Immobilization of enzymes

Enzyme immobilization is defined as confining the enzyme molecules to a distinct phase from the one in which the substrates and the products are present.
It is achieved by fixing the enzyme molecules to or within some suitable material.

1. IMMOBILIZATION OF ENZYMES ALEN SHAJI P1914015

2. ENZYMES Enzymes are proteins that catalyze chemical reactions, both biosynthetic and degradative , occurring in living cells . Enzymes are also known as biological catalyst . Enzyme activities are exploited in almost all biotechnological activities . Enzymes may themselves be present in living cells or in an isolated and purified state . Purified enzymes are employed in industrial processes, medicine, research and recombinant DNA technology. The use of purified enzymes for generating a useful product or service constitutes enzyme technology .

3. Enzymes may or may not have a non protein molecule attached to them. Some enzymes contain covalently bound carbohydrate groups . Many enzymes have metal ions . Some enzymes possess low weight organic molecules, these are called cofactors . An organic cofactor is commonly known as coenzyme . Cofactors and coenzymes may be covalently or non covalently attached to the protein molecule, called apoenzyme . When a cofactor is so tightly bound to the apoenzyme , the cofactor is often called a prosthetic group . The complex of an apoenzyme and the cofactor is known as holoenzyme . Both coenzymes and cofactors generally contribute to enzyme activity as well as stability .

4. MECHANISM OF ENZYME ACTION An enzyme (E) has a highly specific binding site / active site to which its substrate (S) binds to produce enzyme-substrate complex ( ES ). The reaction proceed at the binding site to produce the products (P), which remain associated briefly with the enzyme ( enzyme-product complex , EP ). The product is then liberated and the enzyme molecule is freed in an active state to initiate another round of catalysis. Apparantly , the affinity of binding site for the product is much lower than that for the substrate.

5. IMMOBILIZATION OF ENZYMES Enzyme immobilization is defined as confining the enzyme molecules to a distinct phase from the one in which the substrates and the products are present. It is achieved by fixing the enzyme molecules to or within some suitable material . Imprisonment of enzyme in a distinct support or matrix . The matrix allows the exchange of medium containing substrate / effector / inhibitor molecules. The first immobilized enzyme was amino acylase of Aspergillus oryzae for the production of L-amino acids in Japan.

6. It is critical that the substrates and the products move freely in and out of the phase within the matrix. Immobilization of enzyme molecules does not necessarily render them immobile. In some methods of immobilization, eg ; entrapment and membrane confinement, the enzyme molecules move freely within their phase. In methods like adsorption and covalent bonding enzymes are in immobile state.

7. CARRIER MATRICES Carrier matrices are the materials used for immobilization of enzymes. They are inert polymers / inorganic materials . The ideal carrier matrix has the following properties; Low cost. Inertness. Physical strength. Stability. Regenerability after the useful lifetime of the immobilized enzyme. Enhancement of enzyme stability. Reduction in product inhibition. A shift in the pH optimum for enzyme action to the desired value for the process. Reduction in microbial contamination and non specific adsorption.

8. EFFECTS OF IMMOBILIZATION ON ENZYME Kinetic behaviour of an immobilized enzyme may differ significantly from that of its free molecules. Different enzymes respond differently to the same immobilization protocol. Therefore, a suitable immobilization protocol has to be worked out for a given sample. The effects on enzyme kinetics may be due to the influence of matrix or due to the conformational changes in the enzyme molecules induced by the procedure of immobilization . Immobilization protocol may increase or decrease enzyme stability . However, enzymes, whether free or immobilized, loose activity with time due to denaturation .

9. MATRIX USED IN IMMOBILIZATION TECHNOLOGY The matrix immobilizes the enzyme by holding it permanently or temporarily for a brief period of time . Matrix should be cheap and easily available . Their reaction with the components of the medium or with the enzyme should be minimum as possible . Matrices for immobilization of enzymes are; Natural polymers. Synthetic polymers. Inorganic polymers.

10. 1. NATURAL POLYMERS ALGINATE : Calcium / magnesium alginate is most common; inert; good water holding capacity. CHITOSIN AND CHITIN : The various functional groups in enzymes can bind to the –OH group of chitin and can form covalent bonds. COLLAGEN : The side chains of the amino acids in the collagen and that of enzyme can form covalent bonds to permanently hold the enzyme to the support. CARRAGEENAN : Their good gelling properties together with its high protein holding capacity makes it good support for immobilizing enzymes. GELATIN : It is the partially hydrolyzed collagen with good water holding capacity. CELLULOSE : The hydroxyl group of the monomer (glucose) can form covalent bonds with that of the amino acids of enzyme. STARCH : It has good water holding capacity. PECTIN : It is a gelling agent with good water holding capacity.

11. 2. SYNTHETIC POLYMERS They are ion exchange resins or polymers . They are insoluble supports with porous surface . Their porous surface can trap and hold the enzymes or whole cells . Examples; Diethylaminoethyl cellulose / DEAE cellulose . Polyvinyl chloride / PVC . UV activated Polyethylene glycol / PEG .

12. 3. INORGANIC MATERIALS ZEOLITES : They are microporous , aluminosilicate minerals with good adsorbing properties and extensively used for immobilizing enzymes and whole cells. CERAMICS : They are non metallic solids consisting of metal and non metal atoms held in ionic and covalent bonds. The composition and bonding pattern varies with different types. DIATOMACEOUS EARTH : They are silicious sedimentary rocks formed by fossilized accumilations of the cell wall of diatoms. Celite is the trade name of diatomaceous earth. It is a good adsorbent and are resistant to high pH and temperature. SILICA . GLASS . ACTIVATED CARBON . CHARCOAL .

13. ADVANTAGES OF IMMOBILIZATION Enzymes are costly items and can be used repeatedly only if they can be recovered from the reaction mixtures. Immobilization permits their repeated use since such enzyme preparations can be easily separated from the reaction system . The product is readily freed from the enzyme . This saves on the cost of downstream processing of the product. Immobilized enzymes can be used in non aqueous systems as well, which may be highly desirable in some cases. Continuous production systems can be used , which is not possible with free enzymes. Thermostability of some enzymes may be increased . For example, glucose isomerase denatures at 45 degree C in solution, but is stable for about 1 year at 64 degree C when it is suitably immobilized. Recovery of enzyme may also reduce effluent handling problems . Enzymes can be used at much higher concentrations than free enzyme .

14. Use of continuous processes , which reduces the plant size to nearly on-half. Easy , cheap , off-site fabrication of plants . Greatly increased productivity/unit of enzyme used . Increased functional efficiency of enzyme . Enhanced reproducibility of the process they are undertaking. Continuous use of enzyme . Minimum reaction time . Less chance of contamination in products. More stability of products. Stable supply of products in the market. Improved process control. High enzyme substrate ratio.

15. DISADVANTAGES OF IMMOBILIZATION Immobilization means additional costs . Therefore, it should be used only when there is a sound economic, safety or process advantages over soluble enzymes. Immobilization often adversely affects the stability and activity of the enzymes . In such cases, suitable immobilization protocols should be developed. This approach cannot be used when one of the substrates is insoluble . Some immobilization strategies present large problems in diffusion of the substrate to reach the enzyme . A number of changes in the production process may become necessary when immobilizes enzymes are used . Immobilized enzymes are particularly vulnerable to contamination .

16. High cost of isolation , purification and recovery of active enzyme . Industrial applications are limited and only very few industries are using immobilized enzymes. Catalytic properties of some enzymes are reduced or completely lost after their immobilization on support or carrier. Some enzymes become unstable after immobilization . Enzymes are inactivated by the heat generated in the system .

17. APPLICATIONS OF IMMOBILIZED ENZYMES 1. INDUSTRIAL PRODUCTION Industrial production of antibiotics. Industrial production of beverages. Industrial production of amino acids. In production of High Fructose Corn Syrup. Glucose isomerase obtained from Actinoplanes missouriensis , Bacillus coagulans and Streptomyces spp is used in immobilized state obtained by cross-linking with glutaraldehyde . Immobilized Raffinase . Raffinase activity is provided by immobilized cells of the mould Mortierella vinacea var. raffinoseutilizer . Immobilized Invertase . Invertase is the first enzyme used during 1940s in an immobilized state. The enzyme obtained by autolysis of yeast cells was immobilized by adsorption onto bone char. At present, the purified enzyme-char mix is stabilized by cross-linking.

18. Immobilized Lactase. Lactase is used to remove lactose from milk. Yeast ( Kluyveromyces lactis ) lactase is immobilized in cellulose triacetate fibres which are cut up and used in batch STR. Fungal ( Aspergillus niger ) lactase is immobilized on porous silica and is used in a PBR for treatment of acid. Antibiotic Production. Penicillin amidases obtained from E.coli are immobilized in a variety of ways. Preparation of Acrylamide. The reaction which forms acrylamide is achieved by nitrile hydratase activity obtained from Rhodococcus rhodochrous , an organism rich in nitrile hydratase . The bacterial cells are immobilized in a polyacrylamide gel to keeping the substrate concentration below 3%. About 4000 tons of acrylamide is produced every year using the process.

19. 2. BIOMEDICAL APPLICATIONS Used in the diagnosis and treatment of many diseases. Used to overcome inborn metabolic disorders by the supply of immobilized enzymes. Immobilization techniques are effectively used in drug delivery systems especially to oncogenic sites. 3. FOOD INDUSTRY Enzymes like pectinases and cellulases immobilized on suitable carriers are successfully used in the production of jams, jellies and syrups from fruits and vegetables. 4. RESEARCH A research activity extensively uses many enzymes. The use of immobilized enzyme allow research to increase the efficiency of different enzymes such as Horse Radish Peroxidase (HRP) in blotting experiments and different Proteases for cell or organelle lysis . 5. PRODUCTION OF BIO-DIESEL : From vegetable oils. 6. WASTE WATER MANAGEMENT : Treatment of sewage and industrial effluents. 7. TEXTILE INDUSTRY : Scouring, bio-polishing and desizing of fabrics. 8. DETERGENT INDUSTRY : Immobilization of lipase enzyme for effective dirt removal from cloths.

20. REFERENCE B.D. Singh, 2003, Biotechnology, Expanding Horizons, Kalyani Publishers, India. U. Satyanarayana , 2017, Biotechnology, Books & Allied (P) Ltd, India. www.easybiologyclass.com.

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