enzyme immobilization

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TOPIC – ENZYME IMMOBILIZATION Prepared by: Ankita Gupta M.Pharm (1 st Year ) Psit - kanpur

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What I s An Immobilized Enzyme? An immobilized enzyme is one whose movement in space has been restricted either completely or to a small limited region . Enzyme immobilization may be defined as a process of confining the enzyme molecules to a distinct phase from the one wherein the substrates and the products are present . What Is Enzyme Immobilization ?

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Benefits Of Immobilizing An Enzyme Protection from degradation and deactivation. Retention of enzyme, enzyme-free products. Recycling, repetitive use. Cost efficiency. Enhanced stability. Use as controlled release agents. The ability to stop the reaction rapidly by removing the enzyme from the reaction Solution (or vice-versa) Allows development of multienzyme reaction system. Reduces effluent disposal problems.

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An Ideal Carrier Matrices For Enzyme Immobilization Inert. Physically strong and stable. Cost effective. Regenerable. Enhance specifity of enzyme. Reduction in product inhibition.

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CLASSIFICATION OF CARRIERS Inorganic Carriers High pressure stability. May undergo abrasion Examples : Commercialy SiO2 available materials- Porous glass. Silica. 2. Mineral materials -(clays) Celite ,Centonite Organic Natural Carriers Favourable compatibility with proteins. Examples: cellulose derivatives- DEAE-cellulose CM-cellulose. 2. Dextran. Polysacharides Agarose, Starch Pectine ,Chitosan Organic Synthetic Carriers High chemical and mechanical stability. Examples: Polystyrene 2.Polyvinylacetate 3. Acrylic polymers

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1.Surface Immobilization/Carrier Binding According to the binding mode of the enzyme, this method is further sub classified into: 1(a) Physical Adsorption : Enzyme molecules get adhered to the surface of carrier matrix. Driving force is hydrophobic intxn and salt bridge Binding onto silica, clay or ion-exchange materials by weak interaction (e.g., ionic, electrostatic, hydrophobic) Carriers: silica, carbon nanotube, cellulose, etc. e.g.,catalase & invertase - activated charcoal

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PROCEDURE Enzyme mixed with adsorbent Appropriate pH & desired ionic strength Incubation for a stipulated duration Carrier matrix washed thoroughly to get rid of unabsorbed enzyme molecules Immobilized enzymes

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1(b) - Ionic Binding : Ionic bonds similar to physical adsorption. Carriers : polysaccharides and synthetic polymers having ion-exchange centers. Advantages: Easily desorbed, simple and cost-effective method. H igh enzyme loading (nearly 1 g enzyme/ g matrix). Limitation: Dependent on process conditions (e.g., pH, temperature, ionic strength, hydrophobicity)

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1(c). Covalent Binding The covalent binding method is based on the binding of enzymes and water-insoluble carriers by covalent bonds. Principle (1. derivatization, 2. activation, 3. binding of enzyme). Maximum 0.2 g enzyme/g matrix α-amylase: DEAE-cellulose Glucose isomerase : polyurethane

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Polymeric Supports which are widely used: Hydroxyl groups of polysaccharide, PVA, Polymethylacetate Amino ethyl coated polysaccharides, silica gels Aldehyde and acetyl groups of polymers Amide groups of polypeptides The Protein Functional Groups used for the covalent coupling NH2-lysine COOH- α and β Aspertic acid,Glutamic acid OH- Phenol ring on tyrosine SH- Cysteines

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Lysine residues are the most generally useful groups for covalent bonding of enzymes to insoluble supports due to their widespread surface exposure and high reactivity, especially in slightly alkaline solutions. They also appear to be only very rarely involved in the active sites of enzymes . The most commonly used method for immobilizing enzymes on the research scale involves Sepharose (poly-{b-1,3-D-galactose-a-1,4-(3,6-anhydro)-L- galactose}), activated by Cyanogen bromide, because it is acommercially available beaded polymer which is highly hydrophilic and generally inert to microbiological attack .

Covalent coupling:

Covalent coupling Advantages Very little leakage , prevents elution of proteins in to the production stream Stable method (not reversed by pH ionic strength, substrate) The wide range of choices is possible by selecting carrier materials and binding method. This allows flexibility in designing an immobilized enzyme with specific physical and chemical properties Disadvantages Relatively expensive and complicated in procedures. Low enzyme activity due to exposure of the enzymes to harsh environments and toxic reagents. Active site may be modified through the chemical reactions used to create covalent bonding

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2 . CROSS-L I NK I N G This method is based on the formation of covalent bonds between enzyme molecules ,by means of bi-or multi-functional reagent,leading to 3 dimensional crosslinked aggregates

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Advantages Very little desorption(enzyme strongly bound ) Cross- linking is best use in conjunction with one of the other methods. It is used mostly as a means of stabilizing adsorbed enzymes and also for preventing leakage . Disadvantages Cross -linking may cause significant changes in active site of the enzymes , and also severe diffusion limitation may lead to significant loss of activity. Loss of enzyme activity during preparation .

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3 . ENTRAPMENT The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix ,gels or capsule(micro encapsulation) . It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into lattice and micro capsule types . The support should have very small size pores which facilitates the movement of substrate inside the compartment.

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Inclusion in gels: Poly acrylamide gel,Poly vinyl alcohol gels Inclusion in fibers : Cellulose and Poly - acrylamide gels. Inclusion in micro capsules : Polyamine, Polybasic acid chloride monomers .

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3(a). Lattice-Type Entrapment E ntrapment involves entrapping enzymes within the interstitial spaces of a cross-linked water-insoluble polymer. Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc... and natural polymer (starch) have been used to immobilize enzymes using this technique. ENTRAPPED IN LATTICE ENTRAPPED IN POLYMER NETWORK

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3(b). MicrocapsuleType E ntrap met/ Encapsulation/Membrane Confinement It involves enclosing the enzymes within semi - permeable polymer membranes . Liposome can be used for this purpose e.g.,semipermeable collodion or nylon membranes in the shape of spheres are utilized for microencapsulation of Aminocyclase enzyme Encapsulation of enzyme

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Immobilization Procedure Enzyme blended with polymer solution Polymerization Extrusion/Shape the particles Enzyme entrapped within the microcapsules

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ENTRAPMENT Challenges- Enzyme leakage into solution Diffusional limitation Reduced enzyme activity and stability Lack of control micro-environmental conditions. It could be improved by modifying matrix or membrane.

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Comparison Between The Methods

LIMITATIONS OF ENZYME IMMOBILIZATION:

LIMITATIONS OF ENZYME IMMOBILIZATION Cost of carriers and immobilization. Changes in properties (selectivity). Mass transfer limitations. Problems with cofactor and regeneration. Problems with multienzymes systems. Activity loss during immobilisation.

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REFERENCES Pharmaceutical Biotechnology: Vyaas SP and Dixit VK (page-13-159). A Textbook of Biotechnology: Dubey RC (page 238-240). IMMOBILIZED ENZYMES by M. K. Goel, 1994. http//:www.scribd.com/doc/14657530/applications-0f-enzyme-immobilization.pd f

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