Oriented Immobilization Of Alcohol Dehydrogenase By Magnetic Nanoparticles And Its Application

Magnetic nanoparticles(MNPs)have become one of the research emphases in recent years.Conventional covalent immobilization method is to link the amino acid residues(as immobilization sites)of enzymes with the surface functionalized MNPs.It was usually to randomly select the immobilization active site,which would greatly increase the uncertainty of the immobilization,and made it difficult to control the immobilization effects.Therefore,it is necessary to explore an oriented immobilization method with simple operation and high feasibility.Compared with free enzymes,the immobilized enzymes have the advantages of high stability,easy separation and reusability.In addition,the immobilized enzymes can also be used for Ligand fishing(LF)that is a new method for identifying and screening enzyme active ligands.Tea saponins(TSs)are mixtures of triterpenoid saponins,which have been reported that could inhibit the activity of alcohol dehydrogenase(ADH).But the troubling thing is that TSs include nearly 200 kinds of molecular structures reported in Camellia.The activity of different TSs monomers varies greatly,even though their polarities are very similar.Thus,it makes the separation,purification and structure identification of active compound in TSs complicated.In this study,protein bioinformatics and 3D visualization software VMD were combined to analyze the optimal immobilization active site of ADH,and successfully applied in immobilized alcohol dehydrogenase(IADH)by MNPs materials with the surface amino functional modification.And then IADH was employed to magnetic fishing active ligands from TSs.The conclusions of this paper are as follows:(1)Preparation and characterization of surface amino functionalized magnetic nanoparticles(AMNPs).MNPs with particle size of 105.13±4.67 nm were synthetized via a solvothermal method,and then the surface was coated with Si O₂shell,finally APTES was used to prepare amino groups terminated MNPs.It was revealed by FT-IR spectrum that AMNPs had characteristic absorption peaks of Fe-O bond,Si-O bond and N-H bond,indicating that AMNPs was successfully prepared and could be used as carrier materials for ADH immobilization.The zeta potential increased from 7.81±0.10MV(MNPs)to 40.37±0.45 MV(AMNPs)also confirmed the successful aminated modification of MNPs.The micro-morphology of MNPs observed by SEM and TEM had uniform particle size distribution and good dispersion.In the TEM image,the MNPs core and Si O₂shell could be clearly distinguished,and the thickness of the Si O₂shell was about 15 nm.The results obtained by DLS,SEM and TEM were consistent.(2)Determination of the optimal immobilization active site and oriented immobilization of ADH.The amount and three-dimensional distribution of different active groups(hydroxyl,carboxyl,amino,phenyl,imidazolyl,sulfydryl,guanidyl,and indolyl)in ADH were analyzed by using protein bioinformatics and 3D visualization software VMD.The free?-NH₂ groups were concentrated on the outer surface of the ADH structure and were far away from the active catalytic center besides the number of medium proportion(10.37%),which met the basic requirements of immobilization sites.Hence,the free?-NH₂ groups were selected as the best active group for immobilization of ADH.Based on these,two different immobilization active sites were selected for experimental verification.The results showed that the relative activity of IADH obtained by free?-NH₂ groups reaction was(65.53±1.31)%,while that obtained by free?-COOH groups reaction was(45.35±3.73)%,which the relative activity of the former was 1.45 times of the latter.The experimental results were consistent with the theoretical prediction,which indicated that the research of enzyme bioinformatics could guide the oriented immobilization of enzyme.(3)The performance and characterization of IADH.The optimum catalytic p H of IADH was p H 8.8,which was consistent with that of FADH;the optimum catalytic temperature of IADH was 30?,which was 5?higher than that of FADH.The Michaelis constant K_m and activation energy Ea of IADH were 41.23 mmol/L and 17.58k J/mol,respectively.After immobilization,the p H stability,thermal stability and storage stability of IADH were significantly improved.The relative activity of IADH treated at p H 10.0 for 60 min,was 1.58 times higher than that of FADH,and the relative activity of IADH treated at 55?for 60 min was 4.43 times higher than that of FADH.Under the room temperature(25?),the activity of FADH decreased to 0 on the 5th day,while the relative activity of IADH still retained(10.07±0.11)%on the 10th day.The saturation magnetization of IADH was 34.54 emu/g,which indicated it possessed superparamagnetism.And the relative enzyme activity of IADH remained(46.88±2.70)%after continuous use for 10 batches,which showed IADH could achieve rapid magnetic separation and recycling.(4)Using IADH magnetic fish active ligands(LTSs)from TSs and investigating the interaction between LTSs and ADH.The LTSs were analyzed by LC-TOF-MS/MS,and three kinds of tea saponin monomers were obtained.Two of them were confirmed to be C₅₉H₉₂O₂₆.LTs exhibited ADH inhibitory ability with IC₅₀ was 4.97×10^-5 mol/L and it was mixed type ADH inhibitors.The fluorescence spectrum showed that the interaction between LTSs and ADH could result in static fluorescence quenching of ADH,and the value of static quenching constant K_SV and binding constant K_bwas found to be 6.17×10³L/mol and 2.26×10³L/mol at 25?,respectively.Negative Gibb's free energy change(?G^?)and positive entropy change(?S^?)were suggested that binding process between LTSs and ADH were spontaneous and entropy driven.The analysis of 3D fluorescence indicated that the interaction between LTSs and ADH could lead to the change of ADH conformation,which might be the reason for the decrease of ADH activity.