Immobilization of enzymes

2.  In a solution, biocatalysts behave as any other solute in that they are
readily dispersed in solution or solvent and have complete freedom of
movement in solution.
 Immobilization may be viewed as a procedure specifically designed to limit
freedom of movement of a biocatalyst.
Immobilisation separates a biocatalyst from the
bulk solution phase to produce a
heterogeneous two-phase mixture
Soluble Enzyme + Substrate = Product
(Single time usage)
Immobilied enzyme + Substrate = Product
(Repeated usage of enzyme)

3.  The term “immobilized enzymes” refers to “enzymes
physically confined or localized in a certain defined region /
space with retention of their catalytic activities, and which
can be used repeatedly and continuously.”

4. HISTORY
 The first industrial use of immobilized enzymes was
reported in 1967 by Chibata and co-workers, who
developed the immobilization of Aspergillus oryzae
aminoacylase for the resolution of synthetic racemic D-L
amino acids.

5.  Enzymes are expensive.
 As catalytic molecules, enzymes are not directly used up. After the reaction
the enzymes cannot be economically recovered for re-use and are
generally wasted.
 Separation of enzyme and product using a two-phase system;
a. One phase containing the enzyme
b. The other phase containing the product
• The enzyme is imprisoned within its phase allowing its re-use or
continuous use. The separation prevents the enzyme from contaminating
the product

6. Advantages of enzyme immobilization:-
1. Multiple or repetitive use of a single batch of enzymes.
2. Immobilized enzymes are usually more stable.
3. Ability to stop the reaction rapidly by removing the enzyme from the reaction
solution.
4. Product is not contaminated with the enzyme.
5. Easy separation of the enzyme from the product- saves cost of downstream
processing
6. Allows development of a multienzyme reaction system.
Disadvantages of enzyme immobilization:-
1. It gives rise to an additional bearing on cost.
2. It affects the stability and activity of enzymes.
3. The technique may not prove to be of any advantage when one of the substrate is
found to be insoluble.
4. Certain immobilization protocols offer serious problems with respect to the diffusion
of the substrate to have an access to the enzyme.

7. Entrapment Microencapsulation
Physical retention Chemical bonding
Inclusion in fibers
Inclusion in gels
Inclusion in microcapsules
Liposomal entrapment
Reverse micelle entrapment
Adsorption Cross linking Covalent binding Ionic binding

8. Entrapment
 The entrapment method is based on the occlusion of an enzyme within a
polymeric network (polyacrylamide, alginate etc.) that allows the substrate
and products to pass through (which ensures continuous transformation)
but retains the enzyme.
 The porosity of a gel lattice is controlled to ensure that the structure is tight
enough to prevent leakage of enzyme or cells and at the same time allow
free movement of substrate and product.

10. E
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Inclusion in gels
Inclusion in microcapsules
Entrapment contd....
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Inclusion in fibres
1. Inclusion in gels: Enzymes trapped in gels
(Polyacrylamide, Polyvinyl alcohol and Polyvinyl
pyrrolidone gel). Eg; Glucose oxidase, urease
immmobilized in PA gel.
2. Inclusion in fibres: Enzymes supported on fiber format
made up of polyacetate, collagen, cellulose etc.. Eg;
collagen fiber for immobilizing LDH.
3. Inclusion in microcapsules: Enzymes entrapped in
microcapsules formed by monomer mixtures such as
polyamine and polybasic acid chloride, polyphenol and
polyisocyanate.
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11. Amphipathic lipids…..(Polar heads and Non- polar tails).
Liposomes are small artificial vesicles of spherical shape
that can be created from cholesterol, Phosphatidyl choline,
Phosphatidyl serine, Sterylamine and Sphingomyelin
Sizes ranging from 30 nm to several micrometers
Reverse micelles are nanometer-
sized (1-10 nm) water droplets
dispersed in organic media
obtained by the action of
surfactants.
Surfactant molecules organize
with the polar part to the inner side
able to solubilize water and the
apolar part in contact with the
organic solvent.
Entrapment contd....

12. Advantages
1. Simplicity,
2. No change in intrinsic enzyme properties,
3. Involves no chemical modification,
4. Minimal enzyme requirement and
5. Matrices are available in various shapes.
Disadvantages
1. Enzyme leakage,
2. Only small sized substrate/products can be used,
3. Requires delicate balance between mechanical properties of the matrix and its
effect on enzyme activity and
4. Presence of diffusional constraints

13. Microencapsulation
 Enzymes are immobilized by enclosing them within spherical semi-permeable
polymer membranes with controlled porosity (1–100 μm) .
 The term “microcapsule” is defined, as a spherical particle with the size varying
between 50 nm to 2 mm (2 x 106 nm) containing a core substance.
 Enzymes immobilized in this manner are physically contained within the
membrane, whilst substrate and product molecules are free to diffuse across the
membrane. membranes are made of cellulose nitrate,1,6-diaminohexane,
polystyrene.

14. The techniques used to produce the semi permeable microcapsule
membranes are classified as phase inversion, polyelectroyte
coacervation, and interfacial precipitation .
 Phase inversion involves the induction of phase separation in a
previously homogeneous polymer solution by a temperature change or
by exposing the solution to a non-solvent component.
Microencapsulation contd….

15.  Polyelectrolyte coacervation process, results from mixing of oppositely charged polyelectrolytes
;membrane is formed by the complexation of oppositely charged polymers to yield an
interpenetrating network with poor solvent affinity.
Microencapsulation contd….

16. Different combinations of polyionic species used are
 Cellulose sulphate with poly(diallyl dimethyl ammonium chloride)
 Carboxymethylcelluose with chitosan
 Gelatin with gum arabic/polyphosphate
Microencapsulation contd….

17. Advantages
1. Economic and simple method
2. Extremely large surface area due to which they have higher catalytic
efficiency.
Disadvantages
1. Occassional inactivation of enzyme during microencapsulation
2. Higher concentration of enzyme is required
3. Possibility of enzyme incorporation into the membrane wall
4. Enzyme leakage

19. Carrier binding methods
• Binding of enzymes to water insoluble carriers
• The oldest and most prevalent method.
• The materials used for immobilization of enzymes are called carrier matrices -
usually inert polymers.
Types of carriers
a) Naturally occurring
b) Synthetic organic
c) Inorganic
The different types of carrier binding methods are
1. Adsorption
1. Cross-linking
2. Covalent binding
3. Ionic binding

20. 1. Low cost
2. Inertness towards enzymes
3. Physical strength,
4. Stability,
5. Biocompatibility
6. Reduction in product inhibition,
7. A shift in the pH optimum for enzyme action to the
desired value for the process, and
8. Reduction in microbial contamination and non-
specific adsorption

21. Naturally occurring-Biopolymers
 They are water-insoluble polysaccharides (e.g., cellulose, starch, agarose, chitosan) and
proteins such as gelatin.
Synthetic organic polymers
 Easily and artificially designed.
 Can adjust the porosity, ionic and hydrophobic or hydrophilic properties.
 Mechanical strength and longevity -Superior to those from natural polymers.
Eg; Eupergit-C (acrylic resin)
 It is prepared by using compounds: N,N′-methylene-bis-(methacrylamide),methacrylamide,
allyl glycidyl ether and glycidyl methacrylate.
Other egs; Sepa beads FP-EP, Amberlite XAD-7 Sepa beads

22. Inorganic solids
• Cheapest matrix being used for the immobilization
• Eg; alumina, silica, zeolites and mesoporous silicas
Silica
Alumina
Zeolites

23. Adsorption
 Earliest method of enzyme immobilization
 Physical adsorption of enzyme molecules onto the surface of solid
matrices.
 Enzyme is attached to the support material by (weak) non-covalent
linkages including ionic or hydrophobic interactions, hydrogen bonding,
and van der Waals forces.
 It can be carried out by contacting between the enzyme solution and
polymer support in a stirred reactor
 Therefore, the adsorbed enzymes can be easily removed by minor
changes in pH, ionic strength or temperature
Adsorption contd…..

24.  The method is simple and mild with a vast variety of
carriers helpful for simultaneous purification as well as
enzyme immobilization without any conformational change.
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Adsorption by Van der waal forces
Adsorption by hydrogen bonding
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Adsorption contd…..

25. 1. Static process (enzyme is immobilized on the carrier simply by allowing the solution
containing the enzyme to contact the carrier without stirring)
2. The dynamic batch process (carrier is placed into the enzyme solution and mixed by
stirring or agitated continuously in a shaker.
3. The reactor loading process (carrier is placed into the reactor, then the enzyme
solution is transferred to the reactor by agitating the carrier and enzyme solution.
4. The electrodeposition process (Carrier is placed close to one of the electrodes in an
enzyme bath, the current put on, the enzyme migrates to the carrier and gets
deposited on the surface)
ENZYME CARRIER
α-amylase Ca3(PO4) 2
Catalase Charcoal
Invertase Agarose gel, DEAE - Sephadex
Adsorption contd…..

26. Advantages
1. Because no reactive species are involved, there is little or no
conformational change in the enzyme on immobilization
2. Easy and economic method for preparing immobilized enzymes
3. Easily reversed to allow regeneration of catalyst-Can be Recycled,
Regenerated & Reused (R3)
Disadvantage
1. Desorption of protein from the carrier during use owing to the weakness of
the involved binding forces , with subsequent loss of catalytic activity and
contamination of products
2. Limited reliability when absolute immobilization of an enzyme is desired

27.  Special chemicals used for promoting intermolecular linkage -cross linking
agents- help in formation of covalent bonds between enzyme molecules.
 Cross linking is accomplished using bi- or poly-functional reagents
Toxicity of such reagents is a limiting factor in applying this method to living
cells and many enzymes.
 This type of immobilisation is support free and involves joining cells (or enzymes)
to each other to form a large three-dimensional complex structure.
 Eg; Glutaraldehyde, Diazobenzidine, Disuccinimidyl suberate, Bismaleimide,
Toluene diisocyanate, Hexamethylene isocyanate
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28. Glutaraldehyde is a bifunctional crosslinking agent which effectively hooks
up the amino group of the enzyme.
Eg; Glucose isomerase

29. Advantages
1. Strong linkage leads to low enzyme leakage while use.
2. Higher stability.
Disadvantages
1. Partially or wholly inactivation by active site modification.
2. Not cost effective.

30. Covalent linkage
 The enzyme is attached to the matrix by means of covalent bonds.
 The immobilization of an enzyme by covalent attachment to carrier/matrix must
involve functional groups of the enzyme that are not essential for catalytic action.
 No reagents must be used, which could affect the binding and active sites of the
enzymes
A covalent linkage between the carrier and the enzyme can be established by
different methods.
- Cyanogen bromide activation.
- Carbodiimide coupling.
Covalent linkage contd….

31. Cyanogen bromide activation
Inert support materials (cellulose, PVA, sephadex) containing glycol groups
are activated by CNBr.
The activated carrier is then covalently linked with the amino group of
enzymes- ISOUREA LINKAGE .
Eg; Ascorbic acid oxidase
Covalent linkage contd….

32. Carbodiimide coupling - Peptide bond formation
In carbodiimide activation, a support material should have a
carboxyl (-COOH) functional group and an enzyme and support
are joined via a peptide bond.
Covalent linkage contd….

33. Some of the reagents such as glutaraldehyde can be used to
create bonds between amino groups of enzymes and amino
groups of support (eg: aminoethylcellulose, albumin,
aminoalkylated porous glass)
Activation by bi- or polyfunctional reagents
Covalent linkage contd….

34. Diazotization
Some of the support materials (aminobenzyl cellulose, aminosilanized
porous glass) are subjected to diazotization on treatment with NaNO2 and HCl.
They inturn bind covalently to tyrosyl or histidyl groups of enzyme.
Covalent linkage contd….

35. Advantages
1. The strength of binding is very strong, so, leakage of
enzyme from the support is absent or very little.
2. This is a simple, mild and often successful method of wide
applicability
Disadvantages
1. Enzymes are chemically modified and so many are
denatured during immobilization.
2. Only small amounts of enzymes may be immobilized
(about 0.02 grams per gram of matrix).

36. Ionic binding
 This is based on ionic interactions between enzyme molecules with a charged
matrix.
 Higher the surface charge density on the matrix, the greater would be the amount
of enzyme being bound to the matrix.
 Enzyme binding via ionic interactions during immobilization depends on the pH
of the solution, the concentration of the enzyme and temperature.
Ionic binding contd…

37. Commonly used matrices are:
1. Polysaccharide derivatives (e.g., DEAE cellulose, dextran, CMC,
chitosan),
2. Synthetic polymers (e.g., Polystyrene derivatives, polyethylene glycol,
polyvinyl alcohol) and
3. Inorganic materials (e.g., Amberlite, alumina, silicates, bentonite).
This method of immobilization leads to minimal
changes in enzyme conformation.
Ionic binding contd…

38. Advantages:
1. Low cost.
2. Regeneration is possible.
3. Easy preparation.
Disadvantages:
1. Not fit for industrial use as effectiveness gradually
decreases.

39. Applications of Immobilized Enzymes
1. Immobilized enzyme-aminoacylase used for the production of L-amino acids
2. In food industry, fructose syrup is produce from glucose by use of immobilized enzyme
glucose-isomerase.
3. Immobilized enzyme used in biosensors.
4. Accurate analysis of sample done with the help of specific immobilized enzymes.
5. Immobilized Enzyme or Cells used in industry for the production of various industrial products.
Enzymes Transformation of organic compounds
Aminoacylase Optical resolution of DL-amino acids
α-amylase and glucoamylase Conversion of starch to glucose
Glucose isomerase Conversion of glucose to fructose
Penicillin amidase Production of 6-aminopenicillanic acid from
penicillin
β-Galactosidase Hydrolysis of lactose in milk or whey

40. Let’s Summarize……

41.  With the vast array of research on enzyme immobilization, we can
conclude that it is one of the most promising techniques for highly
efficient and economically competent biotechnological processes in
the field of environmental monitoring, biotransformation, diagnostics,
pharmaceutical and food industries.
 Enzyme-based strategies are increasingly replacing conventional
chemical methods in both laboratories and industries with attributes
like efficiency, quicker performance and multifarious use.
 However, commercialization of immobilized enzymes is still at a lower
pace because of their costs and storage problems.