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

通过蛋白质工程和基因工程改进凝乳酶毛霉凝乳酶。

• 批准号: 06556014
• 负责人: HORINOUCHI Sueharu
• 金额: $ 9.15万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06556014/
• 关键词: 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; 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

(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.

（1）进行真菌根瘤菌Pusillus的诱变，该诱变是一种称为根瘤菌胃蛋白酶（MPP）的天冬氨酸蛋白酶的生产商，以降低热稳定性，以降低的热稳定性，可用于实用性用作酶作为奶油制造剂的突变酶。分离了两种不同的突变菌株Ala94THR和Gly169ASP，它们以明显降低的热稳定性产生突变酶。（2）我们获得了突变酶，Tyr75ASN/TRP190PHE，具有降温性和降低的耐热性，并通过鉴于Mppppppedsite的突变型的底物特异性提高了底物特异性。我们还获得了突变酶Thr218ser和Asn303Ala，具有不同的最佳pH值与野生MPP的最佳pH值相比。（3）用于开发Zygomycete真菌，r.pusillus，R.Pusillus，R.Pusillus，R.Pusillus，Isomopylymalate（Leua）Gene r.pusillus the Leu的IFO 457575.4575.45.4575.45。紫外线诱变获得的R.pusillus的Leua突变剂的原生质体。使用此系统，我们成功地过表达MPP基因。