Enzymes are manufactured in bioreactors for commercial use. These enzymes are in the crude form and have to be purified for further use. The extraction methods are followed by the purification processes. There are mainly three major purification methods depending on the technique or property of enzyme.
1. Based on ionic properties of enzymes
2. Based on the ability to get adsorbed
3. Based on difference in size of molecules
1. Techniques depending on the ionic properties of enzymes
a. Salting out
It is done by varying the pH of the solution or by addition of chemical agents which carry out precipitation.
At the isoelectric pH (pI), proteins have minimum solubility and hence get precipitated out as pure crystals. For example the digestive enzyme pepsin is purified by this process.
Salting out is also possible by addition of chemicals such as Ammonium sulphate, Acetone etc.
Normally used ions based on the decreasing order of ability to precipitate is given below
Anions - Citrate, Tartarate, Sulphate, Acetate, Chloride etc.
Cations - Th4+, Al 3+, Ba2+, Sr2+, Ca2+, Mg2+, NH4+
The series is also known as the Hofmeister series.
As the chemical agent gets dissolved in the aqueous phase 'salting in' takes place. Proteins get soluble during this phase. Once reaching a critical limit, the protein-protein interaction overcomes protein-water interactions as the agent displaces the water molecules. This results in salting out or precipitation of proteins/enzymes.
The major variable that determines the salting out is the ionic strength of the material given by
Î¼i = Â½ âˆ‘ CiZi2
Where Ci - concentration of ion; n - number of different species of ions; and Zi - charge of the ion.
It is the movement of charged particles under the influence of an electric field. The ions migrate based on the electric charge and strength of the field. The velocity of migration of ions in the electric field is given by
Ve = qE / 6Ï€ Î¼crp
q- Charge on the particle
E- Electrical potential
Î¼c - viscosity of the liquid
rp - radius of the particle
Hence the velocity of migration of charged particle is proportional to the charge since all other values of the equation are constants.
c. Ion exchange chromatography
Selective adsorption and exchange of ions take place in the adsorption sites of ion exchange columns filled with ion exchange resins.
Eg: DEAE (Diethyl Amino Ethyl) Cellulose is an Anion exchanger and DM (Dimethyl) Cellulose is a Cation exchanger.
2. Techniques depending on the adsorbing properties of enzymes
a. Adsorption chromatography
It is based on the principle of adsorption. Solute/ enzyme get adsorbed onto the particular sites of the adsorption chromatographic column depending on the effective distribution coefficient. These are then eluted using various solvents. The packing materials used include starch, diatomaceous earth etc.
Effective distribution coefficient is the ratio of distribution of solute across the different phases of chromatography. The process is widely used for initial recovery of extra cellular enzymes manufactured on a large scale.
b. Affinity chromatography
This technique makes use of enzyme substrate interactions. A matrix with a ligand is packed in the column. As the enzyme solution pass through the column, the solute/enzyme molecules get attached to the ligand. These are then eluted by suitable eluants/solvents. In effect the Matrix ligand enzyme complexes remain adsorbed to the column till it gets eluted.
3. Techniques depending on the size of enzymes
a. Molecular sieve/ Gel filtration/ Gel permeation Chromatography
This makes use of gel material as the supporting matrix. Smaller particles get entrapped in the pores whereas larger particles get through the interfacial space. Hence the larger particles get separated first followed by smaller particles
b. Ultra filtration (Dialysis)
It is the process of passage of solvent molecules from a region of higher concentration to a region of lower concentration through a semi permeable membrane. The technique is used for purification of crude enzymes. It is used for enzyme concentration as well as enzyme purification.
Enzymes when present in aqueous solutions cannot be retained in the bioreactor. They can be retained in the system by the process of immobilization. It is the process of attaching a cell/ enzyme to an insoluble inert support. The purity of such immobilized enzymes is higher than free enzymes. The turn over number is high and enzyme activity can be retained for a longer duration.
Other sophisticated methods such as electro dialysis, electro filtration, isoelectric focusing, forced flow electrophoresis, electro decantation and isotachophoresis are also developed and used. Membrane based processes such as reverse osmosis and pervaporation are also gaining popularity. For membrane processes, retained species tend to concentrate on the upstream of the membrane and reduce the permeation flux in a process called concentration polarization.
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