A

REPORT

ON

METHODS OF ENZYME
IMMOBILIZATION AND ITS
APPLICATION IN INDUSTRIAL
SECTOR

SUBMITTED BY:

RAMANI PRADHAN

ROLL NO.: 2078BCH034

SUBMITTED TO:

DR. POONAM BAIDYA

DEPARTMENT OF APPLIED SCIENCES AND CHEMICAL

ENGINEERING

IOE, PULCHOWK CAMPUS

AUGUST 6, 2023
 ACKNOWLEDGMENT

First, I would like to thank our Biochemistry teacher, Dr. Poonam Baidya for giving us this
wonderful opportunity to do this report on the topic, ‘Methods of Enzyme Immobilization and
Its Application in Industrial Sector’ whose invaluable guidance, support, and expertise have
been instrumental in shaping the direction and content of this report.

I would also like to thank the researchers and scholars whose pioneering work in the field of
enzyme immobilization has laid the foundation for our study. As chemical engineering
students, it is important that we learn about the industries of various sectors to gain vast
knowledge about various technologies that can be applied for a sustainable future. Therefore,
I would like to thank each and everyone that directly or indirectly helped to enrich my
understanding of enzyme immobilization and its industrial application.

1
ABSTRACT
Enzymes are biocatalysts that accelerate biological and chemical processes. Enzymes have a
significant role in traditional food processes, medicine, pharmaceuticals and chemicals.
However, their industrial applications often face challenges related to stability, shelf life, and
reuse. Immobilization is a technique that attaches enzymes to support material such that it
provides easy recovery and reusability with efficient enzyme activity for a prolonged time
period reducing the cost of production. It emerged as a solution to these challenges. This report
provides an overview of various enzyme immobilization methods and their applications in
industries such as food, pharmaceuticals, and textiles. Additionally, the report discusses the
impact of COVID-19 on the enzyme production industry. The use of immobilized enzymes has
proven to be cost-effective and efficient, promoting greener and more sustainable processes.
With increasing demand and growing awareness of the advantages of using enzymes, the global
market for enzymes is expected to continue to grow. The future of immobilized enzymes in
industrial processes looks promising, with ongoing research and development in protein
engineering contributing to their widespread application.

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INTRODUCTION
Enzymes may be defined as biocatalysts that are synthesized by living cells to perform various
metabolic processes in the body (Satyanarayana & Chakrapani, 2021). Enzymes are proteins
that accelerate a variety of biological and chemical processes. They are natural catalysts that
are found in plants, animals, and microbes and catalyze metabolic processes that are essential
to living organisms. The advancement in protein extraction and purification has resulted in the
development of various enzymes with analytical grade purity for research and biotechnological
applications. Enzymes play an important role in a wide range of traditional food processes,
including cheese production, beer brewing, and the wine industry (Homaei, Sariri, Vianello, &
Stevanato, 2013). The extensive research in the field of biotechnology has led to the
development of enzymes that are shaped as per the needs.

Enzymes are used in baking, dairy, starch metabolism and beverage processing (beer, wine,
fruit and vegetable juices). Enzymes have an especially prominent role in the textile industry
because of their influence on final products. Many other essential industries, such as health
care and pharmaceutical and chemical, are increasingly benefiting from nature's wonderful
catalysts. In recent years, enzymes have been utilized in the making of biofuels like biodiesel
and ethanol. One of the best applications of enzymes in modern life is their application in
waste treatment in general and particularly in solid waste treatment and wastewater purification
(Homaei, Sariri, Vianello, & Stevanato, 2013).

However, all these desirable properties of enzymes and their wide industrial applications are
often hampered by the lack of long-term operational stability and shelf life as well
as cumbersome recovery and reuse. These disadvantages can often be overcome by enzyme
immobilization. Indeed, a major challenge for industrial biocatalysis is the development of
stable, robust and preferably insoluble biocatalysts.

Enzyme immobilization is a unique technique that involves attaching enzymes to a dormant

substrate, resulting in enhanced stability and the ability to reuse them to their fullest potential.
By using this technique, enzymes are made more efficiently and cost-effectively for industrial
use. Immobilization is often regarded as ‘a goose with a golden egg’. Immobilized enzyme
retain in structure such that they can be used for a prolonged time period.

The topic of immobilizing enzymes has been of great interest since the 1960s (Razzaghi,
Homaei, Vianello, Azad, & Sharma, 2022). The idea of enzyme immobilization was initiated
by Nelson and Griffin in 1916 after finding out that invertase could hydrolyze sucrose after

3
being absorbed into charcoal (Kim, Grate, & Wang, 2008). Since then, many methods of
immobilization have been researched and put into use on an industrial scale. Reversible
immobilization of enzymes involves adsorption, metal binding and ionic bonding. However,
the irreversible immobilization of enzymes involves entrapment and covalent bonding which
will be discussed in detail in this report (Liu & Dong, 2020).

This report aims to provide an overview of various enzyme immobilization methods, its
applications in the industrial sector, and the world market of these immobilized enzymes. It
also provides a brief discussion on the impact of Covid-19 on the enzyme production industry.

4
DISCUSSION

In the human body, there are about 75,000 enzymes that help in metabolic and digestive
reactions. In a similar manner, there are millions of other use of enzymes that benefit humans
through various processes. So the use of enzymes is not just limited inside the human body.
The enzymes can be used as therapeutic agents, analytical reagents, in genetic manipulations
and for industrial applications. For instance, Streptokinase, as a therapeutic agent prepared
from streptococcus is utilized in clearing the blood clots. The use of glucose oxidase and
peroxidase for the estimation of plasma glucose is another example of an enzyme being used
as an analytical reagent. If we look into the use of enzymes in the industrial sector, enzymes
such as rennin are used for cheese production.

The use of enzymes for industrial use proved to be quite wasteful and not economical as the
process of their recovery and reuse is quite cumbersome. This gave rise to the immobilization
of enzymes. The use of immobilized enzymes proved to be cost-effective and more efficient in
the industrial sector. Figure 1 extracted by Maghraby et.al. shows the various problems
associated with the traditional use of enzymes and the various advantages that come with the
usage of immobilized enzymes (Maghraby, El-Shabasy, Ibrahim, & Azzazy, 2023).

The main idea of the immobilization of enzymes is based on fixing or entrapping enzymes
inside the support materials. This supports the structure of the enzyme and provides
environmental resistance increasing its efficiency and prolonged usability.

Figure1 Problems associated with traditional use of enzyme (left), and the advantages of immobilization (right) (Maghraby,
El-Shabasy, Ibrahim, & Azzazy, 2023).

Based on this idea of using support materials, enzymes are immobilized in various ways, some
being reversible while others, irreversible.

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 Figure 2 Major methods for immobilization of enzymes

Figure 2 shows various methods of enzyme immobilization. The reversible methods include
adsorption, ionic binding, and the irreversible methods include covalent binding, entrapment,
encapsulation and cross-linking.

1. Adsorption

Adsorption is a reversible immobilization technique that is based on physical adsorption.

Materials such as activated carbon, alumina, ion-exchange resins are used for this method.

The enzymes are physically bound on the surface of these materials through weak forces
such as van der Waals forces and hydrogen bonds. This method requires only few reagents,
is cheap and can be easily implemented. However, one disadvantage would be that the
adsorbed enzymes could easily be removed by slight changes in pH or temperature,
therefore being a drawback for the industrial use of this method.

2. Ionic Bonding

This method is an inexpensive and reversible immobilization technique based in ionic

interaction between the enzymes and the support materials (Maghraby, El-Shabasy,
Ibrahim, & Azzazy, 2023). The materials used in this method are charged that are opposite
to the charge of the enzymes in order to form an ionic bond. This bonding can be reversed
simply by changing the pH or ‘salting out’ the enzyme. However, an optimum pH can be
maintained by controlling the acidity or alkalinity of the mixture. It must also be taken into

6
consideration that due to the charged support, there is a possibility of distortion of enzyme
structure and change in enzyme kinetics that hinder in the high product yield.

3. Covalent binding

Covalent binding is one of the most useful techniques of enzyme immobilization. This
method involves the formation of covalent bond between the enzyme and the support
material. The support materials used in this method include porous glass, porous silica,
polyacrylamide, agarose, etc. this method has numerous advantages which are listed below:

i. It increases the enzyme's durability with sufficient recovery of the enzyme to reuse
it.
ii. It increases the stability of the enzyme due to the increase in its stereospecificity.
iii. There is a very less possibility of enzyme leakage in a high concentration of
substrate in strong buffer solutions which usually results in the denaturation of the
enzyme.
iv. The leaking of the enzyme into the reaction mixture is prevented due to the strong
covalent bond which eliminates the risk of contamination and hence reduces the
cost of production.

A few limitations arise while applying this method such as it requires complicated steps
and a longer incubation period for the immobilization of the enzymes. Also, the amino
acids of the enzyme may affect the activity by denaturing the enzyme. Nevertheless, the
advantages outweigh the drawbacks and therefore this method is widely used in the
industrial sector.

4. Cross-linking

This is a unique technique of immobilizing enzymes, in the sense that there is the absence
of a solid support material. The enzyme molecules are immobilized by interconnection
among them through covalent bonding between enzymes by linker agents. These linker
agents are used as a bridge between the adjacent enzyme molecules. The reagenst that are
used in this method include diazobenzidine, glutaraldehyde, hexamethylene diisocyanate,
and toluene di-isothiocyanate.

One of the most commonly used cross-linking reagents is glutaraldehyde. The cross-links
created between the enzyme and glutaraldehyde are permanent and can withstand extreme
conditions, such as high temperatures and pH levels. Glutaraldehyde cross-linking has

7
proven successful in immobilizing a variety of industrial enzymes, including glucose
isomerase and penicillin amidase (Bhatia, 2018). The process of cross-linking is relatively
simple and cost-efficient. However, a potential drawback is the risk of enzyme denaturation
due to the use of a poly-functional reagent.

5. Entrapment

Entrapment is an irreversible immobilization technique in which the enzymes are simply

caged or trapped in a network of fibres or support material through covalent or non-covalent
bonds. The enzymes are physically trapped inside a polymer or a gel matrix. There are
different methods for enzyme entrapment, including photopolymerization, sol-gel
technique, and electropolymerization (Nguyen & Kim, 2017). The size of the pores of
matrix can be controlled such that the enzyme is retained while the substrate and product
molecules are allowed to pass through. This helps in avoiding enzyme leakage. As the
enzymes do not react with polymers, denaturation is effectively prevented. This technique
has many advantages including high loading capacity of the enzymes, low fabrication costs,
btter mechanical stability of the entrapped enzymes, and lower mass transfer (Costa,
Azevedo, & Reis, 2004). “Gelation of polycationic or polyanionic polymers after adding
multivalent counterions is the most used technique for the entrapping of enzymes”
(Mohamad, Marzuki, Buang, Huyop, & Wahab, 2015).

6. Encapsulation

Encapsulation is the term used to describe the creation of spherical particles by enclosing
a liquid or suspension in a semipermeable membrane. The membrane can be made of
polymers, lipids, lipoproteins, or non-ionic materials (Jha, n.d.). Encapsulation maintains
biological systems in a fine film preventing any contact with the environment that might
damage their efficiency. Therefore, encapsulation allows biocatalysts to remain active for
extended periods of time. The ionotropic gelation technique, which involves using alginates
and silica-based nanoporous sol-gel glasses, has been found to be effective for
encapsulating enzymes. This method is known for its simplicity in encapsulating enzymes.
Moreover, recent advancements in material sciences have led to the development of
encapsulation with improved properties such as better morphological stability, controlled
physicochemical permeability, and reduced enzyme leakage (Patil, Kamalapur, Marapur,
& Kadam, 2010).

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INDUSTRIAL APPLICATION OF IMMOBILIZED ENZYMES

In the case of using immobilized enzymes in the industrial sector, simple and cost-efficient
methods are preferred. The methods involve either physical immobilization or chemical
immobilization which have already been discussed above. In industrial processes, the cost of
using an immobilized enzyme depends on its reaction kinetics, specificity, and the number of
times it can be reused. This is indirectly related to the total productivity on a per-kilogram
product basis relative to the amount of biocatalyst used (Basso & Serban, 2019).

When scaling up processes using immobilized enzymes, the major challenge is the cost
involved. Before using an immobilized enzyme, a thorough economic estimation and
evaluation should be done, considering all the associated costs such as the cost of the native
enzyme, support material for enzyme immobilization, downstream processing, reactors,
disposal of the immobilized enzyme after use and regeneration of carriers (Basso & Serban,
2019). The applications of immobilized enzymes in various industries are briefly discussed
below:

Immobilized enzymes in the food industry:

Unlike pharmaceutical and chemical industries, the food industry produces in large quantities
in continuous operation rather than batch processes. This particular industry being cost-
sensitive, requires for the cost of the immobilized enzymes to also be low. So the widely used
immobilized enzymes for food production considering this cost requirement include the
following:

Table 1: Showing the use of immobilized enzymes in the food industry

Product Application Immobilized enzyme Type of immobilization

High fructose corn syrup Sweetener D-glucose/xylose Adsorption on silica

isomerase

Vitamin C ester Antioxidant Lipase Adsorption on polymer

Hydrolysed lactose Lactose-free dairy Beta-galactosidase Adsorption on cellulose

products fibre

Source: (Basso & Serban, 2019)

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Immobilized enzymes in the chemical industry

The main aim of the chemical industry has been to direct it towards green and sustainable
processes. Like the food industry, production is on a ton scale and the key goal is the
recyclability of the products. The following table gives an overview on the chemical industry
and the use of immobilized enzymes in the production of various chemicals.

Table 2: showing use of immobilized enzymes in the chemical industry

Product Application Immobilized enzyme Type of immobilization

Organosilicone polyamide Polymers Lipase B Adsorption on

copolymer

Outlook (Dimethenamid-P) Herbicide Lipase B Adsorption on

copolymer

Myristyl myristate Surfactant Lipase B Adsorption on

copolymer

Source: (Basso & Serban, 2019)

Immobilized enzymes in the pharmaceutical industry

It has been demonstrated through protein engineering that enzymes can function with unnatural
molecules as substrates in organic solvents, resulting in the production of highly selective
compounds with abundant yields. This particular find out has been very appealing to the
pharmaceutical industry (Basso & Serban, 2019). The extensive research in the use of
immobilized enzymes that cater to the pharmaceutical industry has resulted in the synthesis of
various enzymes that are used for various medicinal purposes. The following table shows the
active pharmaceutical ingredients (API), the immobilized enzymes used and their applications.

Table 2: showing use of immobilized enzymes in the chemical industry

API Application Immobilized enzyme Type of immobilization

Odanacatib Osteoporosis treatment Lipase B Adsorption on octadecyl

polymethacrylate resin

Sofosbuvir Treatment of hepatitis c Lipase B Adsorption on

copolymer

Sitagliptin Antidiabetic Transaminase Adsorption

Source: (Basso & Serban, 2019)

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Immobilized enzymes in the textile industry

In recent years, the use of immobilized enzymes in the textile industries has increased
significantly, mainly due to the stability and prolonged shelf life that it provides to the enzyme.

One prime example would be that of the wool processing industries. The wool industries
perform chlorination in order to make wool products shrink-resistant. The chlorination
procedure degrades the environment. So the use of proteases instead of chlorination does the
same work of shrink resistivity while also augmenting the wool whiteness, ease of handling
and dye ability and most importantly, this process does not harm the environment (Maghraby,
El-Shabasy, Ibrahim, & Azzazy, 2023). Similarly, there are many other immobilized enzymes
that are used in the textile industry that increase thermal stability, dye decolourization, tensile
strength and less fibre felting.

The use of immobilized enzymes has not been limited to the industrial sector only. It has been
extensively used in the treatment of wastewater as well. The untreated wastes from industries
are the major cause of water pollution. The use of enzymes such as proteases, reductases, etc.
to treat wastewater has been in effect for a long time. However, their decreased activity with
time led to the use of immobilized enzymes. From extensive studies on dye removal from the
wastewater, it has been found that “A cross-linked laccase chitosan bound to a metal ligand
Cu(II) ion has been applied to degrade three types of dyes in wastewater where the removal
efficiencies were reported at 69%, 87%, and 43% for cibacron blue, methyl orange, and reactive
black 5, respectively” (Maghraby, El-Shabasy, Ibrahim, & Azzazy, 2023). Other research and
reports have proved that in a short time, immobilized enzymes can extract phenolic compounds
from the wastewater efficiently.

The effect of coronavirus and world market for industrial enzymes

With the advancements in biotechnology, the market for enzymes has broadened more than
ever. Also, there is growing awareness in people to replace synthetic chemicals and
petrochemicals with natural and biodegradable ones (Maghraby, El-Shabasy, Ibrahim, &
Azzazy, 2023). There has been a very high demand from chemical, pharmaceutical, textile and
other various industries for enzymes. It was reported that the global market for enzymes was
about $11.5 billion in 2021 and is expected to rise with a growth rate of 6.5% from 2022
onwards. With the wide applications and the continuous search for new enzyme technologies,
the global market for enzymes can only go uphill (Maghraby, El-Shabasy, Ibrahim, & Azzazy,
2023).

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With COVID-19 declared as a global health emergency, it had severely affected the economic,
social, mental, and environmental aspects. It was reported that the highest enzyme market share
was that from the food and beverage sectors and remarkably, from the house care products and
cleansers. The market for enzymes can be expected to only grow from here onwards as the use
of enzymes in the field of medicine has increased more than ever due to the coronavirus.

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CONCLUSION

The advancements in the field of biotechnology have led to this level of development of
immobilized enzymes. The immobilization of enzymes not only serves as a crucial
breakthrough in the field of science but also in humanity. The lives of humans have been
significantly upgraded due to the advancement of this development. The various methods of
immobilization of enzymes have helped better understand the usability of enzymes and their
application in numerous ways. Their economic, ecological and technical advantages including
operational stability and reusability make them extremely suitable for industrial use
(Maghraby, El-Shabasy, Ibrahim, & Azzazy, 2023). Hence, the development of sustainable and
economic processes, in addition to extensive research in the field of protein engineering shows
a bright future for the use of immobilized enzymes in industrial processes (Basso & Serban,
2019).

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Catalysis.

Bhatia, S. (2018). Immobilization of enzymes. In Introduction to Pharmaceutical Biotechnology.

Costa, S., Azevedo, H., & Reis, R. (2004). Enzyme immobilization in biodegradable polymers for
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Homaei, A. A., Sariri, R., Vianello, F., & Stevanato, R. (2013). Enzyme immobilization: an update. J
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Jha, N. (n.d.). Immobilization of Enzymes and Cells: Methods, Effects and Applications. Retrieved
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