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 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. - 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. 1. Minimal enzyme leaching from the support results
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

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. Immobilized Enzymes Advantages Disadvantages 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

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. immobilized enzymes free (soluble)
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)

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. Where kd: enzyme denaturation rate constant; Ea: deactivation energy
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

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

52. Far more useful is the Observable Modulus:
and

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

54. The most common definition for immobilized enzymes is that proposed by Katchalski-Katzir in the 1960s:
-“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:

55. 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

56. 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.

57. 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.

58. 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.

59. 4. Mass transfer limitations for enzymes on a support may cause problems when adjusting of the pH 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 carrier-fixed enzymes.
To prevent microbial contamination, these processes are quite often operated at higher temperatures.

60. 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.

61. 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.

62. Comparison of Processes Using Soluble or Carrier-Fixed Enzymes

63. 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.

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

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

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

67. 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

68. 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.

69. 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.

70. 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.

71. 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.

72. 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.

73. 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.

74. 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.

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

76. 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

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

78. 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.

79. 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 mL/min and sample injection volumes of 10–100 mL 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.

80. 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.

81. Diagram of an open enzyme reactor system

82. 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.

83. Diagram of a closed enzyme reactor system.

84. 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.

85. 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.

86. 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 NHþ4 produced by the reactor to be detected as NH3 at an ammonia gas-sensing electrode placed in a flow cell.

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

88. 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.

89. Stopped-flow enzyme reactor with absorbance detection

90. 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 k2 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).

91. 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.