Tianxiang Chen

Optimization of adsorption conditions of papain on dye affinity membrane using response surface methodology.

The adsorption of papain on Reactive Blue 4 dye-ligand affinity membrane was investigated in a batch system. The combined effects of operating parameters such as initial pH, temperature, and initial papain concentration on the adsorption were analyzed using response surface methodology. The optimum adsorption conditions were determined as initial pH 7.05, temperature 39 degrees C, and initial papain concentration 11.0mg/ml. At optimum conditions, the adsorption capacity of dye-ligand affinity membrane for papain was found to be 27.85 mg/g after 120 min adsorption. The papain was purified 34.6-fold in a single step determined by fast protein liquid chromatography. More than 85% of the adsorbed papain was desorbed using 1.0M NaCl at pH 9.0 as the elution agent. The purification process showed that the dye-ligand immobilized composite membrane gave good separation of papain from aqueous solution.

Comparison: Adsorption of papain using immobilized dye ligands on affinity membranes

Novel membranes that involve the immobilization of Reactive Red 120 or Reactive Brown 10 as dye ligands were prepared. These were used in the purification of papain from papaya powder extracts. Papain adsorption capacities for the Red 120 and Brown 10 membranes were 143.6 mg/g and 107.3mg/g, respectively. The effectiveness of adsorption was demonstrated by Freundlich isotherm proficiency. The enzyme was eluted from the respective dye membranes using 1.0M NaCl at pH 6.0 and yields of over 80% were found for the Red 120-CS (chitosan)-nylon membrane whereas only a 50% recovery was possible using the Brown 10-CS-nylon membranes. It is concluded that Red 120-CS-nylon membranes could play an active role in the separation and purification of papain from crude extracts. This system has the potential to be developed for the commercial isolation of the protein.

Papain Immobilization on Chitosan-Coated Nylon Membrane: Preparation and Its Application in Cystatin Separation

Nylon membranes are first hydrolyed with dilute HCl and then treated with chitosan before being used as the affinity carrier. Papain as a ligand has been immobilized on the activated membranes with glutaraldehyde as a crosslinking agent. The factors involving with the activity of immobilized papain, such as concentration of glutaraldehyde, pH, temperature, reaction time, and the amount of added papain have been studied. The results show that the optimum conditions for the preparation of immobilized papain nylon affinity membranes are as follows: pH 9.0, 0.5% glutaraldehyde solution, the concentration of added papain is 10 mg/ml, the reaction time is 6 h at 45degC. The nylon membrane prepared is then used to purify cystatin from potato juice, and shows that they are high efficiency affinity membranes base for cystatin separation.

Novel Affinity Membrane Chromatography Technology: Purification of Pepsin Using Immobilized Reactive Dye Ligands

Novel membranes were prepared through covalently coupling of chitosan (CS) to activate nylon membrane after the reaction of the microporous nylon membrane with formaldehyde. Non-specific adsorption on the CS-coated nylon membrane decreased greatly. Yellow 3G-P/Reactive Red 120 were then covalently immobilized onto composite membrane as dye ligands. These Red 120/Yellow 3G-P-CS-membranes were used in the Pepsin adsorption studies. According to the results, it appears that Yellow 3G-P-CS-nylon membranes could be applied for separation and purification of Pepsin without causing any denaturation, much better than Red 120-CS-nylon membrane. Keywords-affinity chromatography; pepsin; adsorption;