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Enzyme Immobilization
Enzyme immobilization refers to the localisation or physical confinement of the molecule to a matrix such that it retains its catalytic properties. The first industrial use of this process was in 1967 by Chibata and co-workers who immobilized aminoacylase of Aspergillus oryzae in the production of L-amino acids. These immobilized enzymes have widespread application, such as, reusable biocatalysts, selective adsorbents and as a tool in solid-phase protein chemistry. The process which results in such an enzyme is known as enzyme immobilization. An immobilized enzyme should contain two basic functions:
 * Non-catalytic functions: Related to the physical and chemical properties of the non-catalytic part of the enzyme, such as, geometric properties including shape size etc. Designed to aid separation of the catalysts and reusability.
 * Catalytic functions: Related to the properties such as activity, selectivity etc., that deal with transforming substrates.

Classification of Immobilized Enzymes
Classified into carrier-bound immobilized enzymes and carrier-free immobilized enzyme. Carrier-bound immobilized enzymes involve the globular proteins being embedded in a polymer matrix either covalently or non-covalently while carrier-free involves enzyme aggregates and enzyme crystals cross linked to form cross linked products.

Classification of matrix

 * Natural polymers: For example, alginate, carragenan, cellulose, gelatin and starch.
 * Synthetic polymers: For example, amberlite and polyvinylchloride.
 * Inorganic materials: For example, zeolites, ceramics, silica and glass.

Methods of Immobilization
There are four fundamental immobilization techniques:

Adsorption:
A simple method where enzyme is dissolved in a solution and solid support is placed in contact with this solution under optimum conditions of pH, temperature and ionic strength. After incubation there is one round of washing to remove the partially bound and unbound enzymes producing the usable product. This method has high enzyme loading values of about 1g per gram of matrix. The binding is due to weak interactions such as Van der Waal's forces, electrostatic forces and hydrophobic interactions. Examples of adsorbents are ion exchange matrices and porous carbon. It is a reversible immobilization process. The following are the types of adsorption: The advantage of this type of technique is that it is easy to carry out without any reagent requirement, economical and is not destructive to the activity of the enzyme but the downside is that the weak interactions are easily broken down with change in pH and temperature.
 * Physical adsorption
 * Electrostatic adsorption: The solution pH and the isoelectric point of enzymes are taken into consideration. Based on the charge on the surface of the enzyme it is immobilized on an oppositely charged surface through ionic interactions.
 * Hydrophobic adsorption: The hydrophobic interactions between the enzyme ands support is due to displacement of water molecules from both.

Covalent binding:
Binding occurs through the side chains of the functional groups (Eg. Thiol group of cysteine) of the enzyme and the support matrix. The enzyme binding occurs by diazotisation or peptide bond formation. It is important that the functional group undergoing covalent bond formation should not be essentisl to activity of enzyme. The direction of enzyme binding is also of importance with respect to stability. The supported matrix can either be modified prior to addition of enzymes to possess more activating groups, this is achieved with the use of glyceraldehyde or carbodiimide(these are linkers). There are three types of interactions through which the bond is formed: This method is the most widely used irreversible process. Examples of matrices used in this type of immobilization are agarose and poly vinyl chloride. The advantage of such a technique is strong linkage due to bonding and relatively low desorption rates. The disadvantage is the possibility of enzyme inactivation due to conformationa change.
 * -COOH of support and -NH2 of the enzyme.
 * -NH2 of support and -COOH of enzyme.
 * Chemisorption: Interaction of -SH of enzyme and gold support.

Entrapment:
This is an irreversible method that involves the entrapment of the enzymes within a polymeric network of the support. There is no chemical interaction between the two, which is why it is known as a physical process. The substrate and products move through while the enzyme is held tight. The first step of this method is to mix enzyme in a monomer solution, the monomer solution is then polymnerised by the following ways: This can also bind upto 1g per gram of matrix, however, the chances of enzyme leakage are higher. The pore size of the matrix is very important and should be chose accorsingly to avoid loss of enzyme. Examples of matrices are alginate and collagen. The advantages of this method are that it can improve the stability of the enzyme by avoiding direct contact with environment conditions however, the limitation of inability of substrate to penetrate deep enough to reach the enzyme exists.
 * Electrochemical polymerisation: Current is applied to the monomer solution containing enzymes. There is generation of free radicals of the monomer which then form dimers and go on to form polymers at the electrode surface. Enzymes in the vicinity of the electrode are entrapped thereby giving rise to immobilized enzymes.
 * Photopolymerization: Here, the exposure to UV light initiates free radical formation leading to propagation, elongation and termination steps of chain growth polymerisation thereby giving rise to immobilized enzymes.
 * Microencapsulation: It is a type of entrapment where there is a semi permeable membrane within which the enzyme is present. The pore size of the membrane is such that it allows only passage of substrates and products but not the enzyme.

Cross-Linking:
This is yet another irreversible method of immobilization that does not require a support. Carrier-free immobilized enzymes are produced using this technique. Here, the enzyme acts as its own support thereby leading to highly pure immobilized enzymes. Intermolecular cross linkages between enzymes are produced through covalent bonds. There is use of linkers (also mentioned in covalent binding) such as glutaraldehyde to mediate this process. Types of cross linked enzymes produced are: The advantage of this method is that there is strong bonding but there is the huge disadvantage of altering enzyme activity.
 * Cross-linked dissolved enzymes (CLE) which involves the direct cross-linking of enzyme solutions.
 * Cross-linked enzyme crystals (CLEC) where enzyme crystals are made prior and then cross linking is carried out with glutaraldehyde.
 * Cross-linked enzyme aggregates (CLEA) which involves pre-made enzyme aggregates which are cross linked using glutaraldehyde.
 * Cross-linked spray dried enzymes (CSDE) where spray dried enzymes are cross linked.

In addition to these main techniques certain pre-immobilization and post-immobilization modifications are carried out to increase efficiency with higher product production.

Advantages and Disadvantages of Immobilized enzymes
Advantages:
 * 1) Reusability of enzymes.
 * 2) Increased functional efficiency.
 * 3) Increased enzyme stability.
 * 4) Less labour input in process.
 * 5) Enhanced thermal stability.

Disadvantages
 * 1) High cost of immobilization of enzyme.
 * 2) Chances of instability and reduced functionality post immobilization.

Applications of Enzyme Immobilization

 * 1) Highly important in continuously operated processes. Currently used in simple stirred tank reactors, fluidized bed and packed bed reactors.
 * 2) Extremely important in the pharmaceutical industry, since certain of enzymes pharamaceutical importance are very expensive to isolate and purify. For example, 6-APA from peniciliin V/G by action of penicillin amidase. 6-APA is important precursor to synthetic ampicillin and amoxicillin production.
 * 3) Pectinases and cellulases are immobilized and used in jams, syrup production.
 * 4) Used in detection and treatment of diseases. Important due to ability to efficiently deliver drugs to sites of action.
 * 5) Used in biosensors.