Chapter 3:

Enzyme Immobilization
Technology
Course title: Pharmaceutical Biotechnology
Course code: PHR 403
 Enzymes
• Enzymes are biological catalysts that enhance the rate
of biochemical reactions from 10 to 1012 times when
compared to those of uncatalyzed reactions.
• Each enzyme is highly specific and catalyzes one or a
few reactions. That is, the enzymes 'select' their
substrate molecules and trigger a particular type of
reaction.
• One of the costliest enzymes is ornithine carbamyl
transferase which costs 215,000 USD per g.
Sources of Enzymes
Enzymes are usually obtained from three main sources
namely: plants, animals and microorganisms. The
advent of fermentation technology led to the preparation of
purer forms of enzymes from microbes.
Advantages of microbes over other sources of
enzymes
• Microbes have a short generation time and hence the
time required for enzyme production in large quantities
is less than plants and animals.
• Enzymes can be easily extracted from microbes. If the
enzymes are extracellular, they will be secreted into the
medium and can be purified easily.
• The plants and animals show variations in the
amount of enzymes produced. These variations
are seasonal. But microorganisms produce the
enzymes without seasonal variations.
• The enzymes obtained from microbes are usually
stable under extreme environmental
conditions.
• Microorganisms can produce large varieties of
enzymes which is not possible with plants and
animals.
• Microorganisms can easily be manipulated by
mutation or by genetic engineering to give high
yield of enzymes.
Microorganisms and enzymes produced by them
Bacteria
Bacillus cereus ----------------- Penicillanase
B. coagulans -------------------- Amylase
B. megnterium------------------- Penicillin acylase
Escherichia coli ----------------- Penicillin acylase
Fungus
Candida lipolytica --------------- Lipase
Neurospora Crassa -------------Trypsinase
Penicillium funiculosum ------- Dextranase
P. notatum ------------------------ Glucose oxidase
Saccharomyces cerevisae ----Invertase
Types of Enzymes
Production of Enzymes
 Medical Use of Enzymes

Enzyme Medical uses

Asparaginase Leukemia
Collagenase Skin ulcers
Glutaminase Leukemia
Lysozyme Antibiotic
Ribonuclease Antiviral
b-Lactamase Penicillin allergy
Urokinase Dissolve blood clots
Rhodanase Cyanide poisoning
Uricase Gout
Streptokinase Dissolve blood clots
Restriction enzymes, DNA ligase,
Genetic engineering
reverse transcriptase
 Definition of Enzyme Immobilization:

➢ The immobilization involves the conversion of water soluble

enzyme into a solid form of catalyst, which is insoluble.
➢ Immobilization is defined as "imprisonment of an enzyme in
a distinct phase that allows exchange with, but is separated
from the bulk phase in which the substrate, effector or
inhibitor molecules are dispersed and monitored".
➢ Imprisonment refers to the physical or chemical trapping of
the enzyme either onto or into the polymer matrix.

First commercial application of this technique was done in 1969

in Japan with the use of amino acylase from Aspergillus oryzae
for industrial production of amino acids.
Why enzyme immobilization is necessary?
as
➢ Most of the enzymes are expensive and hence they should
be used as efficiently as possible.
➢ An important property of enzymes is their capacity to perform
same function again and again.
➢ But if the enzymes are used either in pure form or in crude
form after completion of catalysis, it is not possible to
separate them from the product since both are in dissolved
state. If they are separated, the process becomes costly and
difficult.
➢ Some enzymes are too costly and the process of enzymatic
catalysis becomes cost effective only if they are reused.
Immobilization techniques helped the biotechnologists to
solve the problem.
Advantages of Immobilized Enzymes
There are many advantages of using immobilized
enzymes. These are:
• Reuse: The same enzyme can be used repeatedly,
since they are not lost at the end of a batch.
• Continuous use: Continuous production system
can be designed easily.
• Cost effectiveness: If the enzymes are immobi-
lized, they can be used repeatedly and hence cost-
effectiveness is achieved. Moreover, enzyme
substrate ratio is very high in immobilized enzymes
and this further increases the cost-effectiveness.
• Less contamination: Since the immobilized
enzyme remains within the polymer matrix, it will
not contaminate the final product.
• Stability: Immobilization increases the thermal
stability of the enzyme. For example, immobilized
glucose isomerase is stable at 65°C for almost
one year, whereas the pure enzyme is denatured
within few hours at a temperature of 45°C.
• Process: Since the immobilized enzymes have a
standardized activity, the process control
becomes very easy.
Disadvantages of enzyme immobilization:

Even though there are many advantages of immobilized

enzymes, there are some disadvantages also.
• High cost for the isolation, purification and recovery of
active enzyme
• Industrial applications are limited and only very few
industries are using immobilized enzymes or
immobilized whole cells.
• Catalytic properties of some enzymes are reduced or
completely lost after their immobilization on support or
carrier.
• Some enzymes become unstable after
immobilization.
• Enzymes are sometimes inactivated by the heat
generated in the system
Supports or matrix used in immobilization
technology:

➢ The matrix or support immobilizes the enzyme by holding it

permanently or temporarily for a brief period of time.
➢ There are a wide variety of matrices or carriers or supports
available for immobilization.
➢ The matrix used should be cheap and easily available.
Their reaction with the components of the medium or with
the enzyme should be minimum as possible.

➢ The matrices or supports for immobilization of enzymes or

whole cells are grouped into three major categories:
1. Natural polymers
2. Synthetic polymers
3. Inorganic materials
(A) Natural polymers: 1 2
1. Alginate: A natural polymer derived from the cell wall
of some algae. Calcium or magnesium alginate is the
most commonly used matrix. They are inert and have
good water holding capacity.
2. Chitosan and chitin: They are structural
polysaccharides occurring naturally in the cell wall of
fungi and the exoskeleton of arthropods. The various
functional groups in enzymes can bind to the –OH
group of chitin and can form covalent bonds.
3. Collagen: It is the protein support with good porosity
and water holding capacity. The side chains of the
amino acids in the collagen and that of enzyme can
form covalent bonds to permanently hold the enzyme
to the support.
Carrageenan Pectin

Chitosan
4. Carrageenan: It is a sulfated polysaccharide obtained
from some red algae. Their good gelling properties
together with its high protein holding capacity makes it
good support for immobilizing enzymes.
5. Gelatin: Gelatin is the partially hydrolyzed collagen
with good water holding capacity.
6. Cellulose: Most abundant polymer of nature and it is
the cheapest support available as carrier of enzymes.
The hydroxyl group of the glucose can form covalent
bonds with that of the amino acids of enzyme.
7. Starch: A natural polymer of amylose and
amylopectin. It has good water holding capacity.
8. Pectin: It is a structural polysaccharide of plants found
in their primary cell wall and they also acts as the inter-
cellular cementing material in plant tissues. Pectin is a
gelling agent with good water holding capacity.
(B) Synthetic polymers:
They are ion exchange resins or polymers and are
insoluble supports with porous surface. Their
porous surface can trap and hold the enzymes or
whole cells. Example: Diethylaminoethyl cellulose
(DEAE cellulose), Polyvinyl chloride (PVC), UV
activated Polyethylene glycol (PEG)
(C) Inorganic materials: 1 2
1.Zeolites: They are microporous, aluminosilicate
minerals with good adsorbing properties and extensively
used for immobilizing enzymes.
2.Ceramics: They are nonmetallic solids consisting of
metal and nonmetal atoms held in ionic and covalent
bonds. The composition and bonding pattern varies with
different types.
3.Diatomaceous earth: They are silicious sedimentary
rocks formed by fossilized accumulations diatoms. Celite
is the trade name of diatomaceous earth.

Other materials include: silica, glass, activated carbon.

Methods of Enzyme Immobilization
Different methods have been discovered for
immobilization of enzymes. All these methods can be
categorized into two different techniques, namely:
1. Immobilization in a support
2. Immobilization on a support
1. Immobilization in a Support
This technique involves entrapment and
encapsulation of enzymes.
Entrapment: In this technique, the enzyme is
entrapped within a cross-linked polymer-matrix.
Enzyme is dissolved in a solution of precursors of the
polymer and then polymerization is initiated.
• The enzyme is physically entrapped within the
matrix and it cannot escape by permeation.
• Substrate molecules can diffuse in can be acted
upon by the enzyme, and product molecules can
diffuse out of the matrix.
• The common polymers used include poly-
acrylamide gel, starch gel, silicone rubber gel,
cellulose triacetate, alginate, gelatin and agar.
But, immobilization of enzymes by entrapment
technique has the following problems:
1. Due to wide pore size distribution in the gel,
there will be continuous leakage of enzyme.
2.Since enzyme is bound within the matrix,
substrate accessibility is reduced.
3.Some of the enzyme activity is lost or reduced
due to free radicals produced during
polymerization.
Encapsulation:
•In this method, the enzyme is immobilized by
enclosing a droplet of enzyme in a semi-permeable
membrane capsule.
•This method has wide applications in pharmaceutical,
and medical fields. Once encapsulation is done, the
enzyme cannot escape, whereas the low molecular
weight substrates and products can diffuse
through the membrane.
•The capsule may be made up of either permanent
materials like nylon or collodion or 'biodegradable'
materials' like polylactic acid or phospholipid liposomes.
2. Immobilization on a Support
The techniques involved are adsorption, covalent
binding and cross linking of enzyme itself.
Adsorption: Adsorption of enzymes onto a
polymeric matrix is the easiest method of
immobilization. The adsorption occurs due to
nonspecific bonding like electrostatic,
hydrophobic, or affinity bonding to a specific
ligand. The disadvantage of this method is the loss
of activity of enzyme during adsorption.
Some of the adsorbents that are used for
immobilization are as follows:
• Alumina
• Amberlite CG-50
• Bentonite
• Calcium phosphate gel
• Carboxymethyl cellulose (CMC)
• Carboxymethyl sephadex (CMS)
• Collagen
• Glass
• Silica gel
Procedure of enzyme immobilization:
The adsorbent is packed in a water jacketed column and
washed with a pre-conditioning solution
↓
The enzyme solution is prepared and buffered fairly close to
the isoelectric point of the enzyme
↓
The enzyme solution is then circulated through the column for
several hours
↓
Then the solution is drained, and column is washed with
water followed by 0.5 M sodium chloride and again with water
During immobilization, the ionic concentration and pH should
be controlled carefully, because a change in pH may lead to
desorption.
 Enzyme Immobilization
•Imprisonment of an enzyme

•Physical or chemical trapping onto or into the

polymer matrix

Methods of enzyme immobilization

1. Adsorption Limitation

I
Immobilization ‘on' a Support 2. Covalent binding Chemical method
3. Crosslinking
4. Entrapment Physical method
Immobilization ‘in' a Support
5. Encapsulation
 Entrapment
•Enzyme polymerized in a
matrix
•Substrate and product can
diffuse in and out, enzyme
can’t.

Limitation:
• leakage of enzyme
• Substrate accessibility less
• Enzyme activity lost or reduced
Encapsulation

•Droplet of enzyme enclosed within a semi

permeable polymeric membrane capsule. The
capsule is usually made up of cellulose nitrate and
nylon.

•Substrate and product can diffuse in and out,

enzyme can’t.
 Adsorption

• Enzymes are adsorbed on a water-insoluble

solid support
• Bond between the enzyme and adsorbent
involve hydrogen bonds, hydrophobic bonds,
ionic bonds and van der Waals forces.

• Easiest
• Disadvantage - loss of activity of enzyme
• Adsorbent choice important e.g. alumina,
carbon, calcium carbonate, hydroxyapatite,
glass or alginate beads, clay-exchange resins,
Diethylaminoethyl (DEAE) cellulose
• Controlled pH
 Covalent binding

• Formation of covalent interactions

between the functional groups
present on the surface of the carrier
and those present on the amino acid
residues of the enzyme surface.

• Support material of enzyme includes

polyacrylamide, porous glass,
agarose and porous silica.
Covalent binding

The enzyme can be covalently bound to the polymer by

many methods. The enzyme forms a covalent bond with
the active groups on a polymer support. This can be done
by three ways:
1. Through the reactive groups on the side chains of its
amino acids such as lysine, arginine and tyrosine.
2. Through the terminal amino and carboxyl groups of
polypeptide chains.
3. The covalent binding of the enzyme with the support can
also be achieved by the use of a bifunctional reagent,
which attaches the enzyme to the polymer.
The polymers used include celluloses, polyacrylamides and
other hydrogels. These can be activated directly by using
cyanogen bromide or by incorporating reactive groups such
as diazo, azide arid carbodiimide on the polymer surface to
which the enzyme is attached.
 Crosslinking
• Formation of covalent bond between the enzyme
molecules leading to three-dimensional cross-
linked aggregates.

• Both intramolecular and intermolecular cross

linking are used for immobilization of enzymes

• The most common reagent used for cross-linking

is cellulose or glutaraldehyde
Cross-linking:

Cross-linking of the enzyme to itself by the use of a bifunctional reagent

with the inclusion of an inert protein is another important technique of
immobilization. The most commonly used bifunctional reagent is
glutaraldehyde. The method is cheap and simple. Three techniques are
used for immobilization of enzymes by cross-linking:

• Cross-linking of the enzyme with glutaraldehyde to form an insoluble

aggregate.
• Adsorption of the enzyme onto a surface followed by cross-linking.
For example, the enzyme can be cross-linked and adsorbed onto the
surface of particles like colloidal silica.
• Impregnation of porous matrix material with the enzyme and cross-
linking the enzyme.
Applications of enzyme immobilization
1. Industrial production: Industrial production of
antibiotics, amino acids etc. uses immobilized
enzymes or whole cells.
2. Biomedical applications: Immobilized enzymes
are widely used in the diagnosis and treatment of
many diseases. Immobilized enzymes can be
used to overcome inborn metabolic disorders.
Immobilization techniques are effectively used in
drug delivery systems especially to oncogenic
sites.
3. Food industry: Enzymes like pectinases and
cellulases immobilized on suitable carriers are
successfully used in the production of jams,
jellies and syrups from fruits and vegetables.
4. Research: A Research activity extensively uses
many enzymes. The use of immobilized enzyme
allow researcher to increase the efficiency of
different enzymes such as Horse Radish
Peroxidase (HRP) in blotting experiments and
different Proteases for cell or organelle lysis.
5. Production of bio-diesel from vegetable oils.
6. Waste water management: treatment of sewage
and industrial effluents.
7. Textile industry: scouring, bio-polishing and
desizing of fabrics.
8. Detergent industry: immobilization of lipase
enzyme for effective dirt removal from cloths.
 Summary: Enzyme immobilization

• Enzymes when used in soluble form cannot be recovered from the medium
for reuse
• A new and valuable area of enzyme technology is concerned with
immobilization of enzymes ‘in’ or ‘on’ insoluble polymers
• After use, the enzymes are recovered from reaction mixture and reused again

Advantages of immobilized enzymes

a. It permits the reuse of the enzyme
b. It is ideal for continuous operation
c. Immobilized enzymes are usually more stable to temperature or pH changes
d. It saves the stability cost
Case Study

• Background: Enzyme immobilization is a technique used to attach

enzymes to a solid support or matrix, enhancing their stability,
reusability, and catalytic activity. This case study focuses on the
immobilization of the enzyme urease for its application in urea
hydrolysis, which is a key process in wastewater treatment to remove
nitrogen pollutants.
• Objective: The objective of this case study is to showcase the
benefits of immobilizing urease for wastewater treatment by
answering specific questions related to the immobilization process
and its advantages.
 Questions and Answers
Q1: Why is enzyme immobilization important in wastewater treatment?
Enzyme immobilization enhances the enzyme's stability, allowing it to
withstand harsh conditions commonly encountered in wastewater
treatment processes. Immobilized enzymes also enable their reuse,
reducing the need for enzyme replenishment and minimizing operational
costs.
Q2: How is urease immobilized onto a solid support?
Urease can be immobilized using various methods such as adsorption,
covalent binding, or entrapment. For this case study, covalent binding will
be employed
Q3: Explain the covalent binding method for immobilizing urease.
Covalent binding involves chemically linking urease to a solid support
through the formation of stable covalent bonds. This method ensures
strong attachment and prevents enzyme leaching. For example, urease
can be modified to introduce amino groups, which can then react with
activated functional groups on the matrix surface.
Q4: What advantages does immobilized urease offer over free urease in
wastewater treatment?
Immobilized urease offers several advantages:
• Stability: Immobilization enhances urease stability, enabling it to
maintain activity under varying pH and temperature conditions.
• Reusability: Immobilized urease can be separated from the treated
wastewater and reused, reducing the need for enzyme replacement.
• Enhanced Catalytic Efficiency: Immobilization concentrates urease at
the reaction site, leading to higher substrate conversion rates.
• Improved Process Control: Immobilized urease allows for better control
over the reaction, making it easier to manage urea hydrolysis.
Questions and Answers

Q5: What is the overall impact of immobilized urease on

wastewater treatment?
Immobilized urease significantly improves the efficiency of urea
hydrolysis in wastewater treatment. The enhanced stability,
reusability, and catalytic efficiency lead to a more sustainable and
cost-effective process for nitrogen pollutant removal.
Q6: Are there any challenges associated with enzyme
immobilization?
Yes, challenges include optimizing the immobilization method,
preserving enzyme activity during the process, and ensuring that
the immobilized enzyme retains its effectiveness over time.
Additionally, the choice of matrix and immobilization method
depends on the specific application.
Questions

1. What do you understand by enzyme immobilization?

2. List the sources of enzymes with an example of each
source
3. Explain three advantages of microbes over other sources
of enzymes.
4. Discuss the importance of enzyme immobilization.
5. Classify enzymes based on the basis of secretion.
6. Write a short note on the types of support or matrices
used in enzyme immobilization technology.
7. Using a flowchart differentiate between immobilization in
a support and immobilization on a support