玉米半胱氨酸蛋白酶突变体W308C的原核表达和酶学性质表征

Prokaryotic Expression and Characterization of Cysteine Protease Mutant W308C from Zea mays

通过对玉米半胱氨酸蛋白酶(cysteine protease from Zea mays,zmCP1)同源模建和底物对接研究发现,W308位点在木瓜蛋白酶家族(C1家族)高度保守,且位于zmCP1催化三联体残基N306位点附近,与绝大多数底物形成π-π键,可参与底物和酶活性中心的结合,是该酶的关键位点;结合Rosetta design程序设计,对W308位点进行突变,获得W308C突变体,并在大肠杆菌BL21中表达。酶学性质表征实验结果表明:突变体W308C的最适温度为55℃,最适pH 6.0;在70、80、90℃下的半衰期为75.33、57.24、40.29 min,分别是野生型的1.21、1.19、1.01倍;突变体W308C和野生型的特异性常数Kcat/Km分别为11.02、5.92 L/(μmol·min),催化活性提高了0.86倍。

Through homologous modeling and docking studies of cysteine protease from Zea mays(zmCP1), W308 was highly conserved in papain super family(C1 family), near the Asn306 which was the residues of catalytic triad, had π-πinteraction with most of the ligands, and was the key residue for zmCP1. By using Rosseta design program, the mutant W308 C was obtained and expressed in E.coli BL21. The characterization of enzymatic properties indicated that the optimal temperature and pH were 55 ℃ and 6.0, respectively. T50 of W308 C at 70, 80, and 90 ℃ were 75.33, 57.24, and 40.29 min, respectively, which were 1.21, 1.19 and 1.01 times higher than wild type. Kcat/Km of W308 C and wild type was 1.02 and 5.92 L/(μmol·min), respectively, showing an activit y level 1.86 times higher than that of wild type.