摘要
【目的】开发厚壳贻贝(Mytilus coruscus)乙醇脱氢酶(Alcohol dehydrogenase,ADH)激活肽,并探究其与ADH作用的分子机制。【方法】以体外ADH激活率为指标,通过凝胶柱层析色谱、反向液相色谱法(RP-LC)分离纯化分子质量小于1 ku的厚壳贻贝活性肽(Mytilus coruscus peptides,MCP),利用液相色谱串联质谱技术(LC-MS/MS)对高ADH激活性的肽组分进行鉴定,并通过分子对接技术探究其与ADH间的相互作用。【结果】MCP经色谱分离得到活性组分Y-I-I,鉴定并筛选出其中3条与ADH对接结合能较低的活性肽序列PPLYE、PPLYQ和APPLYQ,其中活性肽PPLYE体外ADH激活作用最佳,在PPLYE质量浓度为2.0 mg/mL时,ADH激活率达到77.35%,且呈剂量依赖性。分子对接结果显示,PPLYE可结合至ADH活性中心附近的疏水空腔内,通过疏水作用和氢键与受体蛋白ADH形成稳定复合物,其结合能最低,为-35.58 kJ/mol。【结论】PPLYE作为最佳的ADH激活肽,可通过疏水作用和氢键与ADH相互作用形成稳定的复合物来提高ADH活性,本研究为厚壳贻贝资源的开发利用及ADH激活肽的开发提供依据。
【Objective】To develop the alcohol dehydrogenase(ADH) activating peptide from Mytilus coruscus and to investigate the molecular mechanism of its binding with ADH.【Method】Using the in vitro ADH activation rate,M.coruscus peptides(MCP) with molecular less than 1 ku was separated and purified by gel column chromatography and reversed phase liquid chromatography(RP-LC).The peptides with high ADH activation were identified by liquid chromatography tandem mass spectrometry(LC-MS/MS),and the interaction between ADH was explored by molecular docking technology.【Result】The active fraction Y-I-I was separated by chromatography from MCP,and three active peptide sequences PPLYE,PPLYQ and APPLYQ with lower docking binding energy with ADH were identified and selected.Among them,the active peptide PPLYE had the best effect in ADH activation in vitro,reaching 77.35% at 2.0 mg/mL in a dose-dependent manner.Molecular docking results showed that PPLYE could be docked to the hydrophobic cavity near the active center of ADH,and form a stable complex with the receptor protein ADH through hydrophobic interaction and hydrogen bonding,with a lowest binding energy of-35.58 kJ/mol.【Conclusion】PPLYE,as the best ADH activating peptide can interact with ADH through hydrophobic interaction and hydrogen bonds to form a stable compound and increase ADH activity.This study provides a basis for the exploitation and utilization of M.coruscus resources and the development of ADH activating peptide.
作者
刘昱璇
冯子齐
王震
余方苗
LIU Yuxuan;FENG Ziqi;WANG Zhen;YU Fangmiao(College of Food and Pharmacy of Zhejiang Ocean University/Zhejiang Provincial Engineering Technology Research Center of Marine Biomedical Products,Zhoushan 316000,China)
出处
《广东海洋大学学报》
CAS
CSCD
北大核心
2024年第4期11-18,共8页
Journal of Guangdong Ocean University
基金
浙江省属高校基本科研业务费(2021JZ011)。
关键词
乙醇脱氢酶
厚壳贻贝活性肽
分离纯化
分子对接
alcohol dehydrogenase
Mytilus coruscus peptides
separation and purification
molecular docking
