enzyme immobilization, pharmaceutical biotechnology, b pharam 6th sem, PCI. history, why immobilise advantages and disadvantages of enzyme immobilization methods of enzyme immobilization physical retention entrapment microencapsulation chemical bonding adsorption cross linking covalent binding ionic binding Applications of Immobilized Enzymes Enzyme utilization in Industry

1. ENZYME
IMMOBILIZATION
Himanshu Kamboj
Assistant Professor

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
reported
industrial
in 1967
use of immobilized
by Chibata and co-workers,
enzymes was
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
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
and are
• 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 oenzyme immobilizationf :-
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. Entrapme
nt
 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.

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Inclusion in gels
Inclusion in microcapsules
Entrapment
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Inclusion in fibres
1. Inclusion in gels: Enzymes trapped
(Polyacrylamide, Polyvinyl alcohol and
oxidase,
in gels
Polyvinyl
urease
pyrrolidone gel). Eg; Glucose
immmobilized in PAgel.
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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10. 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
in organic media
by the action of
dispersed
obtained
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

11. 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

12. 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.

13. The techniques
 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.
used to produce the semi permeable microcapsule
membranes are classified as phase inversion, polyelectroyte
Microencapsulation contd….

14.  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.

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

16. 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

18. 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

19. 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

20. 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.

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

22. Adsorption
 Earliest method of enzyme immobilization
 Physical adsorption of enzyme molecules onto
matrices.
the surface of solid
 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

23.  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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24. 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 Calcium phosphate
Catalase Charcoal
Invertase Agarose gel, DEAE - Sephadex

25. 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

26.  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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27. Glutaraldehyde is a bifunctional crosslinking agent which effectively hooks
up the amino group of the enzyme.
Eg; Glucose isomerase
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.

28. 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.

29. 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….

30. 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.

31. Activation by bi- or polyfunctional reagents
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)

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

33. 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
immobilized
denatured during immobilization.
2. Only small amounts of enzymes may be
(about 0.02 grams per gram of matrix).

34. Applications of Immobilized Enzymes
General Concepts:
• The immobilized enzyme system - should fit the requirements in terms
of stability, activity, pH optimum and other characteristics should all be
considered
• The property of immobilized enzymes - greatest industrial importance
is the ease with which they can be separated from reaction mixtures
• Hence, in contrast to systems involving soluble enzymes - the reaction
can be stopped by physical removal of the immobilized enzyme -
without requiring such procedures as heat inactivation which might
affect the products of the reaction
• Furthermore, the enzyme will still be active and largely
uncontaminated, so can be used again.
• For these reasons, immobilized enzymes are ideal for use in
continuously operated processes
• Currently, continuous industrial processes involving immobilized
enzymes – carried out in –
(a)Simple stirred tank reactors or
(b)Packed bed reactors

35. Enzyme utilization in Industry
Food and Drink Industry:
1. Use of yeasts(e.g. Saccharomyces carlsbergensis) in the baking and
brewing industries - because they contain the enzymes for alcoholic
fermentation; metabolize hexose sugars to produce pyruvate, but,
whereas animals convert this to lactate under anaerobic conditions, the
anaerobic end-product in yeasts is ethanol, with carbon dioxide being
evolved
2. 2. The clarification of cider, wines and fruit juices (e.g. apple) is usually
achieved by treatment with fungal pectinases Pectinases are a group of
enzymes including polygalacturonases, which break the main chains of
pectins, and pectinesterases, which hydrolyse methyl esters. Their action
releases the trapped particles and allows them to flocculate (pectins of
fruit and vegetables play an important role in jam-making and other
processes by bringing about gel formation)

36. 3. Cheese production involves the conversion of the milk protein,
K-casein, to para-casein by a defined, limited hydrolysis catalysed
by chymosin (rennin) Since chymosin - only be extracted from
calves killed before they are weaned (pepsin is produced instead of
chymosin after weaning) - the enzyme is in short supply - also
ethical issues, there has been a large-scale search for an acceptable
substitute Proteases from animals (pepsin), plants (ficain and
papain) and over a thousand micro-organisms have been tried,
either on their own or mixed with calf chymosin
4. Papain is sometimes used as a meat tenderizer; some South
American natives have traditionally wrapped their meat in leaves of
papaya, the fruit from which papain is extracted Papain (and other
proteases) may also be used in the brewing industry to prevent
chill hazes, caused by precipitation of complexes of protein and
tannin at low temperatures

37. • Washing powders incorporating bacterial proteases Commercial
importance waned because of fears about the effect of enzyme
dust on the respiratory system, but this problem was overcome by
containment in granules which rupture only on contact with water
The enzymes in question, subtilisins from Bacillus subtilis mutants,
are stable to alkali, high temperature (e.g. 65°C), detergents and
bleaches. They will attack blood and other protein stains.
• Bacterial proteases are also used in the leather and textile
industries to loosen hair (or wool) and enable it to be separated
from hide.
• Immobilized enzymes have become an important tool in the
pharmaceutical industry. Among other things, they are used to
manufacture penicillin and other antibiotics. They also form the
basis of various processes in the food industry, for example in the
production of fructose-enriched syrup.

38. THANK YOU