基于高活性血管紧张素转化酶抑制寡肽代谢和构效机制分析的蛋白食品可控串联酶解新策略研究

The project is focus on the relationship between human angiotensin-converting enzyme (ACE) and C-terminal proline oligopeptide which is a possible functional component of food-protein digestion products. According to the recent literature and our previous study, the C-terminal proline oligopeptide showed high ACE inhibitory activity. However, the proteases in human gastrointestinal (GI) tract present poor recognition of PRO. It caused a low digestion rate of C-terminal proline oligopeptide even for pro-rich food-protein, because PRO located in the middle rather than the C-terminal of oligopeptide. Consider the high potential bioactivity of C-terminal proline oligopeptide, the nutritional value of food-protein was limited significantly. This phenomenon was noted by many researchers recently, but neither sufficient knowledge nor effective strategy had been reported. In this project, three main research contents are involved to solve this problem step by step. Firstly, the ACE inhibitory activity of C-terminal proline oligopeptide with no more than five amino acids is comprehensively evaluated by the combination of computational and experimental methods. Subsequently, the inhibitory mechanism of C-terminal proline oligopeptide with high inhibitory activity will be elucidated by thermodynamics analysis and dynamic evaluation of binding conformation. That will achieve a full-depth understanding of the C-terminal proline oligopeptide. Secondly, research will be conducted to investigate the directional metabolism of C-terminal proline oligopeptide through the development of the computer aided protein bioactivity evaluation and highly active CP oligopeptide metabolic spectrum analysis. Last but not least, the rational design will be used to obtain protease with high specificity for PRO in P1 position and the technology for controlling enzymatic hydrolysis will be studied. Above all, a precise directional metabolism strategy based on the tandem enzymatic hydrolysis of food-protein in vitro (controlled) and in vivo (gastrointestinal tract) will be explored in the project. With this strategy, the value of food-protein will be enhanced obviously by the enrichment of C-terminal proline oligopeptide in the digestion products. Through multidisciplinary cross research, the depth and breadth of this project has been greatly improved. This project not only has high academic value in the nutritional mechanism of protein foods but also can provide an effective tool and demonstration for high-quality food research.

项目以蛋白食品消化代谢产物中的C末端脯氨酸寡肽(CP寡肽)与人体血管紧张素转化酶(ACE)的作用关系为主线，针对最新研究发现的“CP寡肽具有较好ACE抑制活性”现象，及人体消化道蛋白酶对脯氨酸识别差，CP寡肽代谢率低的问题展开。首先采用计算与实验相结合的手段，全面评价较易吸收的全部CP寡肽的ACE抑制活性，并从结合热力学及动态结合构象等角度深入分析CP寡肽的高活性机制。其次基于计算机辅助的蛋白整体活性评价和高活性CP寡肽代谢谱分析，设计可特异性提高CP寡肽量的定向代谢途径。最终通过理性设计P1位脯氨酸高专一性蛋白酶，并研究利用其可控酶解食品蛋白获得CP寡肽的方法，探索基于体外可控-体内消化道串联酶解的精确定向代谢策略，从而实现蛋白食品的高质利用。项目通过多学科交叉协作，大幅提高了研究的深度与广度，不仅在蛋白食品的营养机理认知上具有较高学术价值，亦可为其他食品高质化研究提供示范。

项目以蛋白食品消化代谢产物中的C末端脯氨酸寡肽(CP寡肽)与人体血管紧张素转化酶(AC E)的作用关系为主线，针对前期研究发现的“CP寡肽具有较好ACE抑制活性”现象，及人体消化道蛋白酶对脯氨酸识别差，CP寡肽代谢率低的问题展开。首先采用计算与实验相结合的手段，全面评价较易吸收的全部CP寡肽的ACE抑制活性，通过对所有16万8千条三肽和四肽与ACE的对接和结合能研究，成功揭示了C末端为Trp的多肽为ACE抑制效果最佳多肽，其理论性能要超越传统研究方法确定的CP寡肽，项目结合热力学及动态结合构象等角度深入分析，阐明了Trp的侧链环较之Pro更大，因此能够产生更强的亲和力的结合机制。基于计算机辅助的蛋白整体活性评价和高活性CP寡肽代谢谱分析，设计了可特异性提高CP寡肽量和C末端Trp寡肽的定向代谢途径。克隆了两个目前报道具有较高P1位Pro选择性的脯氨酸内肽酶，并通过同源建模和分子模拟解析了其结构特征以及催化机制，建立了“基于完美构象下能量优化”的设计改造策略，成功实现了脯氨酸内肽酶选择性的提高，获得了目前报道的最高选择性脯氨酸内肽酶。同时，针对设计的C末端Trp寡肽代谢策略，构建了Trp高选择性的胃蛋白酶表达纯化系统，最终通过理性设计的P1位脯氨酸高专一性蛋白酶和Trp高选择性的胃蛋白酶的对比研究，证明了采用Trp选择性蛋白酶加工后的蛋白食品降压效果最佳。本项目成功建立了基于体外可控-体内消化道串联酶解的精确定向代谢策略，从而实现蛋白食品的高品质的加工途径。本项目在蛋白食品加工和食品酶的研究方法上均取得了突破性的成果，并且通过多学科交叉协作，大幅提高了研究的深度与广度，不仅在蛋白食品的营养机理认知上具有较高学术价值，亦为其他食品高质化研究提供示范。