羧甲基纤维素调控溶菌酶抑菌活性的分子机制

Lysozyme in eggs is a sort of natural and safe preservative. However, lysozyme is limited to use in foods because of its unstable activity and small range of antibacterial spectrum. There were a number of studies which proved that the activity of enzymes was easily affected by the interaction between the enzymes and other macromolecules. Therefore, it is a valuable work that assesses the residual activity of lysozyme after interacting with other macromolecules in foods. However, this work is seldom intensively studied because of the intricacy of food ingredients and environmental conditions. In our previous studies, the spatial structure and activity of lysozyme was easily affected by the electrostatic interaction between lysozyme and other negative charged polysaccharides. However, the further regulatory mechanism is not clear. Consequently, lysozyme and carboxymethylcellulose were used as the object of study based on electrostatic assembly. Our studies include: ① The influence of the activity and the regulatory effect of the range of antibacterial spectrum of lysozyme at various electrostatic assemble conditions; ②The mechanism of structure-activity of lysozyme at various electrostatic assemble conditions. It is expected to explain the influence of lysozyme activity after interaction with other macromolecules. Our work is wished to provide the theory reference for lysozyme to be more effectively used in food, as well as it will help to enrich the study of interactions between complex food compositions.

禽蛋中的溶菌酶为安全的天然防腐剂，但因酶活不稳定、抑菌谱范围窄等因素限制了其在食品中的使用。已有诸多研究证实高分子间相互作用可对蛋白质活性产生影响，因此溶菌酶与食品组分相互作用后的抑菌活性值得评估，但因食品组分和影响条件复杂而鲜见深入研究。申请人前期研究证明溶菌酶极易与带负电荷的多糖产生静电相互作用而影响溶菌酶分子空间结构，并影响其活性，但具体调控机制尚不清楚。因此，在此基础上申请人以羧甲基纤维素与溶菌酶作为静电组装对象，①研究不同环境条件下此种组装行为对溶菌酶活性的影响，分析组装条件对溶菌酶抑菌谱范围的调控作用；②揭示组装行为对溶菌酶高级结构和抑菌调控之间的构效关系机制。以阐明高分子间的相互作用力对酶活性影响，为溶菌酶能更有效的在食品中应用提供理论参考，丰富食品复杂体系中组分相互作用的研究。

阴离子多糖可影响溶菌酶（Ly）的抑菌活性。本项目以羧甲基纤维素（CMC）作为阴离子多糖模型，考查改变体系环境条件（例如，热处理、二者质量比等），CMC调控Ly活性分子机制。CMC可掩盖Ly的抑菌活性位点，中和Ly表面电荷，并影响Ly分子空间构象而抑制其抑菌活性。Ly与CMC质量比可显著影响二者的相互作用，在Ly与CMC质量比为5：1的时候，二者的静电相互作用最强，形成较大的团聚，从而掩盖了Ly的抑菌活性位点。同时，Ly空间结构发生最显著变化，从而使其热稳定性变得更差。因此，在此质量比条件下，Ly的活性最低。在质量比为1：1附近，为二者相互作用最薄弱的条件，此时Ly活性部分恢复。在热处理条件下，CMC可加速Ly变性。当温度高于80℃时，Ly酶活显著降低，但Ly抑菌性几乎不受影响，尤其在CMC存在的条件下，对革兰氏阴性大肠杆菌的抑制还有增加的效果。CMC加速了蛋白质的热变性，从而暴露更多的疏水性基团，与细菌细胞膜的亲和能力增加。因此，虽然CMC可以通过掩盖Ly的抑菌活性位点而达到抑制其生物活性的效果，热处理使CMC增加了Ly对细菌细胞膜的破坏能力。CMC的取代度越高，与Ly的静电相互作用越强，Ly的抑菌能力随着CMC的取代度提高而逐渐降低。在低盐浓度条件下，可以使复合物发生少量聚集。在高盐浓度条件下，由于离子对高分子物质表面的电荷中和与屏蔽作用，二者的静电相互作用减弱，复合物解离，酶活部分恢复。本项目为解释因Ly与阴离子多糖相互作用而导致的Ly在食品中的不稳定性提供理论依据。

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