Elaboration, Characterization And Study Of A Novel Affinity Membrane Made From Electrospun Nanofibers For Papain Purification

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.Non-woven polymer membrane with nano- or micro-scaled fiber diameter prepared by electrospinning technique is a potential material for affinity membrane application. In our study, composite membranes of nylon-6/chitosan nanofibers with different weight ratio of nylon-6 to chitosan were fabricated successfully using electrospinning. At first, the history, the method, the theories, the influencing factors and the applications of electrospinning were introduced. In the experiments, the mixture of 1,1,1,3,3,3 hexafluoro-2-propanol(HFP) and formic acid (FA) (v/v 9/1) was found as an appropriate solvent for electrospinning of nylon-6 and chitosan blends, and the nylon-6/chitosan nanofibers were obtained by electrospinning for the first time. Moreover, the dependence of the fibers diameter on the fibers diameter on the electrospinning parameters (voltage, feed rate and distance), the concentration of solution and the ratio of nylon-6 to chitosan in the electrospun blends was studied.Morphologies of the nanofibers were investigated by scanning electron microscopy (SEM) and the intermolecular interactions of the nylon-6/chitosan complex were evaluated by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) as well as mechanical testing. We found that morphology and diameter of the nanofibers were influenced by the concentration of the solution and weight ratio of the blending component materials. Furthermore FT-IR analyses on interactions between components demonstrated an IR band frequency shift that appeared to be dependent on the amount of chitosan in the complex. Observations from XRD and DSC suggested that a new fraction of y phase crystals appeared and increased with the increasing content of chitosan in blends, this indicated that intermolecular interactions occurred between nylon-6 and chitosan. Results from performance data in mechanical showed that intermolecular interactions varied with varying chitosan content in the fibers. It was concluded that a new composite product was created and the stability of this system was attributed to strong new interactions such as hydrogen bond formation between the nylon-6 polymers and chitosan structures.The primary objective of this study is to create novel affinity membranes with ultrahigh protein binding capacities. In this study, nanofibrous membranes were prepared by nucleophilic reaction of the chitosan's hydroxyl and amidocyanogen with the triazinyl chloride of Cibacron Blue F3GA (CB) ligand. This system was used to study the purification of papain. Physical and chemical properties of the affinity membrane were characterized by scanning electron microscopy (SEM), fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimtry (DSC), contact angle (CA) and element analysis (EA). The equilibrium adsorption capacity (from Langmuir isotherm data) for papain was 93.46 mg/g affinity membrane. Fifteen layers of the composite affinity membrane were packed into a spin column to separate papain from raw material. Significant amount of the adsorbed papain (about 90.4%) was eluted by 1.0M NaSCN at pH 9.0, and 4.8-fold purification was achieved in a single step. Experiments on regeneration and dynamic adsorption were also performed. It is shown that this system has the potential to be developed for the industrial purification of the papain.