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

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(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.