Immobilization of enzymes and cells
Traditionally enzymes in free solutions that is in soluble or free form react with substrates to result in products. Such use of enzymes is wasteful particularly for industrial purposes since enzymes are not stable and they cannot be recovered for reuse. Immobilization of enzymes or cells refers to the technique of confining or anchoring the enzymes or cells in or on an inert support for their stability and functional reuse. By employing these techniques enzymes are made more efficient and cost effective for their industrial use. Some workers regard immobilization as a goose with a golden egg in enzyme technology.
Immobilized enzymes retain their structural confirmation necessary for catalysis. There are several advantages of immobilized enzymes as below.
• Stable and more efficient in function.
• Can be reused again and again.
• Products are enzyme free.
• Ideal for multi enzyme reaction systems.
• Control of enzyme function is easy.
• Suitable for industrial and medical use.
• Minimise affluent disposal problems.
There is however certain disadvantages also associated with immobilization of enzymes.
• The possibility of loss of biological activity of an enzyme during immobilization or while it is in use.
• Immobilization is an expensive affair often requiring sophisticated requirements.
Immobilized enzymes are generally preferred over immobilized cells due to specificity to yield the product in pure form. However there are several advantages of using immobilized multi enzyme system such as organelles and whole cells over immobilized enzymes. The immobilized cells possesses the natural environment with cofactor availability and also it regeneration capability and are particularly suitable for multiple enzymatic reaction.
Methods of Immobilization
The commonly employed techniques for immobilization of enzymes are adsorptions, entrapment, covalent binding and crosslinking.
Adsorptions involves the physical binding of enzyme or cells on the surface of an inert support. The support materials may be inorganic that is alumina, silica gel, calcium phosphate gel, glass or organic starch carboxymethyl cellulose, DEAE-cellulose, DEAE-sefadex.
Adsorptions of enzyme molecules on the inert support involves weak forces such Van der Walls forces and hydrogen bonds. Therefore the adsorped enzymes can be easily removed by minor changes in pH, ionic strength or temperature. These are the disadvantages of using adsorped enzymes for industrial uses.
Enzymes can be immobilized by physical entrapment inside a polymer or a gel matrix. The size of the matrix pores is such that the enzyme is retained while the substrates and product molecules pass through. In these techniques commonly refer to as lattice entrapment. The enzyme or cells is not subjected to strong binding forces and structural distortions. Some deactivation may however occur during immobilization process due to changes in pH or temperature or addition of solvents. The matrix used for entrapping enzymes includes polyacrylamide gel, collagen, gelatine, starch, cellulose, silicon and rubber.
Enzymes can be entrapped by several ways.
1. Enzyme inclusion in gels: This is the entrapment of enzymes inside the gels.
2. Enzyme inclusion in fibres: The enzymes are trapped in a fibre format of the matrix.
3. Enzyme inclusion in microcapsules: In this case the enzymes are trapped inside a microcapsule matrix. The hydrophobic and hydrophilic forms of the matrix polymerize to form a microcapsule containing enzyme molecules inside.
The major limitations for entrapment of enzymes are their leakage from the matrix. Most workers prefer to use the technique of entrapment for immobilization of whole cells. Entrapment cells are used for industrial production of amino acids such as L-isoleucine, L-aspartic acid, L-malic acid etc.
Microencapsulation is a type of entrapment. It refers to the process of spherical particle formation where in a liquid or suspension is enclosed in a semipermeable membrane. The membrane may be polymeric, lipoidal, lipoprotein based or non-ionic in nature.
There are three distinct ways of microencapsulation.
1. Based on special membrane reactors.
2. Formation of emulation.
3. Stabilization of emulation to form microcapsules.
Microencapsulation is recently being used for immobilization of enzymes and mammalian cells. For example, pancreatic cells grown in cultures can be immobilized by microencapsulation. Hybridoma cells have also been immobilized successfully by these techniques.
Immobilization of the enzymes can be achieved by creation of covalent bonds between the chemical groups of enzymes and the chemical groups of the support. This technique is widely used. However covalent binding is often associated with loss of some enzyme activity. The inert support usually requires pre-treatment to form pre activated support before it binds to enzymes.
The common methods for covalent binding are cyanogen bromide activation, diazotation, peptide bond formation, activation by polyfunctional reagents.
In crosslinking the enzyme molecules are immobilized by creating crosslinks between them through the involvement of polyfunctional reagents.
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