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  • Syllabus

    Various techniques used for immoblized enzyme, Chemical modifications.

    Application of immobilized enzyme in biotechnology

    Kinectics of immobilized enzyme,

    Kinectics of inhibition of immobilized enzyme.

    Mass transfer effects on enzyme kinetics both in free and immobilized enzyme system

  • Immobilized Enzyme SystemsEnzyme Immobilization:To restrict enzyme mobility in a fixed space.

  • Immobilized Enzyme SystemsEnzyme Immobilization:

    - Easy separation from reaction mixture, providing the ability to control reaction times and minimize the enzymes lost in the product.

    - Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions.

    - Ability of enzymes to provide pure products.

    - Possible provision of a better environment for enzyme activity

    - Diffusional limitation

  • Methods of Enzyme ImmobilizationThree major Methods of Enzyme Immobilization are :

    - Entrapment

    - Surface Immobilization

    - Cross-linking

    These are further broadly divided as in next slide

  • Classification of Immobilization Methods for Enzymes

  • Selecting an Immobilization TechniqueIt is well recognized that no one method can be regarded as the universal method for all applications or all enzymes. Consider,widely different chemical characteristics of enzymesdifferent properties of substrates and productsrange of potential processes employed

  • Entrapment Immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. - retain enzyme - allow the penetration of substrate.

    It can be classified into matrix and micro capsule types. Immobilization by Entrapment

  • Gel entrapment places the enzyme within the interstitial spaces of crosslinked, water-insoluble polymer gels. Polyacrylamide gels:Polysaccharides: The solubility of alginate and k-Carrageenan varies with the cation, allowing these soluble polymers to be crosslinked upon the addition of CaCl2 and KCl, respectively.

    Variations of pore size result in enzyme leakage, even after washing. The effect of initiator used in polyacrylamide gels can be problematic. cont.

  • Immobilization by Entrapment in microcapsule Microencapsulation encloses enzymes within spherical,semi-permeable membranes of 1-100 mm diameter.

    Urethane prepolymers, when mixed with an aqueous

    enzyme solution crosslink via urea bonds to generate membranes of varying hydrophilicity.

    Alternatively, photo-crosslinkable resins can be gelled by UV-irradiation.

    Advantage of EntrapmentEnzymes are immobilized without a chemical or structural modification. A very general technique.Disadvantage of EntrapmentHigh molecular weight substrates have limited diffusivity, and cannot be treated with entrapped enzymes.

  • Entrapment - Matrix Entrapment- Membrane Entrapment(microencapsulation)

  • Matrix Materials used in Entrapment : Organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and polyamides. e.g. Ca-alginate, agar, K-carrageenin, collagenImmobilization procedures:Enzyme + polymer solution polymerization extrusion/shape the particles

    Inorganics: activated carbon, porous ceramic.

    Shapes: particle, membrane, fiber

  • Challenges in Entrapment Method - 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.

  • Immobilization by Carrier Binding or Surface ImmobilizationAttachment of an enzyme to an insoluble carrier creates an active surface catalyst. Modes of surface attachment classify carrier methods into physical adsorption, ionic binding and covalent binding.

    Physical Adsorption: Enzymes can be bound to carriers by physical interaction such as hydrogen bonding and/or van der Waals forces.the enzyme structure is unmodifiedcarriers include chitosan, acrylamide polymers and silica-aluminabinding strength is usually weak and affected by temperature and the concentration of reactants.

    Ionic Binding: Stronger enzyme-carrier binding is obtained with solid supports containing ion-exchange residues.cellulose, glass-fibre paper, polystyrene sulfonatepH and ionic strength effects can be significant

  • Surface immobilizationAccording to the binding mode of the enzyme, this method can be further sub-classified into: - Physical Adsorption: Van der WaalsCarriers: silica, carbon nanotube, cellulose, etc. Easily desorbed, simple and cheap, enzyme activity unaffected.

    - Ionic Binding: ionic bonds Similar to physical adsorption.Carriers: polysaccharides and synthetic polymers having ion-exchange centers.

  • Covalent attachment of soluble enzymes to an insoluble support is the most common immobilization technique.Amino acid residues not involved in the active site can be used fix the enzyme to a solid carrier

    Advantages:1. Minimal enzyme leaching from the support resultsin stable productivity2. Surface placement permits enzyme contact with large substrates

    Disadvantages:1. Partial modification of residues that constitute the active site decreases activity2.Immobilization conditions can be difficult to optimize (often donein the presence of a competitive inhibitor)

  • Covalent Binding: covalent bonds Carriers: polymers contain amino, carboxyl, sulfhydryl, hydroxyl, or phenolic groups.

    - Loss of enzyme activity - Strong binding of enzymes

  • Most Convenient Residues for Covalent BindingAmino acid residues with polar and reactive functional groups are best for covalent binding, given that they are most often found on the surface of the enzyme.

    The data shown in next slide is the most convenient residues for binding in descending order.

    The average percent composition of proteins (reactive residues only) is shown, along with the number of potential binding reactions in which the amino acids partake.

  • Abundance(%)Reactions

    7.027

    3.431

    3.416

    2.213

    4.84

    4.84

    3.86

    1.27

  • Covalent Attachment TechniquesCyanogen bromide activates supports with vicinal hydroxyl groups (polysaccharides, glass beads) to yield reactive imidocarbonate derivatives:

    Diazonium derivatives of supports having aromatic amino groups are activated for enzyme immobilization:

    Under the action of condensing agents (Woodwards reagent K), carboxyl or amino groups of supports and amino acid residues can be condensed to yield peptide linkages.

    Other methods include diazo coupling, alkylation, etc.

  • Immobilization by CrosslinkingBi- or multi-functional compounds serve as reagents for intermolecular crosslinking of enzymes,

    creating insoluble aggregates that are effective heterogeneous catalysts.

    Reagents commonly have two identical functional groups which react with specific amino acid residues.

    Common reagents include glutaraldehyde, carbodimide and diisocyanates,

    Involvement of the active site in crosslinking can lead to great reductions in activity, and the gelatinous nature of the product can complicate processing.

  • Cross-linking is to cross link enzyme molecules with each other using agents such as glutaraldehyde.

    Features: similar to covalent binding.

    Several methods are combined.

  • Immobilized EnzymesAdvantagesRetention in reactorSeparation from reaction components is facilitatedUsable in a wide range of reactor configurationsHigh catalytic loadingsEnhanced stability toward T, pH, solvent, etc.Modified selectivitiesDisadvantagesMass-transfer limitationsLoss of activity upon immobilizationImpractical for solid substrates

  • Application of Immobilized-Enzymes

    1-High-fructose corn syrups (HFCS)2-GLUCOSE ISOMERASEa Treatment with activated carbon.3-Use of immobilised raffinase4-Use of immobilised Invertase5-Production of amino acids6- Use of immobilised lactase7- Production of antibiotics

  • Effect of Immobilization on Operational StabilityGiven that activity of enzymes is dictated by structure and conformation, the environmental change resulting from immobilization affects not only maximum activity, but the stability of the enzyme preparation.The factors that inactivate enzymes are not systematically understood, and depend on the intrinsic nature of the enzyme, the method of immobilization, and the reaction conditions employed.In general, immobilized enzyme preparations demonstrate better stability

  • immobilized enzymes

    free (soluble) enzymesNote that the immobilized preparation is ften more stable than the soluble enzyme and displays a period during which no enzyme activity appears to be lost.

  • Effects of Immobilization on Enzyme Stability and UseDesign of enzymatic processes requires knowledge of:reactant and product selectivitythermodynamic equilibria that may limit product yieldreaction rate as a function of process conditions ([Enzyme], [substrate(s)], [Inhibitors], temperature, pH, )

    Two design issues that we have not considered are:enzyme stabilityefficiency losses associated with the use of homogeneous (soluble) catalysts

    Immobilization of an enzyme allows it to be retained in a continuous reactor, but its initial activity and its stability directly influence its usefulness in industrial applications.

  • Effects of Enzyme Immobilization on Activity

  • Enzyme StabilityAlthough enzyme storage stability is important, it is the operational stability of an enzyme that govern

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