Preparation Of New Affinity Absorbents And Study On Its Adsorption Mechanism Based On Papain Model

Affinity chromatography is a technology of separation and purification, by taking advantage of specific affinity between biological macromolecules and the solid phase surface. With the development of high-performance affinity chromatography, it has been applied in organic chemistry, biochemistry, molecular biology, bioengineering, genetic engineering and other fields. However, due to its expensive affinity ligands, high operating pressure, small handling capacity and other shortcomings, it is difficult to play a much more significant role under the premise of low-cost and high efficiency. To overcome this limit, the developments of the small molecules similar to affinity ligands and new low-cost carrier materials have been widely investigated. More and more scholars are pay attention on screening ligands through a combination method and improving the existing materials, but the former method greatly increases the workload and the cost, and the latter method is unable to overcome the dilemma of complicated operation and small handling capacity. There are good solutions to these problems by adopting dye and metal ion as affinity ligand, or screening for the ligands by biopanning, which could separate the targets proteins rapidly and efficiently and keep the targets high activity. In this regards, the dye and biomimetic affinity chromatography are be concerned by lots of scholars.Using papain as model protein, two affinity chromatography medium were prepared by chemical and biological methods:dye affinity membrane and cross-linking affinity phage oligopeptide pellet. Adsorption capacity and adsorption mechanism had been studied and the effectiveness of the separation and purification of model protein had been evaluated.Firstly, using nylon membranes with good mechanical strength as the carrier, covalent coupling of chitosan (CS) to activated nylon membrane was performed after the reaction of the microporous nylon membrane with formaldehyde. The dye Reactive Red120as a ligand was then covalently immobilized on the CS-coated membranes. The dye affinity membranes were characterizied by SEM, UV spectroscopy, FT-IR and elemental analysis. The results showed the chitosan content of dye affinity membranes was determined of118.5±7.5mg/g and Reactive dye Reactive Red120was successfully bond to the modified nylon membrane with the load capacity of163.4±10.3μmol/g. SEM showed the pore sizes of affinity membrane were distributed in the range of about0.5-3.0μm, which facilitated mass transfer. So the dye affinity membrane could be used as an ideal separation and purification media.The protein adsorption behavior and capacity of the new dye affinity membrane absorbent were studied by static and dynamic method, using papain as a model protein. By testing the different concentrations of ligand, pH, Na ionic strength, temperature and adsorption time, a nice result was achieved:at the circumstances of pH8.5,0M Na ionic strength,37℃, maximum static adsorption of papain was reached to37mg/g after3h. Dynamic testing included elution rate, pH, Na ionic strength and other factors. It showed in the condition of pH6.0,1M ionic strength, elution rate of1mL/min, the papain with the highest activity (1035.53U/mg) would be eluted with20-fold purification.Adsorption mechanisms of the dye affinity membrane were described by adoption of adsorption isotherm model, adsorption kinetics model, adsorption thennodynamic model and computer simulation study. Adsorption isotherm analysis was identified by Langmuir model and Freundlich model. The linear fit of Freundlich model indicating that the adsorption type is a3-dimensional structure of multi-molecular layer adsorption model. The adsorption kinetics models studies were combined by Pseudo-first-order kinetic model, Pseudo-second-order kinetic model, Intra-particle diffusion model and the Elovich kinetic model. It was found that the Pseudo-second-order kinetic model can better explain the adsorption behavior, indicating that the adsorption capacity was always proportionate to the amounts of active sites on dye affinity membrane surface, once the active sites covered, the adsorption capacity decreased. The values of the standard enthalpy changes, the entropy changes and Gibbs free energy were18.52kJ/mol,0.077kJ/(mol. K),-4.43kJ/mol (298K), respectively. ΔH0value of18.52kJ/mol indicates that the adsorption process is an endothermic reaction, the adsorption capacity increased when temperature elevated and the adsorption force including chemical and physical effects. ΔS0value of0.077kJ/(mol. K) showed the desorption of water molecules occured before adsorption of papain, which named "solvent replacement role", and also confirmed the adsorption process of the system increased disorder. ΔG0value of-4.43kJ/mol (298K) proved that the adsorption behavior was spontaneous, and confirmed that the physical and chemical effects also exist in the adsorption behavior. Computer simulations using the Maestro9.0software also confirmed both the existing of physical (hydrophobic interactions) and chemical effects (hydrogen bonding).Papain as a model was purified by dye Reactive Red120affinity membrane. Papain was purified22.6-fold in a single step using Red120-CS-membrane, as determined by protein content and enzyme activity assays, meeting papain demands of leather, feed and other areas.Using7-mer peptides (Ile-Gln-Ser-Pro-His-Phe-Phe) from phage display library as ligand, glutaraldehyde as crosslinking agent and cross-linked phage as matrix, cross-linking affinity phage-oligopeptide pellet (CAPOP) was prepared. Qualitative analysis of enzyme-linked immunosorbent assay confirmed the7-mer peptides ligand have an affinity for papain with OD492value of1.9. Particle size of CAPOP was determined5-6μm by DLS and TEM, indicating the cross-linking successfully. The CAPOP absorbent have the advantages of specific adsorption, low cost and simple preparation.The adsorption capacity of CAPOP was studied by static adsorption, response surface methodology and dynamic desorption. The static adsorption mainly concerned on the buffer pH, Na ionic strength, the adsorption temperature and adsorption time. The maximum static adsorption capacity reached50mg/g with the optimization of conditions of pH7.0, Na ionic strength0M,25℃and2h. The RSM analysed the relationship between a variety of factors and adsorption capacity as well as establish a model. The maximum papain adsorption capacity reached55.4229mg/g under the conditions of the phage display amount of ligand1011PFU/mL, pH6.9,32℃and papain crude enzyme solution concentration7.51mg/mL. The dynamic desorption concerned about type, pH, Na ionic strength of eluent and the elution speed. A purification of200-fold with its enzyme activity4647.73U/mg was achieved under the dynamic desorption conditions of NaSCN, pH9.0, Na ionic strength1.0M, the elution speed1.0mL/min.Adsorption mechanisms of CAPOP were studied by adoption of adsorption isotherm model, adsorption kinetics model, adsorption thermodynamic model and computer simulation study. Adsorption isotherm analysis was identified by Langmuir model and Freundlich model. The study showed that the Freundlich model could better explain the adsorption behavior, indicating the adsorption type was a3-dimensional structure of multi-molecular layer adsorption model, which was consistent with the structure of cross-linked phage. The adsorption kinetics models studies were analysed by Pseudo-first-order kinetic model, Pseudo-second-order kinetic model and the Elovich kinetic model. Results showed that the Pseudo-second-order kinetic model could better explain the adsorption behavior, indicating that the adsorption capacity was always proportionate to the amounts of active sites on CAPOP. The values of the standard enthalpy changes, the entropy changes and Gibbs free energy were7.97kJ/mol,0.044kJ/(mol. K),-5.14kJ/mol (298K), respectively. ΔS0value of0.044kJ/(mol. K) showed that the desorption of water molecules occurs before adsorption of papain, which named "solvent replacement role", and also confirmed the adsorption process of the system increased disorder. ΔG0value of-5.14kJ/mol (298K) proved the adsorption behavior was spontaneous, and confirmed that the physical and chemical effects also exist in the adsorption behavior and play a major role. Above25℃, ΔH0value of-12.02kJ/mol, ΔS0value of-0.023kJ/(mol K), indicated that there was a "transition temperature" in adsorption system, which may be due to the new chemical bonds in adsorption process. But ΔG0value of-5.17kJ/mol (298K) still proved the adsorption behavior was spontaneous. Computer simulations using the Maestro9.0software also confirmed both the existing of physical (hydrophobic interactions) and chemical effects (hydrogen bonding).Papain was purified by CAPOP. Papain was purified240.94-fold in a single step using CAPOP, as determined by protein content and enzyme activity assays. It was confirmed CAPOP was an effective separation and purification absorbents with higher separation efficiency, which have the potential to meet the demands of medical, biological and biochemical engineering, antibody engineering and other fields.Inhibition mechanism and inhibition behavior of7-mer peptide affinity ligands for papain was studied. Finally it was confirmed the delay time was increasing with the concentration of7-mer peptide increasing, and the7-mer peptide molecule concentration (IC50) was14.2mM by Lineweaver-Burk double reciprocal model. When the catalytic system didn’t contain7-mer peptide molecules, the enzyme reaction delay time was165s, whiled the lag time was421s when the concentration of7-mer peptide molecular came to20mM. The results also showed that with the increasing of the7-mer peptide molecules concentration, the catalysis efficiency decreased, indicating that the activity was inhibited by the7-mer peptide molecules, rather than by reducing the amount of enzyme.7-mer peptide molecules on the activity of papain inhibition were identified as irreversible inhibition process.In summary, we prepared two kinds of affinity chromatography absorbents by chemical and biological methods. It could be expanded any other field of protein separation process including ligand screening, matrix preparation and the design of separation process. Through the study of this thesis, we wish to prodive fundamental data and theory guidence for the application of affinity chromatograph.