ENZYME

IMMOBILIZATION
GROUP 2
PAN, JOHN • SANTOS, NICCOLE • TORLAO, JULIUS
Enzyme Immobilization
• enzymes are 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.
Advantages of Immobilized
Enzymes
• can be easily separated from the reaction mixture
containing any residual reactants and reused in subsequent
conservations.
• more stable over broad ranges of pH and temperature.
• absent in the waste-stream
• immobilized systems specially lend themselves to
continuous processes.
• reduced costs in industrial production.
• greater control of the catalytic effect.
• greater ease of new applications for industrial and medical
purposes.
Methods of Immobilization
Methods of Immobilization
• Covalent cross-linking
• Adsorption
• Micro-encapsulation
• Entrapment
Methods of Immobilization
Covalent Cross-Linking
• achieved between a functional group on the
enzyme (not essential for catalytic activity) and a
reactive group on a solid water-insoluble support.
• amino and carboxyl groups, hydroxyl groups,
imidazole groups, indole groups, phenolic groups
and sulphydryl groups.
Advantages of Covalent Cross-
Linking
• easy to conduct and consists of allowing support and
enzyme to interact and therefore facilitates centrifuging
and washing off any enzymes not bound.
• enzyme-support derivative is easy to manipulate and
adapt because of the great physical and chemical
variation in the available support.
• has been widely described and methods for carrying it
out are readily available in the literature.
• the supports themselves are widely available
commercially.
Disadvantages of Covalent Cross-
Linking
• some preparations are tedious to make.
• the chemical bonding may inactivate the enzyme in
some cases.
• covalently-bound water-insoluble enzyme-
substrate derivatives act poorly on high molecular
weight substrates.
Adsorption
• consists of bringing an enzyme solution in contact
with a water-insoluble solvent surface and washing
off the unadsorbed enzyme.
• extent of the adsorption depends on a number of
factors including the nature of the support, pH,
temperature, time, enzyme concentration.
• alumina, bentonite, calcium carbonate, calcium
phosphate, carbon, cellulose, charcoal, clay,
collagen, diatomaceous earth, glass, ion-exchange
resins, sephadex, and silica gel.
Advantages of Adsorption
• enzymes are unlikely to be inactivated because the
system is mild.
Disadvantages of Adsorption
• in cases of weak binding the enzyme may be easily
washed away.
Micro-encapsulation
• consists in packaging the enzyme in tiny (usually
spherical) capsules ranging from 5-300 μin.
diameter in semi-permeable (permanent) or liquid
(nonpermanent) membranes.
Micro-encapsulation
• interfacial polymerization technique - the enzyme
solution contains the enzyme as well as one
component of the membrane that will form round
the micro-capsule.
• coacervation-dependent method - the added
organic solvents contain all the components of the
polymer.
Advantages of Micro-
Encapsulation
Permanent (semi-permeable) micro-capsulation:
• an extremely large surface is provided by the tiny
bubbles of enzymes. A micro-capsule with 20m
diameter would for example have a surface area
of2,500 cm^2/ml.
• the specificity of the micro-capsule is increased by
the possibility of using a membrane which will
favor the diffusion of substrates of certain types.
Advantages of Micro-
Encapsulation
Permanent (semi-permeable) micro-
capsulation/Non-permanent liquid membranes:
• an extremely large surface is provided by the tiny
bubbles of enzymes. A micro-capsule with 20m
diameter would for example have a surface area
of2,500 cm^2/ml.
• the specificity of the micro-capsule is increased by
the possibility of using a membrane which will
favor the diffusion of substrates of certain types.
Disadvantages of Micro-
Encapsulation
Permanent (semi-permeable) micro-capsulation:
• a high concentration of enzyme is needed and only
low molecular weight substances pass through.
Non-permanent liquid membranes:
• leakage.
Entrapment
• no reaction occurs between support and the
enzyme.
• a cross-linked polymeric network is formed around
the enzyme; alternatively the enzyme is placed in a
polymeric substance and the polymeric chains
cross-linked.
• Polyacrylamide gels have been widely used for this
purpose.
Advantages of Entrapment
• the small amount of enzyme used.
• the unlikelihood of damage to the enzyme.
• applicability to water-insoluble enzymes.
Disadvantages of Entrapment
• leakage of enzymes.
• some chemical and thermal enzyme damage during
gel formation.
Industrial Application of Enzymes