Enzyme ImmobilizationEnzyme immobilis ation is a technique of restraining the enzymes in or on an inert support for their stability and functional reuse. The immobilisation matrix or the support allows exchange with the bulk phase in which the substrate, effect or inhibitor molecules are dispersed, but r emains separated from it. With the use of enzyme immobilisation, enzymes are made more efficient and cost -effective to be used industrially. Advantages 1) Enzymes are used repeatedly only if they can be completely recovered from the accomplished reaction m ixtures. Thus, immo bilisation allows therepeated usage of enzymes as such enzyme preparation s can be separated from the reaction system involved.2) The final desired product should be from the enzyme. It goes a long way in affecting reduction and saving upon the cost of downstream processing of the end-product.3) Non-aqueous systems ( that utilise organic solvents) and the immobilised enzymes are compatible, and this is extremely desirable in some typical and specific cases.
4) Immobilised enzymes can be used in most continuous production systems, which is not possible with free enzymes.5) Some immobilised enzymes exhibit thermostability of the highest order; forexample, glucose isomerase (free enzyme) denatures at 45°C temperature in solution; however, on immobilisation it remains stable for a year even at65°C temperature.6) The ultimate recovery of immobilised enzyme reduces the high effluent disposable problems (acute in several fermentation industries).7) Immobilised enzymes can be used at a much higher concentration range than the corresponding free enzyme.Disadvantages 1) There is a possibility of the loss of biological activity of an enzyme during immobilisation or while in use.2) It is an expensive technique as it requires sophisticated equipment.3) Immobilisation o f enzymes affects the stability and/or activity. Such conditions can be avoided by strictly following the developed immobilisation protocols.4) If one of the substrates is insoluble, the immobilised enzymes are not considered fit for practical utilisation.5) Some immobilisation protocols give rise to problems related to the diffusion of the resultant substrate to have an access to the corresponding enzyme.1.2.3. Methods of Enzyme ImmobilizationEnzymes can be immobilised by the following four different methods depen ding on the physical relationship of the catalyst being used with the carrier matrix:1) Adsorption method, 2) Covalent bonding,3) Entrapment, and 4) Encapsulation.18.104.22.168. Adsorption MethodAdsorption of an enzyme is brou ght about by allowing the enzyme to come in contact with a polymer support. The enzyme molecules adhere to the surface of the carrier matrix with the help of hydrophobic bonds and by forming several salt-linkages per enzyme molecule. Various specific and n on-specific forces, like hydrophobic bonds, electrostatic interactions, or affinity bondage to some specific ligands attached to the carrier matrix, involve in the adsorption method.Examples of some enzymes that have been immobilis ed by adsorption on different carrier matrices are given below:Enzymes Carrier Matrices-Amylase Calcium phosphateAmyloglucosidase Agarose gel; DEAE-SephadexCatalase Activated charcoal Glucose oxidase Cellophane (followed cross-linking with glutaraldehyde, inorganic adsorbents)Invertase Activated charcoal; DEAE-Sephadex Subtilisin Cellulose
Methods for Immobilisation by AdsorptionThe adsorptive immobilisation of enzymes (figure 1.1) can be carried out by the following ways:1) Static Pores: This is the most efficient technique, but is time consuming. In this process, the enzyme is immobilised on the carrier by bringing the enzyme-containing solution in contact with the carrier without agitation or stirring.2) Dynamic Pores: This technique is frequently used in the laboratories. In this process, the enzyme solution is admixed with the carrier with constant agitation or stirring with a mechanical shaker. The proce ss is effective and results in uniform and high loading provided an adequate concentration of enzymes is used.3) Reactor Loading: This technique is u sed for producing immobilis ed enzymes. In this process, the carrier is placed into the reactor and enzyme solution is transferred to the reacto r loaded with carrier. Immobilis ation is accomplished via dynamic environment by either circulating the enzyme or by agitating the enzyme-carrier solution.4) Electro-Deposition: In this process, the carrier is placed in proximal vicinity of one of the electrodes in an enzyme bath and then electric current is passed. As a result, the enzyme molecules migrate towards the carrier and deposit on its surface. The c arrier system used for immobilis ation by electro-deposition process should be stable in an electric field.22.214.171.124. Covalent BondingIn this method, the enzyme molecules adhere to the carrier matrix by forming covalent bonds (figure 1.2), which forms with the side chains of amino acids present in the enzyme; however, their actual strength of reactivity is entirelyrelated to the status of ‘charge’ present in them as given below:S–> SH > O–> NH2 > COO–> OH >> NH3+
3) Peptide Bond Formation: In this method, peptide bon ds are formed
between the amino (or carboxyl) groups of the support materials and the
carboxyl (or amino) groups of the enzymes ( figure 1.3C). The support
material is previously treated chemically to form active functional groups.
4) Activation by Bi- or Poly-Functional Reagents: In this method, the reagent
(e.g., glutaraldehyde) is used to form bonds between the amino groups of
enzymes and amino groups of support materials ( e.g., aminoethylcellulose,
albumin, and amino alkylated porous glass) (figure 1.3D).