1. CHEE 323J.S. Parent1 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.

2. CHEE 323J.S. Parent2 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, k d : Integrating this expression yields the concentration of active enzyme as a function of time:

3. CHEE 323J.S. Parent3 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 k d 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.

4. CHEE 323J.S. Parent4 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

5. CHEE 323J.S. Parent5 immobilized enzymes free (soluble) enzymes 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. Note that the immobilized preparation is often more stable than the soluble enzyme and displays a period during which no enzyme activity appears to be lost.

6. CHEE 323J.S. Parent6 Classification of Immobilization Methods for Enzymes

7. CHEE 323J.S. Parent7 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  -Carrageenan varies with the cation, allowing these soluble polymers to be crosslinked upon the addition of CaCl 2 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.

8. CHEE 323J.S. Parent8 Immobilization by Entrapment Microencapsulation encloses enzymes within spherical, semi-permeable membranes of 1-100  m 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.

9. CHEE 323J.S. Parent9 Immobilization by Carrier Binding 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

10. CHEE 323J.S. Parent10 Immobilization by Carrier Binding 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)

11. CHEE 323J.S. Parent11 Most Convenient Residues for Covalent Binding Abundance(%)Reactions 7.027 3.431 3.416 2.213 4.84 3.86 1.27 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. Shown are the most convenient residues 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.

12. CHEE 323J.S. Parent12 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.

13. CHEE 323J.S. Parent13 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, 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.

14. CHEE 323J.S. Parent14 Effects of Enzyme Immobilization on Activity

15. CHEE 323J.S. Parent15 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