Enzyme Immobilization

Enzyme Immobilization

• The major handicap in the traditional use of enzymes is that they are used but once. This is mainly because the enzymes are unstable in the soluble form in which they are used and because recovery would be expensive, even if it were possible.
• The immobilization of enzymes and cells provides a basis for the re-use of enzymes and cells. Interest in immobilized enzymes has grown since the 1960s and numerous conferences and papers have been held and given on them.
• An immobilized enzyme may be defined as an isolated or purified enzyme confined or localized in a defined volume of space.

Carrier  Molecules

The Molecules which are used in the Immobilization processes of enzyme is called Carrier molecules. An ideal carrier molecule should contain this characters ;

• It should be stable.
• It Should be Inert.
• Low cost.
• Reduction in product inhibition.
• Reduction in the microbial contamination.

Methods of Immobilizing Enzymes

In 1995 the International Union of Pure and Applied Chemistry (IUPAC) divides methods of immobilized enzymes into four broad groups, based on:

ü  Adsorption

ü  Covalent Binding

ü  Entrapment

ü  Membrane Confinement

The IUPAC groups can be divided into two basic groups, the chemical and the

physical methods as shown in Image.

Ø Immobilization by adsorption

·         This method is both simple and inexpensive and consists of bringing an enzyme solution in contact with a water-insoluble solvent surface and washing off the unadsorbed enzyme.

·         The extent of the adsorption depends on a number of factors including the nature of the support, pH, temperature, time, enzyme concentration.

·         Adsorbents which have been used include alumina, bentonite, calcium carbonate, calcium phosphate, carbon, cellulose,charcoal, clay, collagen etc.

·         The advantage is that the enzymes are unlikely to be inactivated because the system is mild.

·         The disadvantage is that in cases of weak binding the enzyme may be easily washed away.

Ø  Immobilization by entrapment

·         In the entrapment of enzymes, no reaction occurs between support and the enzyme.

·         A cross-linked polymeric network is formed around the enzyme; alternatively the enzyme is placed in a polymeric substance and the polymeric chains cross-linked.

·         The advantages of the entrapping method are:

(i) its simplicity

(ii) the small amount of enzyme used

(iii) the unlikelihood of damage to the enzyme

(iv) applicability to water insoluble enzymes.\

·         The disadvantages include leakage of enzymes and some chemical and thermal enzyme damage during gel formation.

Ø  Immobilization by covalent linkage

·         This is by far the most widely studied method.

·         The covalent linkage is achieved between a functional group on the enzyme not essential for catalytic activity and a reactive group on a solid water-insoluble support.

·         The functional groups available on enzymes for linkage are amino and carboxyl groups, hydroxyl groups, imidazole groups, indole groups, phenolic groups and sulphydryl groups.

·         The advantage of the covalent bonding method of enzyme immobilization are:

                                             i.            The coupling of the enzyme to the support is easy to conduct and consists of allowing support and enzyme to interact and therefore facilitates centrifuging and washing off any enzymes not bound.

                                            ii.            The enzyme-support derivative is easy to manipulate and adapt because of the great physical and chemical variation in the available support: they can be used in a variety of reactors including stirred tank, fluidized bed-reactors and can also be modified into flat sheets fiber.

                                          iii.            Covalent coupling has been widely described and methods for carrying it out are readily available in the literature.

                                          iv.            The supports themselves are widely available commercially.

·         The disadvantages are that some preparations are tedious to make; the chemical bonding may inactivate the enzyme in some cases; and finally covalently-bound waterinsoluble enzyme-substrate derivatives act poorly on high molecular weight substrates.

Ø  Immobilization by Membrane Confinement

·         In this method the enzyme molecule which is soluble in solvent is confine by the semi permeable membrane, which allow the movement of enzyme in one direction to the substrate and enzyme concentration.

·         In this reaction vessel may be partitions into two compartment by the semi permeable membrane, one compartment is contain enzymes and another is contain substrate and product.

·         The enzyme may be enclosed in a liposome.                                                                                                                                                                                         

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      

The advantages of immobilized enzymes

                    I.            They can be easily separated from the reaction mixture

                    II.            Immobilized enzymes are more stable over broad ranges of pH and temperature.

                  III.            Immobilized systems specially lend themselves to continuous processes.

                  IV.            Reduced costs in industrial production.

                   V.            Greater control of the catalytic effect.

                  VI.            Increase the thermo stability of  the enzyme.

                VII.            Enzymes are costly so if we can  recover them we can reused them again.

The Disadvantages of immobilized enzyme

                     I.            It is a costly process.

                    II.            Adversely affect  the enzyme stability and activity. 

                  III.            This approach cannot be use if one of the substances is insoluble.

                  IV.            When life cells are immobilized they may be proliferate.                                                                                                                                                                

The Application of immobilized enzymes

                     I.            The production of energy by biological process.

                    II.            Utilization in manufacturing of new drug.

                  III.            They are used in manufacturing of artificial organ.

                  IV.            Analysis of various compound with high sensitivity and specificity.

                   V.            Selective treatment of pollutant to solve the environmental problem.