海鲈鱼酸性磷酸酶分离纯化及其抑制剂虚拟筛选

Isolation and Purification of Acid Phosphatase from Lateolabrax maculatus and Virtual Screening of Its Inhibitors

为延缓海水鱼类肌苷酸降解,保留鱼类本身的鲜味成分,以新鲜的海鲈鱼为研究对象,经NaAc-HAc缓冲液(pH值为5.0)提取,饱和硫酸铵分级沉淀、阴离子交换柱和葡聚糖凝胶柱分离纯化后,研究海鲈鱼肝中酸性磷酸酶(acid phosphatase,ACP)的酶学特性,采用虚拟筛选和分子对接技术筛选潜在的天然ACP抑制剂。结果表明:分离纯化出ACPⅠ和ACPⅡ2种同工酶;ACPⅠ的比活力为6.57 U/mg,纯化了36.50倍,回收率为9.96%;ACPⅡ的比活力为4.62 U/mg,纯化了25.69倍,回收率为6.07%。对回收率和纯化倍数相对较高的ACPⅠ进一步研究,发现其K_(m)为3.174 mmol/L,v_(max)为0.982μmol/(L·min)。ACPⅠ最适温度为30℃,最适pH值为5.0。ACPⅠ的二级结构中α-螺旋含量为14.10%,β-折叠含量为35.90%,β-转角含量为18.20%,无规则卷曲含量为32.60%。虚拟筛选出5个多酚类化合物可作为ACP抑制剂,与ACP相互作用方式主要是氢键和疏水作用。在一定浓度范围内(0~1 mmol/L),5个多酚类化合物均能有效抑制ACP活性,浓度越大抑制效果越好。研究旨在为贮藏过程中水产品的风味和品质控制提供理论依据。

To reduce the degradation of inosinic acid in marine fishes for maintaining the freshness of the fish,separation and purification of acid phosphatase(ACP)from Lateolabrax maculatus(sea bass)were performed using NaAc-HAc(pH 5.0)buffer extraction,graded precipitation separation by saturated ammonium sulfate,and anion-exchange column and dextran gel column purification.The enzymatic properties of ACP in the liver of sea bass were studied and potential natural ACP inhibitors were screened using virtual screening and molecular docking techniques.The results showed that two isoenzymes(ACPⅠ and ACPⅡ)were isolated and purified.ACPⅠ had a specific activity of 6.57 U/mg and was purified to 36.50 times of the original activity with a recovery rate of 9.96%.The specific activity of ACPⅡ was 4.62 U/mg and it had a recovery rate of 6.07% after being purified to 25.69 times of the original activity.The K m of ACPⅠ with relatively higher recovery rate and purification factor in sea bass was 3.174 mmol/L and v max was 0.982μmol/(L·min).The optimum temperature and pH of ACPⅠ were 30℃ and 5.0.The secondary structure of ACPⅠ had 14.10% α-helix,35.00% β-fold,18.20% β-turn and 32.60% random coil.The five polyphenol compounds as ACP inhibitors were screened virtually,and interacted with ACP mainly by hydrogen bonding and hydrophobic forces.In a certain concentration range(0-1 mmol/L),all the five polyphenol compounds could effectively inhibit the ACP activity,and the higher the concentration,the better the inhibition effect.The research could provide a theoretical basis for flavor and quality control of aquatic products during storage.