地龙胰脂肪酶抑制肽的制备、结构鉴定及活性分析

Preparation, Structural Identification and Activity Analysis of Pancreatic Lipase Inhibitory Peptide from Earthworm Protein

摘要:

为制备具有高效胰脂肪酶抑制活性的地龙蛋白肽,本研究结合地龙内源酶和商业酶的优势,采用两步酶解法对地龙蛋白进行水解,并以胰脂肪酶抑制率为评价指标,采用单因素试验和正交试验对第二步酶解工艺进行优化。此外,对酶解产物进行超滤、凝胶柱分离,并运用LC-MS/MS、分子对接等技术结合PeptideRanker数据库筛选具有较高胰脂肪酶抑制活性的肽段。结果表明,第二步酶解的最优工艺是:料液比为1:2(50%,w/w),碱性蛋白酶用量为6.0× U,温度为50℃,pH为9.5,酶解时间为3 h。在此条件下,胰脂肪酶抑制率可达53.92±0.43%,水解度为41.98±0.42%,可溶性肽得率达到89.17±0.54%。共筛选得到10条亲和力高、生物活性好且无毒的肽段,分别为GDAPPFFP、YPFDPGP、WGPDLP、FDFP、DDFFR、FLDF、SDDGFF、FDDF、SDDFF和WDDFF。小分子肽及氨基酸可能是地龙蛋白发挥胰脂肪酶抑制作用的主要物质基础,本研究为理解地龙蛋白的降脂活性提供了新视角,为后续研究地龙蛋白功能肽的构效关系和胰脂肪酶抑制作用机理机制提供了条件。

Abstract:

In order to obtain earthworm protein peptides with efficient pancreatic lipase inhibition activity, this study combined the advantages of endogenous enzymes and commercial enzymes, employing a two-step enzymatic hydrolysis method to hydrolyze earthworm protein. The pancreatic lipase inhibition rate was used as the evaluation index, and single factor experiments and orthogonal experiments were used to optimize the second step enzymatic hydrolysis process. In addition, the hydrolysates were purified by ultrafiltration and Sephadex G-15 gel chromatography, and peptides with high pancreatic lipase inhibitory activity were screened by LC-MS/MS, molecular docking and other technologies combined with PeptideRanker database. The results showed that the optimal preparation process for the second step enzymatic hydrolysis was a solid-liquid ratio of 1:2 (50%, w/w), alkaline protease dosage of 6.0 x 104U, temperature of 50℃, pH of 9.5, and enzymatic hydrolysis time of 3 hours. Under the conditions, the pancreatic lipase inhibition activity could reached 53.92±0.43%, the hydrolysis degree was 41.98±0.42% and the soluble peptide yield reached 89.17±0.54%. Also, 10 peptide segments with high affinity, good biological activity and non-toxicity were screened, namely GDAPPFFP, YPFDPGP, WGPDLP, FDFP, DDFFR, FLDF, SDDGFF, FDDF, SDDFF and WDDFF. Small molecule peptides and amino acids may be the main material basis for the inhibitory effect of earthworm protein on pancreatic lipase, this study provides a new perspective for understanding the lipid-lowering activity of earthworm proteinconditions, and provides conditions for further research on the structure-activity relationship of earthworm protein functional peptides and the mechanism of pancreatic lipase inhibition.