磁性微球固定化酶分离乳清蛋白源黄嘌呤氧化酶抑制肽及作用机理研究

Food protein hydrolysates are good sources for functional peptides. For example, whey protein hydrolysates are rich in xanthine oxidase (XO) inhibition peptides. However, protein hydrolysates usually have complex composition, which makes it difficult to separate specific peptides from other fractions by traditional ultrafiltration or resin separation techniques. This project aims at the preparation of a magnetic chitosan microspheres-base affinity adsorbents with the immobilization of XO on it. Then, the microsphere with fixed XO will be used to purify specific XO inhibition peptides from whey protein hydrolysates. The affinity strength could be adjusted by the changing of functional group, particle size distribution and thermal property of the microspheres. The sequence of the peptides chain could be identified by Edman degradation analysis, amino acid composition analysis and mass spectrometry assay. The peptide will then be synthesized for enzymatic kinetics test as well as isothermal titration calorimetry assay to clarify the inhibition type. Furthermore, molecular docking techniques will then be applied to better understand the interaction of the peptides with the active site of the enzyme. The 3D structure of the PDB-formatted structures of peptides and XO docking pose will be generated using specific software. The docking sites and related amino acid sequence will be identified to confirm the inhibition mechanism.

食源性蛋白酶解物是功能性肽食品基料的重要来源，如乳清蛋白酶解物中富含可抑制黄嘌呤氧化酶（XO）活性的功能肽段，但因酶解物成分复杂，传统方法如膜过滤、树脂分离等难以从中专一性分离XO抑制肽。本项目通过磁性壳聚糖微球偶联固定XO构建新型“XO-磁性壳聚糖微球”亲和载体，从乳清蛋白酶解物中特异性分离筛选具有XO靶向抑制活性的目标肽，通过调控微球的官能团、粒径分布及热特性等加强载体的亲和力，应用Edman降解、氨基酸分析结合质谱解析目标肽一级序列结构并进行固相合成；通过酶动力学分析及等温滴定量热测定判断目标肽对XO的抑制类型；采用分子对接技术模拟目标肽与XO晶体结构相结合时的诱导—契合效应；借助三维成像软件呈现乳清蛋白肽与XO的对接结果及相互作用力；从分子水平上探讨目标肽与XO酶分子活性中心关键结构域钼喋呤的结合关系，以及与关键氨基酸残基间的键和关系，阐明乳清蛋白肽对XO酶活性的抑制机理和模型。

食源性蛋白酶解物是功能性肽食品基料的重要来源，如乳清蛋白酶解物中富含可抑制黄嘌呤氧化酶（XO）活性的功能肽段。然而，由于蛋白质水解产物的组成复杂，传统的超滤或树脂分离技术很难将多肽从其它组分中分离出来。本项目制备了一种磁性壳聚糖微球偶联固定XO构建新型“XO-磁性壳聚糖微球”亲和载体，从乳清蛋白酶解物中特异性分离筛选具有XO靶向抑制活性的目标肽，通过调控微球的官能团、粒径分布及热特性等加强载体的亲和力，结合质谱解析目标肽一级序列结构并进行固相合成；通过酶动力学分析及等温滴定量热测定判断目标肽对XO的抑制类型；采用分子对接技术模拟目标肽与XO晶体结构相结合时的诱导—契合效应；借助三维成像软件呈现乳清蛋白肽与XO的对接结果及相互作用力；从分子水平上探讨目标肽与XO酶分子活性中心关键结构域钼喋呤的结合关系，以及与关键氨基酸残基间的键和关系；构建高尿酸细胞体外模型，阐明乳清蛋白肽对XO酶活性的抑制机理。