Immobilization Of Enzymes

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  • 1. Immobilization of Enzymes

2. Introduction Immobilization of enzymes can be defined as the confinement of anenzyme (bio-catalyst) in a distinct phase, separated from the bulkphase but allowing it to exchange with the latter. Bulk Phase consists of a substrate, an effecter or inhibitor. Immobilized enzyme is either physically entrapped or covalentlybonded by chemical means to an inert insoluble matrix or carrier. In other words, it involves the restrictive localization of enzymes. Matrix is generally a high molecular weight polymer.Ex : cellulose, polyacrlamide, alginate, etc.S 3. Introduction S 4. Introduction S 5. Introduction S 6. Introduction S 7. Introduction S 8. Need for Immobilization Accelerates the chemical reaction. Specificity & un-modified enzyme. Cost effective. Not difficult to separate. Attachment to polymers/matrix, causes re-use.S 9. Advantages of Immobilized Enzymes Recovered at the end of the reaction thereby can be reused. Economy of the reaction is improved. Easy separation of enzyme from the products occurs. Stability of immoblilised enzyme increases. Enhanced enzyme properties. Efficiency of the catalytic reaction is better in a few cases. Better control of reaction can be achieved. Catalytic process can be operated continuously. Multi enzyme reaction possible.S Potential in industrial & medicinal use. 10. Methods of Immobilization Parameters for Method Selection :- Overall catalytic activity. Effectiveness of the catalytic utilization. Deactivation & Regeneration characteristics. Cost effective. Intended application of immobilized enzyme. Toxicity of immobilized enzyme. Waste disposal (of immobilization process). S 11. Methods of Immobilization Physical Methods Chemical MethodsAdsorption Covalent BondingEntrapping Cross LinkingMembrane confinement Complexation & Chelation S 12. Carrier for Immobilized Enzymes Ideal Characteristics of the Carrier:- Low Cost & of optimum quality Inertness Physical Strength Stability Regenerability Enhancement of enzyme specificity Reduction of product inhibition S 13. Carrier for Immobilized Enzymes Types of Carriers :Naturally occuringStructural proteins (Ex: ceratin, collagen)Globular proteins (Ex: albumin)Carbohydrates (Ex: dextran)Synthetic organicEx: polyvinyls, epoxide,etcInorganicEx: glass, silica gel, bentonite, titania,etc. S 14. A Non-specific binding like electrostatic orDAhydrophobic affinity binding to special ligand. Mostly explained in following terms:S Static pores Dynamic poresO Reactor LoadingR Electro DepositionP NOTE: Adsorbent (mostly polymeric matrix)T Ex: alumina, bentonite, CMC, Silicagel, Titania, etc.ION 15. A Advantages:D Simple & EconomicalS Limited Loss of activity Can be Recycled, Regenerated & Reused (R3)OR Disadvantages:P Relatively Low surface area for binding Exposure of enzyme to microbial attack.T Smaller particles cause high Pressure drop incontinuous packed bed reactor.I Yield are often low due to inactivation &O desorption.N 16. ADSORPTION 17. Enzymes are held or entrapped within theE suitable gels or fibres.N In a gel it may causes: Matrix polymerization orT Precipitation orR Coagulation Entrapment in calcium alginate is the mostA widely used for entrapment for :P Microbial Animal &P Plant enzymes/cellsI Ex: Glucose oxidase + Polyacrlamide (gelentrapment)N NOTE: Adsorbent (mostly commonly used)Ex: polyacrylamides, collagen, silicaG gel, alginates, etc. 18. E Advantages:N No chemical modification Relatively stable forms.T Easy handling & reusage.R Disadvantages: May diffusion of substrate & product occurs.A Substrate accessibility may reduced due toP free radical polymerization of gel. Enzyme in-activation.P Loss of enzyme content.I NOTE: Sometimes covalent bonding may formsN between the entrapped enzyme & the matrix.G 19. E Enzyme + Sod.alginateNMixture is added dropwiseCaCl2 SolutionTR Beads of Calcium alginatesAPPING 20. Membrane Enzyme molecules (usually in aq. form) areCconfined within semi-permeable :Reaction vesselO o Partitioning into two chambers by a semiNpermeable membraneF o One chamber contains the enzyme whileIthe other have substrate & product.Hollow fiber membraneN o Entrapment in semi permeable fibresE(cellulose,triacetate)orspheresM(nylon, collodion).E o In which, the enzyme will be in the lumen/hollowspace, whiletheNfibres/spheres will be submerged in theTsubstrate. 21. MembraneMicro capsulesCo Enzymes are packed in microcapsulesformed by polymerization (like phaseO separation or chemical polymerization).N LiposomesFo Enzymes can be bounded in a concentricI spheres of lipoidal membrane formed byaddition of phospholipid.N Advantages:E No enzyme leakageM No change in enzyme activityE Disadvantages:N Diffusional barrier to the substrate &Tproduct. Not cost effective. 22. MembraneCONFINEMENT 23. MembraneCONFINEMENT 24. Chemical Enzyme forms co-valent link with active group of B the matrix (like terminal -NH2, -COOH,etc,). Support with groups like : O -OH : support activation covalently by CNBr. N -COOH : supports (like CMC) activationD covalently by azide derivatives.I -NH2 : support activation covalently by forming diazonium chlorides on treatmentNwith NaNO2 + HCl. NOTE: The functional group of enzyme which is involved in the linkage, should not affect the active G properties of the said enzyme. 25. Chemical Advantages: B Not affected by pH O Ionic Strength N Disadvantages: Active site may be modifiedD Not cost effective.INOTE: Adsorbent Ex:NAgarose, Cellulose, sepharose, Polyacrlamide,etc. G 26. Chemical B O NDIN G 27. Cross L It involves cross linking of enzyme to a multifunctional reagent without use of any solid Isupport. N Alternatively, chemical bridge of some othermolecule between & with the chemical support K(i.e., reaction of enzyme with reagent bridge orchemical bridge).I Activated carriers are used. NEx : Sepharose by CNBr (most commonlyused)Ex : Sepharose by ethyl chloroformate G 28. Cross Advantages: L Strong linkage leads to low enzyme leakage Iwhile use. Higher stability (i.e., pH, ionic & substrateconcentration. N Disadvantages: K Partially or wholly inactivation by active sitemodification.I Not cost effetcive. N G 29. Cross L I N KI N G 30. Uses of Immobilized Enzymes Biotransformation Secondary metabolite production Biosensors Enzyme-linked immunosorbent assays (ELISAs) Biological washing Powders Food Industry Seed Germination 31. Enzymes in Medicine Glucose oxidaseGlucose Hydrogen peroxideperoxidase Dye:Blue---Green---BrownDye changes according toamount of glucoseEnzyme-linked immunosorbent assays (ELISAs)Sdetect antibodies to infections. 32. Enzymes in biological washingPowders Proteases break down the coloured, insoluble proteins thatcause stains to smaller, colourless soluble polypeptides. Can wash at lower temperatures S 33. Enzymes in Food Industry Pectinase break down substances inapple cell walls and enable greaterjuice extraction. Lactase breaks down lactose in milkinto glucose and galactose.This makes milk drinkable for lactoseintolerant people.S 34. Enzymes in Seed Germinationstarch amylase secretedembryo plantmaltose S 35. ReferencesPharmaceutical Biotechnology By Dr. S.P. Vyas & Dr. V.K. DixitEnymes & its Immobilization Presentation By Dr. S. KhanamInternet Resources : http://www.eplantscience.com http://www.clickbiology.com http://www.lsbu.ac.uk http://www.tech-ceramics.co.uk 36. Thank You !!!S