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

(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)对一种产生天门冬氨酸蛋白酶（Rhizomucor pepsin， MPP）的真菌Rhizomucor pusillus进行诱变，获得了热稳定性降低的突变酶，这是实际使用该酶作为奶酪制造的牛奶凝固剂所需要的。分离出两种不同的突变菌株Ala94Thr和Gly169Asp，产生明显降低热稳定性的突变酶。(2)我们通过MPP亚位点定向诱变获得了Tyr75Asn/Trp190Phe突变酶，其热稳定性降低，底物特异性增加。我们还获得了与野生MPP相比具有不同最佳pH的突变酶Thr218Ser和Asn303Ala。(3)为建立接合菌真菌R.pusillus的同源转化体系，从R.pusillus IFO 4578中克隆了苹果酸异丙酯异构酶（leuA）基因。将leuA基因导入到紫外光诱变获得的一株紫外光突变体原生质体中。利用该系统，我们成功地表达了mpp基因。

项目成果

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)

Y.N.Park、M.Sugiyama、M.Nishiyama、S.Horinouchi 和 T.Beppu：“S_3 亚位点残基在真菌天冬氨酸蛋白酶（Rhizomucor pusillus 胃蛋白酶）催化功能中的作用”蛋白质工程。

M.Yamashita: "Mutation of a fungal aspartic proteinase,Mucor pusillus rennin,to decrease thermostability for use as a milk coagulant" Joural of Biotechnology. 32. 17-28 (1994)

M.Yamashita：“真菌天冬氨酸蛋白酶，毛霉凝乳酶的突变，降低了用作凝乳剂的热稳定性”《生物技术杂志》。

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)

Y. N. Park：“S_3 亚位点残基在真菌天冬氨酸蛋白酶（Rhizomucor pusillus 胃蛋白酶）催化功能中的作用。”

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)

Y.N.Park、K.Takeuchi、M.Nishiyama、S.Horinouchi 和 T.Beppu：“通过定点诱变分析毛霉胃蛋白酶皮瓣上酪氨酸 75 的作用”蛋白质工程。

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)

T. Beppu：“瓣上的酪氨酸 75 有助于提高真菌天冬氨酸蛋白酶（毛霉胃蛋白酶）的催化效率。”