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1, , Biochemistry, , Enzyme immobilization

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Description of Module, Subject Name, , Biochemistry, , Paper Name, Module Name/Title, , Enzyme immobilization, , Dr. Vijaya Khader, Dr. MC Varadaraj, , 2, , Biochemistry, , Enzyme immobilization

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1. Objectives, 1., 2., 3., 4., 5., 6., , What is an immobilized enzyme, Merits and Demerits of Immobilization, Methods of immobilization, Matrix or Solid support used in immobilization, Properties of immobilized enzymes, Applications of enzyme immobilization, , 3, , Biochemistry, , Enzyme immobilization

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2. Enzyme Immobilization, Enzymes are biocatalyst that carries out all the essential biochemical reactions inside the body of an, organism. Their unique feature is that they remain unaltered after the reaction is completed. Therefore,, they can be used again and again. But the limitation of soluble enzymes is their isolation from the, product and the substrate. Most of the Enzymes in the living organism are attached to the cell, membrane or entrapped within the cells. This observation led to the concept that pure isolated enzymes, may actually perform better when they are immobilized on a solid support. The term immobilized, enzyme is used to denote “enzymes physically confined or localized in a defined region of space with, retention of their catalytic activities and which can be used repeatedly and continuously”., Immobilization is beneficial because it facilitates work up product isolation. Some of the potential, advantages and disadvantages of immobilization are highlighted below., Soluble Enzyme + Substrate----------- Product (single time usage of enzyme), Immobilized Enzyme + Substrate---------Product (Repeated usage of enzyme), , Figure 1: Enzyme Immobilization, 4, , Biochemistry, , Enzyme immobilization

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2.1. Advantages:, 1., 2., 3., 4., , Easy recovery of the product, Product is free from enzyme, so there is no cost of purification of enzyme., Enzyme can be used repeatedly, The enzyme generally get stabilized after adsorption, , 2.2. Disadvantages:, 1., 2., 3., 4., , Loss of catalytic properties for some enzymes, Some enzymes become unstable, Additional cost of immobilization, Differential limitations., , Intensive study in the area of enzyme immobilization started in mid 1950 and has since continued. The, first industrial use of immobilized enzymes was reported in 1967 by Chibata and co-workers who, immobilized Aspergillus oryzae amino acylase for the resolution of synthetic , DL amino acids into the, corresponding optically active enantiomers. At present, the use of immobilized enzyme is well, established in various industries., Number of important points should be kept in mind while immobilizing an enzyme, 1., 2., 3., 4., , The biological activity of the enzyme should be retained, The enzyme should be more stable as compared its soluble counterpart., The cost of immobilization should not be too high, It should be used repeatedly., , 5, , Biochemistry, , Enzyme immobilization

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3. Enzyme immobilization Techniques, The various methods of enzyme immobilization are broadly classified as, 1. Reversible immobilization, a. Adsorption, 2. Irreversible immobilization, a. Covalent coupling, b. Entrapment and microencapsulation, c. Crosslinking, , Figure 2: Diagrammatic representation of the various methods of immobilization, , The advantage of reversible immobilization over irreversible systems can be summarized as follows:, 1. No chemical modification of the enzyme is required, 2. If the enzyme gets activated during use, it can be replaced in reversible immobilization., 3. The immobilization of enzyme by adsorption of bioaffinity can be accomplished rapidly, 6, , Biochemistry, , Enzyme immobilization

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3.1. Reversible immobilization, 3.1.1. Adsorption, Immobilization by adsorption is the easiest and fastest method. The adsorption is dependent on the, experimental variables such as pH, nature of solvent, ionic strength, quantity of enzyme and adsorbent,, the time and temperature. A close control of these variables is required owing to the relatively weak, binding forces between protein and adsorbent (hydrogen bonds, van der Waals forces, hydrophobic, interactions, etc.). Enzymes can be immobilized by simply mixing the enzymes with the matrix, under, appropriate conditions of pH and ionic strength. Adsorption process is based on vander Waal forces,, ionic and hydrogen bonding as well as hydrophobic interactions, which are very weak forces, but in large, number, impart sufficient binding strength. Adsorbed enzymes can be protected from agglomeration,, proteolysis and interaction with hydrophobic interfaces. In order to prevent chemical modification and, damage to enzyme, the existing surface properties of enzymes and support are need to be considered., The adsorption through physical method generally involves multipoint protein adsorption between a, single protein molecule and a number of binding sites on the immobilization surface. The main, disadvantage of this method is that the enzyme is easily desorbed by factors like pH, temperature, fluctuations, changes in substrate and ionic concentrations. Few advantages of adsorption methods are,  Easy to carry out,  No reagent are required,  Minimum activation step involved,  Comparatively cheap method,  Less disruptive to protein than chemical methods, , Figure 3: Adsorption, 7, , Biochemistry, , Enzyme immobilization

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3.2. Irreversible immobilization, 3.2.1. Covalent coupling, Covalent coupling is the most frequently used approach of immobilization in which covalent bonds are, formed between surface amino acids of the enzyme and the matrix. Hydrophilic amino acids which are, likely to be present on the protein surface, can be exploited for this purpose. ε-amino group of lysine, residue, cysteine (via SH), tyrosine, histidine, aspartic and glutamic acids, tryptophan and arginine, mostly takes part in bond formation. A number of chemical reagents and protocols are available for, covalently linking an enzyme to the matrix., , Figure 4: Covalent binding, , 8, , Biochemistry, , Enzyme immobilization

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Most commenly used methods of covalent bonding are:, 1. Diazoation: It is based on the diazo linkage between protein and aryldiazonium electrophilic, groups of the matrix., 2. Formation of Peptide bond: bond formation between amino/carboxyl groups of support, and amino or carboxyl group of the enzyme, 3. Poly functional reagents: use of bi-functional or multifunctional reagent (glutaraldehyde), which forms bonding between the amino group of the support and amino group of the, enzyme, , Figure 5: Glutaraldehyde, 4. Amidination reaction: The matrix containing amido ester functional groups can be used for, immobilization of protein., 5. Thiol–disulphide interchanged reaction: This methods is used for protein bonding via thiol, groups of both carrier and protein., 6. Akylation and arylation: This methods is based on alkylation of amino, phenolic and thiol, groups of protein with reactive matrix containing halides, vinyle, sulphonile etc., Advantage of Covalent bonding method:,  Strong linkage of enzyme to the support,  No leakage or desorption problem,  Comparatively simple method,  A variety of support with different functional group available,  Wide applicability, Disadvantage of Covalent bonding method:,  Chemical modification of enzyme leading to functional conformational loss,  Enzyme inactivation by change in the conformational when undergoes reactions at active, site,  This can be overcome through immobilization in the presence of enzyme substrate or a, competitive inhibitor, 9, , Biochemistry, , Enzyme immobilization

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3.2.2. Entrapment, Entrapment and encapsulation is based on the occlusion of an enzyme within a constraining structure,, but tight enough to release an enzyme while allowing penetration of a substrate. However, due to, diffusion limitations, such methods are often unsuitable for the immobilization of enzymes hydrolyzing, macromolecular substrates., , Figure 6, Methods of Entrapment,  Inclusion in the gels: enzymes trapped in gels,  Inclusion in fibers: enzymes supported on fiber formate,  Inclusion in microcapsules: enzymes entrapped in microcapsules formed by monomer, mixtures such as polyamine, calcium alginate, Advantage of Entrapment method:,  Fast,  Cheap (low cost matrix available),  Mild conditions are required,  Less chance of conformational change in the enzyme, Disadvantage of Entrapment method:,  Leakage of enzyme,  Pore diffusion limitation,  Chance of microbial contamination, 10, , Biochemistry, , Enzyme immobilization

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3.2.3. Cross linking, This method involves attachment of biocatalysts to each other by bi- or multifunctional reagents or, ligands. In this way, very high molecular weight typically insoluble aggregates are formed. Cross-linking, is a relatively simple process. It is not a preferred method of immobilization as it does not use any, support matrix. So they are usually gelatinous and not particularly firm. Since it involves a bond of the, covalent kind, biocatalyst immobilized in this way frequently undergoes changes in conformation with a, resultant loss of activity. Still it finds good use in combination with other support dependent, immobilization technologies, namely to minimize leakage of enzymes already immobilized by, adsorption., , Figure 7: Cross-linking, , 11, , Biochemistry, , Enzyme immobilization

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Recent advancement to crosslinking method is the formation of CLEC, CLEA and Spherezyme, , Figure 8: Formation of CLEC, CLEA and Spherezyme by crosslinking, Advantage of Cross linking method:, , , It is used mostly as a means of stabalizing adsorbed enzyme and also for preventing leakage, , Disadvantage of Cross linking method:, , , Cross linking may cause significant change in the active site of enzyme which may lead to, loss of activity, , 12, , Biochemistry, , Enzyme immobilization

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4. Charcteristic for a Solid support, one of the major component of immobilized enzyme is the support materials. Ideal support material, should posses following characteristic:, 1., 2., 3., 4., 5., 6., 7., 8., 9., , Large suface area, Permeability, Hydrophilic character, Insolubility, Chemical mechanical and thermal stability, High rigidity, Soutable shape and particle size, Resistance to microbial attack, Regeneratibilty, , 4.1. Support material are classified into two categories based on their morphology, 1. Non porus support: eg glass, 2. Porus support: eg silica, Porus support have high surface area as compare to non porus support., 4.2. Support material are classified into two categories based on their chemical nature, 1. Organic, 2. Inorganic, 4.2.1. Organic support material are further classified as:, 1. Natural, 2. Synthetic, , 13, , Biochemistry, , Enzyme immobilization

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Natural organic supports include Polysacchrides and proteins., Some of the commonly used polysaccharides are, , , Alginate : are polymers of n-acetyl glucouronic acid, having hydroxyl group which can be, derivatized easily, , Figure 9: Alginic acid, , , Chitosan and chitin: chitosan is deacetylated chitin, , (a), , (b), , Figure 10, , 14, , Biochemistry, , Enzyme immobilization

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, , Carrageenan: Carrageenan, a linear sulfated polysaccharide., , Figure 11: The molecular structures of carrageenan, , , , Cellulose: a linear polymer of D-glucose units (two are shown) linked by β(1→4)-glycosidic, bonds, , Figure 12, ,  Starch: linear polymer of glucose,  Pectin: A heteropolysaccharide made of galacturonic acid present in plant cell wall., Some of the commonly used Proteins are:, , , , Collagen, Gelatin, , 15, , Biochemistry, , Enzyme immobilization

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Synthetic support are the largest number of support materials avalaible for protein immobilization due, to their physical and chemical characteristic., , , , , , , DEAE cellulose, polyvinyl chloride (PVC), UV-activated polyethylene glycol (PEG), glutaraldehyde-activated nylon, cyclodextrin glucosyltransferase, , 4.2.2. Inorganic materials as supports: the surface of most inorganic support is mainly composed of, oxide and hydoxil group, such as silanol group in glass, provide mild reactive surface for activation and, protein binding., , , Zeolites: Zeolites are microporous crystalline solids with well-defined structures., , Figure 13: The microporous molecular structure of a zeolite, ZSM-5, , 16, , Biochemistry, , Enzyme immobilization

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, , , , , , , Ceramics: inorganic , non metal, Celite: Celite is highly porous diatomaceous material., Silica: oxide of silicon, Glass: Glass is a highly viscous liquid, Activated carbon: Activated carbon is produced from carbonaceous source materials, such, as coal, coconuts, nutshells, peat, wood, and lignite., Charcoal: allotrope of carbon, has excellent adsorption capacity, , 5. Biochemical Properties of the Immobilized Enzymes, The biochemical properties of the immobilized enzyme are different when compared to the free, enzyme, this is due to the change in environment of the immobilized enzyme, , , , the physical and chemical properties of the support matrix and interactions of the matrix, with substrates or products changes the kinetics of the immobilized enzyme., , , , Generally there is a decrease in the rate of enzyme catalyzed reaction because the matrix, restric the diffusion of the substrate towards the enzyme., , 17, , Biochemistry, , Enzyme immobilization

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, , The Km of the enzyme also changes after immobilization because of diffusion limitations. If, the matrix is positively charged and substrate is also positively charges due to electrostatic, repulsion the substrate will not come in the viccinity of the enzyme hence Km is altered., , , , Sometimes the 3D structure of the enzyme is also changed which also result in altering the, kinetic properties of enzyme., , , , The performance of the immobilized enzyme can be improved further by studying the, structural changes of the immobilized enzyme, , Figure 14: Structural studies improves the performance of the immobilized enzyme, , 18, , Biochemistry, , Enzyme immobilization

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6. Applications of enzyme immobilization, , Figure 15: Applications of immobilized enzyme., , 19, , Biochemistry, , Enzyme immobilization

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6.1. Biomedical Application, Biosensor: Biosensor are electronic monitoring devices that make use of an enzyme’s specificity and the, technique of enzyme immobilization, , Figure 16: Biosenser, A biosensor has been developed for detecting glucose in the blood of diabetics., , Figure 17: Detecting glucose in blood, , 20, , Biochemistry, , Enzyme immobilization

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6.3. Immobilized enzymes in food industry, Immobilized enzymes are used in the processing of food samples and its analysis., , Figure 18: Removal of lactose from milk by Immobilized enzyme, , 23, , Biochemistry, , Enzyme immobilization

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Figure 19: Conversion of glucose syrup into fructose corn syrups, , 24, , Biochemistry, , Enzyme immobilization

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Table 3 shows the processing of various food substrates using respective immobilized enzymes., Table 3: Immobilized enzymes used in food industry, Enzyme, , Immobilization support, , Food substrate, , β-galactosidase and, amyloglucosidas, , Bone powder, , Lactose,, Whey,, Whey, permeates, skimmed milk, , Pectinase, , Amino exchange resin, , Pectin, , Laccase, , Silica gel, , Ine, fruit, processing, , Trypsin, , cellulose, , Β-lactoglobulin, , Tyrosinase, , Polyacrylic, nanotubes, , Pectinase, , Amino exchange resin, , acid, , juice, , and, , beer, , carbon Phenolic in red wine, , Pectin solution, , 25, , Biochemistry, , Enzyme immobilization

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6.5. Immobilized enzymes for bioremediation, Bioremediation is a technique that involves the use of enzyme and biological organism to remove, pollutants from a contaminated site., , Figure 21: Peroxidase immobilized on support and used for continuous palm oil mill effluent (POME), treatment, , 27, , Biochemistry, , Enzyme immobilization

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Summary, , , Immobilization means restricted movement of the enzyme., , , , Immobilization can be carried out by attaching the enzyme to a solid support., , , , The enzyme is linked by noncovalent interaction or by covalent linkage to the solid support, , , , The immobilized enzyme is more stable in terms of thermal stability, pH etc as compared to free, enzyme, , , , The major advantage of immobilization is repeated use of the enzyme., , , , Nowadays immobilized enzymes are used in all the sectors like medicine, food industry,, pharmaceutical industry and for bioremediation etc., , 29, , Biochemistry, , Enzyme immobilization