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STB3142
Lecture 11
Applications of enzymes
Enzyme immobilization
Usage of enzyme in a technological process depends
on their expense
Recombinant technology makes cheaper cost of
producing enzymes
Enzymes are catalytic molecules (not directly used up
by the processes) so they can be reused when possible
The solution is to separate the enzyme and product
during the reaction in a two phase system
Enzyme is imprisoned in its phase allowing reuse or
continuous use
Also prevent enzyme from contaminating the product
Methods of immobilization
a) Adsorption
b) Covalent bonding
c) Entrapment
d) Membrane confinement
Adsorption
Very simple method of wide applicability and
capable of high enzyme loading (1 g of protein
per 1 g of matrix)
Involves simple mixing of enzyme with suitable
adsorbent, under appropriate pH and ionic
strength after a sufficient incubation period
Combination of hydrophobic effects and
formation of several salt links immobilize enzyme
molecules
Examples of suitable absorbents are ion-exchange
matrices, porous carbon, clays, hydrous metal
oxides glasses and polymeric aromatic resins
Adsorption limit of enzyme
Support type
DEAE-Sephadex CM-Sephadex
% bound at
anion exchanger cation exchanger
pH 2.5 0 100
pH 4.7 100 75
pH 7.0 100 34
Covalent bonding of enzymes
Enzymes are covalently coupled to insoluble matrices
Only small amount of enzymes may be immobilized
(0.02 g per 1 g of matrix)
Strength of binding is very strong with very little
leakage
Lysine residues are found to be most useful for
covalent bonding of enzymes to insoluble matrices
Example: Sepharose, activated by cyanogen bromide
Disadvantage of this method is high cost of Sepharose
Covalent bonding of enzymes
Covalent bonding of enzymes
Effects of covalently bound enzymes
Entrapment of enzymes
A convenient method when involving low
molecular weight substrates
More than 1 g of enzyme per g of matrix can
be entrapped
May involve purely physical caging of enzyme
or covalent binding
Difficult for high molecular weight substrates
to approach enzymes
Example: Enzymes in cellulose acetate fibres
Membrane confinement
Utilizes the semipermeable nature of the membrane
Membrane must confine enzyme while allowing free
passage to the reaction products and substrates
Simplest form is by placing enzyme on one side of the
semipermeable membrane while reactant and product
stream is on the other side
Easy to use for wide variety of enzymes but expensive
method
Enzymes can also be encapsulated in droplets or
liposomes (example: enzyme in aqueous solution of
1,6-diaminohexane)
Comparison of different enzyme
immobilization techniques
Covalent Membrane
Characteristics Adsorption Entrapment
binding confinement