Enzyme and Cell Immobilization

and
its Applications
 Introduction
• Enzymes are the biological catalysts that promote
chemical reactions in living organisms
• They have the ability to catalyze reactions under
very
mild conditions with high degree of substrate
specificity thus decreasing the formation of
byproducts
• Enzymes can catalyze reactions in different
states, individual molecules in solution, in
aggregates with other entities and as attached to
surfaces
 Immobilized enzymes
• “Immobilized enzyme” refers to “enzymes physically
confined or localized in a certain defined region of
space with retention of their catalytic activities, and
which can be used repeatedly and continuously” OR
• Imprisonment of enzyme in a distinct support/matrix.
The support/matrix allows exchange of medium that
contains substrate of effecter or inhibitor molecules.
• The substrate passes over the immobilized enzyme
and is converted to products
 Technological properties of
Immobilized enzymes
• Advantages Disadvantages
• Increased functional efficiency • Limited industrial applications
• Enhanced reproducibility • Loss or reduction in activity in
• Catalyst Reuse some enzymes
• Easier Reactor Operation • Some enzymes become
• Easier Product Separation unstable
• Wider choice of Reactor • Diffusion limitations
• Minimum reaction time • Additional Cost
• Less chance of contamination • High cost for isolation,
in products purification and recovery of
• High stability active enzyme.
• • Does not give required results if
High enzyme substrate ratio
one of the substrate is insoluble
• Enzyme-free products • There may be diffusion problems
• The ability to stop the reaction for the substrate to access the
rapidly by removing the enzyme under certain conditions
enzyme form the reaction
solution
 Applications of immobilization
technique
• Industrial production: e.g., antibiotics, amino acids,
beverages etc.
• Biomedical applications: treatment, diagnosis, drug
delivery
• Food industry: production of jams, jellies and syrups
• Waste water management: treatment of sewage and
industrial effluents
• Production of biodiesel: from vegetable oils
• Textile industry: bio-polishing, desizing of fabrics
• Detergent industry: immobilization of lipase for
effective dirt removal
 Applications of Immobilized
Enzymes
• The greatest industrial importance of immobilized
enzymes 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
 Supports/Matrix used in
immobilization
• The matrix that holds the enzyme should be:
• cheap and easily available.
• Should not react with medium and enzyme.
• Three types of matrix are used:
1. Natural polymers: alginate, chitosan and chitin, collagen,
carrageenan, gelatin, cellulose, starch, pectin
2. Synthetic polymers: ion exchange resins/polymers
[polyvinyl chloride (PVC), UV activated Polyethylene
glycol (PEG)]
3. Inorganic materials: ceramics, silica, glass, activated
carbon, charcoal
 Types / methods of immobilization
1. Adsorption
2. Covalent bonding
3. Entrapment
4. Copolymerization
5. Encapsulation
Types/methods of immobilization
 1. Adsorption (Non
Covalent
Interactions)
• Oldest and simplest method used for
enzyme immobilization
• Enzymes are adsorbed to external
surface of support
•Support /carrier may be:
•Mineral support: aluminium oxide, clay
•Organic support: starch
• Ion exchange resins
• Weak bonds are formed between the
support and the enzyme (ionic
interactions, hydrogen bonds, vand der
waals forces) stabilize enzymes to the
support
•Carrier particle size must be small for
appreciable surface bonding
 Carrier binding: physical adsorption

Cross linking

Adsorption

Porous solid support Enzyme Adsorbed enzyme on support immobilized enzyme

 Methods of adsorption
• Static process: the solution containing enzyme is
allowed to contact the carrier (no stirring)
• Dynamic batch process: carrier is placed in the
enzyme solution and mixed by stirring/agitation.
• Reactor loading process: carrier is placed in the
reactor and enzyme solution is transferred to
reactor.
• Electrode position process: carrier is placed
proximal to an electrode in an enzyme bath and
current is passed. The enzyme migrates to the
carrier and deposited on the surface.
 2. Covalent Bonding
• Involves the formation of stable covalent bonds between
enzyme and support.
• Chemical groups in enzymes that form covalent bonds with
support are amino groups (alpha amino group at N terminal),
hydroxyl, carboxyl (alpha carboxyl group at C terminal), thiols
and phenol rings.
• The method is widely employed when there is a strict
requirement for the absence of the enzyme in the product.
• The enzyme is not released into the solution upon use.
• The matrix has to be discarded together with the enzyme
once the enzymatic activity decays.
• The disadvantage of this type of immobilization is high cost
in terms of generally low yield of immobilized activity and by
the nonreversible character of this binding
• Enzymes attached covalently by disulfide bonds to solid
supports represent one way to avoid this problem.
 Carriers/support used for covalent
bonding
• Carbohydrates:
cellulose, agarose
• Synthetic agents:
polyacrylamide
• Protein carriers
• Amino group bearing
carriers: amino benzyl
cellulose
• Inorganic carriers:
porous glass, silica
 Covalent bonding (contd.)
• Advantages • Disadvantages
Strong linkage of enzyme to the Chemical modification of
support enzyme leading to functional
No leakage or desoprtion conformation loss
problem Enzyme inactivation by changes
Comparatively simple method in conformation during
A variety of support materials reactions at active sites (can
be overcome through
with different functional immobilization in the
groups available presence of enzyme
Wide applicability substrate or a competitive
inhibitor)
 3. Entrapment
• The enzymes are physically entrapped inside a
matrix.
• The entrapment method refers to the occlusion of
an enzyme within a polymeric network that allows
the substrate and products to pass through but
retains the enzyme.
• Bonds involved may be covalent or non-covalent.
• The enzyme is not bound to the matrix or
membrane
• There are different approaches to entrap enzymes
such as gel or fiber entrapping and micro-
encapsulation.
• The practical use of these methods is limited by
mass transfer limitations through membranes or gel
 Types of matrix Methods of entrapment
used Inclusion in gels: enzyme are trapped
 Polyacrylamide in gels
gels Inclusion in fibers: enzymes supported
 Cellulose on the fiber format
triacetate Inclusion in microcapsules: enzymes
 Agar entrapped in microcapsules formed of
 Gelatin polyamines or calcium alginate
monomers
 Carrageenan
 Alginate
 Entrapment (contd.)
• Form and nature of matrix varies
• Pore size of the matrix is adjusted to prevent
enzyme loss
• There might be a possibility of leakage of low MW
enzymes
• Agar and carrageenan have large pore size
• Pore size can be adjusted by changing the
concentration of polymer
• The method is not commonly used in industrial
process
• Easy to practice at small scale
 Entrapment (contd.)
Advantages Disadvantages
• Loss of enzyme activity  Leakage of enzyme in
upon immobilization is surrounding medium
minimized
 Pore diffusion limitation
• Fast
 Substrate cannot diffuse
• Cheap due to availability of
deep into the gel matrix
low cost matrix
• Mild conditions are required  Mass transfer resistance
to substrates and
• Less chances of
conformational changes in
products
enzymes  Chance of microbial
contamination
 4. Cross linking
(copolymerization)
• Cross linking: covalent
bonding between various
groups of enzymes via
polyfunctional reagents
• No matrix or support are
involved
• Cheap and simple technique
but not often used with pure
proteins
• Widely used in commercial
preparations
• However, polyfunctional
reagents can denature the
enzyme
 5. Encapsulation
Enclosing
enzymes in a
semi permeable
membrane
capsule
Capsule is made
up of nitro
cellulose or nylon
Effectiveness
depend upon the
stability of
enzymes
 Encapsulation (contd.)
Advantages Disadvantage
 Cheap and simple  Pore size limitation
method  Only small substrate
 A large quantity of molecules are able to
enzyme can be cross the membrane
immobilized by this
method
 Immobilization of cells
• It is an alternative to enzyme immobilization
• Well developed method for utilization of enzymes from
microorganisms
• The method is effectively used when:
• Individual enzymes become inactive due to
immobilization
• Isolation and purification of enzyme is not cost effective
• In this method enzymes remain stable and active for
longer periods of time
• Methods of cell immobilization are essentially the same
as for enzyme immobilization
 Immobilization of cells
Advantages Disadvantages
• Multiple enzymes can be  Concentration of
introduced to a single step enzymes is less
• Extraction and purification  Production of unwanted
of enzymes are not required enzymes and products
• Enzymes remain stable for
 Modification of end
a long period of time
products by other
• Native conformation of
enzymes
enzyme is best maintained
• Can be used to immobilize
mitochondria and
chloroplasts
Methods of whole cell immobilization

 Methods of cell immobilization are same as

for enzyme immobilization and include
 Adsorption
 Covalent bonding
 Cell to cell cross linking
 Encapsulation
 Entrapment
 Immobilization of cells: methods,
support materials, cells and reaction
 REFERENCES

 http://enzymeimmobilization.blogspot.in/
2011/02/enzymeimmobilization.html
 https://en.wikipedia.org/wiki/
Immobilized_enzyme
 https://www.google.co.in/search?
q=enzyme+immobilization&espv=2&biw=1600&
bih=766&source=lnms&tbm=isch&sa=X&ved=0
CAgQ_AUoAmoVChMIxuqanKDOxwIVE3GOC
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