Enzyme immobilization and applications

Dr. Mahesh Kumar Kataria
Professor, Seth G. L. Bihani S. D. College of
Technical Education, Sri Ganganagar
Dr. Mahesh Kumar Kataria 1

Definition:
Enzymes are proteins that act as biological catalysts to accelerate chemical
reactions. Enzymes are not consumed in chemical reactions. The molecules
upon which enzymes may act are called substrates, and the enzyme converts the
substrates into different molecules known as products.
Traditionally, enzymes in free solutions (i.e. in soluble or free form) react with
substrates to result in products. Such use of enzymes is wasteful, particularly for
industrial purposes, since enzymes are not stable, and they cannot be recovered
for reuse.
Immobilization of enzymes refers to the technique of confining/anchoring
the enzymes in or on an inert support for their stability and functional
reuse. By employing this technique, enzymes are made more efficient and
cost-effective for their industrial use. Immobilized enzymes retain their
structural conformation necessary for catalysis.
Dr. Mahesh Kumar Kataria 2

Why Immobilize Enzymes?
 Protection of enzyme from degradation and deactivation.
 Re-use of enzymes for many reaction cycles, lowering the
total production requirement of the enzyme.
 Low cost of enzyme mediated reactions.
 Ability to stop the reaction rapidly by removing the enzyme
from the reaction solution.
 Enhanced stability.
 Easy separation of the enzyme from the product.
 Product is not contaminated with the enzyme.
Characteristics of immobilization of enzymes:
 Safe homing (can be separated from the reaction mixture)
 Reusability (can be reused again and again).
3Dr. Mahesh Kumar Kataria

Advantages of Enzyme Immobilization:
 Stable and more efficient in function.
 Can be reused again and again.
 Products are enzyme-free.
 Ideal for multi-enzyme reaction systems.
 Control of enzyme function is easy.
 Suitable for industrial and medical use.
Disadvantages also associated with immobilization.
 The possibility of loss of biological activity of an enzyme during
immobilization or while it is in use.
 Immobilization is an expensive affair often requiring
sophisticated equipment.
 Changes in properties(selectivity).
 Mass transfer limitations.
 Problems with cofactor and regeneration.
 Problems with multi enzymes system.

Classification of Enzyme Immobilization Techniques:
There are various methods to classify the techniques of enzyme
immobilization.

Classification of Enzyme Immobilization
Techniques

A. On the Surface Enzyme immobilization
1.Covalent Binding
2.Adsorption
3.Complexation and Chelation
B. Within Surface Method Enzyme Immobilization
1.Entrapment
2.Encapsulation
Classification of Enzyme Immobilization
Techniques

A. On the Surface Immobilization
The enzyme is immobilized on the surface of the support.
1. Covalent Binding:
Based on the binding of enzymes and water-insoluble
carriers by covalent bonds, Stable complexes between
functional groups on enzyme molecules and a support
matrix are formed through covalent bondings.
The functional group present on enzyme, through which a
covalent bond with support could be established, should be
non-essential for enzymatic activity.
The support must be Nontoxic, Non reactive, inert, stable
at the specific pH, Temp.
The enzyme functional groups that could be utilized in
covalent coupling include: Amino group, carboxylic group,
phenolic group, sulfhydryl group, thiol group, imidazole
group, indole group and hydroxyl group.
The functional groups of support that could be utilized in
covalent coupling include: hydroxyl group, Amino group,
carboxylic group, phenolic group, thiol group etc.

Polymeric supports include:
 Amino and related groups of polysaccharides and silica gel etc.
 Carboxylic acid and related groups of polyglutamic acid, carboxy methyl cellulose.
 Aldehyde and acetal groups of polymers.
 Amide group of polypeptide.
Advantages of covalent coupling:-
 Binding force between enzyme and carrier is so strong that no leakage of the
enzymes occurs, even in the presence of substrate or solution of high ionic
strength.
 This is a simple, mild and often successful method of wide applicability
Disadvantages of covalent coupling:-
 The major problem with covalent bonding is that the enzyme may be inactivated
by bringing about changes in conformation structure and active centre of the
enzyme, resulting in major loss of activity and/or changes of the substrate when
undergoes reactions at active sites.
 Only small amounts of enzymes may be immobilized (about 0.02 grams/gram
matrix).

Different methods of covalent bonding are:
(i) diazoation (bonding between the amino group of the
support e.g. aminobenzyle cellulose, aminosilanised porous glass,
aminoderivatives and a tyrosyl or histidyl group of the enzyme),
(ii) formation of peptide bond (bond formation between the amino or carboxyl
group of the support and amino or carboxy group of the enzyme),
(iii) group activation-coupling reaction (use of cyanogen bromide to a support
containing glycol group i.e. cellulose, syphadex, sepharose, etc), and
(iv) poly functional reagents (use of a bifunctional or multifunctional
reagent e.g. glutaraldehyde which forms bonding between the amino group of
the support and amino group of the enzyme).
The polymers may be engaged in direct coupling as well as could be modified by
other coupling groups or activating groups. The most commonly used polymers
are polysaccharides, polyvinyl alcohol, silica and porous glasses.

Fig: Coupling reaction
Fig: Glutaraldehyde based enzyme coupling reaction

2. Adsorption
 This method is based on the physical adsorption of
enzyme protein on the surface of water-insoluble
carriers. Carrier may be organic or inorganic. Examples
of suitable adsorbents are ion-exchange matrices,
porous carbon, clay, hydrous metal oxides, glasses and
polymeric aromatic resins.
 The bond between the enzyme and carrier molecule
may be weak vander waal forces, ionic, covalent,
hydrogen, coordinated covalent or even combination of
any of these.
 Immobilization can be brought about by coupling an
enzyme either to external or internal surface of the
carrier.
 Simply mixing the enzyme with a suitable adsorbent,
under appropriate conditions of pH and ionic strength,
followed, after a sufficient incubation period, by
washing off loosely bound and unbound enzyme will
produce the immobilised enzyme in a directly usable
form. The driving force causing this binding is usually
due to a combination of hydrophobic effects and the
formation of several salt links per enzyme molecule. 12Dr. Mahesh Kumar Kataria

Carriers used in adsorption;
 Mineral based support: Aluminium oxide or Clay
 Organic bimolecular based ions based: Starch, cellulose
 Modified ion exchange resins: Sepharose
Advantages of adsorption:-
 Little or no confirmation change of the enzyme.
 Simple and cheap.
 No reagents are required.
 Wide applicability and capable of high enzyme loading.
 High enzyme loading (about one gram per gram of matrix).
Disadvantages of adsorption:-
 Desorption of enzyme due to changes in temperature, pH and ionic strength.
 Slow method.
 Exposure of enzymes to microbial attack.
 Physical abrasion of enzyme due to turbulence associated with the bulk
solution.

Methods of immobilization by adsorption:-
1. Static Process:-
This is most efficient technique but requires maximum time. In this technique,
enzyme is immobilized by allowing it to be in contact with the carrier without
agitation.
2. Dynamic Process:-
This process typically involves the admixing of enzyme with the carrier under
constant agitation using mechanical shaker.
3. Reactor loading:-
This process is employed for the commercial production of immobilized
enzymes. The carrier is placed into the reactor and enzyme solution is transferred
to the reactor with agitation of the whole content in the reactor.
4. Electro-Deposition:-
In this technique, carrier is placed in the vicinity of one of the electrode in an
enzyme bath and electric current is applied leading to migration of enzyme
towards the carrier. This results in deposition of enzyme on the surface of the
carrier.

3. Complexation and chelation
 Transition metal salts or hydroxides deposited on the surface of organic carriers
become bound by coordination with nucleophilic groups on the matrix
 Mainly titanium and zirconium salts have been used due to their non toxic
nature and the method is known as “metal link immobilization”
 Metal salt or hydroxide is precipitated onto support by heating or neutralization
 These metal chelated supports – named as – Immobilized Metal-ion
Affinity(IMA) adsorbents

B. Within Surface Immobilization
1. Entrapment
 Entrapment is defined as an irreversible method of enzyme immobilization
where enzymes can be physically entrapped inside a matrix (support) of a
water soluble polymer such as polyacrylamide type gels and naturally derived
gels eg. cellulose triacetate, agar, gelatin, carrageenan, etc. that allows the
substrate and products to pass through but retains the enzyme.
 Entrapment is also described as physical restriction of enzyme within a
confined space or network.
Advantages:
 Improve mechanical stability
 Minimize enzyme leaching
 Avoid denaturation of enzyme as it does not chemically interact with the
polymer.
There are several methods for enzyme entrapment:
(i) inclusion in gels (enzyme entrapped in gels),
 In situ gels: poly acrylamide gel
 polycondensation: Urethane
 Gelatin 16Dr. Mahesh Kumar Kataria

(ii) inclusion in fibers (enzyme entrapped in fiber format),
 Three dimensional gels: hydrogel

2. Encapsulation
 This Microencasulation involves the formation of spherical particle called as
“microcapsule” / “Beads” in which a liquid or suspension of biocatalyst is
enclosed within a semi permeable polymeric membrane.
 Encapsulation is the enclosing of a droplet of solution-of enzyme in a
semipermeable membrane capsule. The capsule is made up of cellulose nitrate
and nylon. The method of encapsulation is cheap and simple but its
effectiveness largely depends on the stability of enzyme although the catalyst is
very effectively retained within the capsule.
 In this method a large quantity of enzyme is immobilized but the biggest
disadvantage is that only small substrate molecule is utilized with the intact
membrane.
 The extrusion method involves the drop-wise addition of cells suspended in
sodium alginate (2–4% w/v) into calcium chloride (20–100 mM) hardening
solution.

Salts of sodium of alginic acid (Sodium alginate) are
water soluble, whereas the salts of polyvalent cations,
e.g., calcium (Calcium alginate), are water insoluble.
Polyvalent cations are responsible for the cross-linking
of both different polymer molecules and different parts
of the same polymer chain. The process of gelation,
simply the exchange of calcium ions for sodium ions, is
carried out under relatively mild conditions.
2 Na(Alginate) + Ca++ -------> Ca(Alginate)2 + 2 Na+
The gel of sodium alginate containing enzyme can be
transferred drop-wise with constant stirring into a
beaker containing high concentration of calcium. The
beads are formed in the beaker due to conversion of
water soluble sodium alginate into water insoluble
calcium alginate beads contains encapsulated enzyme as
core material.
Alginate is currently widely used in food,
pharmaceutical, textile, and paper products. The
properties of alginate utilized in these products are
thickening, stabilizing, gel-forming, and film-forming.
Alginate polymers isolated from different alginate 19Dr. Mahesh Kumar Kataria

Applications of Enzyme immobilization
 There are several application of enzyme application in
different fields.
 Broadly the applications of immobilized enzyme can be
subdivided into three major areas of pharmaceutical
sciences;
A. Industrial applications
B. Analytical Applications
C. Therapeutic applications

A. Industrial Applications
1. Immobilized Penicillium acrylase (obtained from E.coli.) may be used for
conversion of Pencililin G (Produced by fermentation) to 6-APA (6-Amino
Penicillanic acid), a basic nucleus required to produce various semi synthetic
penicillins such as carbeniciilin, ampicillin, amoxycillin etc.
2. Production of high fructose rich syrup: fructose is sweeter than glucose, so
glucose is conrted to fructose in several syrup for palatability by the use of
immobilized enzyme glucose isomerase.
3. Use of yeasts in the baking and brewing industries - because they contain
the enzymes for alcoholic fermentation; metabolize hexose sugars to
produce pyruvate, but, whereas animals convert this to lactate under
anaerobic conditions, the anaerobic end-product in yeasts is ethanol, with
carbon dioxide being evolved.
4. The clarification of cider, wines and fruit juices (e.g. apple) is usually
achieved by treatment with fungal pectinases. Pectinases are a group of
enzymes including poly galacturonases, which break the main chains of
pectins, and pectin esterases, which hydrolyse methyl esters. Their action
releases the trapped particles and allows them to flocculate (pectins of fruit
and vegetables play an important role in jam-making and other processes by
bringing about gel formation)

5. Cheese production involves the conversion of the milk protein, K-casein, to
para-casein by a defined, limited hydrolysis catalysed by chymosin (rennin)
Since chymosin - only be extracted from calves killed before they are weaned
(pepsin is produced instead of chymosin after weaning). The enzyme is in
short supply and also an ethical issues, these enzymes are used as
immobiized enzyme to improve the reusability and safe homing.
6. Papain (immobilized) is sometimes used as a meat tenderizer; some South
American natives have traditionally wrapped their meat in leaves of papaya,
the fruit from which papain is extracted Papain (and other proteases) may
also be used in the brewing industry to prevent chill hazes, caused by
precipitation of complexes of protein and tannin at low temperatures
Other Industrial Applications:
1. Washing powders incorporating bacterial proteases containment in granules
which rupture only on contact with water The enzymes subtilisins from
Bacillus subtilis mutants, are stable to alkali, high temperature (e.g. 65°C),
detergents and bleaches. They will attack blood and other protein stains.
2. Bacterial proteases are also used in the leather and textile industries to loosen
hair (or wool) and enable it to be separated from hide.

B. Analytical applications
1. Enzyme electrode and Biosensors (Discussed in another chapter)
2. ELISA (Enzyme linked immuno sorbent assay):
It is a plate-based assay technique designed for detecting and quantifying
soluble substances such as peptides, proteins, antibodies, and hormones.
(Discussed in another chapter).
3. Immobilized haptens as immunopurifier): Various supports/surfaces are
capable to immobilize different proteins/enzyme.
4. Affinity chromatogaraphy and purification.: One species may have high
affinity for the material to be removed from solution purification. For eg;
Concavalin A (plant protein) can be purified by passing crude extract
through a column of beads containing covalently attached glucose residue.
Concavalin A has affinity to glucose and thus bind to the bead while other
proteins pass through the column.
5. Immobilized avidin biotin based system: Avidin (egg protein) have avidity
for biotin (Cell vitamin). Biotin may bind with antibodies/protein whereas
avidin may carry support. Thus biotinylated antibodies may be used to
separate specific antigen/protein from the system by column 23Dr. Mahesh Kumar Kataria

3. Therapeutic Applications
1. Preparation of immobilized enzyme for eg. Fibronolysis, streptokinase, urokinase
in microgranules of sephadex can be used for the treatment of thrombus and
thromboemboli of any vessels (atherosclerosis in coronary arteries ocuurs in
myocardial infaraction).
2. Treatment of phenylketonutria: it is a mental retardation state, where deficiency of
enzyme (phenyl alanin hydroxylase -PAH) leads to hindrance of conversion of
phenylalanin to tyrosin. The immobilized PAH may be used for treatment of the
disease.
3. Triggered drug delivery: This system contain the active agent placed subdermally or
in other appropriate body sites where it remain passive until a specific molecular
appears in tissue surrounding the device. Eg. Naltroxen (narcotic antagonist).
4. Artificial Organs: The specific enzyme may be immobilized and implanted in the
body may work as artificial organs
eg; Artificial pancreas may immobilize B-islet cells of pancreas to release insulin,
Artificial liver, may immobilize several metabolic enzyme
Artificial kidney may be used as blood purifier by used several adsorbents etc.

References:
 Vyas S. P. and Dixit V.K., Pharmaceutical Biotechnology, First Edition,
2005 (reprint), C.B. S. Publishers and Distributors, New Delhi.
 Patil A.S. et al., A Text Book of Pharmaceutical Biotechnology, First
Edition, 2019, S. Vikas and Company, Jalandhar.
 Rajesh Gollapudi and Sujitha Paladugu, Concise course in
Pharmaceutical Biotechnology, First Edition, 2020, S. Vikas and
Company, Jalandhar.
 Sikander Ali et. al., “Enzymes Immobilization: An Overview of
Techniques, Support Materials and its Applications”, International
Journal of Scientific & Technology Research, 6 (9), 2017
 Viet T.Q. et al., “Immobilization of Cellulase Enzyme in Calcium
Alginate Gel and its Immobilized Stability”, American Journal of
Research Communication, 1(12), 2013

Important Questions
 What do you mean by term “enzyme immobilization”? Enlist different
advantages and disadvantages of enzyme immobilization.
 Explain different techniques of enzyme immobilization.
 Write various analytical applications of enzyme immobilization.
 Discuss different therapeutic applications of enzyme immobilization.
 Describe various industrial applications of enzyme immobilization.
 Write a brief note on immobilization of enzyme by covalent binding or
adsorption.

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