Study On Mechanism Of Site-specific Immobilization Of Acetylcholinesterase Based On Protein Surface Recognition

In recent years,environmental pollution and food safety are among the most important issues worldwide increasingly drawing attention.Enzymatic Biosensors(EBS)is a newly developed rapid detection method for organic contaminants.It has become a research focus for its high sensitivity,fast response and high timeliness.The key to design of enzyme biosensors is the method of enzyme immobilization on carriers.In the light of the previous research of enzyme immobilization,here we selected Acetylcholinesterase(AChE)as the object of study,developed a whole new site-specific immobilization method based on protein surface recognition,and elucidate the mechanism in site-specific immobilization of AChE,which will be described in detail below:1.Design of site-specific immobilization of AChE.We developed complete three dimensional structural model for AChE.Using MOLCAD's multi-channel,we discovered 6 potential immobilization sites on the surface of AChE.Site1 was selected as the binding site of site-specific immobilization of AChE based on the hydrophobicity,electrical property,hydrogen bond donor/receptor and cavity structure on the protein surface.Based on the structural data of Site1 and the active site of AChE,molecular docking was applied for virtual screening of 54159 small molecules from ZINC database.Through three steps,13 functional immobilized ligands of different molecular structure were obtained,which are(+)-catechin,(-)-epicatechin,(-)-gallocatechin,(R)-hesperetin,(S)-hesperetin,(R)-naringenin,(S)-naringenin,quercetin,taxifolin,(-)-epicatechin gallate,flupirtine,atropine,and hyoscyamine2.Interaction between AChE and the functional immobilized ligands.To test and verify the binding ability and affinity of the selected functional immobilized ligands to AChE,fluorescence spectrometry and enzyme activity assay were applied and results showed the binding affinity was in the order of NAR>HES>QUE>>EPG,TAX>CAT,EPI,FLU>GLL>>ATR,HYO.The catalytic activity of AChE wouldn't be affected when binding with the functional immobilized ligands.3.Construction,characterization and stability of site-specific immobilized AChE.NAR,CAT,GLL were selected as functional immobilized ligands and amine-functionalized magnetic nanoparticles was selected as carrier to design,prepare and characterize the three different immobilized AChE.The stability to pH and temperature and reusability of immobilized AChE was also investigated using enzyme activity assay.Comparing to free enzyme and traditional immobilization methods,many characteristics were improved evidently for site-specific immobilized AChE,especially in maintaining the activity(all above 90%)and stability(maximum increase of stability to pH reached 70%and to temperature about 130%).This further proved the validity of site-specific immobilization method developed in this study.4.Mechanism of site-specific immobilization of AChE.In order to elucidate the mechanism and identify the factors affecting site-specific immobilized AChE,we studied the binding affinity,stability and the interaction in the site-specific immobilization of functional immobilized ligands and AChE using multiple molecular simulation methods(molecular dynamics and accelerated molecular dynamics).The dynamic process of the interaction between AChE and ligands was discovered through massive simulation,analysis and validation.It was confirmed that the binding between functional immobilized ligands and AChE would potentially improve the stability of the enzyme,rather than change its structure and function.The mechanism of stability improvement of site-specific immobilization AChE was clarified by combining with experiment data.