【摘要】 在溶菌酶催化机理以及α 乳清蛋白和溶菌酶同源比较的基础上 ,构建了含 3个突变点的α 乳清蛋白突变体 (H32L ,T33E ,Y10 3A) ,并将其克隆到具有T7启动子的表达质粒pET2 8a(+ )中 ,在大肠杆菌BL2 1(DE3) /pLys中获得高效表达 .存在于包涵体中的目标蛋白经变复性并通过DEAESepharoseStreamline和SephacrylS2 0 0层析柱获得纯化蛋白 .合成底物 p Nitrophenyl β D N’ ,N’’ ,N’’’ Triacetyl chitotriose [pNP (NAcGlc) 3]与突变体蛋白的反应时间曲线证明突变体蛋白获得了部分溶菌酶生物活性 ,为重组鸡蛋清溶菌酶 (HEWL)的 5 .2 6 % .利用等温滴定量热法测定突变体蛋白与五糖底物chitopentaose的平均热结合能为 71.34kJ/mol,重组HEWL为 10 5 .4 7kJ/mol.实验表明 ,通过有限的几个突变就可以使α 乳清蛋白获得溶菌酶的活性 ,这在分子水平上有力地佐证了两者的高度同源性和进化关系更多还原

【Abstract】 We bulit an α lactalbumin mutant with three mutation sites:H32L, T33E, Y103A based on the catalytic mechanism of lysozyme and the homological comparison between lysozyme and α lactalbumin. The mutant gene was cloned into the expression vector pET28a(+) and got a high level expression in E. coli. BL21(DE3)/pLys. After renaturation from the inclusion body, the target protein was purified by DEAE Sepharose Streamline and Sephacryl S200 column. Synthesized substrate PNP (NAcGlc) 3 [p Nitrophenyl β D N’,N’’,N’’’ Triacetyl chitotriose] was used to measure the lysozyme activity of the mutant. The result showed that the mutant acquired 5.26% lysozyme activity of recombinant HEWL. Using the Isothermal Titration Calorimeter, we got the average binding energy of the mutant with the substrate chitopentaose as 71.34 kJ/mol, against that of lysozyme as 105.47 kJ/mol. These results showed that some limited mutation sites could make α lactalbumin acquire lysozyme activity, which was the strong evidence in molecular level that these two proteins had high homology and were evolved from a common ancestor.更多还原