Enzyme immobilization

Immobilization of enzymes

1. Enzyme Immobilization By, Jessy N & Akash Mahadev S 2 - M.Sc Biochemistry Department of Biochemistry University of Kerala Kariyavattom Campus

2. In a solution, biocatalysts behave as any other solute in that they are readily dispersed in solution or solvent and have complete freedom of movement in solution . Immobilization may be viewed as a procedure specifically designed to limit freedom of movement of a biocatalyst. Immobilisation separates a biocatalyst from the bulk solution phase to produce a heterogeneous two-phase mixture Soluble Enzyme + Substrate = Product (Single time usage) Immobilied enzyme + Substrate = Product (Repeated usage of enzyme)

3. The term “ immobilized enzymes ” refers to “ enzymes physically confined or localized in a certain defined region / space with retention of their catalytic activities, and which can be used repeatedly and continuously .”

4. HISTORY The first industrial use of immobilized enzymes was reported in 1967 by Chibata and co-workers, who developed the immobilization of Aspergillus oryzae aminoacylase for the resolution of synthetic racemic D-L amino acids.

5. WHY IMMOBILISE??? Enzymes are expensive. As catalytic molecules, enzymes are not directly used up. After the reaction the enzymes cannot be economically recovered for re-use and are generally wasted. Separation of enzyme and product using a two-phase system; One phase containing the enzyme The other phase containing the product The enzyme is imprisoned within its phase allowing its re-use or continuous use. The separation prevents the enzyme from contaminating the product

6. Advantages of enzyme immobilization:- Multiple or repetitive use of a single batch of enzymes. Immobilized enzymes are usually more stable . Ability to stop the reaction rapidly by removing the enzyme from the reaction solution. Product is not contaminated with the enzyme. Easy separation of the enzyme from the product- saves cost of downstream processing Allows development of a multienzyme reaction system . Disadvantages of enzyme immobilization:- It gives rise to an additional bearing on cost . It affects the stability and activity of enzymes. The technique may not prove to be of any advantage when one of the substrate is found to be insoluble. Certain immobilization protocols offer serious problems with respect to the diffusion of the substrate to have an access to the enzyme.

7. I M M O B I L I Z A T I O N Entrapment Microencapsulation Physical retention Chemical bonding Inclusion in fibers Inclusion in gels Inclusion in microcapsules Liposomal entrapment Reverse micelle entrapment Adsorption Cross linking Covalent binding Ionic binding

8. Entrapment The entrapment method is based on the occlusion of an enzyme within a polymeric network (polyacrylamide, alginate etc.) that allows the substrate and products to pass through (which ensures continuous transformation) but retains the enzyme. The porosity of a gel lattice is controlled to ensure that the structure is tight enough to prevent leakage of enzyme or cells and at the same time allow free movement of substrate and product.

9. E E E E E E E E E E E E E E E E E E E E E E E E E E Inclusion in gels Inclusion in microcapsules Entrapment contd.... E E E E E Inclusion in fibres Inclusion in gels : Enzymes trapped in gels (Polyacrylamide, Polyvinyl alcohol and Polyvinyl pyrrolidone gel). Eg; Glucose oxidase, urease immmobilized in PA gel. Inclusion in fibres : Enzymes supported on fiber format made up of polyacetate, collagen, cellulose etc.. Eg; collagen fiber for immobilizing LDH. Inclusion in microcapsules : Enzymes entrapped in microcapsules formed by monomer mixtures such as polyamine and polybasic acid chloride, polyphenol and polyisocyanate. E E E E E E E E E E E E E E E E E E

10. Amphipathic lipids …..(Polar heads and Non- polar tails). Liposomes are small artificial vesicles of spherical shape that can be created from cholesterol, Phosphatidyl choline , Phosphatidyl serine, Sterylamine and Sphingomyelin Sizes ranging from 30 nm to several micrometers Liposomal encapsulation Reverse micelle entrapment Reverse micelles are nanometer-sized (1-10 nm) water droplets dispersed in organic media obtained by the action of surfactants. Surfactant molecules organize with the polar part to the inner side able to solubilize water and the apolar part in contact with the organic solvent. Micelle Reverse micelle Entrapment contd....

11. Advantages Simplicity, No change in intrinsic enzyme properties, Involves no chemical modification, Minimal enzyme requirement and Matrices are available in various shapes. Disadvantages Enzyme leakage, Only small sized substrate/products can be used, Requires delicate balance between mechanical properties of the matrix and its effect on enzyme activity and Presence of diffusional constraints

12. Microencapsulation Enzymes are immobilized by enclosing them within spherical semi-permeable polymer membranes with controlled porosity (1–100 μm) . The term “ microcapsule ” is defined, as a spherical particle with the size varying between 50 nm to 2 mm (2 x 10 6 nm) containing a core substance. Enzymes immobilized in this manner are physically contained within the membrane, whilst substrate and product molecules are free to diffuse across the membrane. membranes are made of cellulose nitrate,1,6-diaminohexane, polystyrene.

13. The techniques used to produce the semi permeable microcapsule membranes are classified as phase inversion, polyelectroyte coacervation , and interfacial precipitation . Phase inversion involves the induction of phase separation in a previously homogeneous polymer solution by a temperature change or by exposing the solution to a non-solvent component. Microencapsulation contd….

14. Polyelectrolyte coacervation process, results from mixing of oppositely charged polyelectrolytes ;membrane is formed by the complexation of oppositely charged polymers to yield an interpenetrating network with poor solvent affinity. Polyelectrolyte coacervation Microencapsulation contd….

15. Different combinations of polyionic species used are Cellulose sulphate with poly(diallyl dimethyl ammonium chloride) Carboxymethylcelluose with chitosan Gelatin with gum arabic/polyphosphate Microencapsulation contd….

16. Advantages Economic and simple method Extremely large surface area due to which they have higher catalytic efficiency. Disadvantages Occassional inactivation of enzyme during microencapsulation Higher concentration of enzyme is required Possibility of enzyme incorporation into the membrane wall Enzyme leakage

17. CHEMICAL METHODS

18. Carrier binding methods Binding of enzymes to water insoluble carriers The oldest and most prevalent method. The materials used for immobilization of enzymes are called carrier matrices -usually inert polymers. Types of carriers Naturally occurring Synthetic organic Inorganic The different types of carrier binding methods are Physical Adsorption Chemical Cross-linking Covalent binding Ionic binding

19. The ideal carrier matrix has the following properties : Low cost Inertness towards enzymes Physical strength, Stability, Biocompatibility Reduction in product inhibition, A shift in the pH optimum for enzyme action to the desired value for the process, and Reduction in microbial contamination and non-specific adsorption

20. Naturally occurring-Biopolymers They are water-insoluble polysaccharides (e.g., cellulose, starch, agarose, chitosan) and proteins such as gelatin. Synthetic organic polymers Easily and artificially designed. Can adjust the porosity, ionic and hydrophobic or hydrophilic properties. Mechanical strength and longevity - Superior to those from natural polymers. Eg; Eupergit-C (acrylic resin) It is prepared by using compounds: N,N′-methylene-bis-(methacrylamide),methacrylamide, allyl glycidyl ether and glycidyl methacrylate. Other egs; Sepa beads FP-EP, Amberlite XAD-7 Sepa beads

21. Inorganic solids Cheapest matrix being used for the immobilization Eg; alumina, silica, zeolites and mesoporous silicas Silica Alumina Zeolites

22. Adsorption Earliest method of enzyme immobilization Physical adsorption of enzyme molecules onto the surface of solid matrices. Enzyme is attached to the support material by (weak) non-covalent linkages including ionic or hydrophobic interactions, hydrogen bonding, and van der Waals forces. It can be carried out by contacting between the enzyme solution and polymer support in a stirred reactor Therefore, the adsorbed enzymes can be easily removed by minor changes in pH, ionic strength or temperature Adsorption contd…..

23. The method is simple and mild with a vast variety of carriers helpful for simultaneous purification as well as enzyme immobilization without any conformational change. E E E E E E E E E E + + + + + + + + + + + Adsorption by Van der waal forces Adsorption by hydrogen bonding E Adsorption contd…..

24. There are four procedures for immobilization by adsorption Static process (enzyme is immobilized on the carrier simply by allowing the solution containing the enzyme to contact the carrier without stirring) The dynamic batch process (carrier is placed into the enzyme solution and mixed by stirring or agitated continuously in a shaker. The reactor loading process (carrier is placed into the reactor, then the enzyme solution is transferred to the reactor by agitating the carrier and enzyme solution.   The electrodeposition process (Carrier is placed close to one of the electrodes in an enzyme bath, the current put on, the enzyme migrates to the carrier and gets deposited on the surface) ENZYME CARRIER α -amylase Ca 3 (PO 4 ) 2 Catalase Charcoal Invertase Agarose gel, DEAE - Sephadex Adsorption contd…..

25. Advantages Because no reactive species are involved, there is little or no conformational change in the enzyme on immobilization Easy and economic method for preparing immobilized enzymes Easily reversed to allow regeneration of catalyst-Can be Recycled, Regenerated & Reused (R 3 ) Disadvantage Desorption of protein from the carrier during use owing to the weakness of the involved binding forces , with subsequent loss of catalytic activity and contamination of products Limited reliability when absolute immobilization of an enzyme is desired

26. Cross linking Special chemicals used for promoting intermolecular linkage - cross linking agents- help in formation of covalent bonds between enzyme molecules. Cross linking is accomplished using bi- or poly-functional reagents Toxicity of such reagents is a limiting factor in applying this method to living cells and many enzymes. This type of immobilisation is support free and involves joining cells (or enzymes) to each other to form a large three-dimensional complex structure. Eg; Glutaraldehyde, Diazobenzidine, Disuccinimidyl suberate, Bismaleimide, Toluene diisocyanate, Hexamethylene isocyanate Cross linking contd….. E E E E E E E E E E E E E E E

27. Glutaraldehyde is a bifunctional crosslinking agent which effectively hooks up the amino group of the enzyme. Eg; Glucose isomerase Cross linking contd…..

28. Advantages Strong linkage leads to low enzyme leakage while use. Higher stability. Disadvantages Partially or wholly inactivation by active site modification. Not cost effective.

29. Covalent linkage The enzyme is attached to the matrix by means of covalent bonds. The immobilization of an enzyme by covalent attachment to carrier/matrix must involve functional groups of the enzyme that are not essential for catalytic action. No reagents must be used, which could affect the binding and active sites of the enzymes A covalent linkage between the carrier and the enzyme can be established by different methods. Formation of isourea linkage - Cyanogen bromide activation. Formation of a peptide bond - Carbodiimide coupling. Activation by bi- or polyfunctional reagents Formation of a diazo linkage Covalent linkage contd….

30. Cyanogen bromide activation Inert support materials (cellulose, PVA, sephadex) containing glycol groups are activated by CNBr. The activated carrier is then covalently linked with the amino group of enzymes- ISOUREA LINKAGE . Eg; Ascorbic acid oxidase Covalent linkage contd….

31. Carbodiimide coupling - Peptide bond formation In carbodiimide activation, a support material should have a carboxyl (-COOH) functional group and an enzyme and support are joined via a peptide bond. Covalent linkage contd….

32. Some of the reagents such as glutaraldehyde can be used to create bonds between amino groups of enzymes and amino groups of support (eg: aminoethylcellulose, albumin, aminoalkylated porous glass) Activation by bi- or polyfunctional reagents Covalent linkage contd….

33. Diazotization Some of the support materials (aminobenzyl cellulose, aminosilanized porous glass) are subjected to diazotization on treatment with NaNO 2 and HCl. They inturn bind covalently to tyrosyl or histidyl groups of enzyme. Covalent linkage contd….

34. Advantages The strength of binding is very strong, so, leakage of enzyme from the support is absent or very little. This is a simple, mild and often successful method of wide applicability Disadvantages Enzymes are   chemically modified and so many are denatured during immobilization. Only small amounts of enzymes may be immobilized (about 0.02 grams per gram of matrix).

35. Ionic binding This is based on ionic interactions between enzyme molecules with a charged matrix. Higher the surface charge density on the matrix, the greater would be the amount of enzyme being bound to the matrix. Enzyme binding via ionic interactions during immobilization depends on the pH of the solution, the concentration of the enzyme and temperature. Ionic binding contd…

36. Commonly used matrices are: Polysaccharide derivatives (e.g., DEAE cellulose, dextran, CMC, chitosan), Synthetic polymers (e.g., Polystyrene derivatives, polyethylene glycol, polyvinyl alcohol) and Inorganic materials (e.g., Amberlite, alumina, silicates, bentonite ). This method of immobilization leads to minimal changes in enzyme conformation. Ionic binding contd…

37. Advantages: Low cost. Regeneration is possible. Easy preparation. Disadvantages: Not fit for industrial use as effectiveness gradually decreases.

38. Applications of Immobilized Enzymes Immobilized enzyme-aminoacylase used for the production of L-amino acids In food industry, fructose syrup is produce from glucose by use of immobilized enzyme glucose-isomerase. Immobilized enzyme used in biosensors. Accurate analysis of sample done with the help of specific immobilized enzymes. Immobilized Enzyme or Cells used in industry for the production of various industrial products. Enzymes Transformation of organic compounds Aminoacylase Optical resolution of DL-amino acids α -amylase and glucoamylase Conversion of starch to glucose Glucose isomerase Conversion of glucose to fructose Penicillin amidase Production of 6-aminopenicillanic acid from penicillin β -Galactosidase Hydrolysis of lactose in milk or whey

39. Let’s Summarize……

40. Conclusion With the vast array of research on enzyme immobilization, we can conclude that it is one of the most promising techniques for highly efficient and economically competent biotechnological processes in the field of environmental monitoring, biotransformation, diagnostics, pharmaceutical and food industries . Enzyme-based strategies are increasingly replacing conventional chemical methods in both laboratories and industries with attributes like efficiency, quicker performance and multifarious use. However, commercialization of immobilized enzymes is still at a lower pace because of their costs and storage problems.

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