利用光谱技术定量研究蛋清溶菌酶结构与功能关系

Spectral detecting approaches to relationship between structure and fucntionality of hen egg white lysozyme

通过对蛋清溶菌酶(hen egg white lysozyme,HEWL)热处理过程中圆二色谱(CD)和紫外差谱(UV diff.)信号测定,利用蛋白质去折叠二态转变模型,对CD和UV diff.信号进行最小二乘法非线性拟合,定量获取蛋白质稳定性热力学数据.实验结果表明:随pH下降,蛋白质构象稳定性降低.功能性质分析发现:随pH下降,HEWL的酶活性降低,但表面活性(乳化性和乳化稳定性)升高.进一步,将蛋白质构象稳定性(ΔG_(25℃,water))数据和酶活性、乳化性和乳化稳定性数据进行相关性分析发现:蛋白构象稳定性与酶活性、乳化活性和乳化稳定性呈较好的相关性.这些结果说明:蛋白质构象稳定性降低引起蛋白质柔性增加,使其铺展到油水两相界面能力提高.该研究为从分子水平定量研究食品蛋白结构和功能关系奠定了基础.

The conformational stability and conformational change induced by heat processing affect significantly on biological functions in the case of enzymes and functional properties of food proteins. By means of measurements on the heat-induced transition curves followed by observation of changes in CD/UV diff. and non-linear least-squares fit for heat-induced denaturation curves based on two-state transition model,a practical method has been developed for determining the stability parameters of proteins,such as ΔHm,Tm and ΔG25 ℃,water. The results of the present study indicate that the thermodynamic parameters,such as ΔHm,Tm and ΔG25 ℃,water,decrease with lower pH value,which show that the stability of protein decrease with the decrease of p H. Furthermore,the enzymatic activity decrease with lower pH value as well,however the surface functional properties( Emulsifying activity and Emulsion stability) increase with lower pH value. At last,good correlations were obtained between enzymatic activity,emulsifying activity,emulsion stability and conformational stability of HEWL,which support the hypothesis that reduced conformational stability( ΔG25 ℃,water) lead to increased protein flexibility and adsorption property to oil/water interface. It is concluded that our research constructs a stable base for the further quantitative research on the structure and function relationship of food protein.