Immobilized enzymes

Enzyme definition, Enzyme immobilization introduction , Enzyme immobilization definition, Explanation about support/ matrix, Examples about immobilized enzymes and their product, Advantages of immobilization, Applications of immobilization, Methods of immobilization in different categories like Adsorption method, Covalent bonding method, Entrapment method, Co polymerization /Cross linking method, Encapsulation method, Applications of immobilized enzymes, Diagrammatic explanation about methods of immobilization.

1. Immobilized / bound Enzymes – Methods of immobilization Jasmine Juliet .R Teaching Assistant Biotechnology Dept AC&RI, Madurai.

2. Enzyme - Introduction Enzymes are biological catalysts promote chemical reactions in living organisms. Enzyme has the ability to catalyze reactions under very mild conditions with high degree of substrate specificity thus decreasing the formation of by-products.

3. Enzyme immobilization - Introduction Enzymes are extensively used in diverse food processing , for e.g. in beer, wine, and cheese production. However, the widespread industrial application of enzymes, which has been going on for a long time, is often hindered by their short shelf-storage life, and inconvenient recovery and reutilization. These downsides can generally be overcome using various metho ds to immobilize the enzymes. Supported materials for immobilization are selected on the basis of enzyme application .

4. Enzyme immobilization - Introduction Enzyme immobilization is a technique specifically designed to restrict the freedom of movement of an enzyme. Immobilization of enzymes is a common practice, to minimize enzyme costs on the process by making it possible to reuse the enzyme many times. T he immobilization technique may be modify the enzyme behavior , thus reducing the enzyme and product costs. Many techniques have been used for enzyme immobilization.

5. Enzyme immobilization - Definition Immobilization is defined as the imprisoned of cell or enzyme in a distinct support or matrix. The support or matrix on which the enzymes are immobilized to allows the exchange of medium containing substrate or effector or inhibitor molecules. The practice of immobilization of cells is very old and the first immobilized enzyme was Amino acylase of Aspergillus oryzae for the production of L- aminoacids in Japan.

6. Examples for immobilized enzymes and their products Enzyme Product Glucose isomerase High-fructose corn syrup Aminoacid acylase Aminoacid production Penicillin acylase Semi-synthetic penicillins Nitrile hydratase Acrylamide Β - Galactosidase Hydrolyzed lactose(Whey)

7. Advantages of immobilization Increased functional efficiency of enzyme Enhanced reproducibility of the process. Reuse of enzyme. Continuous use of enzyme. Less labour input in the processes. Saving in capital cost and investment of the process.

8. Advantages of immobilization 7 ) Minimum reaction time . 8 ) Less chance of contamination in the products. 9) More stability of products. 10) Stable supply of products in the market. 11) Improved process control. 12)High Enzyme substrate ratio.

9. Applications of enzyme immobilization (1 ) Industrial production of antibiotics , beverages, aminoacids etc. Uses immobilized enzymes or whole cells. (2) Biomedical applications : Immobilized enzymes are widely used in the diagnosis and treatment of many diseases. (3) Food industry: Enzymes like pectinases , and cellulases immobilized are successfullyused in the production of jams, jellies and syrups from fruits and vegetables.

10. Applications of enzyme immobilization (4) Research: The use of immobilized enzyme allow researcher to increase the efficiency of different enzymes such as Horse Radish Peroxidase (HRP) in blotting experiments. (5) Production of bio-diesel from vegetable oils. (6) Waste water management: treatment of sewage and industrial effluents.

11. Applications of enzyme immobilization ( 7) Textile industry: Scouring, Bio- polishing , desizing of fabrics. (8) Detergent industry: immobilization of lipase enzyme for effective dirt removal from clothes.

12. Support / matrix used in immobilization technology The matrix or support immobilizes the enzyme by holding it permanently or temporarily for a brief period of time. There are a wide variety of matrixes or carriers or supports are available for immobilization. The matrix used should be cheap and easily available. Their reactions with the components of the medium or with the enzyme should be minimum possible.

13. Support / matrix used in immobilization technology The matrixes or supports for immobilization of enzymes or whole cells are grouped into three categories: Support/matrix Example Natural polymers Alginate, chitosan , chitin, collagen, carageenan , gelatin, cellulose, starch, pectin. Synthetic polymers Diethylaminoethylcellulose , Polyvinylchloride, UV activated polyethylene glycol (PEG). Inorganic materials Zeolites . ceramics, Diacetomaceous earth, silica, glass, Activated carbon, Charcoal.

14. Methods of immobilization

15. Methods of immobilization Based on matrix or support and the types of bond involved, there are five different methods of immobiliztion of enzymes or whole cells: Adsorption Covalent bonding Entrapment Copolymerization Encapsulation

16. (I) Adsorption Adsorption is the oldest and simplest method of enzyme immobilization. Nelson & Griffin used charcoal to adsorb invertase for the first time in 1916. In this method enzyme is adsorbed to external surface of the support . The support or carrier used may be of different type such as: Mineral support e.g. Aluminium oxide, clay Organic support e.g. Starch Modified sepharose and exchanged resin .

17. (I) Adsorption There is no permanent bond formation between the enzyme and carrier in adsorption method. Only weak bonds stabilize the enzyme to the support or carrier. The weak bonds ( low energy bonds ) involved are mainly: Ionic interaction Hydrogen bonds Vander waals force

18. Methods of adsorption Static Process: Immobilization to carrier by allowing the solution containing enzyme to contact the carrier without stirring . Dynamic batch process: Carrier is placed in the enzyme solution and mixed by stirring or agitation. Reactor loading process: Carrier is placed in the reactor , and then the enzyme sloution is transferred to the reactor with continuous agitation.

19. Methods of adsorption Electrode position process: Carrier is placed near to an electrode in an enzyme bathh and then the current is put on, under the electric field the enzyme migrates to the carrier and deposited on its surface.

20. Advantages of adsorption method No pore diffusion limitation. Easy to carry out. No reagents are required. Minimum activation steps involved. Comparatively cheap method of immobilization. Less disruptive to enzyme than chemical methods .

21. (II) Covalent bonding It is one of the widely used method enzyme immobilization. In covalent bonding, the covalent bonds formed between the chemical groups in enzyme and the chemical groups on the support or carrier. Hydroxyl groups and amino groups of support or enzyme form covalent forms quickly.

22. (II) Covalent bonding Carriers or supports are commonly used for covalent bonding are: Carbohydrates e.g. cellulose, DEAE cellulose, Agarose . Synthetic agents e.g. Polyacrylamide . Protein carriers e.g. collagen, gelatin. Inorganic carriers e.g. silica, porous glass.

23. Methods of covalent bonding Diazoation : Bonding between amino groups of support and tyrosyl or histidyl group of enzyme. Peptidyl bond: Bonding between amino or carboxyl group of the support and that of the enzyme. Poly functional reagents: Use of a bi-functional or multifunctional reagent ( glutaraldehyade ) which forms covalent bonds between the amino group of the support and amino group of the enzyme.

24. Advantages of covalent bonding Strong linkage of enzyme to the support. No leakage or desorption problem. Comparatively simple method. A variety of support with different functional groups available. Wide applicability .

25. (III) Entrapment In this method enzymes are physically entrapped inside a porous matrix. Bonds involved in stabilizing the enzyme to the matrix may be covalent or non-covalent. The matrix used will be a water soluble polymer. Examples of commonly used matrixes : Agar, Polyacrylamide gels, cellulose triacetate, gelatin .

26. Methods of Entrapment Inclusion in the gels: Enzymes trapped inside the gels. Inclusion in fibers: Enzymes supported on fibers made of matrix material. Inclusion in microcapsules: Enzymes entrapped in microcapsules formed by monomer mixtures such as polyamine and calcium alginate.

27. Advantages of Entrapment Fast method of immobilization. Cheap. Easy to practice at small scale. Mild conditions are required. Less chance of conformational changes in enzyme. Can be used for sensing applications.

28. (IV) Copolymerization This method is also called as cross linking . Enzymes are directly linked by covalent bonds between various groups of enzymes via polyfunctional reagents. There is no matrix or support is involved in this method. Commonly used polyfunctional reagents are glutaraldehyde and diazonium salt. This method is widely used in commercial preparations and industrial applications.

29. (V) Encapsulation Immobilization is done by enclosing the enzymes in a membrane capsule. The capsule will be made up of semipermeable membrane like nitrocellulose or nylon. In this method the effectiveness depends upon the stability of enzymes inside the capsule.

30. Advantages of Encapsulation Cheap and simple method. Large quantity of enzymes can be immobilized by encapsulation .

31. Immobilized enzymes - Conclusion Enzymes are intimately involved in a wide variety of traditional food processes , such as cheese-, beer- and wine-making. Desirable characteristics of enzymes and their widespread industrial applications are often hampered by their lack of long-term operational stability and shelf-storage life. These drawbacks can be overcome by immobilization of enzymes . In recent years, much attention has been directed towards the potentiality of immobilized enzymes in the food industry. 

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