目的 采用干湿结合策略系统挖掘珍珠Pteria martensii废弃壳蛋白质中潜藏的血管紧张素转化酶（angiotensin converting enzyme，ACE）抑制肽。方法 珍珠废弃壳经脱钙、透析处理得到珍珠层粉蛋白，以ACE抑制活性和水解度为评价指标，优选出最适蛋白酶。采用超滤和凝胶过滤色谱法对酶解物进行分离纯化，通过液质联用技术（LC-MS/MS）对活性组分进行肽段鉴定。采用生物信息学工具筛选预测活性较高的肽段进行固相合成和体外ACE抑制活性验证。通过双倒数作图法（Lineweaver-Burk）对ACE的抑制动力学参数进行分析，以判定活性肽的抑制作用类型。通过体外模拟消化实验分析活性肽的消化稳定性。采用分子对接技术探讨活性肽与ACE的相互作用和与ACE N/C端活性位点作用的差异性。采用分子动力学模拟探究活性肽与ACE复合物体系的结合稳定性与动态作用。结果 珍珠层粉蛋白经8种蛋白酶酶解后，其中木瓜蛋白酶酶解物水解度为（11.77±0.09）%，抑制ACE的半数抑制浓度（half maximal inhibitory concentration，IC₅₀）为（2.03±0.14）μg/mL，酶解产物经分离纯化后得到活性组分F1，IC₅₀为（1.56±0.06）μg/mL，从中共鉴定出294条肽段，最终筛选出9条肽段进行固相合成与活性验证，其中肽段SPAR活性最强，IC₅₀值为（33.46±0.56）μmol/L，对ACE抑制作用表现为混合型抑制。SPAR经体外模拟消化后的保留率为（23.6±4.9）%。分子对接中，SPAR能与ACE S1、S2活性口袋中的关键氨基酸残基Ala-354、Glu-384、Tyr-523、Gln-281、Lys-511形成氢键作用。SPAR与ACE的N/C端均能形成氢键相互作用，对C端的结合能强于N端。SPAR-ACE复合物在分子动力学模拟过程中表现出较好的构象稳定性和持续的相互作用。结论 通过干湿结合策略从珍珠废弃壳蛋白质中共筛选出8条新的活性肽，其中肽段SPAR的ACE抑制活性最强，为珍珠废弃壳的再利用提供了新的研究思路与方向，干湿结合策略协同互补，能够更高效地挖掘中药废弃物蛋白质潜藏的活性肽。

Objective To systematically identify potential angiotensin-converting enzyme (ACE) inhibitory peptides from proteins in waste pearl shells of Pteria martensii using an integrated in silico and experimental (“dry-wet”) strategy. Methods Waste pearl shells were subjected to decalcification and dialysis to obtain nacre proteins. The optimal protease was screened using ACE inhibitory activity and degree of hydrolysis as evaluation indicators. The enzymatic hydrolysates were separated and purified via ultrafiltration and gel filtration chromatography, and the peptide sequences of active fractions were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatic tools were applied to predict and screen peptides with high potential ACE-inhibitory activity; candidate peptides were then prepared by solid-phase peptide synthesis (SPPS) and verified for their in vitro ACE inhibitory capacity. Lineweaver-Burk double-reciprocal plots were used to calculate enzyme kinetic parameters and define the inhibition mode of active peptides. An in vitro simulated gastrointestinal digestion assay was performed to evaluate the digestive stability of target peptides. Molecular docking was performed to investigate the interactions between the active peptide and ACE and to compare its interactions with the active sites in the N- and C-domains of ACE. Molecular dynamics (MD) simulations were conducted to assess the conformational stability and dynamic interaction characteristics of peptide-ACE complexes. Results Eight proteases were used to hydrolyze nacre proteins, among which papain hydrolysate showed a degree of hydrolysis of (11.77 ± 0.09)% and a half-maximal inhibitory concentration (IC₅₀) of (2.03 ± 0.14) μg/mL against ACE. After separation and purification, the active fraction F1 was harvested with an IC₅₀ value of (1.56 ± 0.06) μg/mL. A total of 294 unique peptides were identified from F1, and nine peptides with high predicted activity were selected for solid-phase synthesis and in vitro activity validation. Among all synthetic peptides, peptide SPAR exhibited the strongest ACE inhibitory activity with an IC₅₀ of (33.46 ± 0.56) μmol/L, and its inhibitory pattern against ACE was confirmed as mixed-type inhibition. After in vitro simulated gastrointestinal digestion, the retention rate of SPAR was (23.6 ± 4.9) %. Molecular docking results revealed that SPAR formed hydrogen bonds with key amino acid residues located in the S1 and S2 active pockets of ACE, including Ala354, Glu384, Tyr523, Gln281 and Lys511. SPAR was capable of binding to both the N-domain and C-domain of ACE, and displayed stronger binding affinity for the C-domain. MD simulation results demonstrated that the SPAR-ACE complex maintained favorable conformational stability and sustained intermolecular interactions throughout the simulation period. Conclusions Eight novel ACE inhibitory peptides were successfully screened from waste pearl shell proteins via the integrated dry-wet strategy, among which peptide SPAR possessed the optimal ACE inhibitory activity. This work provides a novel research perspective and technical route for high-value reutilization of discarded pearl shells. The complementary combination of in silico prediction and wet-lab experiments enables more efficient excavation of latent bioactive peptides from traditional Chinese medicine waste proteins..

R284.1

国家自然科学基金资助项目（81960698）；国家自然科学基金资助项目（82560762）；2024年度广西农作物废弃物功能成分研究协同创新中心开放基金项目（CICAR 2024-Z3）；2025年广西中医药大学研究生教育创新计划项目（YCSY2025006）；广西特色中药现代产业学院2024年大学生创新创业训练计划项目（C202411）