Syllabus Various techniques used for immoblized enzyme Chemical

Syllabus ØVarious techniques used for immoblized enzyme, Chemical modifications. ØApplication of immobilized enzyme in biotechnology ØKinectics of immobilized enzyme, ØKinectics of inhibition of immobilized enzyme. ØMass transfer effects on enzyme kinetics both in free and immobilized enzyme system

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

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

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

Classification of Immobilization Methods for Enzymes

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

Immobilization by 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.

• 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 Ca. Cl 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. 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 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.

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, collagen Immobilization 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 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 – p. H and ionic strength effects can be significant

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.

• 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)

- 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 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.

• Abundance(%)Reactions • 7. 0 27 • 3. 4 31 • 3. 4 16 • 2. 2 13 • 4. 8 4 • 3. 8 6 • 1. 2 7

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.

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.

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 Enzymes • 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, p. H, solvent, etc. • Modified selectivities • Disadvantages • Mass-transfer limitations • Loss of activity upon immobilization • Impractical for solid substrates

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

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 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

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, p. H, …) • 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.

Effects of Enzyme Immobilization on Activity

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, p. H, [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:

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

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.

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

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.

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

• Effect of Temperature - on the rate of enzyme catalyzed reaction k 2=A*exp(-Ea/R*T) T k 2 - enzyme denaturation T Denaturation rate: kd=Ad*exp(-Ea/R*T) Where kd: enzyme denaturation rate constant; Ea: deactivation energy

Kinetics of immobilized enzyme

External Mass Transfer

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:

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.

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 p. H; Negligible electrostatic effects.

Derivation of Key Expressions where

In terms of the same dimensionless numbers as for external diffusion;

Far more useful is the Observable Modulus: and

Enzyme Reactors, • Different Types of Enzyme Reactors, • Heterogenous reaction system, transient analysis of Enzyme Reactors • Process design and operational strategies of immobilized Enzyme Reactors

The most common definition for immobilized enzymes is that proposed by Katchalski-Katzir in the 1960 s: -“Enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, which can be used repeatedly and continuously. ” According to this definition, three types of immobilized enzymes can be distinguished:

1. Heterogenization of the soluble enzyme by coupling to an insoluble support by adsorption or covalent binding, by cross-linking of the enzyme or entrapment in a lattice or in microcapsules such as alginate Beads 2. Retention of the enzyme by means of ultrafiltration Membranes 3. Use of whole cells for biotransformations using their enzyme apparatus

Enzyme Reactors Membrane reactors have been used quite recently as have applications of whole cell processes. Retention of the cells within the reactor may be achieved by membrane separation or by the same immobilization methods that are used for isolated enzymes (34). In principle, the cell itself can be regarded as a form of native immobilization of enzymes. Biosensors are a very special form of carrier-fixed biocatalysts.

The major goal behind immobilization is the recovery of the biocatalysts, separation from products and reactants, and subsequent reuse -in batch or continuous processes. Subsequent reuse in batch or continuous processes is especially important for a reduction of the catalyst costs. Enzymes immobilized on a support often show enhanced stability when compared with the soluble form.

When considering the use of soluble or carrier-fixed enzymes, the following topics we have to be addressed: 1. Additional costs for support and chemicals performing the immobilization have to be balanced against the increase of stability. 2. Loss of activity during the immobilization step. 3. When the catalyst is immobilized only by adsorption or entrapment without covalent attachment, its leakage from the carrier support has to be examined and compared with the overall deactivation rate.

4. Mass transfer limitations for enzymes on a support may cause problems when adjusting of the p. H is necessary during the reaction. 5. With soluble enzymes, higher volumetric activities at high catalyst concentrations are possible, enabling conversion of poor substrates at reasonable rates. 6. Whereas membrane reactors can be easily sterilized before use, this is not possible for reactors with carrierfixed enzymes. To prevent microbial contamination, these processes are quite often operated at higher temperatures.

7. When enzymes are to be used together with organic solvents to increase reactant or product solubility or to alter their kinetics, it may become necessary to immobilize them on a support The support will at the same time act as a water pool to maintain the enzymatic activity. In such systems, water-insoluble organic solvents have less effect on the enzyme stability than water-soluble solvents.

Process design and operational strategies of immoblized enzyme reactors • The final decision for a certain reactor design should be based on an • optimization process covering all relevant factors contributing • to the overall costs, including investment, catalyst • consumption, or productivity.

Comparison of Processes Using Soluble or Carrier-Fixed Enzymes

Reactors for Immobilized Enzymes • The methods for the heterogenisation (or localization) of enzymes • – by coupling them to insoluble supports or – by entrapment. • The types of reactors used for immobilized enzymes are summarized in Figure given bellow.

Reactors for immobilized enzymes. (a–c) (a) Batch reactors with complete backmixing; (b) Stirred-tank reactor; (c) Fixed-bed reactor;

Fluidized-bed reactor. (d–f) are Continuously operated reactors with complete back mixing.

(g–h) are the Continuously operated reactors with plug-flow behavior.

(i) Reactor with the enzyme immobilized in or on a membrane that may at the same time separate two phases such as water and organic solvent. (j) reactor with physically separated enzyme and organic solvent in order to prevent denaturation of the protein

The principles developed for general heterogeneous catalysis in synthetic chemistry are valid, resulting in well-known reactor configurations. Differences between enzyme catalysis and other systems result from the nature of the biocatalyst and reaction medium. For example, soft particles containing the biocatalyst, such as alginate beads, may limit the pressure drop in fixed-bed reactors.

The decision as to specific reactor design will be based on a careful analysis of the kinetic properties of the reaction system. For example, if the enzyme shows a strong substrate-surplus inhibition, a continuously operated reactor with complete backmixing working at high conversion is advantageaous.

• A reaction with strong product inhibition may utilize a batch reactor or a plug flow reactor to achieve higher volume and catalyst specific productivities. • An extractive bioreactor may be used if substrates and products show different solubilities. • By using this reactor configuration, the destabilizing effect of organic solvents may also be overcome, because the enzyme is separated from the organic phase, which is used to extract the insoluble product. • The aqueous phase containing the enzyme will be saturated until the maximum solubility of with the substrate is reached.

• Reactions using biocatalysts are normally performed in aqueous solution at temperatures between 10 and 80 C and at ambient pressure. • Due to the inhibition of some enzymes by heavy metals, the materials of construction must not release these elements. • Reactors are operated under conditions that prevent microbial contamination. • The reactor itself as well as the substrate may be sterilized prior to reaction by using chemical agents (ethanol, formaldehyde, ethylenoxide, Velcorin) or steam.

• Ultraviolet rays may be used to sterilize the immobilized enzyme on its support. • Alternatively, the immobilization may be performed under sterile conditions. • Antibacterial agents may be added to the reaction mixture to prevent microbial growth while the reactor is running.

• In some cases, the reactants may act as sterilants or inhibitors of microbial growth, such as ketones or alcohols. • At higher concentrations (more than 500 mmol/L), solutions may become autosterile because of osmotic pressure effects. • Ndustrial processes are often performed at elevated temperatures, above 55 C, reducing the danger of microbial contamination.

• For a constant product quality and reproducibility of downstream processing, the reactor should be operated at constant conversion. • To overcome the deactivation per unit of time that shows all biocatalysts as a result of denaturation processes, either the residence time has to be increased or fresh enzyme has to be supplied. • The latter is especially easy for soluble enzymes. For carrier-fixed enzymes, a combination of both methods is used, as discussed later.

Immobilized Enzyme Reactors Recycle packed column reactor: - allow the reactor to operate at high fluid velocities.

Fluidized Bed Reactor: - a high viscosity substrate solution - a gaseous substrate or product in a continuous reaction system - care must be taken to avoid the destruction and decomposition of immobilized enzymes

- An immobilized enzyme tends to decompose upon physical stirring. - The batch system is generally suitable for the production of rather small amounts of chemicals.

• The immobilization of enzymes onto particulate carriers that may be packed into a column (the ‘‘packed-bed’’ reactor), such as a typical HPLC column, facilitates repetitive use of the enzyme and also allows the automation of enzymatic assays. • Open-tubular reactors have also been constructed by covalently immobilizing an enzyme onto the inner wall of a nylon or polyethylene tube. • Immobilized enzyme reactors are used in conjunction with a pump, to force a buffer, or mobile phase, through the reactor at a steady rate, an injector located between the pump and the reactor to allow the introduction of substrate solutions, and a detector located close to the column exit.

• The mobile phase contains all required cosubstrates and activators required for the enzymatic reaction, but does not contain the analyte substrate. • A typical packed-bed system may use a 25 -cm long reactor with a 5 mm inner-diameter, packed with the carrier-enzyme solid phase at high pressures. • Flow rates of 0. 5– 2 m. L/min and sample injection volumes of 10– 100 m. L are common. • Detection involves the same principles used in homogeneous enzymatic assays, and flow-through optical absorbance and fluorescence detectors, and amperometric and potentiometric electrochemical detectors may be employed, with detector volumes of the order of tens of microliters being standard.

• Enzyme reactor systems may be of the continuous flow or the stopped-flow variety. • Continuous flow systems are further categorized as open or closed systems. • The open system, shown in Figure , continuously pumps fresh buffer through the injector, reactor and detector, ultimately into a waste reservoir for discarding. • This arrangement is preferred for the testing of enzyme reactors, since unreacted substrate, cofactors and the products of the enzymatic reactions will not be reexposed to the column.

Diagram of an open enzyme reactor system

• Closed systems may be employed when buffer recycling is possible, that is when the buffer contains high concentrations of all necessary cosubstrates, when complete consumption of injected substrate occurs within the reactor, and when products of the enzymatic reaction do not inhibit the immobilized enzyme. • A closed system for immobilized oxidase enzymes is shown in Figure below.

Diagram of a closed enzyme reactor system.

• Both open and closed continuous flow systems rely on the fixed time, or endpoint method for the determination of substrate concentrations. • At a fixed and constant flow rate, the injected volume of substrate will spend a fixed time on the column, and this time is related to the volume of the column (that volume not occupied by stationary phase) and the mobile-phase flow rate.

• Indicator reactions that are chemical in nature may be introduced either into the mobile phase or at the end of the column by the method of postcolumn reagent addition. • Postcolumn addition of reagents dilutes the column eluent, so that, when possible, the addition of indicator reagents to the mobile phase is preferable.

• The conditions under which chemical indicator reactions are used often necessitates the use of postcolumn addition, however. • Figure given below shows an experimental setup for urea assays using an immobilized urease reactor. 30 The postcolumn addition of sodium hydroxide allows the NHþ 4 produced by the reactor to be detected as NH 3 at an ammonia gas-sensing electrode placed in a flow cell.

Enzyme reactor system for urea based on immobilized urease and potentiometric detection.

• Stopped-flow enzyme reactor systems have been designed for automated kinetic assays. • A diagram of a stopped-flow reactor that uses a postcolumn chemical indicator reaction is shown in Figure below. • In this system, the flow rate of themobile phase through the reactor dictates the residence time of the analyte on the column.

Stopped-flow enzyme reactor with absorbance detection

THEORETICAL TREATMENT OF PACKED-BED ENZYME REACTORS • • Packed-bed enzyme reactors, those employing enzymes immobilized onto a particulate phase that is subsequently packed into a column, may be characterized by their column capacity, C, and the degree of reaction P. The parameter C is defined by the equation. where k is the decomposition rate constant for the enzyme–substrate complex (either k 2 or kcat), Et is the total number of moles of enzyme immobilized, and the value of β is a constant for a given reactor, and is equal to the ratio of reactor void volume to total reactor volume (i. e. , β is always less then unity). The degree of reaction, P, varies between zero (no product formed) and unity (complete conversion of substrate).

An equation equivalent to the Michaelis–Menten equation has been derived for immobilized enzymes in packed-bed reactor systems, and is given in Eq. where Q is the volume flow rate of the mobile phase. In general, this equation predicts that for a given column capacity, the degree of reaction, P, is inversely related to the mobile-phase flow rate, Q. That is, the faster the analyte plug flows through the reactor, the less likely will be its complete conversion into product.