海参二肽基肽酶Ⅳ抑制肽的酶解制备及结构鉴定

通过抑制二肽基肽酶IV(Dipeptidyl-peptidase IV, DPP-IV)的活性,从而减少GLP-1的降解,提高血液中胰岛素的水平,是控制血糖水平的一种重要手段。本文以海参(Holothariatubulosa)为原料,通过探究蛋白酶种类、加酶量和酶解时间对酶解产物DPP-IV抑制活性、蛋白回收率和水解度的影响,确定了海参DPP-IV抑制肽的制备条件,并进一步测定了其分子量分布和总氨基酸组成,最后通过UPLC-MS/MS鉴定了其潜在的活性肽序列。结果发现,木瓜蛋白酶与复合蛋白酶1:1的复配酶解具有最佳的酶解效果,其产物的得率和DPP-IV抑制活性均最高,且在加酶量为干海参质量的1%,酶解时间为4 h时,海参酶解产物在终浓度为2 mg/mL时的DPP-IV抑制率为66.97%,蛋白回收率为76.25%,水解度为6.10%。酶解产物的分子量大都小于5000 u,且富含脯氨酸(Pro)和丙氨酸(Ala)等与抑制DPP-IV活性有关的氨基酸。将获得的酶解物中的肽段进行液质联用检测,并通过Mascot分析筛选得到28条具DPP-IV抑制肽的肽序列,分子量在500～1936 u。本实验结果为以海参为原料进行降血糖产品的开发奠定了基础。

Preparation and Structure Identification of Dipeptidyl-Peptidase IV (DPP-IV) Inhibitory Peptides from Holotharia tubulosa by Enzymatic Hydrolysis

Dipeptidyl-peptidase IV (DPP-IV) inhibitors are among the newest agents to regulate blood glucose levels by preventing degradation of the gut-derived hormone GLP-1. These inhibitors play an important role in regulating insulin secretion. In this study, DPP-IV inhibitory peptides were prepared from sea cucumber (Holotharia tubulosa) by enzymatic hydrolysis. The types of protease, their concentrations, and hydrolysis times were optimized to maximize DPP-IV inhibition, protein recovery (PR), and degree of hydrolysis (DH). In addition, the molecular weight distribution and the total amino acid composition of each hydrolysate were determined. DPP-IV inhibitory peptide sequences were identified by UPLC-MS/MS. Sea cucumber hydrolysates prepared with a 1:1 combination of papain and Protamex displayed optimal hydrolysis efficiency, and yielded products exhibiting the highest DPP-IV inhibition and PR. Optimal hydrolysis conditions were as follows: the total amount of Protamex added was 1% of the mass of the dry sea cucumber, and hydrolysis time was 4 h. Under these optimal conditions, DPP-IV inhibition, PR, and DH were 66.97%, 76.25%, and 6.10%, respectively. Most peptide molecular weights in the optimal enzymatic hydrolysates were under 5000 u. The peptides were rich in Pro, Ala, and other amino acids related to DPP-IV inhibition. Peptides within the hydrolysates were identified by UPLC-MS/MS. Twenty eight of these polypeptides (with molecular weights ranging from 500 to 1936 u) were found by Mascot analysis to possess features typical of DPP-IV inhibitory peptides. The results of this study lay a foundation for development of hypoglycemic agents from sea cucumber.