1
PHARMACEUTICAL
BIOTECHNOLOGY
SJM College of Pharmacy,
Chitradurga
Prepare By,
AdarshaBP
Ass Professor(Pharmacognosy)
SJM College of Pharmacy
ENZYME BIOTECHNOLOGY - METHODS OF
ENZYME IMMOBILIZATION AND
APPLICATION:
Enzymes: Natural proteins produced by living
organisms( Bacterias, Plants & Animals) &
Functioning as highly selective biochemical catalysts
in converting one molecule into another.
Speed up metabolic reactions.
• As direct pharmaceutical products, such as in the treatment of enzymatic deficiency
caused by various genetic disorders.
• Extraction of medicinally important compounds, such as heparin.
• Manufacture of chemical pharmaceuticals where enzymes are used for the inter
conversion of chemical intermediates or the removal of chiral components.
• Research and development - especially in the field of molecular biology where almost
all of the processes of nucleic acid
• manipulation are performed using enzymes, such as restriction endonucleases and
DNA polymerases.
• Diagnostic procedures also frequently utilise enzymes - such as Enzyme Linked
Immunosorbent Assays (ELISA), pregnancy and diabetic testing kits.
• In the production of synthetic and semi-synthetic hormones and steroids.
Enzyme Immobilization:
Defn- Restriction or prevention of movement or mobility of enzymes in a fixed
space.
Need or Advantages:
• Reusability
• Enhance the stability and efficiency
• Can separate the enzyme after reaction
• Economical
• Can readily stop the reaction
• In bio-catalysts
• Process reduction
Solid Supports:
Methods of Immobilization:
Physical Methods
a)Adsorption
b)Entrapment
c) Micro encapsulation
Chemical Methods
a) Covalent Bonding
b) Cross linking
a) Adsorption/Carrier-bonding
method:
•
Adsorption of enzymes to
outer surface of the support.
•
Simple and widely used one.
•
Physical binding- Weak
forces(Van der wall) &
Hydrogen bonding
• Enzymes can be easily separated from support by some
change i.e Ph, Ionic changes or temperature
• Prepared by easily mixing enzymes & adsorbant, Under
appropriate Conditions
• Supports used,
 Mineral
 Organic
 Modified sepharose & ion exchange resins
•
Examples: Aluminium oxide, Clay & Starch, Coconut fibre
Methods of Adsorption:
1) Static process
2) Dynamic batch process
3) Reactor loading process
4) Electrode position process
Advantages:
• No pore diffusion limitation
• Easy
• No reagents required
• Minimum activation steps
• Cheap
• Less disruptive
• Resistant to Proteolysis & Aggregation
• No Conformational changes.
Disadvantages:
• Desorption
• Less efficient
• Physiological condition weaken the bond
b) Entrapment method:
Enzymes are Physically Entrapped inside the porous matrix
Covalent or Non Covalent bond
Water soluble polymer
Pore size of the polymer-Concentration of polymers
Examples: Gelatin, PAG, Agar, Alginate etc.
Example
• 3-5% Sodium aginate is prepared in H2O
• Yeast cell is added to above solution
• Taken in the Syringe
• 5 % Cal. Chloride is prepared in H2O
• Add the solution taken in Syringe into Calcium
chloride drop by drop
• Beads will form Containing yeast cells
• Yeast contain Invertase enzyme
1. Enzymes in gels
2. Enzymes in Fibres
3. Enzymes in microcapsules- Formed by
monomer such as Polyamide and calcium
alginate
Advantages
• Faster method
• Cheap
• Easy to practice at a small scale
• Less chance of conformational changes in enzyme
Disadvantages
• Leakage of enzymes
• Pore diffusion limitation
• Not much used in Industry
c) Microencapsulation:
• Done by enclosing the enzymes
in membrane capsules.
• Capsule will be made up of semi
permeable membrane like nitro
cellulose or nylon.
• Effectiveness-stability of enzymes
inside capsule.
Advantages:
• Cheap & Simple method
• Large quantity of enzymes can be immobilizes in
encapsules.
Disadvantages:
• Pore size limitation
• Only small substrate molecule could able to cross
the membrane.
a)Covalent bonding:
•
This method involves the formation of covalent bonds between the
chemical groups in enzyme and to the chemical groups on the support or
carrier.
•
It is one of the widely used methods of enzyme immobilization.
•
Hydroxyl groups and amino groups of support or enzyme form covalent
bonds more easily.
•
Chemical groups in the support or carrier that can form covalent bonds
with support are amino groups, imino groups, hydroxyl groups, carboxyl
groups, thiol groups, methylthiol groups, guanidyl groups, imidazole
groups and phenol ring.
• Important functional groups of the enzyme that provide chemical groups to form
covalent bonds with support or carrier are:
1. Alpha carboxyl group at ‘C’ terminal of enzyme
2. Alpha amino group at ‘N’ terminal of enzyme
3. Epsilon amino groups of Lysine and Arginine in the enzyme
4. β and γ carboxyl groups of Aspartate and Glutamate
5. Phenol ring of Tyrosine
6. Thiol group of Cysteine
7. Hydroxyl groups of Serine and Threonine
8. Imidazole group of Histidine
9. Indole ring of Tryptophan
• Carriers or supports commonly used for covalent bonding are:
(a). Carbohydrates: Eg. Cellulose, DEAE cellulose, Agarose
(b). Synthetic agents: Eg. Polyacrylamide
(c). Protein carriers: Collagen, Gelatin
(d). Amino group bearing carriers: Eg. amino benzyl
cellulose
(e). Inorganic carriers: Porous glass, silica
(f). Cyanogen bromide (CNBr)-agarose and CNBr
Sepharose
• Methods of covalent bonding
(1). Diazoation: Bonding between amino group of
support and tyrosil or histidyl group of enzyme.
(2). Peptide bond: Bonding between amino or carboxyl
groups of the support and that of the enzyme.
(3). Poly functional reagents: Use of a bi-functional or
multifunctional reagent (glutaraldehyde) which forms
covalent bonds between the amino group of the support
and amino group of the enzyme.
Advantages of covalent bonding:
(a). Strong linkage of enzyme to the support
(b). No leakage or desorption problem
(c). Comparatively simple method
(d). A variety of support with different functional
groups available
(e). Wide applicability
Disadvantages of covalent bonding (major
problem with covalent bonding):
(a). Chemical modification of enzyme leading to
the loss of functional conformation of enzyme.
(b). Enzyme inactivation by changes in the
conformation when undergoes reactions at the
active site.
b) Cross linking/ Copolymerization:
This method don't uses the supporting material
Instead poly functional reagents are used to
create the cross linking between the enzymes.
These reagents are react with enzymes and
create the bridge or linkage b/w Enzymes
which forms backbone to hold the enzymes.
Ex; Glutaraldehyde, diazobenzidine,
hexamethylene di isocynanate etc