Enzyme Immobilization- Biotechnology- B.Pharm SEM 5

B I O T E C H N O L O G Y
UNIT 1
Introduction to biotechnology and enzyme immobilisation:
• Brief introduction to biotechnology with reference to pharmaceutical
sciences
• Enzyme biotechnology- methods of enzyme immobilisation andapplications,
• Biosensors- working and applications of biosensors in pharmaceutical
industries
PRESENTED BY: Prof. Vedanshu Malviya
Department of Pharmaceutics
Dr. Rajendra Gode Institute of Pharmacy, Amravati

I N T R O D U C T I O N T O B I O T E C H N O L O G Y
•
•
•
•
Biotechnology: It is the branch of science which deals with the principles of bio-science and engineering
for the development of different products prepared by using different biological agents. Biotechnology
states the usage of bio-organisms and techniques to fabricate bio-product in industries. Biotechnology is
technology that utilises biological systems, living organisms or parts of this to develop or create different
products. Brewing and baking bread are examples of processes that fall within the concept of
biotechnology (use of yeast (living organism) to produce the desired product).
Pharmaceutical biotechnology is a relatively new and growing field inwhich the principles
of biotechnology are applied to the development of drugs. A majority of therapeutic drugs in the
current market are bio-formulations, such as antibodies, nucleic acid products and vaccines ( As shown in
figure 1.1)
Figure 1.1One of the application of Pharmaceutical Biotechnology in development of Vaccine
from Plants using bacteria and Fluvirus

I N T R O D U C T I O N T O B I O T E C H N O L O G Y
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6. Pharmaceutical biotechnology:
The principles of biotechnology are utilised for the production of Advanced formulation
or products either for treatment or prevention or diagnosis. Example- development of
monoclonal antibodies
development of vaccines
development of hormones (Humaninsulin, growth hormones)
development of antibiotics enzymes and enzyme immobilisationetc.
-
5. Environmental
biotechnology:
Development of the
specific microbial
strain for the
destruction of
Industrial waste
material. Example- for
sewage treatment
specific anaerobic
bacteria was
developed and for
sewage treatment
rhizobium bacteria
was developed.
-
1. Agricultural biotechnology:
For the development of new high
yielding, disease resistant and cost-
effective plant by r-DNA
(Recombinant deoxyribonucleic
acid) technology or by gene cloning.
Example- development of transgenic
plant (transgenic tomato) and
development of edible vaccines.
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2. Medical biotechnology:
For detection or diagnosis of diseases example in AIDS( Acquired Immune
Deficiency Syndrome) and ELISA (Enzyme-linked ImmunoSorbent Assay)
is a technique used to detect antibodies in blood-related infectious
conditions.
For the detection of Hepatitis B (RIA (Radio ImmunoAssay) uses radio
labelled molecules in a stepwise formation of immunecomplexesmeasure
the presence of antigen.
For correction of the hereditary disorder by gene manipulation or gene
Incorporation
-
4. Engineering
biotechnology:
Development of
biosensors for detection
of pollutant.
-
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3. Textile biotechnology:
It is used for the preparation/
development of specific microbial
strain for development of high
quality fibres.
Example development of transgenic
animal (Silk forming Goat).
• Applications of biotechnology:

• Enzyme are the protein molecules which act as catalyst in biochemical reactions (Bio catalyst).
- The properties are:
1.These are highly efficient catalyst and highly specific.
2.Should be biodegradable and should accelerate the rate of biochemical reactions.
3.It should be sensitive to pHand temperature.
4.During reaction it should be chemically and structurally uncharged.
-
• Nature of enzymes: ( As shownin figure 1.2)the Apoenzyme which is the protein part and inactive +
cofactor which is is a nonprotein part and is activator both combines to give the Holoenzyme which is an
activated whole enzyme.
The nonprotein part is of two types: 1)Coenzyme- heat stable, low molecular weight organic compound
e.g. NAD+/NADHreducing agent. 2)Cofactor- inactive enzyme along with metallic ione.g. Arginase
enzyme in urea cycle, cofactor manganese.
E N Z Y M E B I O T E C H N O L O G Y
Figure 1.2 The complete enzyme is called as Holoenzyme, The Holoenzyme consist of Protein and non-protein group

• Zymogens- These are enzyme precursor , mostly in inactive form which can be activated by 2 ways:
1)By trimming polypeptide chain.
2) By covalent modification
Eg. Trypsinogen Enterokinase. Trypsin, Pepsinogen HCl Pepsin
• Mode of Enzyme action: ( As shownin figure 1.3)the enzyme can activated by workingof two sites
i.e allosteric site and catalytic site. While in figure 1.4shows the action of allosteric molecule which can
either activate or inhibit the enzyme action by increasing or decreasing the affi nity of substrate with
the enzyme.
Figure 1.3Generally enzyme have two sites an active
site (catalytic site) and another is allosteric site(over
this site small molecular structures attaches which
can either stimulate or inhibits theresponse)
Figure 1.4a)allosteric inhibitor , inhibits the substrate
to attach thus inhibits the action.
b)allosteric activator, increases the affinity of
enzyme towards substrate

• THEORIES OF ENZYME ACTION. 1) Lock and Key theory 2) Induced fit model
1)Lock and Key theory (Template model): ( As shownin figure 1.5)
-Emil Fischer proposed this theory in1894.
-According to lock and key hypothesis, the bindingof the substrate into an active site of an enzyme is
equalised into the lock and key mechanism.
-The particular lock can be open using the correct key. Similarly, if the enzyme is the lock, it will be
open only by the correct substrate which is the key.
-Both fit with each other correctly and tightly. Their shapes are complementary with each other.
Hence, this bindingis very specific and cannot be easily broken.
Figure 1.5Lock and key hypothesis: The enzyme is like a lock and substrate is like a key, both are
when fits well to form enzyme substrate complexand results in the formation of product.

Figure 1.6Induced fit model
2) Induced fit model:
- Daniel E Koshland proposed this theory in 1959.The active site of the enzyme is not static
according to this theory.
- The induced fit is a theory that explains the bindingof a substrate into an active site of an
enzyme that does not have a correct conformation with that of the active site.
- According to this theory, confirm
ation of the active site modifies into a correct shape
when the substrate binds. ( As shownin figure 1.6)
- The binding of the substrate induces the modification of the shape of the active site.
Hence, the name ‘Induced fit’ is given to this hypothesis.

• Factors affecting Enzyme action/Kinetics:
9) Enzyme inhibitor
8) Enzyme activator
7) Oxidation state of enzyme 6) Time 4) Enzyme concentration
3) Productconcentration
2) Substrate concentration
5) pH
1)Temperature
FACTORS AFFTECTING
ENZYME KINETICS
Figure 1.7Factors affecting Enzymeaction

1)Temperature
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At low temperature, the enzyme activity is very less. (
As shownin figure 1.8)With increase in temperature ,
the enzyme activity will increase upto a maximum
level (optimumtemperature) then with increase in
temperature , its activity decreases.
At high temperature denaturation of enzyme occurs.
2) Substrate concentration
-
-
The concentration of substrate initially increase the
rate of reaction untomaximumactivity and then it
remains constant. ( As shownin figure 1.9)
Enzyme concentration has to be constant (showing
increasing activity until all the site of enzyme is
been occupied and then remainsconstant).
Figure 1.8effect of temperature onenzyme activity
Figure 1.9effect of substrate concentrationonenzyme activity
3) Productconcentration
- With the increase in product concentration , the rate
of reaction will decrease because the Enzyme-
Product complexis more stable than Enzyme-
Substrate complex. ( As shownin figure 2.0)
Figure 2.0 effect of product concentration onenzyme activity

-
-
- The biochemical reaction influenced by enzyme will increase
initially until the enzyme reach to itsmaximumactivity.
The enzyme activity will increase with increase in enzyme
concentration when substrate concentration is constant.
Rate of reaction will increase with increased enzyme
concentration to attain constant value untilreaches
maximumactivity. ( As shown in figure 2.1) Figure 2.1effect of enzyme concentration onenzyme activity
4) Enzymeconcentration
5) pH
-
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Initially the rate of reaction will increase with
increase in pHunless and until it reaches to its
maximumlevel. (OptimumpH) ( As shown in figure 2.2)
For mostenzymes suitable pHrange is between 5-9
exception include pepsin which requires acidic pHfor
its maximumaction.
Figure 2.2 effect of pHonenzymeactivity
6) Time
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It is totally dependent ontemperature. Time is inversely proportional to temperature.
If the temperature decreases the time will increase for the complete reaction to take place and
vice versa.
Ex. Most of enzymes obtained from humanshave a optimumtemperature of 37℃ and when the
enzyme is isolated for in-vitro studies, it take hours for the process to occur.

7) Oxidation state ofenzyme
- Enzymes with sulphydryl groups are activated by reducing agents and inactivated by oxidising
agents.
- Oxidation causes decreased enzyme activity.
- Example- enzyme like urea, succinic dehydrogenase gets activated by reducing agents like hydrogen
sulphide or cysteine.
8) Enzymeactivator
-
-
Some ions or molecules activates the enzyme activities.
eg. Chlorine ions stimulates the activation of the salivary or pancreatic amylase , Chlorine ions
activates Thrombokinase which converts prothrombin to thrombin, Bile salts activates pancreatic
lipase.
9) Enzyme inhibitor
-
-
These inhibit the activity of enzyme.
Example- maltose decreases the activity of succinic dehydrogenase (required for citric acid
production).

E N Z Y M E I M M O B I L I S A T I O N
• Enzyme immobilisation is a strategy to improve stability of an enzyme, but also a strategy for
easily re-using the enzymes.
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•
- Enzyme immobilisation technology refers to the natural enzyme limited within a certain space or
attached ona solid structure.
Immobilisation is a common,effective, and convenient means for enzymatic modification to improve its
catalytic activity and stability.
Enzyme immobilisation is the process by which the enzyme catalyst is trapped at the bio-anode or
bio-cathode surface.
Immobilised enzymes are the enzymes that are fixed to inert and insoluble carrier. ( As shownin
figure 2.3)
Figure 2.3 Enzyme immobilisation : the enzyme is immobilised at the insert and insolublecarrier.

• Properties of carrier molecules:
-
-
-
-
-
Inert
I nsoluble
Stable at all pH
Carrier should be stable at all ionicstrength
Should be stable in a particular solvent at a particular condition(neither should be unstable nor
insoluble).
• Types of carriers:
Organic natural carriers
- Favourable compatibility with
proteins.
- Example: chitosan, starch,
agar
Inorganic carriers
- Highpressure stability and
may undergo abrasion.
- Example: mineral material-
clay, celite, centonite. Porours
glass, silica.
Organic synthetic carriers
- Highchemical and
mechanical stability.
- Example: polystyrene,
polyvinylacetate, acrylic
polymers.
• Advantages/significance of enzyme immobilisation:
-
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Prevents deactivation/degradation of enzymes.
The enzymes can be recovered at the end of the reaction and can be reused.
Easily separated from the products.
Increases the stability of the enzyme.
Better control on reaction
Potential in preparation of medicine and other products in food and detergent industry.
E N Z Y M E I M M O B I L I S A T I O N

METHODS O F E N Z Y M E I M M O B I L I S A T I O N
Classification On the basis of nature Classification On the basis of surface/support
2.Crosslinking
3.Chelation and
complexation
1.Adsorption
2.Covalent bonding
3.Chelation
On surface
1.Covalent bonding immobilisation
Within support
immobilisation
1.Entrapment
2.Micro-encapsulation
3.Crosslinking
Physical methods
Chemical methods
1.Adsorption
2.Entrapment
3.Micro-encapsulation
Figure 2.4 Enzyme immobilisationmethods
2)
3)
1)
5)
4)

1.Adsorption
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Enzymes are immobilised by adsorbing onto the surface of carrier material. ( As shown in figure 2.4) This
technique is reversible, enzymes can easily be desorbed from carrier molecule due to the change in
substrate and ionic strength.
In this technique, enzymes are goingto attach with the carrier molecule by hydrogen bondingand Van der
Waal force of attraction.
Adsorption immobilisation can be done by 4 different methods:
1) Static process-the enzyme solution is kept in contact with the carrier material without agitation. It is
mostaffection method but timeconsuming.
2) Dynamic process-enzyme solution and carrier material is mixed with constant agitation by using mechanical
stirrer. It is laboratory based enzyme immobilisation technique.
3) Reactor loading-the carrier material is loaded in the bioreactor(used for fermentation process) , than
added enzyme solution and mixed with constant agitation.this is commercially used technique for
producton of immobilised enzymes)
4) Electro deposition- electrodes are introduced in the enzyme bath.The carrier molecule is paced near the
area of the electrodes. When electric field is applied the enzymes migrate towards the carrier molecule
and get absorb onitssurface.
Examples of enzyme immobilisation by adsorption are:
S.no. Enzymes Carrier
1 α-amylase Calcium phosphate
2 Invertase, catalase Charcoal
3 Glucose oxidase Cellophane
Table 1
Examples of enzyme immobilisation by adsorption

2) Entrapment
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-
-
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- By this technique the enzymes are immobilised by entrapping within the pores of carrier matrix. ( As
shownin figure2.4)
The matrix material like polyacrylamide gel, cellulose derivatives, silica and calcium alginate.
Immobilisation by this method can be doneby two ways:
1) Inclusion in gel eg. polyacrylamide gel
2) Inclusion in fibre eg. cellulose derivatives
Enzymes are entrapped within the interstitial spaces which are cross linked water insoluble polymers.
Example- calcium alginate is used for enzymes Immobilisation in plant and microbial cells.
1 α-amylase and Invertase Polyacrylamide gel
1 Lactase Polyacrylamide gel
Table 2 Example of enzymeimmobilisation by entrapment
3) Micro-encapsulation
- By this technique the enzymes are immobilised by encapsulating within the semi permeable
membrane of the carrier. ( As shownin figure 2.4)
- Carrier used for micro-encapsulation includes cellulose derivatives, polystyrene, nylon etc.
- The example is as follows:
Table 3 Example of enzyme immobilisation by micro-encapsulation

4) Covalent bonding
By this technique the enzymes are immobilised by forming the covalent bondswith the carriermatrix.
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The functional group of matrix like carboxylic acid, alcoholic group,suphydryl group,aminogroup,
tyrosyl group,etc attaches with enzyme for immobilisation.
The functional group of carrier, participate in Covent couplingbut would not affect the activity of
enzyme. ( As shownin figure2.4)
- There are three methods by which the immobilisation is done:
1) formation of Diazotization bond-bondis formed between aminogroup of carrier & Histidyl/Tyrosyl
group of the enzyme.
2)Formation of peptide bond-bondis formed between amino/carboxylic group of the carrier by that of
enzyme.
3)By multifunctional/ Di-functional agents- bondis formed between aminogroup by that of enzyme.
Examples are as follows:
1 Lactase Polyacrylamide gel
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By this technique the enzymes are immobilised by cross linking with multi-functional agents which
lead to the formation of 3Dnetwork of enzymes. ( As shownin figure 2.4)
Example of multifunctional agents are Glutaraldehyde, Diazoniumsalts are used in industrial
techniques for enzyme immobilisation.
Increase in concentration of these agents can cause enzyme denaturation.
1 Catalase Glutaraldehyde
Table 4 Example of enzyme immobilisation by covalent bonding
5) Cross-linking(Polymerisation)
Table 5 Example of enzyme immobilisation by crosslinking

METHODS O F EN Z Y M E IMMOBI L I S AT I O N AN D A P P L I C AT I O N S
6) Complexation andchelation
- By this technique the enzymes are immobilised by formation of complexwith transition metal like Titanium,
Zirconium(commercially used), oxide/floride of Titanium, cobalt and manganese are used for immobilisation.
- Example is asfollows:
1 Invertase Zirconium
Applications:
•
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Immobilised enzymes are used in Biotransformation.
Used in development of Biosensors.
Used in production of secondary metabolites.
Used in fermentationtechniques.
Used in diagnoses of disease by Serological technique. eg. ELISA.
Used in preparation of washing powder- immobilised bacterial proteases used in washing powder to
remove heavy stains.
Used in processed food industry.
Used in baking industry (immobilised yeast)- used in baking and brewing industry, as they contain
Maltase-which break maltose into Glucose, Invertase- which break sucrose. These enzymes act upon
simple sugars and produce alcohol and carbon-dioxide.
Used for immobilised pectinase helps in preparation of wine and fruit juice.
Used in immobilisation of chymosin and pepsin used in cheese production.
Table 6 Example of enzymeimmobilisation by complexation

BIOSENSORS - W O R K I N G AND A P P L I C A T I O N S
• Biosensors: A biosensor is an analytical device, used for the detection of an analyte, that combines a
biological component with a physicochemical detector.
- It is an analytical device which converts a biological response into an electrical signal. ( As shownin
figure 2.5)
- It detects, records, and transmits information regarding a physiological change or process.
- It determines the presence and concentration of a specific substance in any test solution.
- It is a device having immobilised biocatalyst which oninteraction with appropriate analyte converts
the presence of desired analyte into physical/chemical/electrical signals that can be measured.
Figure 2.5 The general working of Biosensors

• The components of biosensors are as follows: ( As shownin figure 2.6)
Figure 2.6 The structure of Biosensors
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Analyte (sample): it might be protein, sugar, cholesterols, microbes, toxins etc.
Bioreceptor/Biocatalyst: it must always be immobilised.
Transducer: it is a device that converts one form of signal to another.
Signal: generate and analyse the developed signal.
Detector/Signal processing: the signals are detected and displayed.

• The working of biosensors are as follows: ( As shownin figure 2.7)
-
-
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- Analyte diffuses from the sample preparation and interact with the immobilised bio-elements present
onthe surface of biosensors.
After the interaction, there will be change in physiochemical property which can be read by
transducer.
It leads to change in either optical/electrical, Physical property of the transducer surface & develop
the signals
These signals are measured/analysed and finally detected or displayed by display unit/detector.
Analyte Bioreceptor Transducer
Electro active signal:
Electrode
Antibody
Enzyme
Cell
Microbe
pHchange:
pH electrode
Heat: Transducer
Photon counter:
Light
Figure 2.7 the working of Biosensors
Signal
Detected and
Displayed

ELECTROCHEMICAL
BIOSENSORS
Bioelement- Immobilised enzyme
Transducer- Electricfield
Enzymes- oxygen consumingenzyme
immobilised ona platinum electrode
where the decrease in concentration
of oxygen produce electric current
which co-relates to analyte
concentration. oxygen
concentration is inversely
promotional to analyte conc
Application: Detection of
Glucose, Hyberdised DNA,
DNAbinding drugs.
CALORI METRI C
BIOSENSORS
This particular biosensor is used to
measure the heat generated or
absorbed during Enzyme-substrate
interaction.
Change in temperature of the
sample is preparation either by
thermistor or transistor.
Application: Detection of
cholesterols level in
blood,, detection of amino
acid and sugar in
products.
OPTICAL
BIOSENSORS
and antibodies.
Transducer- optical fibres
Optical fibres are used for
detection of analytes onthe basis
of either absorption,
fluorescence, or light
scattering.
Application: to find
out the concentration
of oxygen,carbon
dioxide and pHof the
blood.
PIEZO-
ELECTRIC BIOSENSORS
Bioelement- Immobilised antibodies.
Transducer-Gold
Gold is used to detect the specific
angle at which electron waves are
emitted when analyte is exposed
to area of light which vibrate
under influence of electric
field.
Application: for the
detection of antigen
present in the sample.
Figure 2.8 Types of Biosensors

• Applications of biosensors:
-
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Analysis of processed food.
Study of new drug development.
Detection of DNA in forensic laboratories.
Diagnosis of a disease.
Detection of an antigen in patient’s blood sample.
• Examples of biosensors:
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Gluco-meter- detection of blood sugar level ( As shownin figure 2.9)
Pregnancy detection kit- used to detect HCG(HumanChorionic Gonadotrophin) protein. ( As shownin
figure 3.0)
Infectious disease biosensor- detect the pathogen present in the blood sample.
Old time coal mines biosensors- used to detect the presence of toxic gas like methane and carbon
mono-oxidein coal mines.
Figure 2.9 Biosensors- Pregnancy detection kit
Figure 3.0 Biosensors- Gluco-meter

R E F E R E N C E S
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https://www.nature.com/subjects/biotechnology
http://dbtindia.gov.in/about-us/introduction
https://www.biotechnologycongress.com/europe/events-list/biotechnology-and-its-applications
https://www.intechopen.com/books/biosensors-for-health-environment-and-biosecurity/biosensors-
for-health-applications
https://www.elprocus.com/what-is-a-biosensor-types-of-biosensors-and-applications/
https://www.sciencedirect.com/topics/engineering/enzyme-immobilization

Enzyme Immobilization- Biotechnology- B.Pharm SEM 5

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