Immobilized enzymes have several advantages over soluble enzymes including easy separation from product streams, potential for reuse, and improved stability. Enzymes can be immobilized through various techniques including entrapment in matrices or membranes, adsorption to surfaces, covalent or ionic binding to surfaces, and cross-linking. Recent research has shown that conjugating enzymes to nanoparticles can help maintain or enhance enzyme activity compared to other immobilization methods. Immobilized enzymes have wide applications in biomedical areas like biosensors, bioreactors, and extracorporeal blood treatment as well as industrial processes such as food production, detergents, biodiesel production, and waste water treatment.

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2. SHABINA MEHMOOD
FA15-ROO-021
ARAFAT YAMEEN
Immobilized Enzymes
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3. CONTENTS
 Definition
 Historical background
 Need of immobilization strategy
 Attributes of immobilized enzymes
 Immobilization techniques
 Application of immobilized enzymes
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4. Enzyme immobilization
 The term "immobilized" means unable to move or
stationary. An enzyme that is physically attached to a
solid support over which a substrate is passed and
converted to product.
 To restrict enzyme mobility in a fixed space.
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5. Historical background
 The first scientific observation that led to the discovery of
immobilized enzymes was made in 1916 . It was
demonstrated that invertase exhibited the same activity
when absorbed on a solid, such as charcoal or aluminum
hydroxide, at the bottom of the reaction vessel . This
discovery was later developed to the currently available
enzyme immobilization techniques.
 During 1950s and 1960s, different covalent methods of
enzyme immobilization were developed.
 Up till now , many enzymes have been immobilized and
example include penicillin G acylase, invertase, lipases,
proteases.
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6. Need of immobilization
strategy
 In most of the industrial, analytical, and clinical
processes, enzymes are mixed in a solution with
substrates and cannot be economically recovered after
the exhaustion of the substrates.
 This process becomes quite wasteful when the cost of
enzymes is considered.
 Thus, there is an incentive to use enzymes in an
immobilized or insolubilized form so that they may be
retained in a biochemical reactor to catalyze further
reaactions
 The use of an immobilized enzyme makes it
economically feasible to operate an enzymatic process
in a continuous mode. 6

7. Attributes of immobilized
biocatalysts
ADVANTAGES DISADVANTAGES
Easy for continuous and
batch formats
Loss of enzyme activity
upon immobilization
Reuse over multiple cycles Unfavorable alterations in
kinetic properties
Improved stability over
soluble enzymes forms
Cost of immobilization
process
Easy separation of enzymes
from product stream
Cost of carrier and fixing
agents
Co-immobilization with other
enzymes possible
Subject to fouling
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8. Immobilization Methods
 Methods of Enzyme immobilization:
- Entrapment
- matrix
- membrane (microencapsulation)
- Surface immobilization
- physical adsorption
- ionic binding
- covalent binding
- Cross-linking
 Conjugation with nano particles
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9. Entrapment Immobilization is the
restriction
or localization of enzyme in a structure which
limits the diffusion of the enzymes based on
the localization of an or membrane.
It can be classified into:
 Matrix
 Micro capsule types.
ENTRAPMENT
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10. 1) Matrix Entrapment :
enzyme within the lattice of a polymer matrix.
2) Membrane Entrapment (microencapsulation)
- Localization of enzyme in membrane.
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11.  Matrix Materials:
1) Organics:
polysaccharides, proteins, carbon, vinyl and alkyl
polymers, and polyamides.
2)Inorganic:
activated carbon, porous ceramic.
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12. CHALLENGES:
1) Enzyme leakage into solution
2) This insoluble substances hinders the arrival of the substrate, and the
exit of products.
3) Reduced enzyme activity and stability
ADVANTANGES :
1) Retain enzyme
2 ) Allow the penetration of substrate.
 It could be improved by modifying matrix or
membrane.
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13. Surface immobilization
 Enzyme is attached to the outside of an inert
material.
 According to the binding mode of the enzyme,
this method can be further sub-classified into:
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14. 1)Physical Adsorption
There is no permanent bond formation
between carrier and the enzyme in
adsorption method. Only weak bonds i.e.,
Vander walls forces stabilize the enzymes
to the support or carrier.
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15. Advantages of adsorption method:
 Easy to carry out
 No reagents are required
 Minimum activation steps involved
 Comparatively cheap method of immobilization
 Less disruptive to enzyme than chemical methods
Easily desorbed, simple and cheap, enzyme activity unaffected
Disadvantages of adsorption method:
 Desorption of enzymes from the carrier
 Efficiency is less
This method is the slowest
The active site of the immobilized enzyme may be blocked by the
matrix or bead, greatly reducing the activity of the enzyme
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16. 2)Ionic bonding
 Ionic bonds are involve.
 Similar to physical adsorption.
 Carrier must have ion exchange centre
 This type of interaction can be easily
reversed by the change in temperature,
polarity and ionic strength condition
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17. 3)COVALENT BONDING
This method involves the formation of covalent bonds between
the chemical groups in enzyme and to the chemical groups on
the support or carrier.
 Advantages of covalent bonding:
 Strong linkage of enzyme to the support
 No leakage or desorption problem
 Comparatively simple method
 DISADVANTAGES
 Loss of enzyme activity
 Strong binding of enzymes
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18. CROSS LINKING
 This method is also called as copolymerization. In this
method of immobilization, enzymes are directly linked by
covalent bonds between various groups of enzymes via
poly functional reagents. Unlike other methods, there is no
matrix or support involved in this method.
 Commonly used poly functional reagents are
glutaraldehyde and diazonium salt.
 This technique is cheap and simple but not often used
with pure enzymes.
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19. Features:
 Similar to covalent binding.
 The reaction ensures that the binding
site does not cover the enzyme's active
site.
 The activity of the enzyme is only
affected by immobility and the
inflexibility of the covalent bonds
precludes the self-healing properties
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21. Immobilized enzymes with
nanoparticle conjugation
 Many of the conventional enzymes immobilization method reduce
enzyme activity.
 However, recent research has shown that the integration of
nanoparticles into enzyme carrier schemes has maintained or even
enhanced immobilized enzyme performance.
 The nanoparticle size and surface chemistry as well as the
orientation and density of immobilized enzymes all contribute to the
enhanced performance of enzyme-nanoparticle conjugates.
 These improvements are noted in specific nanoparticles including
those comprising carbon (e.g., graphene and carbon nanotubes),
metal/metaloxides and polymeric nanomaterials, as well as
semiconductor nanocrystals or quantum dots.
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22. Application of immobilized
enzymes
 Biomedical applications:
 It was demonstrated that the substrate present in
blood can be attacked by the immobilized enzymes
via extracorporeal shunt system. Example include
the use of micro encapsulated urease enzyme to
remove blood urea.
 Immobilized enzymes can be applied directly to the
local lesion to prevent their entry into the blood
circulation .Hence the microencapsulated catalase
is applied to oral lesion in mice with hereditary
catalase deficiency to replace enzyme deficiency
locally 22

23.  The use of immobilized enzymes in biomedical
sciences can be classified in two categories.i.e
the use in biosensors and the use in bioreactors.
• Biosensor involves the example of glucose
sensor in which glucose oxidase is covalently
bound with polyaniline used to measure the
amount of glucose with the response time of
less than 5 min.
• Significant efforts have been made to use
immobilized enzymes in bioreactors. Example
being the use of phospholipase A2 and
heparinase in bioreactor for blood detoxification
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24.  Industrial production: Industrial production of
antibiotics, beverages, amino acids etc, uses
immobilized enzymes or whole cells.
 Food industry: Enzymes like pectinases and
cellulases immobilized on suitable carriers are
successfully used in the production of jams, jellies
and syrups from fruits and vegetables
 Detergent industry: immobilization of lipase
enzyme for effective dirt removal from cloths
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25.  Production of bio-diesel .immobilized lipase is
use for the production of biodiesel
 Waste water management: Treatment of
sewage and industrial effluents by peroxide and
lactase
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