Dye-affinity Membrane Research Articles

Kinetic and Thermodynamic Studies of Lysozyme Adsorption on Cibacron Blue F3GA Dye-Ligand Immobilized on Aminated Nanofiber Membrane.

The polyacrylonitrile (PAN) nanofiber membrane was prepared by the electrospinning technique. The nitrile group on the PAN nanofiber surface was oxidized to carboxyl group by alkaline hydrolysis. The carboxylic group on the membrane surface was then converted to dye affinity membrane through reaction with ethylenediamine (EDA) and Cibacron Blue F3GA, sequentially. The adsorption characteristics of lysozyme onto the dye ligand affinity nanofiber membrane (namely P-EDA-Dye) were investigated under various conditions (e.g., adsorption pH, EDA coupling concentration, lysozyme concentration, ionic strength, and temperature). Optimum experimental parameters were determined to be pH 7.5, a coupling concentration of EDA 40 μmol/mL, and an immobilization density of dye 267.19 mg/g membrane. To understand the mechanism of adsorption and possible rate controlling steps, a pseudo first-order, a pseudo second-order, and the Elovich models were first used to describe the experimental kinetic data. Equilibrium isotherms for the adsorption of lysozyme onto P-EDA-Dye nanofiber membrane were determined experimentally in this work. Our kinetic analysis on the adsorption of lysozyme onto P-EDA-Dye nanofiber membranes revealed that the pseudo second-order rate equation was favorable. The experimental data were satisfactorily fitted by the Langmuir isotherm model, and the thermodynamic parameters including the free energy change, enthalpy change, and entropy change of adsorption were also determined accordingly. Our results indicated that the free energy change had a negative value, suggesting that the adsorption process occurred spontaneously. Moreover, after five cycles of reuse, P-EDA-Dye nanofiber membranes still showed promising efficiency of lysozyme adsorption.

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 °C, and initial papain concentration 11.0 mg/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 1.0 M 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.

Adsorption of papain on dye affinity membranes: Isotherm, kinetic, and thermodynamic analysis

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, and 310 K was investigated. The experimental data were analyzed using two adsorption kinetic models: the pseudo-first-order and the pseudo-second-order. 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: Δ G°, Δ H°, and Δ S° 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.

Rapid neutral protease purification by dye-affinity membrane chromatography

Dye-affinity hollow fibres were prepared by radiation-induced grafting of polymers with different hydrophilicity degree on to the membranes and dye attachment after reaction of the grafted epoxy groups with ammonia to produce amino groups. Membranes grafted with glycidyl methacrylate/dimethyl acrylamide (GMA/DMAA) 1/3 showed better chromatographic properties than those grafted with GMA. The dye Yellow HE-4R was selected for purification of a neutral protease from Flavourzyme™ on the basis of the maximum capacity of the membranes obtained. No leakage of the dye from the membranes was evidenced after incubation in 8 M urea in 0.5 M NaOH for 2 months. From isotherms developed with membranes grafted with GMA/DMAA 1/3, a maximum capacity of 24220 U/ml membrane and a dissociation constant of 91 U/ml was calculated. Elution with 2 M NaCl at pH 4 allowed quantitative recovery of the adsorbed protease. A yield of 72% with a purification factor of 3.7 was achieved by working at 5 ml/min for the load, wash and regeneration steps and 1 ml/min for the elution step. Under these conditions, productivity was 1828 U/h ml, far higher than that attained with the same dye attached to a soft gel: 304 U/h ml [Iannucci NB, Navarro del Cañizo AA, Cascone O. Purification of neutral protease by dye-affinity chromatography. Appl Biochem Biotechnol 2003;104:173–84].

Comparison of Breakthrough Performance Using Dye-Affinity Membrane Disks and Gel Bead Columns

The breakthrough curve performance of lysozyme and bovine serum albumin to immobilized Cibacron Blue 3GA using different solid supports such as gel beads and membrane disks was investigated in this work. The effects of flow rate and different module designs were also studied. Variation in the flow rate was found to be insignificant for the column process in both nonadsorption and single-protein experiments, but it affected the elution peak height for membrane disks. The peak height decreased with increasing flow rate. As for the effect of different designs, a long column and a wide membrane stack induced a broader breakthrough performance. The performance varied with different solid supports in two-protein experiments. Competitive adsorption occurred for a gel bead column, and the breakthrough curve performance resembled the prediction of combining local equilibrium theory and the extra module effect. The affinity strength to the gel bead support is in the order lysozyme BSA dimer BSA monomer. As for membrane performance, two BSA solutes did not adsorb onto membranes, so a simple separation of lysozyme from BSA, instead of displacement phenomena, was observed.

Comparison of Ligand Density and Protein Adsorption on Dye-Affinity Membranes Using Different Spacer Arms

The use of 1,6-diaminohexane, 1,10-diaminodecane, 1,6-diaminohexane + ethylene glycol diglycidyl ether (EGDGE), and 1,10-diaminodecane + EGDGE as spacer arms for the immobilization of Cibacron Blue 3GA onto Immobilon AV membranes was investigated in this work. The dye ligand density was optimized by the employment of 1,6-diaminohexane, followed by 1,10-diaminodecane, 1,10-diaminodecane + EGDGE, and 1,6-diaminohexane + EGDGE. The adsorption properties of lysozyme onto the dye-affinity membranes were also studied. The results show that the use of 1,10-diaminodecane as a spacer molecule appears to offer the best adsorption capacity and hence results in the highest percentage of ligand utilization. Consequently, a 12-atom straight molecule such as 1,10-diaminodecane is a good choice for dye-affinity membrane separations.