低值鱼天然肌球蛋白分子的定向修饰及其热稳定性机理研究

Protein directional modification based on glycosylation for enhanced functional properties has been widely studied recent years in food industry. In our previous study, konjac oligo-glucomannan (KOG) with multi-branched chains, special hydrophilic group and moderate molecular weight was prepared to directionally modify myosin (Ms) from low-quality fish, and the result showed that the thermal stability of Ms was greatly increased. However, the mechanisms of directional modification and thermal stability were unclear. The present study prepares KOG, which is used to directionally modify Ms to form Ms-KOG. The content of lysine and degree of substitution were determined to verify the sites and amount of directional modification, and the physicochemical properties including isoelectric point, molecular weight and apparent viscosity were analyzed, for elucidating the mechanism of directional modification. The spatial structure consisting of microstructure of Ms-KOG, primary, secondary, tertiary and quaternary structure of Ms were characterized. Thermodynamic properties including denaturation temperature, Gibbs free energy, entropy and enthalpy of Ms-KOG were determined, in order to study the increasing way (change in entropy or enthalpy) of energy barrier (ΔGglyco). Based on the multi-factors correlation analysis, the relationship between spatial structure and thermodynamic properties would be revealed to obtain the spatial structure of Ms during change in entropy or enthalpy, with the purpose of elucidating the mechanism of thermal stability. Our study may provide some useful theoretical guidance for the mechanism of directional modification based on glycosylation, and the effective control between the structure of protein-polysaccharide and thermal stability.

糖基化定向修饰蛋白质提高功能特性是食品领域的研究热点。前期实验表明，具有多支链、特异亲水基团和适度分子量的魔芋葡甘露低聚糖（KOG）定向修饰低值鱼肌球蛋白（Ms）后热稳定性极大提高，但定向修饰和热稳定性机理尚不清楚。本项目拟制备KOG定向修饰Ms获得Ms-KOG；通过测定赖氨酸含量和取代度确定定向修饰位点和量，分析Ms-KOG等电点、分子量、表观黏度变化，阐明定向修饰机理；采用现代仪器分析法表征Ms-KOG微观结构、Ms一级、二级、三四级结构变化；测定Ms-KOG变性温度、吉布斯自由能、熵和焓的热力学特性变化，确定定向修饰后Ms能垒（ΔGglyco）增加途径（熵变或焓变）；结合空间结构和热力学特性变化进行多因素相关性分析，确定Ms熵变或焓变发生时的空间结构（折叠态或去折叠态），揭示Ms定向修饰热稳定性机理。本项目将为糖基化定向修饰机理、蛋白质-多糖结构和热稳定性有效调控等研究提供理论借鉴。

本项目从鲢鱼中分离纯化获得肌球蛋白Ms，以魔芋粗粉为底物，采用β-甘露聚糖酶进行酶解制备魔芋葡甘低聚糖KOG，其平均聚合度为5.2。建立糖基化反应成功制备Ms-KOG复合物。随着糖基化反应的进行，复合物中的总糖含量明显增加，赖氨酸与精氨酸的相对含量明显降低，动态黏度和分子量出现不同程度的增加，等电点、总巯基含量及表面疏水性随着反应时间的延长逐渐减低，两种不同的糖供体修饰肌球蛋白糖基化反应，其变性峰值都有不同程度的升高。扫描电镜（SEM）研究发现，Ms的表面结构与其糖基化复合物的明显不同，后者聚集体逐步分散开来，尤其是Ms-KOG糖基化复合物；AFM分析表明，经糖基化过后，所有样品颗粒的表面结构都比原蛋白颗粒大且更加分散；通过DLS分析结果可知，糖基化复合物的粒径分布不一，减少了蛋白质分子的聚集。FTIR分析证明了Ms以共价键形式引入了糖分子。CD分析二级结构表明，与Ms相比，糖基化复合物的α-螺旋与无规则卷曲结构含量降低，β-折叠与β-转角结构含量增加。内源性荧光分析表明，与Ms相比，糖基化复合物的内源性荧光强度随着反应时间的增加而下降，且在反应一定时间后出现红移现象。此外，KOG作为一种多羟基且含一定量的支链的低聚糖在与Ms糖基化后，其空间位阻效应比葡萄糖作用更明显，所以其相对荧光强度下降幅度更大且红移现象出现的更早。从功能性的研究发现，Ms-糖糖基化复合物的溶解性（尤其是在低盐溶液中）、热稳定性较Ms都有了显著地提高，乳化性呈先增后减的态势。Ms的改性复合物比蛋白本身更容易消化。与未发生糖基化反应的Ms膜相比，糖基化反应能够导致Ms膜色泽加深，亲水性降低，延长率增加以及水蒸气渗透率降低。此外，与Ms-葡萄糖糖基化改性膜相比，Ms-KOG糖基化改性膜的色泽较深，延长率较差，然而其在水中的稳定性，拉伸强度和水蒸气阻隔性能均较好。本研究通过糖基化改性显著地改善了Ms功能性质，同时明确了糖基化改善Ms热稳定性的机理，为蛋白的实际应用提供了理论依据。