牛蛙皮源DPP-IV抑制肽的高效制备、稳定性及构效关系分析

Efficient preparation, stability, and structure-activity relationship analysis of DPP-IV Inhibitory Peptides Derived from Lithobates catesbeiana Skin

摘要:

为实现牛蛙皮（Lithobates catesbeiana）的高值化利用并开发新型抗糖尿病活性肽。本文以牛蛙皮胶原蛋白为原料，采用7种蛋白酶进行酶解工艺优化，以水解度、蛋白质回收率及二肽基肽酶IV（Dipeptidyl-peptidase IV，DPP-IV）抑制率为评价指标筛选最佳酶解产物，并测定其分子量分布和加工稳定性，通过nano-LC-MS/MS技术鉴定活性肽序列，最后探究活性肽抑制DPP-IV的分子作用机制。结果表明，当木瓜蛋白酶与菠萝蛋白酶质量比为7:1、酶解时间为6 h、酶添加量为2.4%（m/m）时，酶解产物B76的各项评价指标最优（水解度为36.63%，蛋白质回收率为53.99%，DPP-IV抑制率为58.40%），其分子量小于1000 u的肽段占比达92.61%，并且在高温和极端酸碱条件下，其DPP-IV抑制活性可稳定保留。Nano-LC-MS/MS发现多肽GPSGPSGP、VGPRGPAG与DPP-IV结合能最低。合成验证GPSGPSGP的IC50为0.6812 mmol·L-1，并以底物类似物形式竞争抑制DPP-IV活性位点。本实验为牛蛙皮胶原蛋白在抗糖尿病功能食品的开发提供了理论依据。

Abstract:

In order to achieve high-value utilization of Lithobates catesbeiana skin and develop novel anti-diabetic peptides, L. catesbeiana skin collagen was used as raw material in this study and the enzymatic hydrolysis process was optimized using seven proteases. The optimal hydrolysate was selected based on hydrolysis degree, protein recovery rate, and dipeptidyl peptidase IV (DPP-IV) inhibition rate. The molecular weight distribution and processing stability of the hydrolysate were determined. The peptide sequences were identified using nano-LC-MS/MS technology, and the molecular mechanism of DPP-IV inhibition by the active peptides was explored. The results showed that when the mass ratio of papain to bromelain was 7:1, the hydrolysis time was 6 h, and the enzyme dosage was 2.4% (m/m), the hydrolysate B76 exhibited the best performance (with a hydrolysis degree of 36.63%, protein recovery rate of 53.99%, and DPP-IV inhibition rate of 58.40%). The peptides with a molecular weight less than 1000 u accounted for 92.61% of the total peptides in B76, and its DPP-IV inhibitory activity could be stably retained under high temperature and extreme acidic, alkaline conditions. Nano-LC-MS/MS analysis revealed that the peptides GPSGPSGP and VGPRGPAG were found to exhibit the lowest binding energies to DPP-IV. GPSGPSGP was synthesized and determined to possess an IC50 of 0.6812 mmol·L-1, and the DPP-IV active site was competitively inhibited by this peptide in a substrate-like manner. This experiment provided a theoretical basis for the development of L. catesbeiana skin collagen in anti-diabetic functional foods.