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1. Seminar On
Enzyme Immobilization Techniques
Presented By
Manoj V. Dhoke
Roll No – 516
M. Pharm 2nd Sem
(Pharmaceutical Chemistry)
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2. Content
• Introduction
• Characters Of Ideal Matrix
• Advantages
• Disadvantages
• Methods Of Immobilization
• Applications
• Reference
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3. Introduction
• An immobilized enzyme is an enzyme attached to an inert,
insoluble material—such as calcium alginate (produced by
reacting a mixture of sodium alginate solution and enzyme
solution with calcium chloride).
• They are immobilized on to an inert and insoluble matrix, under
mild and controlled conditions where it can act upon its natural
substrate.
• The matrix is usually a high molecular polymer such as
polyacrylamide, cellulose, starch, glass, beads etc.
• First reported in 1916 by J.M. Nelson and E.G. Griffin.
• Reported the adsorption of invertase on charcoal without loss of
activity.
• Technique was developed in 1960s.
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4. An ideal matrix should have the following characters:
Low cost
Inertness
Physical strength
Stability
Regenarabillity after the useful lifetime of the enzyme.
Enhancement of the enzyme specificity.
Reduction in microbial contamination and non specific
adsorption.
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5. Advantages
Repeated use of the enzyme as the recovery of enzyme
molecules are easy
More stability and reaction potential than free enzymes
Enzyme –substrate ratio is high
Less chance of contamination so pure products are
obtained.
Better efficiency
Minimum reaction time
The cell free reaction system acts as model for studying
the enzyme action of living cells
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6. Disadvantages
Causes additional cost.
Adversely affects activity , stability and the catalytic
properties of enzymes.
Suitable immobilization protocols are to be standardized
for each enzyme.
It becomes difficult when any of the substrates is
insoluble.
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7. METHODS OF
IMMOBILIZATION
Adsorption
Covalent bonding
Entrapment
Membrane confinement
Cross linking
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8. Adsorption
Immobilized by physical adsorption to several types of
materials.
Adsorption due to electrostatic forces, hydrophobic interaction
,hydrogen bonds between atom and ions of carrier and enzyme.
Commonly used matrices are ion exchange matrices ,porous
carbon, clays, hydrous metal oxides, glasses and polymeric
aromatic resins.
Adsorption is done at appropriate pH and ionic strength.
Enzyme loading rate is 1gm enzyme/ 1 gm matrix,
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9. Covalent bonding
Enzyme matrix molecules are attached to the carrier by
formation of covalent bonds so that there is no loss of enzyme
during use.
Covalent bond formation occurs with the side chain of amino
acids of the enzyme.
Hydroxyl and amino groups are the important ones involved in
bonding.
Commonly used matrices are agarose, cellulose and
polyacrylamides.
Enzyme loading is low ie. 0.02 gm /gm matrix.
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10. Entrapment
Enzyme molecules are physically entrapped within a cross
linked matrix of water soluble polymer.
Polyacrylamide gel ,cellulose triacetate ,agar and gelatine are
commonly used.
Enzyme molecules are immobilized by the polymerization of
polymer around the enzymes.
Polymerization happens either with covalent bonding or without
covalent bonding.
Enzyme loading is 1gm /g gel.
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11. Membrane confinement
Membrane confinement or encapsulation is a method of enzyme
immobilization by entrapping method.
A droplet of aqueous solution of enzyme is enclosed in a
semipermeable membrane capsule.
It doesn’t allow escape of enzyme molecules.
The semipermeable membrane capsule is made up of cellulose nitrate
and nylon.
Size of the pores of the membranes are too small , therefore no free
diffusion from membrane takes place.
This method is cheap and simple.
Effectiveness depends on the stability of enzyme.
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12. Co-polymerization
Enzymes are immobilized by cross linking between the enzyme
molecules through a polyfunctional reagent.
Polyfunctional reagents include gluteraldehyde , diazonium salt,
hexamethylene di-isocyanite and ethylene bismaleimide.
There is no support or matrix used in this method.
This method is cheap and simple but are not frequently used as
it may denature the enzymes leading to the loss of catalytic
properties.
This method is used in commercial and industrial applications.
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13. Applications
Immobilized enzymes are used in fermentation industry, agriculture,
environmental protection.
High fructose corn syrup is produced by the immobilized glucose
isomerase from Actinoplanes misouriensis ,Basillus coagulans and
Streptomyces etc.
Raffinase activity is provided by the immobilized cells of the mould
Mortierella vinacea.
Immobilized invertase is used to produce sugar.
Lactase from Kluyveromycus lactis is immobilized in cellulose
triacetate fibers , and are used in processing of milk and sweet whey.
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14. Immobilized Penicillin amides from E.coli are used in the
production of penicillin, cephalosporins and other drugs.
Immobilized microbial enzymes are used in agriculture for
enhancing their activity without being affected by biotic or
abiotic factors.
Immobilizes bacteria, fungi and algae re used for removing
heavy metal and radio nucleotide waste from atmosphere.
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15. Reference
• Beatriz Brena , Paula González-Pombo , and
Francisco Batista-Viera “ Immobilization of Enzymes: A
Literature Survey ”.
• Dr. Sikander ali, wajeeha zafar, sammia shafiq, mehvish
manzoor “ enzymes immobilization: an overview of techniques,
support materials and its applications ” International journal of
scientific & technology research volume 6, issue 07, july 2017
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