As enzymes are biological catalysts that promote the rate of reactions but are not themselves consumed in the reactions; they may be used repeatedly for as long as they remain active. However, in most of the processes, enzymes are mixed in a solution with substrates and cannot be economically recovered after the reaction and are generally wasted. Thus, there is an incentive to use enzymes in an immobilized or insolubilized form so that they may be retained in a biochemical reactor for further catalysis. This is done by Enzyme immobilization which may be defined as-
“The process whereby the movement of enzymes, cells, organelles, etc. in space is completely or severely restricted usually resulting in a water-insoluble form of the enzyme.”
Immobilized enzymes are also sometimes referred to as sound, insolubilized, supported or matrix-linked enzymes.
SALIENT FEATURES OF ENZYME IMMOBILIZATION:-
- The enzyme phase is called as carrier phase which is water insoluble but hydrophilic porous polymeric matrix, e.g. agarose, cellulose, etc.
- The enzyme phase may be in the form of fine particulate, membranous, or microcapsule.
- The enzyme in turn may be bound to another enzyme via cross linking.
- A special module is produced employing immobilization techniques through which fluid can pass easily, transforming substrate into product and at the same time facilitating the easy removal of catalyst from the product as it leaves the reactor.
- The support or carrier utilized in immobilization technique is not stable at particular pH, ionic strength, or solvent conditions. Hence, may be disrupted or dissolved releasing the enzyme component after the reaction.
Advantages of enzyme immobilization:-
- Multiple or repetitive use of a single batch of enzymes.
- Immobilized enzymes are usually more stable.
- Ability to stop the reaction rapidly by removing the enzyme from the reaction solution.
- Product is not contaminated with the enzyme.
- Easy separation of the enzyme from the product.
- Allows development of a multienzyme reaction system.
- Reduces effluent disposal problems.
Disadvantages of enzyme immobilization:-
- It gives rise to an additional bearing on cost.
- It invariably affects the stability and activity of enzymes.
- The technique may not prove to be of any advantage when one of the substrate is found to be insoluble.
- Certain immobilization protocols offer serious problems with respect to the diffusion of the substrate to have an access to the enzyme.
TECHNIQUE OF ENZYME IMMOBILIZATION:-
1. Carrier binding.
· Physical adsorption.
· Covalent bonding.
· Ionic bonding.
2. Cross linking.
· Occlusion within a cross linked gel.
This method is based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers. Examples of suitable adsorbents are ion-exchange matrices, porous carbon, clay, hydrous metal oxides, glasses and polymeric aromatic resins.
The bond between the enzyme and carrier molecule may be ionic, covalent, hydrogen, coordinated covalent or even combination of any of these.
Immobilization can be brought about by coupling an enzyme either to external or internal surface of the carrier.
The external surface binding method is advantageous as it does not involve conditions like pore diffusion. The disadvantages, however, include exposure of enzymes to microbial attack, physical abrasion of enzyme due to turbulence associated with the bulk solution.
The major disadvantage of the internal immobilization method is the pore diffusion.
Advantages of adsorption:-
· Little or no confirmation change of the enzyme.
· Simple and cheap.
· No reagents are required.
· Wide applicability and capable of high enzyme loading.
Disadvantages of adsorption:-
· Desorption of the enzyme protein resulting from changes in temperature, pH, and ionic strength.
· Slow method.
Methods of immobilization by adsorption:-
The absorptive immobilization of enzymes can be done by following methods:
1. Static Process:- This is most efficient technique but requires maximum time. In this technique, enzyme is immobilized by allowing it to be in contact with the carrier without agitation.
2. Dynamic Process:- This process typically involves the admixing of enzyme with the carrier under constant agitation using mechanical shaker.
3. Reactor loading:- This process is employed for the commercial production of immobilized enzymes. The carrier is placed into the reactor and enzyme solution is transferred to the reactor with agitation of the whole content in the reactor.
4. Electro-Deposition:- In this technique, carrier is placed in the vicinity of one of the electrode in an enzyme bath and electric current is applied leading to migration of enzyme towards the carrier. This results in deposition of enzyme on the surface of the carrier.
Covalent binding is the most widely used method for immobilizing enzymes. The covalent bond between enzyme and a support matrix forms a stable complex. The functional group present on enzyme, through which a covalent bond with support could be established, should be non essential for enzymatic activity.
The most common technique is to activate a cellulose-based support with cyanogen bromide, which is then mixed with the enzyme.
The protein functional groups which could be utilized in covalent coupling include:
- Amino group
- Carboxylic group
- Phenol ring
- Indole group
- Imidazole group
· Amino and related groups of polysaccharides and silica gel etc.
· Carboxylic acid and related groups of polyglutamic acid, carboxy methyl cellulose.
· Aldehyde and acetal groups of polymers.
· Amide gr. Of polypeptide.
The polymers may be engaged in direct coupling as well as could be modified by other coupling groups or activating groups. The most commonly used polymers are polysaccharides, polyvinyl alcohol, silica and porous glasses.
Advantages of covalent coupling:-
- The strength of binding is very strong, so, leakage of enzyme from the support is absent or very little.
- This is a simple, mild and often successful method of wide applicability
Disadvantages of covalent coupling:-
· Enzymes are chemically modified and so many are denatured during immobilization.
· Only small amounts of enzymes may be immobilized (about 0.02 grams per gram of matrix).
This method is based on the formation of covalent bonds between the enzyme molecules, by means of multifunctional reagents, leading to three dimensional cross linked aggregates.
The most common reagent used for cross-linking is glutaraldehyde.
Advantages of cross linking:-
· Very little desorption(enzyme strongly bound)
· Best used in conjunction with other methods.
Disadvantages of cross linking:-
· Cross linking may cause significant changes in the active site.
In entrapment, the enzymes or cells are not directly attached to the support surface, but simply trapped inside the polymer matrix. Entrapment is carried out by mixing the biocatalyst into a monomer solution, followed by polymerization initiated by a change in temperature or by a chemical reaction.
Polymers like polyacrylamide, collagen, cellulose acetate, calcium alginate or carrageenan etc are used as the matrices.
Advantages of entrapment:-
- Loss of enzyme activity upon immobilization is minimized.
Disadvantages of entrapment:-
- The enzyme can leak into the surrounding medium.
- Another problem is the mass transfer resistance to substrates and products.
- Substrate cannot diffuse deep into the gel matrix.
In this entrapment method, a highly cross-linked gel is formed as a result of the polymerization which has a fine "wire mesh" structure and can more effectively hold smaller enzymes in its cages.
Amounts in excess of 1 g of enzyme per gram of gel or fibre may be entrapped.
Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc... and natural polymer (starch) have been used to immobilize enzymes using this technique.
This entrapment involves the formation of spherical particle called as “microcapsule” in which a liquid or suspension of biocatalyst is enclosed within a semi permeable polymeric membrane.