卵黄高磷蛋白<i>α</i>-淀粉酶抑制肽的制备、鉴定及酶抑制动力学分析

薛天睿; 吕彬斐; 张铭冉; 李灿; 张晓维

doi:10.13386/j.issn1002-0306.2024010363

卵黄高磷蛋白α-淀粉酶抑制肽的制备、鉴定及酶抑制动力学分析

Preparation, Identification, and Enzyme Inhibition Kinetic Analysis of Phosvitin α-Amylase Inhibitory Peptides

摘要

摘要: 为探索卵黄高磷蛋白肽（phosvitin phosphopeptide，PPP）对α-淀粉酶的抑制作用，使其能够调控血糖水平从而缓解Ⅱ型糖尿病，本研究以α-淀粉酶抑制率为指标酶解卵黄高磷蛋白制备PPP，利用酶抑制动力学实验分析PPP对α-淀粉酶的抑制类型，采用液质联用鉴定后用分子对接筛选出高活性的α-淀粉酶抑制肽，最后验证其活性。结果表明，最优酶解条件为先经胰蛋白酶（加酶量7000 U/g）再经胃蛋白酶（加酶量60000 U/g）分别酶解6 h，制得的PPP在浓度7.81×10⁻³ mg/mL时具有最高α-淀粉酶抑制率（70.69±1.71）%。PPP对α-淀粉酶的抑制类型为混合型抑制。鉴定筛选出了两种新型的高活性α-淀粉酶抑制肽FGTEPDAK和IWGR，经验证其IC₅₀值分别为（0.80±0.14）×10⁻³ mg/mL和（1.80±0.31）×10⁻³ mg/mL，远低于阳性对照阿卡波糖（3.17±0.47）×10⁻³ mg/mL。本研究揭示PPP可作为新型降糖物质，应用于缓解Ⅱ型糖尿病的功能性食品开发中。

Abstract: To explore the inhibiting effect of phosvitin phosphopeptide (PPP) on α-amylase, enabling it to regulate blood glucose levels and alleviate type 2 diabetes mellitus. This study employed enzymatic hydrolysis of phosvitin with the inhibition rate of α-amylase as an index, followed by enzyme inhibition kinetics experiments to analyze the inhibitory type of PPP on α-amylase. LC-MS identification was conducted, and molecular docking was performed to screen for highly active α-amylase inhibitory peptides, which were subsequently validated. The results showed that optimal enzymatic hydrolysis conditions involved initial hydrolysis with trypsin (7000 U/g), followed by pepsin (60000 U/g) for 6 hours each. The prepared PPP exhibited the highest α-amylase inhibition rate (70.69±1.71)% at a concentration of 7.81×10⁻³ mg/mL. PPP acted as a mixed-type inhibitor. Two novel highly active α-amylase inhibitory peptides FGTEPDAK and IWGR were identified and screened, exhibiting IC₅₀ values of (0.80±0.14)×10⁻³ mg/mL and (1.80±0.31)×10⁻³ mg/mL, respectively; both significantly lower than the positive control acarbose's IC₅₀ value (3.17±0.47)×10⁻³ mg/mL. This study highlights the potential use of PPP as a new hypoglycemic substance in developing functional foods for alleviating type 2 diabetes mellitus.