Immobilization of

Enzymes

Present by:- Gauri Singh

Roll no.:- SDT-02/21
Subject:- Food and Industrial
Microbiology
 Contents
• Introduction to Immobilization of Enzymes
• Immobilized enzymes
• Introduction of Carrier Matrices
• Characteristics of Ideal Carrier Matrices
• Advantages of Immobilizations of Enzymes
• Methods of Immobilization of Enzymes
• Adsorption
• Entrapment
• Encapsulation
• Covalent binding
• Cross linking
• Application of immobilization of enzymes in Dairy & Food Industry
 What is Immobilization of Enzymes?

• Enzyme Immobilization is a technique use to attach or fix enzymes to a solid support

material, making them more stable and reusable.

• The material used for immobilization of Enzymes are called carrier materials, this
are usually inert polymers or inorganic materials.

• The support or matrix on which the enzymes are immobilized to allow the exchange
of medium containing substrate or effectors or inhibitors molecules.

• The practice of immobilization of enzymes or cells is very old and the first
immobilized enzymes was Amino acylase of Aspergillus oryzae for production of L-
amino acid in Japan
 Immobilized enzymes
• Immobilized enzymes are enzymes that have been attached or fixed to a solid
support material, such as a polymer, membrane, or particle.

• This attachment makes the enzymes more stable, reusable, and easily separable
from reaction mixtures.

• Examples of immobilized enzymes include:

1. Lipase :- immobilized on a membrane for biodiesel production.
2. Protease:- immobilized on a particle for protein hydrolysis.
3. Amylase:- immobilized on a polymer for starch hydrolysis

Immobilized enzymes have revolutionized various industries by enabling efficient,

sustainable, and cost-effective bio catalysis.
 What is Carrier Matrices?

The materials used for immobilization of enzymes, called carrier matrices,which

are usually inert polymers or inorganic or organic materials.

Classification of Carrier Matrices:-

Organic matrix:-
Polysaccharides (Cellulose, Inorganic matrix:
dextran, agar, agarose, chitin,
alginate etc.), proteins It includes Bentonite, celite,
(Collagen, albumin) and centolite, silica, charcoal,
carbon Polystyrene, Porous glass, metals and
polyamides metal oxides.
 Ideal carrier Matrices for
enzymes Immobilizations
• The ideal carrier matrix has the following properties:
Low cost,
• Inertness,
• Physical strength
• Stability
• Regenerability after the useful lifetime of the immobilized enzyme,
• Enhancement of enzyme specificity,
• Reduction in product inhibition,
• A shift in the ph optimum for enzyme action to the desired value for
the process, and reduction in microbial contamination and nonspecific
adsorption
 Advantages of Immobilizations of Enzymes
1. Protection from degradation & deactivation hence increase the shelf life of enzymes.

2. Reuse of enzymes for many reactions cycles lowering total cost of enzymes mediated
reaction .

3. Enhanced stability, resistant to denaturation, degradation and inhibitions.

4. Easy separation of the enzymes from the products.

5. Prevent the product from the contamination of enzymes.

6. Immobilized enzymes can increase reaction rate and yields due to improved
enzymes –substrate reactions.
 Methods of Immobilization of Enzymes

Physical Method Chemical Method

1. Adsorption 1. Covalent
Binding

2. Entrapment 2. Cross linking

3. Encapsulation
 Adsorption
1. Adsorption involves the physical
binding of enzymes (or cells) on the
surface of an inert support through
forces such as van der wal forces,
hydrogen bonding.

2. The support materials may be

inorganic (e.g. alumina, silica gel,
calcium phosphate gel, glass) or organic
(starch, carboxymethyl cellulose,
DEAE- cellulose, DEAE-sephadex).
 Advantages Disadvantages
• Relatively low surface area of
• This method of immobilization is binding.
simple & Economical.
• Exposure of enzymes to microbial
• Limited loss of activity of enzymes. attack

• Enzymes can be recycled, • Yield are often low due to

Regenerated & reused inactivation & desorption.
 Entrapment
1. Entrapment involves inclusion of an enzyme in a
polymer network (gel lattice) such as an organic
polymer or a silica sol–gel, or a membrane device
such as a hollow fiber or a microcapsule.

2. Entrapment requires the synthesis of the

polymeric network in the presence of the enzyme.

3. In entrapment immobilization, enzymes is not

directly attached to the support surface but
entrapped within a polymeric network.
 Advantages
Disadvantages
• Entrapment allow for high
enzymes loading
• Support materials can degrade,
• It is a relatively simple method results in releasing enzymes and
and performed under mild reducing activity
condition
• Can be challenging to scale up for
• Minimizes enzymes leakage, industrial applications.
maintaining activity on time.
 Encapsulation
• Encapsulation Immobilization represents an
entrapment method in which enzymes are
enclosed in a spherical semi- permeable
membrane.

• The membrane may be polymeric, lipoidal,

lipoprotein based or non-ionic in nature.

• It involves enclosing the enzymes within semi

–permeable polymer membrane eg:- semi
permeable collodion or nylon membrane in
shape of sphere.
 Advantages Disadvantages

Encapsulation protects Enzymes can be inactivated

enzymes from denaturation, during encapsulation .
degradation and inhibitions
Encapsulation material can
It’s minimize enzymes leakage degrade releasing enzymes &
maintaining activity reducing activity.

Encapsulation can be scaled up This process can be complex,

for industrial applications. requiring optimization .
 Covalent Binding
1. In enzyme immobilization technique,
covalent binding is one of the most widely
used methods.

2. Covalent immobilization provides strong

binding between enzymes and support
matrix( polymers , silica or nano particles)
to form a stable complex.

3. Covalent binding immobilization of enzymes

involves chemically attaching enzymes to a
support material using covalent bonds.
Advantages:-

Covalent bonds ensures strong and stable

enzymes attachment. Covalent binding can
enhance stability and resistance to
denaturation.

Disadvantages :-

Covalent binding enzymes can inactive

enzymes if binding occurs at critical sites. It’s
reagents and processes can be costly .
 Cross linking Method

1. Cross linking enzymes are first adsorbed or

attached to support material, the cross – Linking
agents are used to create covalent bonds between
enzymes and molecules or between enzymes
molecules and support molecules.

2. Cross linking of proteins( enzymes) either to other

enzymes or functional groups on a insoluble
support matrix.

3. The most used cross linked reagents are :-

Glutaraldehyde, Formaldehyde.
Advantages:-

Cross linking of enhances enzymes stability and

resistance to denaturation. Cross linkes enzymes
can be reused multiple times. It minimize
enzymes leakage.

Disadvantages:-

Cross linking requires suitable support

materials. It can activate enzymes if excessive
cross linking occurs.
 Applications of Immobilization of Enzymes in Dairy & Food
Industry
1. Cheese production: Immobilized enzymes like rennet, lipase, and lactase improve
cheese quality, texture, and flavor.

2. Lactose-free milk: Immobilized lactase breaks down lactose, making milk suitable
for lactose-intolerant individuals.

3. Yogurt and fermented products: Immobilized enzymes like lactase, lipase, and
protease enhance flavor, texture, and nutritional value.

4. Fruit juice clarification: Immobilized enzymes like pectinase, cellulase, and

hemicellulase improve juice clarity, stability, and quality.

5. Flavor enhancement: Immobilized enzymes like lipase, protease, and oxidase

create natural flavor compounds.