pusillus / ムコールペプシン |
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| 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.
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