基于末端组学深度解析食品碱性蛋白酶的动态水解规律及其位点选择特性

The hydrolysis process of food protein by proteases is complicated and dynamic, and different kinds of food proteases have various site-specificity and hydrolysis rules. However, limited by the technique bottlenecks of analyzing cleavage sites, it is still absent that a clear and detailed profiling of the entire dynamic proteolysis process, including the degradation of substrate and the autolysis of protease. Moreover, the previous studies about the site-specificity of protease based on the hydrolysis of small peptides, which failed to reflect the actual proteolysis characters on proteins. Quantitative analysis techniques of cleavage sites have been developed by terminomics in recent years, making it possible to reveal the entire hydrolysis process of food proteases. The present project, using two representative alkaline proteases trypsin and Subtilisin Carlsberg as research subjects, tries to reveal the site-specificity at the protein substrate level and uncover the entire hydrolysis process of soy protein, including the inherent mechanism of the autolysis of both proteases. This project is expected to strengthen the understanding of food proteases, bring new theory and technology patterns for their application and engineering, and rise their rational application level in the food industry in China.

蛋白酶水解食品蛋白，是一个复杂的动态变化过程，且不同种类蛋白酶具有不同的位点选择特性。长期以来受限于位点分析技术上的瓶颈，无法深入清晰地认识酶解变化的全过程及其内在规律。酶解的全过程涉及酶切位点和肽谱的动态变化，既有底物分子的持续降解，又有酶分子的自切演变。此前对食品蛋白酶位点选择性的认识，多以短肽水解为基础，不能客观反映蛋白酶的实际水解过程。近年来末端蛋白质组学突破性地发展了可定量的酶切位点分析技术，使得深度认识酶解的全过程及其规律成为了可能。本项目拟以代表性的胰蛋白酶和Subtilisin Carlsberg蛋白酶为对象，基于末端组学技术，在复杂蛋白底物层次上，阐明其位点选择特性和自切规律，并深入解析其单独水解和复合水解大豆分离蛋白的全过程，揭示两者协同水解的内在模式。本研究将加深对蛋白酶水解食品蛋白复杂过程的认识，提高食品蛋白酶的理性应用和分子改造水平。

蛋白酶的自切和水解是一个复杂的动态变化过程，且不同种类蛋白酶具有不同的位点选择特性。长期以来受限于位点分析技术上的瓶颈，无法深入清晰地认识自切和酶解变化的全过程及其内在规律。本项目拟以代表性的胰蛋白酶和Subtilisin蛋白酶为对象，基于末端组学技术阐明了其位点选择特性和自切规律，明确了关键的自切位点，如胰蛋白酶倾向于在R57、K77、K97、R107、R115、K133、K157位点进行自切。在此基础上通过自切位点的单点或多点突变改造提高了胰蛋白酶和Subtilisin的抗自切稳定性和比活力，最终获得了性能优异的新型胰蛋白酶和Subtilisin酶分子，胰蛋白酶比活力最高提高1.84倍，抗自切稳定性提高3.07倍。研究还深入解析了大豆分离蛋白水解肽谱的变化过程及其规律。本研究加深了对食品蛋白酶自切规律和水解蛋白复杂过程的认识，为提高蛋白酶的改造水平及其在食品领域的理性应用建立基础。