Preparation of A pH-sensitive Polymer Grafted with Different Metal Ligands and the Purification of Papain
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Abstract:
A pH-sensitive polymer (PMDMN) was synthesized from four types of acrylic monomers. Polymer PMDMN-Cu or PMDMN-Fe was obtained after PMDMN was attached to a metal chelating agent, and then chelated with Cu2+ or Fe3+ via a coordination bond. First, the infrared spectra, isoelectric point, and recovery rate of the polymer were determined. Second, the adsorption conditions of PMDMN-Cu and PMDMN-Fe on the crude papain, including pH, time, and NaCl concentration, were explored. Based on the measurement of enzyme activity recovery rate, the elution efficacies of imidazole, guanidine hydrochloride, and ethylenediaminetetraacetic acid and their combinations were evaluated. Finally, enzyme purity under optimal elution conditions was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the isoelectric points before and after grafting of the metal ligand were 5.15 and 5.08, respectively; the recovery rate was higher than 95% after five adsorption-elution cycles. The optimum pH value for adsorption by PMDMN-Cu or PMDMN-Fe was 7.0, and PMDMN-Cu and PMDMN-Fe reached adsorption equilibrium at 120 and 210 min, respectively, with maximum adsorption capacities of 5.09 and 4.58 mg/g, respectively. The amount of adsorbed papain increased with increasing NaCl concentration to some extent. In all elution methods, the combination of 0.5 M imidazole with 1.0 M guanidine hydrochloride showed the best elution effect, with an elution rate close to 95% and without metal ligand leakage. The electrophoresis results revealed that both PMDMN-Cu and PMDMN-Fe largely improved the purity of papain, with purification folds of 4.29 and 4.48, respectively.
