为了探究石金钱龟低聚肽作为新冠肺炎病毒（SARS-CoV-2）药物开发原料的可能性，该研究用“风味蛋白酶+木瓜蛋白酶”组合对龟肉进行酶解，通过不同酶配比、加酶量、pH和酶解时间的单因素实验和正交实验确定最优工艺条件，制备出石金钱龟低聚肽。再通过HPLC-MS分离、Peak Studio解析、Peptide Ranker活性预测逐步筛选出活性较高的低聚肽，最后进行各低聚肽序列与SARS-COV-2棘突蛋白（S蛋白）的分子对接。结果显示，最佳工艺参数为风味蛋白酶:木瓜蛋白酶=7:3、加酶量7%、pH 5.5、酶解时间4 h，此时的水解度为42.56%；Peak Studio解析出可信度（ALC）>90%的肽共510条，经Peptide Ranker生物活性评估后筛选出6条低聚肽与S蛋白分子对接，低聚肽序列分别为：LDFFK、LDFFKAL、FRVL、AFRVL、AGGKPFQ、SPFRVT，其Docking Score 在-138.50~-169.68之间，其中LDFFKAL的Docking Score最低，为-169.68，说明该低聚肽抑制新冠肺炎病毒的潜能最强。总的来说，经工艺优化后石金钱龟龟肉的水解度大大增加，且制得的石金钱龟低聚肽有较强的抑制新冠肺炎病毒的潜能。

This study aimed to explore the possibility of using Mauremys mutica oligopeptides as raw materials to develop a drug targeting the SARS-CoV-2 virus. M. mutica oligopeptides were prepared through the combined enzymatic hydrolysis of M. mutica meat using flavoenzyme and papain, and the optimal enzyme ratios, enzyme dosage, pH, and enzymatic hydrolysis time were determined using single-factor and orthogonal experiments. After separating the oligopeptide fractions via high-performance liquid chromatography-mass spectrometry (HPLC-MS), Peak Studio and Peptide Ranker were used for peptide sequence analysis and peptide activity prediction, respectively, to screen for oligopeptides with high bioactivity. Finally, the binding ability of oligopeptides with high predicted activity against the SARS-CoV-2 spike protein receptor-binding domain (RBD) was evaluated using molecular docking. The results show that the highest degree of hydrolysis (DH; 42.56%) was obtained when M. mutica meat was treated with a flavoenzyme: papain ratio of 7:3 and an enzyme dosage of 7% at pH 5.5 for 4 h. According to Peak Studio analysis, 510 peptides with an average local confidence (ALC) of > 90% were discovered. Six oligopeptides with high Peptide Ranker scores (LDFFK, LDFFKAL, FRVL, AFRVL, AGGKPFQ, and SPFRVT) were screened to dock with S protein. Their docking scores ranged from -138.50 to -169.68, and the lowest docking score (-169.68) was exhibited by LDFFKAL. Therefore, a high DH of M. mutica meat could be attained after process optimization, and a high inhibitory potential for SARS-CoV-2 was observed in the resulting M. mutica oligopeptides.

广东省教育厅普通高校青年创新人才项目（2020KQNCX108）；广州工商学院校级科研项目（KA202036；KA202037）