菊粉酶降低N-羟乙酰神经氨酸含量机制的分子模拟研究

Molecular Simulation Study on the Mechanism of Inulinase Reducing N-Glycolylneuraminic Acid Content

为探究菊粉酶降低红肉风险物质N-羟乙酰神经氨酸(Neu5Gc)含量的效果和分子机制,本研究首先采用菊粉酶处理Neu5Gc标准品并对其进行液相质谱检测,其次选择外切型菊粉酶(蛋白数据序列号:1Y9G)和内切型菊粉酶(蛋白数据序列号:3SC7)与Neu5Gc进行分子对接和分子动力学模拟,并以氨基酸虚拟突变验证菊粉酶结合Neu5Gc的关键残基。最后利用密度泛函理论(DFT)和独立密度梯度模型(IGM)分析动力学平衡后Neu5Gc和菊粉酶活性残基复合物的分子间弱相互作用强度与分布。结果表明,外切和内切型菊粉酶对Neu5Gc标准品含量的平均降低率分别为55.33%和43.81%;理论计算表明,Neu5Gc主要以羟基氧、酰胺氮原子与菊粉酶活性位点残基发生弱相互作用而稳定结合到活性口袋中,水环境下对外切和内切菊粉酶的结合能分别为-22.71和-8.46 kJ·mol^-1。本研究结果为菊粉酶的理性设计和在红肉安全性控制中的应用提供了一定的参考。

To explore the effect and molecular mechanism of inulinase to reduce the content of N-glycolylneuraminic acid(Neu5 Gc)in red meat.Firstly,Neu5 Gc standard sample was detected by the liquid phase mass spectrometry after treatment with inulinase.Secondly,the exo-inulinase(PDB code:1 Y9 G)and endo-inulinase(PDB code:3 SC7)were selected to perform molecular docking and molecular dynamics simulation with Neu5 Gc,and the inulinase active residues that bind with Neu5 Gc was verified by amino acid virtual mutation.Finally,the intensity and distribution of intermolecular weak interactions between Neu5 Gc and inulinase active residue complexes after dynamic equilibrium was analyzed by density functional theory(DFT)and independent density gradient(IGM)theory.The experimental results showed that the average Neu5 Gc content reduction rates of the exo-and endo-inulinases were 55.33%and 43.81%,respectively.The theoretical calculations showed that Neu5 Gc could stably bind into the enzyme active pocket with inulinase active site residues through intermolecular weak interactions,which mainly related to Neu5 Gc hydroxyl oxygen and amide nitrogen.The binding energy of Neu5 Gc with exo-and endo-inulinase complex in water environment were-22.71 and-8.46 kJ·mol^-1,respectively.The results of this study provided a reference for the rational design of inulinase and application in the control of red meat safety.