An immobilized enzyme is one which has been attached to or enclosed by an insoluble support medium termed as carrier or one where the enzyme molecules have been cross linked to each other, without loss of catalytic activity. Many methods are involved in the preparation of the immobilized enzymes which may include the following.
Physical adsorption on to an inert carrier is a very simple procedure for immobilizing an enzyme, for it requires just the mixing of the enzyme solution with the carrier. Other inorganic materials which may be used are alumina, clay and silica. The weak linkages between the enzyme and carrier have little effect on catalytic activity. However as the bonds are so weak, the enzyme can be desorbed from the carrier: this can be brought about by the changes in pH ionic strength or substrate concentration. Also adsorption is non specific and many other substances get attached to the carrier as immobile enzyme is used.
Ionic bonding provides a slightly more specific way of attaching an enzyme and carrier. However, it is essential that the conditions used for the formation of the covalent bonds are sufficiently mild that even if ant catalytic activity is lost the active site of the enzyme is free from covalent attachments, so it is sometime protected with a substrate during the procedure.
Yet another widely used method for enzyme immobilization was developed, which involves the reagent, cyanogens bromide. This is used for affinity activates polysaccharide hydroxyl groups. Immobilization by cross linking molecules of enzyme is most commonly brought about by the action of glutaraldehydr, whose two aldehyde from Schiff's base links with the free amino groups.
Immobilized enzymes are usually prepared in particle form, but enzyme may be attached or entrapped within, carrier in the form of membranes, tubes or fibers, according to the requirements of given application. Also in case of intracellular enzymes it may be more economical to immobilize the intact cell rather than to perform an extraction step. This is satisfactory provided the appropriate substrates and products can freely pass through the cell membranes.the most common method for immobilizing microbial cells is to trap them in polyacrylamide gel. The use of immobilized cells is particularly advantageous where it is known that the enzyme of importance would be unstable during or after extraction, a not an uncommon situation.
PROPERTIES OF IMMOBILIZED ENZYME
The properties of an immobilized enzyme may be different from those of the same enzyme in free solution, and depend both on the method of immobilization and on the nature of the insoluble carrier. A reduction in specific activity may occur as an enzyme is immobilized, particularly if a chemical process is involved, since conditions might cause some denaturation to take place.Further more, carrier crates a new micro environment for the enzyme and could thus influence its activity in a variety of ways.
The stability of an enzyme on heating or storage can increase, decrease or stay the same when it is immobilized, depending on how the new micro environment affects its tendency to denature: few clear cut trends have been observed .another relevant factor is the ease of attack by substances which might degrade the enzyme, For example, or by forming electrostatic interactions with molecule of substrate or product.
The stability of an enzyme on heating or storage can increase or stay the same when it is immobilized, depending on how: the new micro - environment affects its tendency to denature: few clear cut trends have been observed. Another relevant factor is the case of attack buy the substance which might degrade the enzyme. For example auto digestion of the proteolysis enzyme is reduced if the molecules are protected from each other by immunization . Similarity, steric hindrance protects many 0ther immobilized enzymes from attack by proteolytic enzyme.
The Ph optimum can change by as much as 2 PH units when the enzyme is immobilized. Largely owing to the effect of new micro-environment.
APPLICATIONS OF IMMOBILIZED ENZYMES
The immobilized enzyme system chosen for a given application should fit the requirements in terms of stability, activity, PH optimum and other characteristics.
Sometimes the changes in properties which take place when an enzyme is immobilized may be used to advantage also. For example, if an enzyme catalyzed reaction cannot be linked directly to another because of incompatibly PH activity ranges now overlap, thus allowing them to be used in a single rather than a two step process.
In general, the property of immobilized enzymes which is of greatest industrial importance is the ease with which they can be separated from reaction mixtures. And the enzyme will stay active and highly uncontaminated which can be easily used again. For this reason these enzymes can be used in continuously operated processes. They are also used in components of analytical systems, either in dry - reagent or automated techniques.
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