The document discusses immobilization technology for enzymes, detailing methods, supports, advantages, disadvantages, and applications. Key immobilization methods include adsorption, covalent bonding, entrapment, cross-linking, and encapsulation, each with unique characteristics and usage scenarios. Enzyme immobilization is utilized across various industries, such as pharmaceuticals, food production, and wastewater management, highlighting its significance and diverse applications.

1. prepared by:
Ashwathi Govind

2. INDEX
1. Support or matrix used in immobilization technology
2. Methods of immobilization
3. Enzyme immobilization
4. Advantages of enzyme immobilization
5. Disadvantages of enzyme immobilization
6. Application of enzyme immobilization
7. Reference

3. An immobilized
enzyme is one whose
movement in space
has been restricted
either completely or to
a small limited region
Immobilization is defined as the process of confining the
enzyme molecule to a solid support over which substrate is
passed and converted to product.
1st immobilized enzyme
was amino acylase of
Aspergillus orzyzae

4. The matrix or support
immobilize the enzyme by
holding it permanently or
temporarily for a brief
period of time.

5.  There are wide variety of matrix or supports
available for immobilization.
 The matrix used should be cheap and easily
available.
 Their reaction with the component of the
medium or with the enzyme should be
minimum as possible.
Support or matrix used in
immobilization technology

6. 1. Natural polymer 2. Synthetical polymer
3. Inorganic Polymer
The support or matrix for immobilization
of enzyme

7. •Alginate
•Chitosan And Chitin
•Collagen
•Carrageenan
•Gelatin
•Cellulose
•Starch
•Pectin
Natural
polymers

8. They are ion
exchange resins
or polymers and
are insoluble
supports with
porous surface.
Their porous
surface can trap
and hold the
enzyme or whole
cells
Examples:
Diethylaminoethyl
cellulose (DEAE) ,
Polyvinyl chloride
(PVC) ,UV activated
polyethylene
glycol(PEG)

9. Zeolites
Ceramics
Diatomaceous earth
Silica
Glass
Activated carbon
Charcoal

10. •Adsorption
•Covalent Binding
•Entrapment
•Copolymerization
•Encapsulation
Methods
of
immobiliz
ation

11. Adsorption
Adsorption is the oldest and
simplest method of enzyme
immobilization.
Nelson and Griffin used
charcoal to adsorb invertase
for the first time in 1916.
In this method enzyme is
adsorbed to external
surface of the support.

12. The support or carrier
used may be of
different type such as:
mineral support
E.g. aluminum
oxide, clay
Organic support
E.g. starch
Modified
sepharase and ion
exchange resins

13. There is no permanent bond formation between carrier
and the enzyme in adsorption method.
Only weak bond stabilize the enzyme to the support or
carrier.
The weak bonds
(low energy bond)
involved are
mainly
• Ionic interaction
• Hydrogen bonds
• Vander wall force

14. For significant surface bonding the
carrier particle size must be small
(500 A to 1mm diameter)
 The greatest advantages of adsorption method is that
there will not be “pore diffusion limitation”
 Since the enzymes are immobilized externally on the
support or carrier.

15. 1.Static process: Immobilization to
carrier by allowing the solution
containing enzyme to contact the
carrier without stirring
2.Dynamic batch process:
Carrier is placed in the enzyme
solution and mixed by stirring
or agitation

16. 3.Reactor loading process:
Carrier is placed in the
reactor and then the enzyme
solution is transferred to the
reactor with continuous
agitation.
4.Electrode position process:
Carrier is placed near to an
electrode in an enzyme bath
and then the current is put on
under the electric field the
enzyme migrate to the carrier
and deposited on its surface.

17. 1.No pore diffusion limitation
2.Easy to carry out
3.No reagents are required
4.Minimum activation steps involved
5.Comparatively cheap method of immobilization
6.Less disruptive to enzyme than chemical methods
Advantages of adsorption methods

18. Desorption of
enzyme from the
carrieer
Efficiency is less

19. Methods involve the formation of
covalent bond between the
chemical group on the support or
carrier.

20. Hydroxyl group and amino groups of support or
enzyme form covalent bond more easily.
Chemical group in the support or carrier that can
form covalent bonds with support are amino groups,
carboxyl groups, imino groups, hydroxyl groups,
carboxyl groups thiol groups, methylthiol groups.
guanidyl groups, imidazole groups and phenol ring.

21. Important functional groups of the enzyme that provide chemical
group to form covalent bonds with support or carrier are:
1. Alpha carboxyl group at ‘c’ terminal of enzyme
2. Alpha amino group at ‘N’ terminal enzyme
3. Epsilon amino groups of lysine and arginine in the enzyme
4. Beta and gamma carboxyl groups of aspartate and glutamate
5. Phenol ring of thyrosine
6. Thiol group of cysteine
7. Hydroxyl groups of serine and threonine
8. Imidazole group of histidine
9. Indole ring of tryptophan

22. 1.Carbohydrates: E.g.. Cellulose, DEAE,
cellulose agarose
2.Synthetic agents: E.g.. Polyacrylamide
3.Protein carriers: Collagen, gelatin
4.Amino group bearing carriers: E.g..
Amino benzyl cellulose

23. 5.Inorganic carriers; Porous glass silica
6.Cyanogen bromide (CNBr) Agarose and CNBr
sepharose

24. 1. Diazoation: Bonding between amino group of
support and tyrosil or histidyl group of enzyme
2. Peptide bond: Bonding between amino or
carboxyl groups of the support and that of the
enzyme.
3. Poly functional reagents: Use of a bi-
functional or multifunctional reagent which forms
covalent bonds between the amino group of the
support and amino group of the enzyme.

25. a) Strong linkage of enzyme to the support
b) No leakage or desorption problem
c) Comparatively simple method
d) A variety of support with different functional
group available
e) Wide applicability

26. 1.Chemical modification
of enzyme leading to the
loss of functional
conformation of enzyme
2.Enzyme inactivation by
changes in the conformation
when undergoes reactions at
the active site. This can be
overcome through
immobilization in the
presence of enzyme’s
substrate or a competitive
inhibitor

27. • In this method enzymes are physically entrapped
inside a porous matrix.
Bonds involved in stabilizing the enzyme to the
matrix may be covalent or non-covalent.
Entrapment

28. • The matrix used will be a water soluble
polymer
• The form and the nature of matrix varies with
different enzymes
• Pore size of matrix is adjusted to prevent the
loss of enzyme .
• Pore size of the matrix can be adjusted with the
concentration of the polymer used.
• Agar-agar and carrageenan have comparatively
large pore sizes

30. Inclusion in the gels; Enzymes trapped inside the gels.
Inclusion in fibers: Enzymes supported on fibers made of
matrix material.
Inclusion in microcapsules: Enzymes entrapped in
microcapsules formed by monomer mixtures such as
polyamine and calcium alginate.

31.  Fast method of immobilization
 Cheap (low cost matrixes available)
 Easy to practice at small scale
 Mild conditions are required
 Less chance of conformational changes in
enzyme
 Can be used for sensing application

32. 1.Leakage of enzyme
2.Pore diffusion limitation
3.Chance of microbial contamination
4.Not much success in industrial process

33. Cross linking
(copolymerization)
In this method
immobilization enzymes
are linked by covalent
bonds between various
groups of enzymes, via
polyfunctional reagents

34. Unlike other method there is no matrix or support
involved in this method.
Commonly used polyfunctional reagents are
glutaraldehyde and diazonium salt.
This method is cheap and simple but not often used
with pure enzymes.
This method is widely used in commercial
preparations and industrial applications .

35. The greatest disadvantages or demerit of
this method is that the polyfunctional reagents
used for cross linking the enzyme may denature
or structurally modify the enzyme leading to the
loss of catalytic properties.

36. • The type of immobilization is
done by enclosing the enzymes
in a membrane capsule.
The capsule will made up of
semi permeable membrane like
nitro cellulose or nylon

37. In
In this method the effectiveness
depends upon the stability of
enzyme inside the capsule.

38. Cheap and simple method
Large quantity of enzymes can be
immobilized by encapsulation

39. Pore size limitation
Only small substrate molecule
is able to cross the membrane

40. 1. Protection from
degradation and
deactivation 2.Re- use of
enzyme for many
reaction cycles,
lowering the total
production cost of
enzyme mediated
reaction.
3.Ability to stop
the reaction
rapidly by
removing the
enzyme from
reaction solution.
4.Enhanced
stability.
5.Easy separation
of enzyme from
product.
6.Product is not
contaminated with
the enzyme.

41. 1. High cost for the isolation, purification and recovery of
active enzyme
2. Industrial application are limited, only very few industries
are using immobilized enzymes or immobilized whole cells.
3. Catalytic properties of some enzymes are reduced or
completely lost after their immobilization on support or
carrier.
4. Some enzyme become unstable after immobilization.
5. Enzymes are inactivated by heat generated by the system.

42. 1. Industrial production
Industrial production of antibiotics, beverages, amino acids, etc.; use
immobilized enzyme or whole cells.
2. Biomedical applications:
Immobilized enzyme can be used to overcome inborn metabolic disorder.
Immobilization technique are effectively used in drug delivery system
especially to oncogenic sites.
3. Food industry:
Enzyme like pectinases and celluloses immobilized on suitable carriers are
successfully used in the production of jams, jellies and syrups from fruits
and vegetables.

43. 4. Research:
The use of immobilized enzyme allow researcher to increase the efficiency
of different enzyme such as horse radish peroxidase(HRP) in blotting
experiments and different proteases for cell or organelle lysis.
5. Production of bio diesel: From vegetable oil.
6. Waste water management: Treatment of sewage and
industrial effluents.
7.Textile industry: scouring, bio-polishing and desizing of fabrics.
8. Detergent industry: Immobilization of lipase enzyme for
effective dirt removal from cloths

44. 9.Immobilized enzyme engineering
• Several of the immobilized enzymes are currently
undergoing active investigation in the
pharmaceutical, sugar, brewing, diary and fine
chemical industries.

45.  www.easy biologyclass.com/enzyme cell
immobilization- techniques/amp
 R.L Foster; the nature of enzymology, The
nature of enzymology, A Holsted Press Book