Preparation Of Nylon Affinity Membranes And Their Applications In Purification Of Papain And Cysteine Protease Inhibitors

In recent years, the membrane affinity chromatography has a fast development in the separation, purification and recovery of proteins and enzymes. Compared with the classical column chromatography, the affinity membrane chromatography possesses a number of advantages, such as higher flow rate, faster binding rate, lower pressure drop, higher productivity and easier scale-up.In our study, covalent coupling of chitosan (CS) to activated nylon membrane was performed after the reaction of the microporous nylon membrane with formaldehyde. Compared with plain nylon membrane, non-specific adsorption on the CS-coated nylon membrane decreased greatly. The quantity of CS-attached membranes were 89.6 mg/g nylon membrane.Papain (EC3.4.22.2) is a minor constituent (about 8%) among the papaya endopeptidases. The enzyme has found many application in cell isolation, leather, cosmetic, textiles, detergents, food and pharmaceutical industries. Purification of papain from papaya latex has traditionally been achieved by precipitation methods. However, the methods can not match the requirements of purity and scale. In this paper, the dye Cibacron Blue F3GA(CB F3GA) as a ligand was covalently immobilized on the CS-coated membranes. Physical properties of the composite membrane and its applications in affinity membrane chromatography were examined. The contents of CS and CB F3GA-attached membranes were 89.6 mg/g nylon membrane and 146.1μmol/g nylon membrane, respectively. These CB F3GA-attached composite membranes were used in the papain adsorption studies. Higher papain adsorption capacity, up to 235.3 mg/g affinity membrane, was obtained. Papain was purified from the crued papain by the dye affinity membrane, and the purify ratio is 46.5. Moreover, the adsorption of papain on the dye affinity membranes, the Cibacron Blue F3GA as a ligand and the chitosan-coated nylon membrane as a base, under batch equilibrium experimental conditions at 277, 298, 310 K was investigated. The experimental data was analyzed using two adsorption kinetic models: the pseudo first-order Lagergren and the pseudo second-order kinetics. The pseudo second-order kinetic model provided the better correlation to the experimental results. Studies showed that the adsorption of papain on dye affinity membranes could be described by the Freundlich isotherm. The thermodynamic constants of adsorption phenomena:△G~0,△H~0, and△S~0 were found as -12.58 kJ/mol (at 298 K), 17.81 kJ/mol, and 0.101 kJ/(mol K), respectively. The result shows that the adsorption of papain on the dye affinity membranes was endothermic and spontaneous.According the above method, the papain purified by the dye affinity membrane chromatography was used in affinity membrane chromatography again, which the papain was used as another ligand on the nylon affinity membrane to purify cystatin from potato juice.Cysteine protease inhibitor is a kind of Proteinase inhibitors which is widespread in the animal and plant tissues and body fluids. It can form an effective, non-covalent combination of competitive inhibition with papain, cathepsin Proteinase and restrain their activity, thereby, protecting that the protein's disulfide bonds is not damaged, stopping the degradation of proteins. Studies indicate that cysteine protease inhibitors have good biological defense role participating in various physiological and pathological processes, such as rheumatoid arthritis, kidney failure, osteoporosis and tumor invasion and metastasis. Plant cysteine protease inhibitors can inhibit pathogenic fungi such as Fusarium, Blast chemoheterotroth growth. In this thesis, flat nylon membrane was first used as a base, papain was coupled as a ligand on modified nylon membrane with chitosan, the conditions of preparing nylon affinity membranes was optimized, and physical properties of the composite membrane and its applications in affinity membrane chromatography were examined. The cysteine protease inhibitors with high purity were obtained from potato juice. Through the experiment, it can be concluded that modified nylon membrane with chitosan can be well used as a type of affinity membrane carrier. The method of using glutaraldehyde as a crosslinking agent is feasible. The nylon membrane with high reactivity was finally obtained according to this method. The optimized conditions of the papain immobilization on nylon membrane is as follows: pH 9.0, 0.5% glutaraldehyde solution, the loading capacity of papain was 9 ml (10 mg/ml papain solution) per 0.35 g carrier, the reaction time is 6 h at 45℃. Under the above optimized conditions, the cysteine protease inhibitors was purified from the potato juice. The purification ratio is 7.25.