接枝不同金属配基的pH敏感型聚合物的制备及对木瓜蛋白酶的纯化

本文以四种丙烯酸类单体合成了一种pH敏感型聚合物(PMDMN)。该聚合物连接金属螯合剂(IDA)后与Cu~(2+)或Fe~(3+)通过配位键形成聚合物PMDMN-Cu或PMDMN-Fe。首先对聚合物的红外光谱、等电点及回收率进行了测定。其次,探讨了PMDMN-Cu、PMDMN-Fe对木瓜蛋白酶粗酶的吸附条件,包括pH、时间及NaCl浓度。通过对洗脱液的酶活回收率的测定,考察了咪唑、盐酸胍、EDTA及其浓度配比的洗脱效果。最后通过SDS-GAGE对最优洗脱条件的酶液纯度进行了验证。结果显示:接枝金属配基前后的等电点分别为5.15或5.08,回收率皆大于95%(5个吸附-洗脱循环后);PMDMN-Cu、PMDMN-Fe最适吸附pH值均为7.0,并于120 min和210 min达到吸附平衡,最大吸附量分别为5.09 mg、4.58 mg,NaCl浓度的增加能一定程度增加木瓜蛋白酶的吸附量。在洗脱方案中,0.5 mol/L咪唑+1.0 mol/L盐酸胍具有最好的洗脱效果,洗脱率皆接近95%,同时不会造成金属配基的泄露。SDS-PAGE显示PMDMN-Cu和PMDMN-Fe都能大幅度提高木瓜蛋白酶的纯度,纯化倍数分别为4.29和4.48。

Preparation of A pH-sensitive Polymer Grafted with Different Metal Ligands and the Purification of Papain

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.