Study On The Stabilization Method And Mechanism Of DhaA Adsorbed In Mesocellular Foam

The in-depth understanding of the structure and catalytic performance of the alkylhalide dehalogenase DhaA has made it possible to develop a green enzymatic decontamination pathway of the chemical agent mustard gas?HD?,but the poor stability of DhaA is still a bottleneck problem that restricts the application.The immobilization using silicon-based mesoporous materials is a general technique for the stabilization of biological enzymes.However,due to the large differences in structural characterization among natural enzymes,Universal strategy of porous carrier design,synthesis and modification based on the specificity of different enzymes has not yet been designed and formed,As a result,the research on DhaA immobilization by adsorption and its practical application in the field of chemical agent decontamination has not been carried out.In this thesis,the interaction between mesocellular foam?MCF?and DhaA was investigated.the adsorption process of DhaA into MCF was studied by computer simulation-experimental verification for the first time.The functional group modification of MCF with was guided by this method.The mechanism of DhaA stabilization was discussed.Based on the study of denaturation process and thermodynamic parameters of DhaA after adsorption into modified MCFs,a novel?sandwich-like model?was designed,which further enhanced the stability of DhaA.The main research contents and innovative findings of the thesis are as follows:1.Study on the preparation of MCF and its interaction with DhaAConsidering molecular size of DhaA,2D channel-structured SBAs and 3D cage-structured MCFs serials carriers were prepared by hydrothermal synthesis method.The adsorption behaviors of DhaA in these two types of adsorption carriers like adsorption kinetics,saturated adsorption/desorption amounts,adsorption at different pH values were investigated.The results showed that the adsorption of DhaA by MCF with large pore size and open structure was better.The adsorption process of DhaA in MCF was systematically studied.It was seen that the rate-determining step of this adsorption process was intraparticle diffusion,which was influenced by electrostatic repulsion between DhaA and MCF.The residual catalytic activity of the adsorbed DhaA was only 12.4%.The result of fluorescence spectrum showed that the reduction of catalytic activity was due to the conformational change of DhaA caused by electrostatic repulsion during adsorption.2.Study on the interaction between modified MCF and DhaAParallel tempering Monte Carlo?PTMC?method was adopted to study the predominant adsorption orientations of DhaA on different charged surface.The orientations were further computed by conventional all-atom molecular dynamics simulation.The simulation results showed that conformation or tunnel of DhaA changed after being adsorbed on the functional surfaces of COOH,SO₃ and CH₃,which is not convenient for substrate entry.After being adsorbed on the NH₂ surface with a dissociation degree of 25%,the tunnel of DhaA suffered structural change because of the high binding energy.Meanwhile,the hydrogen bond couldn't be formed between Glu130 and His272,which affected catalytic activity of DhaA.The NH₂ surface with a dissociation degree of 7%adsorbed DhaA through electrostatic attraction with Asp76,Asp78 and Asp156.After adsorption,the tunnel faced the surface.However,the conformation maintained,and the changes of structural tunnel facilitated catalytic reaction.To verify the simulation results,MCF was grafted with amino,methyl and sulfonic acid groups,which made its zeta potential to be+3.2mV,-2.9mV,and-23.8mV respectively.All the modified MCFs kept their structural function for DhaA adsorption.From the intrinsic fluorescence spectra of DhaA,it can be deduced that the conformation of DhaA altered being adsorbed in SO₃H-MCF and CH₃-MCF.The corresponding catalytic activities of DhaA decreased to 5.5%and 7.2%.On the other hand,the native structure of DhaA adsorbed into NH₂-MCF was well preserved,and the catalytic activity largely maintained.These experimental results were consisted with the previous simulation conclusion.More importantly,after being adsorbed into NH₂-MCF,the stability of DhaA had significantly improved in extreme denaturation conditions.3.Study on the stabilization mechanism of DhaA adsorption into NH₂-MCFThe denaturation mechanisms of DhaA induced by urea and DMSO were analyzed by means of fluorescence spectroscopy and molecular dynamics simulation.It was found that urea molecules can replace water molecules in hydrated layer around DhaA and form H-bonds with DhaA,which made main tunnel lengthened,curvature enhanced and bottleneck radius reduced.The denaturation process of DhaA induced by urea conformed to?two-state?model.On the contrary,it can be seen that the DMSO molecules couldn't disrupt the hydrogen bonds between DhaA and water molecules,but can entered into the hydrophobic cavity of DhaA.This caused main tunnel shortened,curvature reduced,bottleneck radius enhanced,conformation changed,which eventually unfolded DhaA.The denaturation process of DhaA induced by DMSO conformed to?three-state?model.Adsorption into NH₂-MCF had not changed the denaturation process of DhaA in urea or DMSO,but influenced the thermodynamic parameters of denaturation.In urea solution,electrostatic force and hydrogen bonds formed by DhaA and NH₂-MCF hindered the binding of urea molecules effectively,finally increased the value of?G?H₂O?.In DMSO solution,the polar microenvironment in channel of NH₂-MCF effectively inhibited the entry of non-polar DMSO molecules,and reduced the solvent-accessible surface area of DhaA in denaturation process.4.Study on the PEGylation of DhaA adsorbed in NH₂-MCFDhaA was firstly absorbed into NH₂-MCF.Then the MCF-absorbed DhaA?MD?was intermolecularly crosslinked with 8-arm PEG N-hydroxysuccinimide ester,enhancing the interactions between DhaA and NH₂-MCF.Then PEGylation by maleimide-thiol chemistry was used to provide a hydrated layer around DhaA.The results indicated after crosslinking and PEGylation,the leakage ratio of DhaA in NH₂-MCF reduced that from 14.6%to 1.1%.The enzymatic activity of DhaA was maintained?83.7%of native activity?.The k_cat/K_m value of DhaA increased from 0.450s^-1·?mmol/L?^-11 to 0.575 s^-1·?mmol/L?^-1.Meanwhile,after crosslinking and PEGylation,DhaA exhibited higher stability in the extreme environmental conditions,as reflected by the remaining activity of 99.8%in 40%?v/v?DMSO for 5 h,87.3%in 3 mol/L urea solution for 1 h,25.9%at pH 3.0,and 51.8%at room temperature for 30 days.Thus,our study was expected to develop an effective immobilized DhaA for practical applications.