Syllabus

1. 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

2. Immobilized Enzyme Systems Enzyme Immobilization: To restrict enzyme mobility in a fixed space.

3. Immobilized Enzyme Systems Enzyme 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

4. Methods of Enzyme Immobilization • Three major Methods of Enzyme Immobilization are : • - Entrapment • - Surface Immobilization • - Cross-linking • These are further broadly divided as in next slide

5. Classification of Immobilization Methods for Enzymes

6. Selecting an Immobilization Technique • It 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 enzymes • different properties of substrates and products • range of potential processes employed

8. Immobilization by Entrapment 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.

9. 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……….

11. 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 Entrapment • Enzymes are immobilized without a chemical or structural modification. A very general technique. • Disadvantage of Entrapment • High molecular weight substrates have limited diffusivity, and cannot be treated with entrapped enzymes.

13. Entrapment - Matrix Entrapment - Membrane Entrapment (microencapsulation)

14. Matrix Materials used in Entrapment : Organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and polyamides. e.g. Ca-alginate, agar, K-carrageenin, collagen Immobilization procedures: Enzyme + polymer solution → polymerization → extrusion/shape the particles Inorganics: activated carbon, porous ceramic. Shapes: particle, membrane, fiber

15. 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.

16. Immobilization by Carrier Binding or Surface Immobilization • Attachment 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 Waal’s forces. • the enzyme structure is unmodified • carriers include chitosan, acrylamide polymers and silica-alumina • binding 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 sulfonate • pH and ionic strength effects can be significant

17. Surface immobilization According to the binding mode of the enzyme, this method can be further sub-classified into: - Physical Adsorption: Van der Waals Carriers: 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.

18. 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 results • in stable productivity • 2. Surface placement permits enzyme contact with • large substrates • Disadvantages: • 1. Partial modification of residues that constitute the active site decreases activity • 2. Immobilization conditions can be difficult to optimize (often done • in the presence of a competitive inhibitor)

19. Covalent Binding: covalent bonds • Carriers: polymers contain amino, carboxyl, sulfhydryl, hydroxyl, or phenolic groups. • - Loss of enzyme activity • - Strong binding of enzymes

20. Most Convenient Residues for Covalent Binding • Amino 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.

21. Abundance(%)Reactions • 7.0 27 • 3.4 31 • 3.4 16 • 2.2 13 • 4.8 4 • 4.8 4 • 3.8 6 • 1.2 7

22. Covalent Attachment Techniques • Cyanogen 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 (Woodward’s 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.

23. Immobilization by Crosslinking • Bi- 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.

24. 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.

26. Advantages Retention in reactor Separation from reaction components is facilitated Usable in a wide range of reactor configurations High catalytic loadings Enhanced stability toward T, pH, solvent, etc. Modified selectivities Disadvantages Mass-transfer limitations Loss of activity upon immobilization Impractical for solid substrates Immobilized Enzymes

27. Application of Immobilized-Enzymes 1-High-fructose corn syrups (HFCS) 2-GLUCOSE ISOMERASE a Treatment with activated carbon. 3-Use of immobilised raffinase 4-Use of immobilised Invertase 5-Production of amino acids 6- Use of immobilised lactase 7- Production of antibiotics

28. Effect of Immobilization on Operational Stability • Given 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

29. Note 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. immobilized enzymes free (soluble) enzymes

30. Effects of Immobilization on Enzyme Stability and Use • Design of enzymatic processes requires knowledge of: • reactant and product selectivity • thermodynamic equilibria that may limit product yield • reaction rate as a function of process conditions ([Enzyme], [substrate(s)], [Inhibitors], temperature, pH, …) • Two design issues that we have not considered are: • enzyme stability • efficiency 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.

31. Effects of Enzyme Immobilization on Activity

32. Enzyme Stability • Although enzyme storage stability is important, it is the operational stability of an enzyme that governs its reactor performance. • Operation stability is a complex function of temperature, pH, [substrate] and the presence of destabilizing agents. • Generally, the rate of free enzyme deactivation is first order with a deactivation constant, kd: • Integrating this expression yields the concentration of active enzyme as a function of time:

33. Yields of the concentration of active enzyme as a function of time:

34. Effect of Thermolysin Instability on APM Production • Recall the rate expression developed for APM synthesis by thermolysin: • If thermolysin deactivation were adequately described as a first order process, the observed reaction rate would have an explicit time dependence, as shown below: • where [E]T,o represents the initial enzyme concentration and kd is the deactivation rate constant. • The conversion versus time profile for aspartame synthesis by a batch process can be developed from this expression by integration.

35. Effect of Thermolysin Instability on APM Production • The evolution of [L-Asp] and conversion with time for a batch process is shown below. • Depending on the relative rates of reaction and enzyme deactivation, the ultimate conversion can be strongly affected

37. Industrial Enzymatic Synthesis of Aspartame • The unique regio and stereoselectivity afforded by enzymes has been exploited on an industrial scale Aspartame production. • The process employs a protease, • thermolysin, to catalyze the • condensation of the modified Asp • and Phe). • The forward reaction is written as: • Note however, that the synthesis reaction is equilibrium limited by the reverse (hydrolysis) reaction for which proteases are known. Furthermore, the equilibrium strongly favours hydrolysis.

38. Factors Affecting Immobilize Enzyme Kinetics • pH effects - on enzymes - enzymes have ionic groups on their active sites. - Variation of pH changes the ionic form of the active sites. - pH changes the three-Dimensional structure of enzymes. - on substrate - some substrates contain ionic groups - pH affects the ionic form of substrate affects the affinity of the substrate to the enzyme.

39. Effect of Temperature • - on the rate of enzyme catalyzed reaction • k2=A*exp(-Ea/R*T) • T k2 • - enzyme denaturation • T Denaturation rate: kd=Ad*exp(-Ea/R*T) Where kd: enzyme denaturation rate constant; Ea: deactivation energy

40. Kinetics of immobilized enzyme

44. External Mass Transfer

45. External Mass Transfer The governing expression is the Nernst equation: ks = Mass transfer coefficient (cm/sec). This is determined from well-established, empirical correlations; S* = Substrate concentration at the solid-liquid interface; So = Substrate concentration in the bulk solution. At steady state, the enzymatic reaction rate cannot exceed the rate of substrate diffusion to the enzyme. This can be written as follows:

48. External Effectiveness Factor The effectiveness factor requires that you know b (=1/v) and Da. This is often difficult as you need to know the intrinsic kinetics of the immobilized enzyme (e.g., V’max and Km). Use the Observable Damkohler Number.

49. Intraparticle Mass Transfer where Assume: Immobilized enzyme is uniformly distributed (e.g.,homogeneously loaded); Transport of solute is described by Fick’s law; Isothermal reaction at constant pH; Negligible electrostatic effects.

50. Derivation of Key Expressions where