ENZYMES are large biological molecules (catalysts) that speed up rate of chemical reaction without being used up in the reaction. (responsible for the thousands of chemical interconversions that sustain life). IMMOBILIZATION means that the biocatalysts are limited in moving due to chemically or physically treatment.

1. IMMOBILIZATION OF
ENZYMES
ISF College of Pharmacy, Moga
Ghal Kalan,GT Road, Moga- 142001, Punjab, INDIA
Internal Quality Assurance Cell - (IQAC)
Ruchika Sharma
Assistant Professor
Dept. of Biotechnology
ISF COLLEGE OF
PHARMACY
Website: - www.isfcp.org

2. INTRODUCTION
 ENZYMES are large biological molecules (catalysts) that speed up
rate of chemical reaction without being used up in the reaction.
(responsible for the thousands of chemical interconversions that
sustain life).
 IMMOBILIZATION means that the biocatalysts are limited in
moving due to chemically or physically treatment.
2

3. Enzyme immobilization can be defined as the attachment of free or soluble enzymes to
different types of supports resulting in reduction or loss of mobility of the enzyme.
Almost all enzymes are proteins.
In enzymatic reactions, the molecules at the begining of the process are called substrates,
and the enzyme converts them into different molecules, the products.
Enzyme + Substrate ↔ Enzyme- Substrate Complex ↔ Enzyme + Products
[ E ] + [ S ] ↔ [ ES ] ↔ [ E ] + [ P ]
3

4. 1
4

5. HISTORY 5

6. 1926:
James B Sumner produced first pure crystalline enzyme
(urease)
and showed enzymes were proteins
1960:
single enzyme immobilized: production of L-amino acids,
isomerization of glucose
1971:
immobilized enzyme was defined in an enzyme
engineering conference held at New Hampshire

7. The first industrial use of an
immobilized enzyme is amino acid acylase
by Tanabe
Seiyaku Company, Japan, for the resolution of
racemic mixtures of chemically synthesized
amino acids.

8. Why we immobilized enzyme
1. The immobilized material can be reused and
gives process economy.
2. The products can be easily separated.
3. The reaction can be controlled and monitored
at will.
8

9. Enzymes lower the activation energy of a reaction
Final energy state of
products
Initial energy state
of substrates
Activation energy
of uncatalysed
reactions
Activation energy
of enzyme catalysed
reaction
Progress of reaction (time)
Energylevelsofmolecules 9

10. Immobilized enzymes are very important for commercial
uses as they possess many benefits to the expenses and
processes of the reaction of which include:
Economical: The immobilized enzymes are easily removed
from the reaction making it easy to recycle the biocatalyst.
10

11. In the past, biological washing powders and detergents would contain many
proteases and lipases which would break down dirt. However, when the
cleaning products would come into contact with the skin, it would create
allergic reactions. This is why immobilization of enzymes are important, not
just economically.

12. IMMOBILIZATION
• Biochemical properties
• Enzyme kinetic parameters
• Chemical characteristics
• Mechanical properties
ENZYME CARRIER
12

13. TYPES OF IMMOBILIZATION
On the basis of the surface of the carrier for immobilization,
it is of two types;
External surface immobilization
Internal surface immobilization
13

14. External surface immobilization:
For external surface immobilization the carrier size must be small in order to
achieve an appreciable surface for binding
Advantages:
 No pore limitations are encountered
Disadvantage:
 Enzymes are more exposed to microbial attacks
14

15. Internal surface immobilization
It involves the use of inner surface of the carrier for immobilization.
Advantages:
 Enzymes are protected from microbial attacks.
Disadvantage:
 Pore diffusion limitations.
15

17. Carrier binding
 In this physical adsorption method enzyme proteins are adsorbed on the surface of
water-insoluble carriers.
 The earliest example of enzyme immobilization using this method is the adsorption of
beta-D-fructo-furanosidase onto aluminium hydroxide.
Adsorption
17

18. EXAMPLES OF SUITABLE ADSORBENTS
 Ion-exchange matrices
 Porous carbon
 Clays
 Hydrous metal oxides
 Glasses
 Polymeric aromatic resins
18

19. ADVANTAGES AND DISADVANTAGES
ADVANTAGES
DISADVANTAGES
• This technique is simple and cheap
• No reagent is required in adsorption
• It may cause change in pH, temperature, ionic strength
• It may cause release of enzyme from carrier
19

20. Ionic binding
Ionic bonding involves the use of attraction between charged ions of
ion exchange matrices and charged functional amino acids of enzymes
The ion exchangers used in ionic adsorption are of two types:
• Polysaccharide polymers
• Synthetic polymers
20

21. Amino group
Carboxyl group
Sulfhydryl group
Hydroxyl group Imidazole group
Phenolic group
Thiol group
Threonine group
Indole group
Different types of functional groups involved in covalent binding depending upon their
degree of reactivity
21

22. ADVANTAGES AND DISADVANTAGES
ADVANTAGES
DISADVANTAGES
Binding force between enzyme and carrier is so strong that no leakage of
enzyme occurs even in the presence of substrate or high ionic strength
solution.
It may cause change in the active site
22

23. Entrapment
It is the physical enclosure of enzymes in a small space.
- Matrix entrapment (matrices used are polysaccharides, proteins, polymeric
materials, activated carbon, porous ceramic and so on)
- Membrane entrapment (microcapsulation or trapped between thin, semi-permeable
membranes)
23

24. Lattice-Type
 Entrapment involves entrapping enzymes within the interstitial spaces of a
cross- linked water-insoluble polymer. Types of polymers used are:
 Synthetic polymers (polyarylamide)
 Natural polymer (starch)
Microcapsule-Type
 Entrapping involves enclosing the enzymes within semi permeable polymer
membranes
24

25. Advantage is enzyme is retained.
Disadvantages are:
- substrate need to diffuse in to access enzyme and product
need to diffuse out
- reduced enzyme activity and enzyme stability owing to the
lack of control of micro environmental conditions
25

26. GENERALISED COMPARISON OF DIFFERENT
ENZYME IMMOBILISATION TECHNIQUES
Characteristics Adsorption Covalent binding Membrane confinement
Preparation Simple Difficult Simple
Cost Low High High
Binding force Variable Strong Strong
Enzyme leakage Yes No No
Applicability Wide Selective Very wide
Running Problems High Low High
Matrix effects Yes Yes No
Large diffusional
barriers
No No Yes
Microbial protection No No Yes
26

27. • Easy separation from reaction mixture, providing the ability to control reaction
times and minimize the enzymes lost in the product
• Re-use of enzymes for many reaction cycles, lowering the total production cost of
enzyme mediated reactions
• Ability of enzymes to provide pure products
• Possible provision of a better environment for enzyme activity
• Protection from degradation and deactivation
• Retention of enzyme, enzyme-free products
• Recycling, repetitive use
• Cost efficiency
• Enhanced stability
• Use as controlled release agents
Advantages of immobilized enzymes:

28. • Problem in diffusional mass transfer
• Enzyme leakage into solution
• Reduced enzyme activity and stability
• Lack of controls on micro environmental conditions
Disadvantages of immobilized enzymes:

29. Factors Affecting Enzyme Kinetics
 pH effects
on enzymes
- enzymes have ionic groups on their active sites.
- Variation of pH changes the ionic form of the active sites.
- pH changes the three-Dimensional structure of enzymes.
on substrate
- some substrates contain ionic groups
- pH affects the ionic form of substrate affects the affinity of
the substrate to the enzyme.
29

30. Applications of Immobilized Enzymes
Production of antibiotics-
Production of amino acids-
Manufacture of golden syrup- immobilized invertase in a packed bed reactor.
High Fructose Corn Syrup production- immobilized glucose isomerase and hexokinase is used.
Detection of glucose in blood and urine
Ethanol production
Baking of bread
Brewing
Production of invert sugar from sucrose by the enzyme β- fructofuranosidase/ invertase. This is
used for the preparation of jam and jellies.
30

31. Future of enzyme immobilization
 As method for the immobilization of enzymes continue to improve and become
commercially wide spread, the availability of immobilized enzymes to industry
has the opportunity to increase significantly.
 Several new types of carriers and technologies have been implemented in the
recent past to improve traditional enzyme immobilization which aimed to
enhance enzyme loading, activity and stability to decrease the enzyme
biocatalyst cost in industry.
31

32.  These include cross-linked enzyme aggregates, microwave-
assisted immobilization, mesoporous supports and most
recently nanoparticle-based immobilization of enzymes.
 Enzymes immobilized on nanoparticles showed a broader
working pH and temperature range and higher thermal
stability than the native enzymes.
32

33. Conclusion
 Immobilized enzymes have been widely used in the processing of variety of products.
 Immobilized enzymes have biomedical and industrial applications and for this reason, this
area has continued to develop into an ever-expanding and multidisciplinary field during the
last couple of decades.
 New strategies are continuously emerging for the formation of diverse immobilized enzymes
having superior efficiency and usage.
33

34. THANK
YOU