Enzyme engineering Conference held at New Hampshire (1971) defined immobilized enzymes as, "Enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities and which can be used repeatedly and continuously".

This technique involves immobilization of enzymes on an inert, insoluble matrix such as calcium alginate. Studies of immobilized enzymes, in which the enzyme is attached to an insoluble matrix, have displayed subtly different kinetic behavior from that of free enzymes. The reasons for this are:

i) The perturbation of the 3-D structure of the enzyme by the immobilizing matrix
ii) Changes in the microenvironment of the active site of the immobilized enzyme.

Kinetic studies of immobilized enzymes are valuable for our understanding of their action in-vivo, especially their regulation, since there is evidence that in-vivo enzymes may be less free to diffuse in solution than was originally thought.

Methods of Immobilization
Immobilization of enzymes can be achieved by any of the following methods:-

i) Physical entrapment
ii) Micro-encapsulation
iii) Adsorption
iv) Covalent cross-linking

However, this is broadly divided into two types, as described below:-

1) Physical immobilization
• It forms no covalent bonds between the enzymes and the supporting matrix.
• Earlier approaches include the adsorption of the enzyme onto animal charcoal or alumina but current advancements make use of ionic adsorption technique onto ion-exchange resins, especially those of the sephadex type and controlled-pore glass.

Advantages
• Simplicity
• General applicability
• High yield
• Confers ability to replace the immobilized enzyme when its catalytic activity has decreased below an acceptable level.

Limitations
• Need to control the working conditions for the use of immobilized enzyme to prevent its desorption.
• Products of enzyme entrapment in liposomes (artificially produced concentric spheres of phospholipids bilayers), polyacrylamide and agarose suffer from poor flow properties, inefficiency and progressive leaching of the enzyme.
• Desorption of the protein resulting from changes in temperature, ionic strength and hydrogen ion concentration.

2) Chemical immobilization
• One covalent bond formation between the enzyme and the matrix.
• Procedures are similar to those used in affinity chromatography.
• A loss of activity would be observed if attachment involves amino acid residues at the active site of the enzyme.
• The matrix may be polysaccharide, polymers (nylon, inorganic carriers such as glass and titanium dioxide).

Applications:
1) The first reported application is the production of L-amino acids from immobilized aminoacylase.
2) Production of a chiral molecule is of principal importance for pharmaceutical and research purposes because isomers formed differ significantly with respect to their biological properties.
3) Use of immobilized glucose isomerase in food industry for the production of high fructose syrup as fructose is sixteen times sweeter than glucose.
4) A number of immobilized enzymes are also becoming an important part of a variety of biosensors. The term 'biosensor' is used for analytical tools or systems, which make use of an immobilized biological material alongwith a suitable transducer device to convert a biochemical signal to a quantifiable chemical signal.
5) Immobilized enzymes have an increasing number of analytical applications, especially in clinical situation where they differ in the potential for fast, sensitive and accurate determinations of analytes such as blood glucose and urea.
6) Most significant development is the combination of immobilized enzymes with high specificity and sensitivity electroanalytical chemistry. The resulting enzyme electrode offers an advantage of accurate analysis without the need of sample preparation.
7) With reference to industrial use, the solutions of enzymes are commonly employed in the synthesis of antibiotics and steroids.
8) Another recent development involves the quantification of enzymes, substrates and other analytes in samples of clinical importance. For this, impregnate the reagents including the enzyme onto an immobile structure. On addition of a drop of test sample to the matrix, the analyte in the sample triggers an enzyme reaction, resulting in the formation of colored product. The intensity of color can be measured with the help of reflectance spectroscopy.


In conclusion, enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation, diagnostics, pharmaceutical and food industries. Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase, lipases, proteases, invertase, etc. and are being currently used as catalysts in various large scale processes.

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