The Effect Of Phosphorylation On The Molecular Characteristics And Functional Properties Of Ovalbumin And Study Of The Mechanism

Ovalbumin(OVA),the most abundant protein in egg white(54-60%),was the only protein contains free sulfhydryl group that resides inside the hydrophobic core of egg white.In this study,ovalbumin was modified by phosphorylation to improve the emulsifying activity,emulsifying stability and foam stability,as well as texture and rheological properties.The microstructures,crystal structures and thermal properties of phosphorylated ovalbumin were studied by SEM,XRD and DSC.The molecular conformation and intermolecular interactions of phosphorylated ovalbumin were explored by FTIR,FT-Raman,CD and fluorescence spectroscopy.The effects of phosphorylation on the molecular composition and structure of ovalbumin were investigated by ¹³C NMR,³¹P NMR,XPS and MALDI-TOF/MS.During this research,the mechanism of phosphorylation was investigated at atomic and molecular level in order to evaluate the quality improvements in ovalbumin.The results of aforementioned reseacrh provided theoretical basis and experimental support for the application of ovalbumin in food industry.The main results of this research were as follows:1.Based on the principle of ion exchange,two-step purification method was used to separate ovalbumin from egg white without using the chromatographic column.In order to significantly shorten the time required for purification,and increase the processing capacity of egg white and throughput of ovalbumin,the optimal conditions were as follows:The volume ratio of Q-Sepharose FF gel to the solution was saturated at 1:3,and the shortest time for Q-Sepharose FF gel to complete the adsorption of protein in egg white was 20 min.The protein adsorbed on Q-Sepharose FF gel was eluted with Tris-HCl buffer containing 0.08 mol/L Na Cl for 10 min followed by eluted with Tric-HCl buffer containing 0.15 mol/L Na Cl for 10 min.The purity of ovalbumin product was 97.84%and the recovery rate was 79.4%.Western Blot and ELISA showed high immunological activity for purified ovalbumin suggesting that the purification method was mild and retained the structural integrity of ovalbumin.2.The molecular characteristics of phosphorylated ovalbumin were studied.The effect of different reaction p H on the phosphorylation degree of OVA was studied.The phosphorylation of OVA showed the highest phosphorus content at p H 7.0.DSC and TGA results indicated that phosphorylation improved the thermal stability of ovalbumin.By grafting the phosphate group,the p I of OVA shifted to lower values from 4.723(N-OVA)to 4.398(P-OVA7)as the degree of phosphorylation increased.The result of SDS-PAGE showed that the electrophoretic mobility of P-OVA increased.The surface hydrophobicity of P-OVA5 was significantly improved due to the expose of internal hydrophobic group,while the surface hydrophobicity of P-OVA7 and P-OVA5 was lower than that of N-OVA.The molecular average particle size D[4,3]of P-OVA7 and P-OVA9 decreased,and SEM further demonstrated that the particle size of the ovalbumin was reduced after phosphorylation,and the spatial three-dimensional structure of the natural ovalbumin was well maintained by phosphorylation.3.The structure-function relationship of phosphorylated ovalbumin was systematically analyzed.FTIR and Raman spectra indicated that the phosphate group was successfully grafted onto the OVA molecule and formed P-O bond,and Raman spectroscopy indicated that due to the grafting of negatively charged phosphate groups,the polarity of microenvironment of tyrosine residues and carbon chain backbone of OVA was increased.Moreover,the amide I band of the infrared spectrum was subjected to Gaussian fitting and second derivative,and it was found that the secondary structure of solid-state N-and P-OVA was mainly?-sheet,and the random coil content was low.The circular dichroism indicated that the secondary structure of N-and P-OVA in the solution was mainly random coil.The UV absorption of P-OVA7 and P-OVA9 at?280nm decreased due to the shielding effect of the phosphate group.In addition,the UV second-order spectroscopy of P-OVA7 and P-OVA9 were blue-shifted,indicating that the tyrosine and tryptophan residues were more hydrophilic.XRD analysis showed that the number of diffraction peaks after phosphorylation were decreased and the peak intensity increased,indicating that the OVA molecules were rearranged and directional alignment,resulting in larger crystalline grain.4.The interfacial properties of phosphorylated ovalbumin were studied.The absolute value of the zeta potential of the P-OVA emulsion increased and the average particle size D[4,3]decreased.Moreover,the stability of the emulsion was examined by storage at 60min,the D[4,3]of N-OVA emulsion increased significantly during the 60-min storage.The corresponding particle size distribution showed that the water and oil separation occurred due to the destabilization of emulsion,while the D[4,3]of P-OVA5 emulsion did not increase during the 60-min storage.The particle size distribution showed single peak with uniform distribution,indicating that P-OVA5 had better emulsion stability,corresponding to higher ESI values.In addition,P-OVA exhibited a higher EAI value than N-OVA,indicating an increased emulsifying activity of ovalbumin after phosphorylation.Futhermore,the observation of the emulsion microstructure showed that the particle size of the P-OVA decreased,showing a more dense and uniform distribution compared with N-OVA emulsion.On the other hand,P-OVA9 has a lower interfacial tension at the oil/water interface compared with N-OVA.OVA phosphorylated at different p H showed obviously different interfacial behavior in the air/water interface,but the foaming ability and the foam stability of OVA were both improved by phosphorylation.5.The texture and rheological properties of N-OVA and P-OVA heat-induced gels were improved with increased protein concentration.The results showed that the heat-induced gel with the highest degree of phosphorylation(P-OVA7)demonstrated the greatest hardness,stickiness,viscoelastic properties and viscosity,and the weak frequency-dependence of P-OVA7 gel showed a stable network structure.The increase in gel point of P-OVA5 indicated that phosphorylation promoted the formation of the protein gel network at lower temperatures.Moreover,creep-recovery tests showed that the compliance of heat-induced gel was decreased and the stiffness was increased by phosphorylation,which indicated increased resistance of P-OVA heat-induced gel against mechanical deformation.These resulted in the decrease of maximum mechanical deformation(JMax)and irreversible deformation(J?),and the recovery of gels was improved by phosphorylation.In addition,SEM showed that the structure of N-OVA heat-induced gel had larger pores and was prone to irreversible deformation,whereas P-OVA7 and P-OVA9 heat-induced gels exhibited firm gel networks and therefore had better mechanical properties.On the other hand,the results of LF-NMR showed that the T2 relaxation time of P-OVA7 heat-induced gel was reduced than that of N-OVA,which indicated the gel structure was enhanced with the formation of a more dense network by phosphorylation,thereby improving the water holding capacity of the OVA gel.And the proton density-weighted image of the heat-induced gels further confirmed the improvement of water holding capacity by phosphorylation.6.The effect of phosphorylation on the specific binding of ovalbumin to IgG was explored.Amino acid composition analysis showed that the amino acid content of OVA changed after phosphorylation,leading to the change of OVA epitope.The optimum reaction conditions of indirect ELISA were as follows:the working concentrations of antibody and anti-antibody were 1:17500 and 1:250,respectively,and the antigen coating concentration was determined to be 0.4?g/m L.The results of indirect ELISA showed that the antigenicity of P-OVA7 and P-OVA9 decreased compared to N-OVA,while the absorbance of P-OVA5 increased.Western Blot showed that the P-OVA band narrowed and the corresponding antigenicity decreased slightly.On the other hand,the OVA biosensors were prepared by modified glassy carbon electrode with oxidized graphene,and the surface of the electrode was then activated by EDC/NHS and covalently coupled with rabbit anti-OVA IgG.The results of cyclic voltammetry,differential pulse voltammetry and AC impedance test showed that the specific binding of phosphorylated ovalbumin to IgG was reduced and the antigenicity was decreased.7.The molecular mechanism of OVA phosphorylation was revealed by ¹³C NMR,³¹P NMR,XPS and MALDI-TOF/MS.The results of ³¹P NMR,XPS and MALDI-TOF/MS showed that the phosphate group was successfully grafted onto the OVA molecule through one-step method.The surface phosphorus element content of OVA molecule increased by phosphorylation,and the formation of the P-O bond led to a change in O_1Sbinding energy.In addition,the phosphorylation sites of P-OVA5,P-OVA7 and P-OVA9were 23,21 and 18,respectively.The STPP directed to the-OH of Ser and Thr residues in OVA molecules through esterification,and phosphorylation occured mainly in Ser residues.Futhermore,the phosphorylation of Ser353 and Ser355 on the natural phosphopeptides of OVA was first identified by MALDI-TOF/MS.The precise localization of phosphorylation by ¹³C NMR suggested that phosphorylation mainly occured on the C atom of?CH2 of the Ser residues of the OVA molecule.Since the negatively charged phosphate group was introduced into the OVA molecule,the isoelectric point of the OVA molecule was shifted to the low p H value,the surface tension was decreased and the intermolecular repulsion was increased,which resulted in the increase of emulsifying activity and the foam stability of the phosphorylated OVA,thus improving the interface performance and gel properties.