Affinity Chromatography based on Cellulose Membrane
  
 

 

  
 

 

  
   
   
   
   
   
   
   
   
   
   
   
   
   
     
     
     
 
  •  Membrane Preparation

    Mercerized macroporous cellulose membranes with large pore sizes (0.5~1 micron) and high porosity (about 55%) were prepared from filter paper by Mercerization (caustic treatment) followed by chemical crosslinking.
 
     
     
 
  •  Effect of Mercerization

    During Mercerization, swelling occurred, and some crystalline I domains were converted into crystalline II and amorphous domains. The following shows that during Mercerization the diameter of the cellulose fiber increased and that its smoothness was reduced. The figure also shows that the thickness of a Mercerized membrane expanded. However, when the membranes were packed into a cartridge under pressure, the thickness of the Mercerized membrane became even somewhat smaller than that of the original non-Mercerized membrane. This indicates that Mercerization increased the softness of the fibers.
  
 
 
Micrographs of crosslinked cellulose membranes
(a) (c) before Mercerization, (b) (d) after Mercerization
  
     
 

Stabilities of Immobilized trypsins on Mercerized and non-Mercerized Membranes
 
     
     
 
  •  Effect of Crosslinking

    The woven fibers of cellulose became by this means linked each other and fixed firmly. Compared with its precursor, the product possesses a tighter structure with less free fibers.
 
 

(A)

(B)
 
 
 The SEM Photos of Cellulose Membrane (B) and Its Precursor (A).
  
     
     
 
  • Biocompatibility of Prepared Membrane

    The stability of the immobilized trypsin, determined as indicated in the experimental part, was investigated and the results are presented in the following figure. The experiments were performed in severe conditions for the immobilized enzyme, namely, the room temperature. The immobilized trypsins, prepared using the three activation methods, were very stable during at least one week, indicating that the Mercerized crosslinked cellulose membrane provided a good matrix for affinity chromatography. The immobilized trypsin prepared by the epoxy method possessed the highest stability, most likely because it also exhibited the lowest activity and hence was less subjected to change.
  
 

Stability of Immobilized Trypsin on Cellulose Membrane
  
     
     
 
  • Immobilization of Concanavalin A
  
 

Immobilization of Concanavalin A by (a) Triazine, (b) Glutaraldehyde and (c) Diazotization Methods
  
     
     
 
  • Immobilization of Enzyme
  
 

Immobilization of Enzyme by (a) Epoxy, (b) Glutaraldehyde and (c) Diazotization methods
  
     
     
 
  •  Immobilization of Maltose
  
 

Immobilization of Maltose via Spacers of
(a) 5-atom, (b) 7-atom, (c) 10-atom and (d) 12-atom
  
     
     
 
  • Purification of Ovamucoide from Egg White

    Trypsin immobilized affinity membrane was used for the separation of trypsin inhibitor (ovomucoide) from the egg white, and a product with high purity was obtained. This procedure is advantageous because the membranes possess fairly large pore sizes and porosities, and only a simple pretreatment (centrifugation) was necessary to remove the insoluble from the solution of egg white.
 
 


 
     
     
 
  •  Separation of Papain Inhibitor from Potato Tuber

    A wide elution peak of the papain inhibitors is present in following chromatogram, from which three fractions at 70, 80 and 90 min were selected to examine their purity by electrophoresis (Sample B, C and D in electrophoretic result). As shown in electrophoresis, the elution peak involves at least three types of papain inhibitors. Because the amount of impurities became smaller in the later stages of elution, the final purity of the papain inhibitors was enhanced.
 
 
 
  
     
     
 
  • Separation of Peroxidase from Horseradish 

    Compared to column chromatography, the method using concanavalin A affinity membranes has three main advantages: (1) A single chromatographic procedure is involved; (2) The affinity membranes employed are more stable; and (3) A higher loss of enzyme activity is expected to occur during the ion exchange chromatography than during dialysis.
 
 

Separation of Peroxidase from Horseradish
by Concanavalin A Affinity Membrane
  
     
     
 
  • Purification of Concanavalin A 

    As an application, the commercially available crude con A was purified. Con A is a common affinity ligand that is mostly used to separate saccharides containing molecules, such as glycoproteins, some enzymes, IgGs, interferons and some protein hormones. In this paper, the crude con A was purified by using a membrane activated with 1,4-butanediol diglycidyl ether, upon which maltose was immobilized. The chromatogram of con A is presented in the following figure, where the shape of con A peak indicates a good separation efficiency. The quality of the purification achieved was determined by electrophoresis. The results show that the purified con A exhibits a single band.
 
 


 
     
     
 
  • Purification of Phosphatase

    Triazine dyes can serve as an analogue for nucleotide so that a protein will bind them at nucleotide binding sites. They have been used successfully in the purification of many enzymes, especially kinases and dehydrogenases. The phosphatase can be purified on dye immobilized medium. In this work, commercial alkaline phosphatase from calf intestine was purified on the affinity membrane cartridge containing 80 sheets red cellulose membranes. The phosphatase was eluted by 1 M NaCl, and an unknown component with stronger affinity to the ligand was eluted by 60% glycol.The recovery of alkaline phosphatase activity was 60% and a 40-fold purification was achieved.
 
     
     
 
  • Selfcleaning of Trypsin Affinity Membrane
  
  Since the immobilized trypsin exhibits a high activity and stability, the affinity membranes can self-clean themselves, after their permeability was decreased by fouling. In this paper the self-cleaning could be easily carried out by incubating the cartridge in a suitable buffer, which, activating the immobilized enzyme, could hydrolyze the proteins retained inside the membrane. The permeability of the cartridge could be thus restored to its original level.