ENZYME IMMOBILIZATION What,Why & How

1. TOPIC: ENZYME
IMMOBILIZATION
By,
ARKADEB BHUINYA
Department of Microbiology
UG-3rd year
RAMAKRISHNA MISSION VIDYAMANDIRA

2. A Pre-talk
Enzymes are well-known as highly effective and efficient catalysts
of a wide variety of processes characterized by high selectivity and
activity. Additionally, enzymes may reduce the number of reaction
steps and quantities of hazardous solvents needed and thus make
a process more inexpensive and environmentally friendly . For
these reasons enzymes have become extremely important catalysts
which exhibit great potential in many practical applications in
industries ranging from food to pharmaceuticals . The use of
enzymes in multiple catalytic processes has resulted in studies
leading to significant improvement of the enzyme properties. One
of the most important and widely used techniques is enzyme
immobilization in which catalysts are attached to a solid support
that is insoluble in the reaction mixture . The greatest advantage of
immobilization is that it significantly improves the stability of the
biomolecules under various reaction conditions and enhances the
reusability of biomolecules over successive catalytic cycles .

3. IMMOBILIZED ENZYME SYSTEM
• An immobilized enzyme is
an enzyme attached to an inert, insoluble
material.It also lets enzymes be held in place
throughout the reaction, following which they
are easily separated from the products and
may be used again.
• It is actually localization of enzyme in a
distinct support or matrix.

4. WHY ENZYME IMMOBILIZATION?
Advantages!!
• Stable and more efficient in function.
• Multiple or repetitive use of a single batch of
enzyme.
• Products are not contaminated by
enzymes.(specially useful in food & pharmaceutical
industries)
• Control of enzyme function is easy.
• High enzyme substrate ratio
• Minimize effluent disposal problems

5. DISADVANTAGES
• It gives rise to additional bearing on costs of
support materials & techniques
• Causes high stress on enzymes
• Alterations in enzymatic conformation &
activity
• Reduction on accessibility of biocatalyst to it’s
respective substrate
• Specific reactor system & high engineering
design

6. APPLICATION OF IMMOBILIZED
ENZYME SYSTEM
• Industrial application: antibiotics, beverages,
amino acids etc.
• Biomedical applications: treatment, diagnosis
and drug delivery.
• Food industries: production of jams, jellies
and syrups.
• Textile industries: scouring, bio-polishing .
• Detergent industries: immobilization of lipase
for effective dirt removal.

7. Carrier Matrix/Support materials
• A very broad variety of materials of various origins can be used as supports for enzyme
immobilization. These materials may, in general, be divided into organic, inorganic and
hybrid or composite. The support should protect the enzyme structure against harsh
reaction conditions and thus help the immobilized enzyme to retain high catalytic activity .
• Moreover, use of a suitable material, for example hydrophobic carriers in lipase
immobilization, may additionally increase the activity of the biocatalyst .
• However, there are some limitations in this area, because the matrix must not have a
negative effect on the structure of the enzyme and should not disturb the enzyme more
than is required to create stable enzyme–matrix interactions. Additionally, there should be
affinity between the functional groups of the two materials to allow the formation of these
enzyme–matrix interactions and effective binding of the enzyme to the support.
• The carrier should expose the active sites of the catalyst for easy attachment of substrate
molecules and to reduce diffusional limitations of the substrates and products .

8. An Ideal Carrier Matrix For Enzyme
Immobilization
Enzyme immobilization is quite impossible without
carrier matrices. They are actually inert polymers or
inorganic materials. Thus for being an ideal one a
matrix should contain following features:
Inert
Physically strong & stable
Cost effective
Regeneration & reusability
Enhancement of enzyme specificity
Reduction in product inhibition

9. Carrier characteristics:
Chemical (e.g. hydrophobicity , chemical & microbial stability)
Morphological ( e.g-particle diameter,pore size,(inner) surface
for adsorption)
Mechanical (e.g-resistance to pressure, compressibility)
General (e.g- food or pharma grade, cost)

10. Carrier matrices can be classified in two modes.
Let’s check this out……

12. There remains another type of support material-
Hybrid & composite materials:
*high stability
*properties of support materials designed for
selective enzyme & catalytic process
e.g- silica-magnetite, silica-zinc oxide

13. IMMOBILIZATION TECHNIQUES
IMMOBILIZATION
Carrier Binding
Physical
Adsorption Mode
Ionic Binding
Mode
Covalent Binding
Mode
Cross linking Entrapment

14. Carrier Binding Type
1. PHYSICAL ADSORPTION MODE
 Involves the physical binding of the enzyme
on the surface of water insoluble carrier
matrix.
 The process of adsorption involves the weak
interactions like van der Waal’s,multiple salt
linkages & hydrogen bonds.
 Polystyrene carrier coated with DEAE -
cellulose etc.

15. Advantages & Disadvantages of
Adsorption Technique
Advantages:
 Little or no conformation change in the enzyme
 Simple & chip
 No reagents are required
 Wide applicability & capable of high enzyme loading
Disadvantages:
 Slow method
 The adsorbed enzyme may leak from the carrier during use
due to a weak binding force between the enzyme and the
carrier

16. Use of adsorption-
Vinegar production- adsorption of Acetobacter sp. onto wood
chips

18. Carrier Binding Type
2. IONIC BINDING MODE
 Ionic binding of the enzyme protein to water-insoluble carriers containing ion-
exchange residues.
 Polysaccharides & synthetic polymers having ion-exchange centers are usually
used as carriers.

19. Carrier Binding Type
3.Covalent Binding Mode
 This method is based on the binding of enzymes ans water-
insoluble carriers by covalent bonds. The functional groups
that may take part in this binding are listed below,
Amino group Carboxyl group Sulfhydryl group
Hydroxyl group Imidazole group Phenolic group
Thiol group Threonine group Indole group

20. Advantages:
 No leakage of the enzyme occurs.
Disadvantages:
 Covalent binding may alter the conformational structure &
active center of the enzyme, resulting in major loss of activity.

21. Cross Linking Type
 Intermolecular cross-linking of the protein, either to other
protein molecules or to functional groups on an insoluble
support matrix
 The most common reagent used for cross-linking is
Glutaraldehyde.

22. Enzyme Entrapping Method
 The localization of an enzyme within the
lattice of a polymer matrix or membrane
The enzyme itself does not bind to the gel
matrix or membrane

23. So,Entrapment means physical enclosure of a bio-molecule in
a small space.
Entrapment technique is of two types:
 Lattice-Type Entrapment:- involves entrapping enzymes
within the interstitial spaces of a cross-linked water-insoluble
polymer. Some synthetic polmers such as polyacrylamide,
polyvinylalcohol etc…..& natural polymer (starch) have been
used to immobilize enzymes using this technique.
 Microcapsule-Type Entrapment:- involves enclosing the
enzymes within semi permeable polymer membranes. Types
of membranes used include cellulose acetate, polycarbonate,
collagen, and Teflon.

25. There lies some disadvantages also regarding this technique:
1.Enzyme leakage may occur(can be reduced by lowering the
MW cutoff/by decreasing the radius of pores.
2.Lack of control of micro environmental conditions
3.Reduced enzyme stability & activity

26. Characterization of immobilized
enzyme
Activity
✓ Presented in International Units (IU)
✓ IU defines as micromoles substrate converted per min per gm
immobilized enzyme
Activity of immobilized enzymes is highly sensitive to
➢ Initial substrate concentration
➢ Concentration of immobilized enzyme
➢ Temperature
➢ pH
➢ Reaction time
➢ Agitation or flow rate
➢ Physical dimension of the carrier

27. Characterization of immobilized
enzyme
Bound protein
✓ It is nothing but amount of protein bound to the carrier
✓ Unit of bound protein is expressed as mg protein per
gram of carrier
Specific activity of bound protein
✓ Expressed as micromoles substrate converted per
minutes per mg bound protein
✓ It provides some idea of the effectiveness of the
immobilization procedure

29. Thank you