2. ENZYME :
A substance produced by a living organism which acts as a
catalyst to bring about a specific biochemical reaction & shows its
activity even when it is independent.
ENZYME IMMOBILIZATION :
It is a process by which the enzyme catalyst is
trapped at the bio – anode or bio – cathode surface .
• Immobilized enzyme are the enzyme that are fixed to inert &
insoluble carrier .
3. BENEFITS OF IMMOBILISING AN ENZYME :
1. Prevention from degradation and deactivation .
2. Cost efficiency
3. Enhanced stability .
4. Better control on reaction .
5. Allows development of multienzyme reaction system .
6. The enzyme can be recovered at the end of the reaction
and can be reused .
7. The ability to stop the reaction rapidly by removing the
enzyme from the solution .
8. Easily separated from the products .
4. CARRIERS / SUPPORTS / MATEIX :
The substances use for immobilizing of enzymes are termed as
“CARRIERS” .
PROPERTIES OF CARRIER MOLECULES :
1. Inert
2. Insoluble
3. Stable at all pH
4. Carrier should be stable at all ionic strength
5. Should be stable in particular solvent at a particular condition
TYPES OF CARRIERS :
2. ORGANIC NATURAL
CARRIERS
• Favourable compatibility with
proteins .
• Example : Cellulose , Dextrin ,
Starch , Agar , Gelatine
1. INORGANIC CARRIERS
• High pressure stability & may
undergo abrasion .
• Example : Mineral material (Clays) ,
commercially sio2 available material
(Porous glass or Silica )
3. ORGANIC SYNTHETIC
CARRIERS
• High chemical & mechanical
stability .
• Example : Polyvinylacetate ,
Polystyrene , Acrylic
polymers
5. THE IDEAL CARRIRE FOR IMMOBILIZATION
1. Low cost , easily available
2. Inertness towards enzyme
3. Physical strength
4. Stability
5. Biocompatibility
6. Reduction in product inhibition
7. A shift in the pH optimum for enzyme action to the desired
value for the process
8. Reduction in microbial contamination & non-specific
adsorption
7. PHYSICAL METHODS :
1. ADSORPTION :
• It is oldest simplest method of enzyme immobilization .
• Physical adsorption of enzyme molecules onto the surface of solid carrier
.
• Enzyme is attached to the support carrier by (weak) non-covalent
linkages including Ionic or Hydrophobic interactions , Hydrogen bonding
, & , Vander Waals forces .
• It can be carried out by contacting between the enzyme solution &
polymer support in a stirred reactor .
• Therefore , the adsorbed enzymes can be easily removed by minor
changes in pH , Ionic strength or Temperature .
• The method is simple & mild with a vast variety of carriers helpful for
simultaneous purification as well as enzyme immobilization without any
conformation change .
8. • ADVANTAGES :
1. Simple & economical
2. Limited loss of activity
3. Can be recycle , regenerated , reused
• DISADVANTAGES :
1. Relatively low surface area for binding
2. Exposure of enzyme to microbial attack
3. Smaller partials cause high pressure drop in continuous packed bed
reactor
4. Yield are often low due to inactivation & desorption
9. 2. IONIC BINDING :
• This is based on ionic interactions between enzyme
molecules with a charged carrier .
• Higher the surface charge density on the carrier , the
greater would be the amount of enzyme being bound to
the carrier .
• Enzyme binding via ionic interactions during
immobilization depends on the pH of the solution , the
concentration of the enzyme & temperature .
COMMONLY USED CARRIERS ARE :
1. Polysaccharide derivatives (cellulose , dextran)
2. Synthetic polymers (polyethylene glycol)
3. Inorganic materials (alumina , silicates)
11. CHEMICAL METHODS :
1. COVALENT BONDING :
• By this technique the enzyme are immobilize by forming the
covalent bonds with the carrier .
• The functional group of matrix like carboxylic group , amino
group , etc. attach with enzyme for immobilization .
• The functional group of carrier , participate in Covent
coupling but would not affect the activity of enzyme .
• SUPORT USE FOR COVALENT BONDING :
• Carbohydrates : cellulose, agarose
• Synthetic agents : polyacrylamide
• Amino group bearing carriers : amino benzyl cellulose
• Inorganic carriers : porous glass, silica
12. COVALANT BONDING
ADVANTAGE
1) Strong linkage of enzyme to the
support
2) No leakage or desorption
problem
3) Comparatively simple method
4) A variety of support materials
with different functional groups
available
5) Wide applicability
DISADVANTAGE
1) Chemical modification of
enzyme leading to functional
conformation loss
2) Enzyme inactivation by
changes in conformation
during reactions at active
sites (can be overcome
through immobilization in the
presence of enzyme substrate
or a competitive inhibitor)
13. 2. CROSS LINKING :
• The crosslinking method is based on the
formation of covalent bonds between the enzyme
by means of β or multifunctional reagents .
• The individual enzymes are joined to one
another with help of bi- or multifunctional
reagents (e.g., Glutardialdehyde , glyoxal , etc.) .
• Enzyme crosslinking involves normally the
amino groups of the lysine but , in occasional
cases , the sulfhydryl groups of cysteine
,phenolic OH groups of tyrosine , or the
imidazole group of histidine can also be used for
binding .
14. ADVANTAGES
1. Strong linkage leads a low enzyme
leakage while use .
2. Higher stability(pH , ionic &
substrate concentration) .
DISADVANTAGES
1. Partially or wholly inactivation by
active site modification .
2. Not cost effective .
15. MECHANICAL METHODS :
1. ENTRAPMENT :
• By this technique the enzymes are immobilised by
entrapping with the pores of carrier matrix .
• 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
• The matrix material like Polyacrylamide gels , Cellulose
triacetate , Agar , Gelatine , Carrageenan , Alginate
16. Methods of entrapment :
1. Inclusion in gels : enzyme are trapped in gels
2. Inclusion in fibres : enzymes supported on the fibre format
3. Inclusion in microcapsules : enzymes entrapped in
microcapsules formed of polyamines or calcium alginate
monomers
17. • 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
18. ENTRAPMENT
ADVANTAGE
1. Loss of enzyme activity upon
immobilization is minimized
2. Fast
3. Cheap due to availability of low
cost matrix
4. Mild conditions are required
5. Less chances of conformational
changes in enzymes
DISADVANTAGE
1. Leakage of enzyme in
surrounding medium
2. Pore diffusion limitation
3. Substrate cannot diffuse deep
into the gel matrix
4. Mass transfer resistance to
substrates and products
5. Chance of microbial
contamination
19. 2. MICRO – 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
ADVANTAGES
1. Cheap and simple
method
2. A large quantity of
enzyme can be
immobilized by
this method
DISADVANTAGES
1. Pore size limitation
2. Only small
substrate molecules
are able to cross the
membrane
20. APPLICATIONS OF IMMOBILIZED TECHNIQUE :
1. Industrial production :
antibiotics, amino acids, beverages
2. Biomedical applications :
treatment, diagnosis, drug delivery
3. Food industry :
production of jams, jellies and syrups
4. Waste water management :
treatment of sewage and industrial effluents
5. Production of biodiesel :
from vegetable oils
6. Textile industry :
bio-polishing, designs of fabrics
7. Detergent industry : immobilization of lipase for effective dirt removal
