UNIT III

ENZYME IMMOBILIZATION
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
(or cells) refers to the technique of confining/anchoring the enzymes (or cells) 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. Some workers regard immobilization
as a goose with a golden egg in enzyme technology. Immobilized enzymes retain their
structural conformation necessary for ca

talysis.
Adsorption, Covalent bonding, Entrapment, Encapsulation and crosslinking
BIOSENSOR
A biosensor is an analytical device containing an immobilized biological material (enzyme,
antibody, nucleic acid, hormone, organelle or whole cell) which can specifically interact with
an analyte and produce physical, chemical or electrical signals that can be measured. An
analyte is a compound (e.g. glucose, urea, drug, pesticide) whose concentration has to be
measured.
Biosensors basically involve the quantitative analysis of various substances by converting their
biological actions into measurable signals. A great majority of biosensors have immobilized
enzymes. The performance of the biosensors is mostly dependent on the specificity and
sensitivity of the biological reaction, besides the stability of the enzyme.

General Features of Biosensors:

A biosensor has two distinct components (Fig. 21.13).

1. Biological component—enzyme, cell etc.

2. Physical component—transducer, amplifier etc.

The biological component recognises and interacts with the analyte to produce a physical
change (a signal) that can be detected, by the transducer. In practice, the biological material is
appropriately immobilized on to the transducer and the so prepared biosensors can be
repeatedly used several times (may be around 10,000 times) for a long period (many months).

Principle of a Biosensor:
The desired biological material (usually a specific enzyme) is immobilized by conventional
methods (physical or membrane entrapment, non- covalent or covalent binding). This
immobilized biological material is in intimate contact with the transducer. The analyte binds
to the biological material to form a bound analyte which in turn produces the electronic
response that can be measured.

In some instances, the analyte is converted to a product which may be associated with the
release of heat, gas (oxygen), electrons or hydrogen ions. The transducer can convert the
product linked changes into electrical signals which can be amplified and measured.

Types of Biosensors:
There are several types of biosensors based on the sensor devices and the type of biological
materials used. A selected few of them are discussed below.

Electrochemical Biosensors:
Electrochemical biosensors are simple devices based on the measurements of electric current,
ionic or conductance changes carried out by bio electrodes.

Amperometric Biosensors:
These biosensors are based on the movement of electrons (i.e. determination of electric current)
as a result of enzyme-catalysed redox reactions. Normally, a constant voltage passes between
the electrodes which can be determined. In an enzymatic reaction that occurs, the substrate or
product can transfer an electron with the electrode surface to be oxidised or reduced (Fig.
21.14).
This results in an altered current flow that can be measured. The magnitude of the current is
proportional to the substrate concentration. Clark oxygen electrode which determines reduction
of O2, is the simplest form of amperometric biosensor. Determination of glucose by glucose
oxidase is a good example.
In the first generation amperometric biosensors (described above), there is a direct transfer of
the electrons released to the electrode which may pose some practical difficulties. A second
generation amperometric biosensors have been developed wherein a mediator (e.g. ferrocenes)
takes up the electrons and then transfers them to electrode. These biosensors however, are yet
to become popular.

Blood-glucose biosensor:
It is a good example of amperometric biosensors, widely used throughout the world by diabetic
patients. Blood- glucose biosensor looks like a watch pen and has a single use disposable
electrode (consisting of a Ag/AgCI reference electrode and a carbon working electrode) with
glucose oxidase and a derivative of ferrocene (as a mediator). The electrodes are covered with
hydrophilic mesh guaze for even spreading of a blood drop. The disposable test strips, sealed
in aluminium foil have a shelf-life of around six months.

An amperometric biosensor for assessing the freshness of fish has been developed. The
accumulation of ionosine and hypoxanthine in relation to the other nucleotides indicates
freshness of fish-how long dead and stored. A biosensor utilizing immobilized nucleoside
phosphorylase and xanthine oxidase over an electrode has been developed for this purpose.

Potentiometric Biosensors:
In these biosensors, changes in ionic concentrations are determined by use of ion- selective
electrodes (Fig. 21.15). pH electrode is the most commonly used ion-selective electrode, since
many enzymatic reactions involve the release or absorption of hydrogen ions. The other
important electrodes are ammonia-selective and CO2 selective electrodes.

The potential difference obtained between the potentiometric electrode and the reference
electrode can be measured. It is proportional to the concentration of the substrate. The major
limitation of potentiometric biosensors is the sensitivity of enzymes to ionic concentrations
such as H+ and NH+4.
Ion-selective field effect transistors (ISFET) are the low cost devices that can be used for
miniaturization of potentiometric biosensors. A good example is an ISFET biosensor used to
monitor intra-myocardial pH during open-heart surgery.

Conduct Metric Biosensors:

There are several reactions in the biological systems that bring about changes in the ionic
species. These ionic species alter the electrical conductivity which can be measured. A good
example of conduct metric biosensor is the urea biosensor utilizing immobilized urease. Urease
catalyses the following reaction.

The above reaction is associated with drastic alteration in ionic concentration which can be
used for monitoring urea concentration. In fact, urea biosensors are very successfully used
during dialysis and renal surgery.

Thermometric Biosensors:
Several biological reactions are associated with the production of heat and this forms the basis
of thermometric biosensors. They are more commonly referred to as thermal biosensors or
calorimetric biosensors. A diagrammatic representation of a thermal biosensor is depicted in
Figure. It consists of a heat insulated box fitted with heat exchanger (aluminium cylinder).
The reaction takes place in a small enzyme packed bed reactor. As the substrate enters the bed,
it gets converted to a product and heat is generated. The difference in the temperature between
the substrate and product is measured by thermistors. Even a small change in the temperature
can be detected by thermal biosensors.

Thermometric biosensors are in use for the estimation of serum cholesterol. When cholesterol
gets oxidized by the enzyme cholesterol oxidase, heat is generated which can be measured.
Likewise, estimations of glucose (enzyme-glucose oxidase), urea (enzyme-urease), uric acid
(enzyme-uricase) and penicillin G (enzyme-P lactamase) can be done by these biosensors. In
general, their utility is however, limited. Thermometric biosensors can be used as a part of
enzyme-linked immunoassay (ELISA) and the new technique is referred to as thermometric
ELISA (TELISA).

Optical Biosensors:
Optical biosensors are the devices that utilize the principle of optical measurements
(absorbance, fluorescence, chemiluminescence etc.). They employ the use of fibre optics and
optoelectronic transducers. The word optrode, representing a condensation of the words optical
and electrode is commonly used. Optical biosensors primarily involve enzymes and antibodies
as the transducing elements.

Optical biosensors allow a safe non-electrical remote sensing of materials. Another advantage
is that these biosensors usually do not require reference sensors, as the comparative signal can
be generated using the same source of light as the sampling sensor. Some of the important
optical biosensors are briefly described hereunder.
Fibre optic lactate biosensor:

Figure represents the fibre optic lactate biosensor. Its working is based on the measurement of
changes in molecular O2 concentration by determining the quenching effect of O2 on a
fluorescent dye. The following reaction is catalysed by the enzyme lactate mono-oxygenase.

The amount of fluorescence generated by the dyed film is dependent on the O2. This is because
O2 has a quenching (reducing) effect on the fluorescence. As the concentration of lactate in the
reaction mixture increases, O2 is utilized, and consequently there is a proportionate decrease in
the quenching effect. The result is that there is an increase in the fluorescent output which can
be measured.
Optical Biosensors for Blood Glucose:
Estimation of blood glucose is very important for monitoring of diabetes. A simple technique
involving paper strips impregnated with reagents is used for this purpose. The strips contain
glucose oxidase, horse radish peroxidase and a chromogen (e.g. toluidine). The following
reactions occur.

The intensity of the colour of the dye can be measured by using a portable reflectance meter.
Glucose strip production is a very big industry worldwide.
Colorimetric test strips of cellulose coated with appropriate enzymes and reagents are in use
for the estimation of several blood and urine parameters.

Luminescent biosensors to detect urinary infections:

The microorganisms in the urine, causing urinary tract infections, can be detected by employing
luminescent biosensors. For this purpose, the immobilized (or even free) enzyme namely
luciferase is used. The microorganisms, on lysis release ATP which can be detected by the
following reaction. The quantity of light output can be measured by electronic devices.

Other Optical Biosensors:

Optical fibre sensing devices are in use for measuring pH, pCO2 and pO2 in critical care, and
surgical monitoring.
Piezoelectric Biosensors:
Piezoelectric biosensors are based on the principle of acoustics (sound vibrations), hence they
are also called as acoustic biosensors. Piezoelectric crystals form the basis of these biosensors.
The crystals with positive and negative charges vibrate with characteristic frequencies.
Adsorption of certain molecules on the crystal surface alters the resonance frequencies which
can be measured by electronic devices. Enzymes with gaseous substrates or inhibitors can also
be attached to these crystals.

A piezoelectric biosensor for organophosphorus insecticide has been developed incorporating

acetylcholine esterase. Likewise, a biosensor for formaldehyde has been developed by
incorporating formaldehyde dehydrogenase. A biosensor for cocaine in gas phase has been
created by attaching cocaine antibodies to the surface of piezoelectric crystal.

Limitations of Piezoelectric Biosensors:

It is very difficult to use these biosensors to determine substances in solution. This is because
the crystals may cease to oscillate completely in viscous liquids.

Whole Cell Biosensors:

Whole cell biosensors are particularly useful for multi-step or cofactor requiring reactions.
These biosensors may employ live or dead microbial cells. A selected list of some organisms
along with the analytes and the types of biosensors used is given in Table 21.8
Advantages of microbial cell biosensors:
The microbial cells are cheaper with longer half-lives. Further, they are less sensitive to
variations in pH and temperature compared to isolated enzymes.

Limitations of microbial cell biosensors:

The whole cells, in general, require longer periods for catalysis. In addition, the specificity and
sensitivity of whole cell biosensors may be lower compared to that of enzymes.

Immuno-Biosensors:
Immuno-biosensors or immunochemical biosensors work on the principle of immunological
specificity, coupled with measurement (mostly) based on amperometric or potentiometric bio-
sensors. There are several possible configurations for immuno-biosensors and some of them
are depicted in Fig. 21.18, and briefly described hereunder.
1. An immobilized antibody to which antigen can directly bind (Fig. 21.18A).

2. An immobilized antigen that binds to antibody which in turn can bind to a free second antigen
(Fig. 21.18B).

3. An antibody bound to immobilized antigen which can be partially released by competing

with free antigen (Fig. 21.18C).

4. An immobilized antibody binding free antigen and enzyme labeled antigen in competition
(Fig. 21.18D).

For the biosensors 1-3, piezoelectric devices can be used. The immuno-biosensors using
enzymes (4 above, Fig. 21.18D) are the most commonly used. These biosensors employ
thermometric or amperometric devices. The activity of the enzymes bound to immuno-
biosensors is dependent on the relative concentrations of the labeled and unlabeled antigens.
The concentration of the unlabeled antigen can be determined by assaying the enzyme activity.

APPLICATION OF BIOSENSORS
The advantages of biosensors include low cost, small size, quick and easy use, as well
as a sensitivity and selectivity greater than the current instruments. Biosensors have many uses
in clinical analysis, general health care monitoring. The most popular example is glucose
oxidase-based sensor used by individuals suffering from diabetes to monitor glucose levels in
blood. Biosensors have found potential applications in the industrial processing and
monitoring, environmental pollution control, also in agricultural and food industries. The
introduction of suitable biosensors would have considerable impact in the following areas:
A. Clinical and Diagnostic Applications: Among wide range of applications of biosensors,
the most important application is in the field of medical diagnostics. The electrochemical
variety is used now in clinical biochemistry laboratories for measuring glucose and lactic acid.
One of the key features of this is the ability for direct measurement on undiluted blood samples.
Consumer self-testing, especially self-monitoring of blood components is another important
area of clinical medicine and healthcare to be impacted by commercial biosensors. Nowadays
reusable sensors also permit calibration and quality control unlike the present disposable sticks
where only one measurement can be carried out. Such testing will improve the efficiency of
patient care, replacing the often slow and labour intensive present tests. It will bring clinical
medicine closer to bedside, facilitating rapid clinical decision-making.
B. Environmental Monitoring: Environmental water monitoring is an area in which whole
cell biosensors may have substantial advantages for combating the increasing number of
pollutants finding their way into the groundwater systems and hence into drinking water.
Important targets for pollution biosensors now include anionic pollutants such as nitrates and
phosphates. The area of biosensor development is of great importance to military and defense
applications such as detection of chemical and biological species used in weapons.
Biosensors are used for environmental qualitative monitoring of both inorganic and organic
priority pollutants through physical, chemical, and biological assessments. Pollutants are
classified into various groups depending on the chemical structure, the mode of action and their
effects. A wide variety of compounds of environmental concern are considered.
HEAVY METALS Heavy metals are the most dangerous environmental contaminants, which
present a threat to human health, even in trace quantity (Silva, et al. 2011) because they are
nonbiodegradable. The metal contaminants largely observed in the environment are: Lead,
Chromium, Zinc, Mercury, Cadmium and Copper. (Brian, R.et al. 2000) Heavy metals are
released in the ecological system in form of waste water, commercial fertilizers and pesticides.
These are known for their bioaccumulation and toxicity in the food chain. Existing techniques
for analysis of heavy metals such as spectroscopic, volumetric and chromatographic methods
are precise but have the limitations such as high cost and lack of qualified technicians. Bacteria
biosensors are currently used for the determination of heavy metals in different environmental
samples. They make use of enzyme and DNA as bio-receptors, optical and electrochemical
transduction systems
BIOCHEMICAL OXYGEN DEMAND (BOD) Biochemical oxygen demand (BOD or BOD5)
is an important parameter mostly used in the estimation of the amount of biodegradable organic
pollutant in water. This process is time consuming and considerably not suitable for online
process monitoring. Based on this fact, BOD biosensor methods are used to achieve rapid
determination of waste-water samples. An optical biosensor for parallel multi-sample
determination of BOD in effluent samples is developed. The biosensor monitors the BOD
concentration of effluent sample by oxygen sensing film immobilized at the end of glass sample
vials. The rate of oxygen consumption is determined. Recently, BOD biosensor has been
developed using yeast with oxygen probe which can detect organic contaminants within 15
minutes.
NITROGEN COMPOUNDS Nitrogen compounds (Nitrites) are commonly used as food
preservatives (increase shelflife) and soil fertilizers (increase soil fertility). These chemical
compounds in continuous consumption can cause serious effects on human health. They
contaminate ground and surface water destroying the aquatic environment. Their harmful effect
is due to irreversible reaction with haemoglobin leading to severe health issues. Various
biosensor devices are used for the determination of Nitrogen compounds in water samples.
POLYCHLORINATED BIPHENYLS (PCBs) Polychlorinated biphenyls (PCBs) are
extremely toxic organic compounds. They exist everywhere even when their production has
been banned in several countries across the globe. Large portion of PCB accumulates in the
food chain due to their highly lypophilic nature. More than 209 polychlorinated biphenyl
congeners persist worldwide in the environment and food-chain. Gas chromatography and
mass spectroscopy (GC-MS) were used for determination of PCBs. Overtime, immunosensor
, a class of biosensor resulted to be a better approach. It has an advantage of direct extraction
without any additional purification steps. Immunosensor has a successful application for
constructing low cost sensors for environmental monitoring by the use of sol-gel silica
entrapment of viable Pseudomonas species.
PHENOLIC COMPOUNDS Phenolic compounds are organic pollutants with high toxicity
distributed commonly in the environment as industrial effluent. Phenolics are used in the
production of drugs, antioxidants, polymers, pesticides, detergents, dyes, etc. Substituted
phenols have toxic effects because they can easily penetrate the skin and cell membrane which
affects the rate of biocatalyst reactions and the processes of respiration and photosynthesis.
Electrochemical DNA sensors have been identified for environmental screening of toxic
aromatic compounds and for molecular interaction existing among pollutants and DNA.
Amperometric biosensor with tyrosinase have been developed for the determination of the
phenol index in environmental samples.
C. Industrial Applications: Along with conventional industrial fermentation producing
materials, many new products are being produced by large-scale bacterial and eukaryotes cell
culture. The monitoring of these delicate and expensive processes is essential for minimizing
the costs of production; specific biosensors can be designed to measure the generation of a
fermentation product.
D. Agricultural Industry: Enzyme biosensors based on the inhibition of cholinesterases have
been used to detect traces of organophosphates and carbamates from pesticides. Selective and
sensitive microbial sensors for measurement of ammonia and methane have been studied.
However, the only commercially available biosensors for wastewater quality control are
biological oxygen demand (BOD) analyzers based on micro-organisms like the bacteria
Rhodococcus erythropolis immobilized in collagen or polyacrylamide.

E. Food Industry: Biosensors for the measurement of carbohydrates, alcohols, and acids are
commercially available. These instruments are mostly used in quality assurance laboratories or
at best, on-line coupled to the processing line through a flow injection analysis system. Their
implementation in-line is limited by the need of sterility, frequent calibration, analyte dilution,
etc. Potential applications of enzyme based biosensors to food quality control include
measurement of amino acids, amines, amides, heterocyclic compounds, carbohydrates,
carboxylic acids, gases, cofactors, inorganic ions, alcohols, and phenols. Biosensors can be
used in industries such as wine beer, yogurt, and soft drinks producers. Immunosensors have
important potential in ensuring food safety by detecting pathogenic organisms in fresh meat,
poultry, or fish.
UNIT IV
PRODUCTION OF ENZYME
Enzymes are the biocatalysts synthesized by living cells. They are complex protein molecules
that bring about chemical reactions concerned with life. It is fortunate that enzymes continue
to function (bring out catalysis) when they are separated from the cells i.e. in vitro. Basically,
enzymes are nontoxic and biodegradable. They can be produced in large amounts by
microorganisms for industrial applications.

Enzyme technology broadly involves production, isolation, purification and use of enzymes (in
soluble or immobilized form) for the ultimate benefit of humankind. In addition, recombinant
DNA technology and protein engineering involved in the production of more efficient and
useful enzymes are also a part of enzyme technology.

The commercial production and use of enzymes is a major part of biotechnology industry. The
specialties like microbiology; chemistry and process engineering, besides biochemistry have
largely contributed for the growth of enzyme technology.

Applications of Enzymes:
Enzymes have wide range of applications. These include their use in food production, food
processing and preservation, washing powders, textile manufacture, leather industry, paper
industry, medical applications, and improvement of environment and in scientific research.
As per recent estimates, a great majority of industrially produced enzymes are useful in
processes related to foods (45%), detergents (35%), textiles (10%) and leather (3%). For details
on the applications of individual enzymes, Tables 21.1-21.3 must be referred.

Commercial Production of Enzymes:

Microbial enzymes have been utilized for many centuries without knowing them fully. The
first enzyme produced industrially was taka-diastase (a fungal amylase) in 1896, in United
States. It was used as a pharmaceutical agent to cure digestive disorders.

In Europe, there existed a centuries old practice of softening the hides by using feces of dogs
and pigeons before tanning. A German scientist (Otto Rohm) demonstrated in 1905 that
extracts from animal organs (pancreases from pig and cow) could be used as the source of
enzymes-proteases, for leather softening.

The utilization of enzymes (chiefly proteases) for laundry purposes started in 1915. However,
it was not continued due to allergic reactions of impurities in enzymes. Now special techniques
are available for manufacture, and use of enzymes in washing powders (without allergic
reactions). Commercial enzymes can be produced from a wide range of biological sources. At
present, a great majority (80%) of them are from microbial sources.

The different organisms and their relative contribution for the production of commercial
enzymes are given below:
Fungi – 60%

Bacteria – 24%

Yeast – 4%

Streptomyces – 2%

Higher animals – 6%

Higher plants – 4%

A real breakthrough for large scale industrial production of enzymes from microorganisms
occurred after 1950s.
Enzymes from animal and plant sources:
In the early days, animal and plant sources largely contributed to enzymes. Even now,

A selected list of plant (Table 21.1) and animal (Table 21.2) enzymes with their sources
and applications are given:

Animal organs and tissues are very good sources for enzymes such as lipases, esterases and
proteases. The enzyme lysozyme is mostly obtained from hen eggs. Some plants are excellent
sources for certain enzymes-papain (papaya), bromelain (pineapple).

Limitations:
There are several drawbacks associated with the manufacture of enzymes from animal and
plant sources. The quantities are limited and there is a wide variation in their distribution. The
most important limitations are the difficulties in isolating, purifying the enzymes, and the cost
factor. As regards extraction of industrial enzymes from bovine sources, there is a heavy risk
of contamination with bovine spongiform encephalopathy (BSE is prion disease caused by
ingestion of abnormal proteins). For these reasons, microbial production of enzymes is
preferred.

Enzymes from mammalian cell cultures:

There exists a possibility of producing commercial enzymes directly by mammalian cell
cultures. But the main constraint will the cost factor which will be extremely high. However,
certain therapeutic enzymes such as tissue plasminogen activator are produced by cell cultures.

Enzymes from microbial sources:

Microorganisms are the most significant and convenient sources of commercial enzymes. They
can be made to produce abundant quantities of enzymes under suitable growth conditions.
Microorganisms can be cultivated by using inexpensive media and production can take place
in a short period.

In addition, it is easy to manipulate microorganisms in genetic engineering techniques to

increase the production of desired enzymes. Recovery, isolation and purification processes are
easy with microbial enzymes than that with animal or plant sources.

In fact, most enzymes of industrial applications have been successfully produced by micro-
organisms. Various fungi, bacteria and yeasts are employed for this purpose. A selected list of
enzymes, microbial sources and the applications are given in Table 21.3.
Aspergillus niger— A unique organism for production of bulk enzymes:
Among the microorganisms, A. niger (a fungus) occupies a special position for the manufacture
of a large number of enzymes in good quantities. There are well over 40 commercial enzymes
that are conveniently produced by A. niger. These include a-amylase, cellulase, protease,
lipase, pectinase, phytase, catalase and insulinase.
The Technology of Enzyme Production—General Considerations:
In general, the techniques employed for microbial production of enzymes are comparable to
the methods used for manufacture of other industrial products .The salient features are briefly
described.

1. Selection of organisms

2. Formulation of medium

3. Production process

4. Recovery and purification of enzymes.

An outline of the flow chart for enzyme production by microorganisms is depicted in Fig. 21.1.

Selection of organism:
The most important criteria for selecting the microorganism are that the organism should
produce the maximum quantities of desired enzyme in a short time while the amounts of other
metabolite produced are minimal. Once the organism is selected, strain improvement for
optimising the enzyme production can be done by appropriate methods (mutagens, UV rays).
From the organism chosen, inoculum can be prepared in a liquid medium.

Formulation of medium:
The culture medium chosen should contain all the nutrients to support adequate growth of
microorganisms that will ultimately result in good quantities of enzyme production. The
ingredients of the medium should be readily available at low cost and are nutritionally safe.
Some of the commonly used substrates for the medium are starch hydrolysate, molasses, corn
steep liquor, yeast extract, whey, and soy bean meal. Some cereals (wheat) and pulses (peanut)
have also been used. The pH of the medium should be kept optimal for good microbial growth
and enzyme production.

Production process:
Industrial production of enzymes is mostly carried out by submerged liquid conditions and to
a lesser extent by solid-substrate fermentation. In submerged culture technique, the yields are
more and the chances of infection are less. Hence, this is a preferred method. However, solid
substrate fermentation is historically important and still in use for the production of fungal
enzymes e.g. amylases, cellulases, proteases and pectinases.

The medium can be sterilized by employing batch or continuous sterilization techniques. The
fermentation is started by inoculating the medium. The growth conditions (pH, temperature,
O2 supply, nutrient addition) are maintained at optimal levels. The froth formation can be
minimised by adding antifoam agents.
The production of enzymes is mostly carried out by batch fermentation and to a lesser extent
by continuous process. The bioreactor system must be maintained sterile throughout the
fermentation process. The duration of fermentation is variable around 2-7 days, in most
production processes. Besides the desired enzyme(s), several other metabolites are also
produced. The enzyme(s) have to be recovered and purified.

Enzyme purification
Selection of organism
It is always preferable to select a source enriched in that particular enzyme.
To check from the literature whether the enzyme occurs universally (in animals, plants as
well as microbes) or confined to a particular Kingdom.

Working with microbial and animal enzymes is easier compared to plant enzymes since
plants are generally rich in phenolics, which on exposure with air get converted into quinones
and quinones bind with enzyme protein and makes it in active.
 On the other hand, it is easier to get a plant tissue provided plants are grown in plenty in the
surrounding compared to get animal tissue or a pure microbe

For animal tissue, either one will have to sacrifice the animal in the laboratory or will have
to bring the tissue from a slaughterhouse. In case of microbes, one will have to grow microbe
in pure form on a suitable growth medium under aseptic conditions after getting inoculum of
the microbe.
Since almost all the enzymes (with few exceptions) are heat labile and not much stable
at room temperature, the entire process of enzyme isolation, purification is carried out at 0-4°C
using a cold room. The component of the homogenization technique like pestle and mortar,
bowl of the Waring blender should also be in chilled condition. While homogenizing in a pestle
and mortar, it should be surrounded by the ice flakes. In case of Waring blender bowl, many
people also wrap a cloth wet with chilled water. Distilled water is used as isolating
(homogenizing) medium, but generally a buffer of a suitable ionic concentration and pH is
preferred in order to maintain enzyme activity Every enzyme is stable in a particular pH range
only.
Techniques used for enzyme isolation
Once a promising source material has been identified the next step is to extract the protein from
this source. The objective in extracting proteins is to get them from the site where they occur
in the tissue, into solution where they can be more easily manipulated and separated out. Most
tissue proteins occur within cells, and possibly within organelles in the cells, and in these cases
it is necessary to break open the cells and organelles, to release their protein contents. The
methods chosen to disrupt the cells and organelles should be such that the proteins themselves
are minimally damaged.
Osmotic shock
It is a sudden change in the solute concentration around a cell, causing a rapid change in the
movement of water across its cell membrane. Water will tend to flow into the cells and
organelles by osmosis, promoting their lysis and release of their proteins. To further promote
the disruption of cell membranes, a low concentration of organic solvent, e.g. 2% n-butanol, is
often added to the extraction buffer.
Pestle and mortar
Pestle and mortar is a moderate technique for tissue homogenization. Mechanical breakdown
occurs during the process. Sometimes, grinding is done in the presence of purified sand or glass
beads for aberration. Pestle and mortar is considered to be a moderate grinding technique and
rupturing of the cell organelles does not occur if isotonic grinding medium without detergent
is used.
Blenders
Waring blender (commonly called as mixie) is comparatively harsh technique of grinding the
tissue compared to pestle and mortar and is mostly used for homogenizing the harder tissues
(generally the plant tissues). If the worker is interested in isolating intact cell organelles, then
Waring blender is not a preferred technique. Waring blender is first operated at low speed
forfew seconds and then at medium speed(s) for few seconds before bringing it at high speed.
Time of grinding at various speeds is decided according to the nature of the tissue being ground.
If homogenization has to be done for a little longer time, then it is generally done after few
seconds interval after every minute of grinding at high speed to avoid heating during operation
of the Waring blender.
Ultra- Sonicator
This technique of rupturing the cells is generally used for microbial/ bacterial cells. Ultra-
sonicator generates low as well as high wavelength ultrasonic waves. For the purpose, a
suitable probe depending on the volume of the homogenizing medium is selected and
connected with the ultra-sonicator. The container having cells and homogenizing (isolating)
medium is put in chilled condition by covering the container with ice. There is much generation
of heat during ultra-sonication, therefore, ultrasonic waves are thrown in the sample after few
seconds interval, every 10 to 15 seconds ultrasonication. Fig 1 Ultra- Sonicator
Freeze-Thaw
With certain susceptible microbes and eukaryotic cells, repeated freezing and thawing results
in extensive membrane lesions with release of periplasmic and intracellular proteins.
Acetone powder
Drying with acetone is a good method for rupturing the cell membrane. Using acetone, powder
of the tissue may be prepared which may be stored in a Deep freezer for a long time. It forms
a convenient starting material from which the enzyme may be extracted with the isolating
medium, whenever required. However, one has to take much precautions of low temperature
(generally –20oC), otherwise, acetone may denature the enzyme protein. Isolation of enzymes
from sub-cellular organelles requires rupturing of the organelle. Generally for the purpose,
organelle is isolated in intact form thus removing the contaminating proteins of the cytoplasm
and other cell organelles. Afterwards, cell organelle is ruptured in the presence of a suitable
detergent like tween, teepol, digitonin etc.
Methods of enzyme purification
The purification of a particular enzyme involves removal of other substances (proteins as well
as non-proteins) present in the preparation. Purification of an enzyme protein is generally a
multi-step process exploiting a range of biophysical and biochemical characteristics such as
Its relative concentration in the source

Solubility

Charge

Size (molecular weight)

Hydrophobicity/ hydrophilicity of the target protein.

In general, design of the purification technique/ protocol should be focused on

High recovery

Highly purified enzyme protein

Reproducibility of the methods

Economical use of the chemicals (reagents) and

Shorter time for complete purification.

Proteins are relatively labile and get denatured at high temperatures and variation in pH. Each
protein has its own physico-chemical characteristics. The techniques selected for enzyme
purification should be moderate and native conformation of the enzyme protein should not
change as a result of purification. During purification, degree of purity and percent of recovery
should be checked after each step of purification. In general, it has been observed that enzymes
are more unstable in dilute solutions. Therefore, while designing the purification procedure,
initially emphasis is given on concentrating the protein concentration in the sample rather than
purification. After concentration, emphasis is given to purification (removal of unwanted
proteins) and lesser loss of enzyme activity of the targeted enzyme. Commonly, the first step
in enzyme purification is based on fractionation of proteins on the basis of solubility of proteins
in aqueous solutions of salts or organic solvents.
Fractionation of the proteins on the basis of solubility in aqueous solutions of salts or
organic solvents
The solubility of a protein is the result of polar interactions with the aqueous solvent, ionic
interactions with salts and repulsive electrostatic interactions between alike charged molecules.
The properties of water may be changed by changing the ionic concentration and pH. The
addition of miscible organic solvents, other inert solutes and polymers with temperature
variation can be manipulated to cause selective precipitation. Isoelectric precipitation may also
be used since protein is least soluble when net charge on it is zero.
Salting out
Generally, salting out of proteins using ammonium sulfate is used as the first step in the enzyme
purification. However, other salts like sodium sulfate may also be used but ammonium sulfate
is most common.
A large number of water molecules bind with the salt reducing the amount of water available
to interact with the protein molecules. Precipitation of proteins using salt also removes non-
protein impurities present in the enzyme homogenate (crude extract). At a particular
concentration of the salt, unbound water will keep the protein in the soluble form. Generally,
solid salt is added in the range of 0 to 30%, 30 to 60% and 60 to 90 % with continuous gentle
stirring keeping the pH constant (near neutrality or slightly alkaline by the addition of dilute
ammonia dropwise). Care is taken that no local precipitation of protein occurs. On the other
hand, care is taken that no denaturation of protein should occur due to stirring. Stirring is done
either with the help of a glass rod (if volume is not too much) or with the help of a motorized
mechanical stirrer. After addition of the salt, the suspension is stored for few hours in cold
condition for complete precipitation. The precipitate is collected by centrifugation in cold
condition and thereafter the precipitate is dissolved in suitable medium (which may be same as
the isolating medium or may be little different).
Protein fractionation using organic solvents
Generally, acetone is used for fractionation of the enzyme protein. While using acetone,
extreme care is to be taken otherwise acetone will denature the enzyme protein. Chilled acetone
at –20oC is used and continuous stirring is done to avoid denaturation of the proteins. Like salt
fractionation, here also fractionation of proteins is done by using different concentrations of
acetone like 10%, 20%, 30% and so on. The suspension is stored for few hours before
centrifugation at –20oC to collect the precipitate. The precipitate is dissolved in suitable
medium.
Protein fractionation using nonionic polymers
Nonionic polymers can be used for precipitation of enzyme proteins. Polyethylene glycol
(PEG) is commercially available in different molecular weight ranges like 6000, 40,000,
360,000. Lower molecular weight PEG is more soluble in aqueous solvents compared to high
molecular weight, therefore, PEG of 6000 molecular weight is preferable. Different ranges of
PEG (like 0-3%, 3-10%, 10-20%, 20-30%, 30-40%, 40-50%) are used for fractionation of
proteins.
Protein fractionation by heat treatment
In many cases, the enzyme protein to be purified is fairly stable at temperature like 55oC or
60oC. At this temperature, many unwanted proteins get denatured and precipitated out with
little or no loss of enzyme activity. The protein solution is kept for 5 to 10 minutes at this
temperature and afterwards immediately chilled by keeping in ice. In few cases of allosteric
enzymes, the enzyme is stable at high temperature in the presence of an effector molecule.
However, this method is not in much use since there is always a possibility of getting the
conformation of the enzyme protein changed at higher temperature.
Three-phase partitioning (TPP)
Three-phase partitioning (TPP) is a method in which proteins are salted out from a solution
containing a mixture of water and t-butanol. t-Butanol is infinitely miscible with water but
upon addition of sufficient ammonium sulfate the solution splits into two phases, an underlying
aqueous phase and an overlying t-butanol phase. If protein was present in the initial solution,
three phases would be formed, protein being precipitated in a third phase between the aqueous
and t-butanol phases. The amount and type of protein precipitated is dependent upon the
ammonium sulfate concentration, as in conventional salting out. Unlike in conventional salting
out, however, the protein precipitate is largely dehydrated and has a low salt content.

Dialysis
Dialysis is typically used to desalt protein solutions, or to effect a buffer exchange, i.e. to get
the protein from one buffer solution into another
Dialysis can be done in various ways, but in the laboratory, it is most commonly done using
tubing. This is a cellulosic material reconstituted into tubular form, dried, and supplied in rolls.
A length can be cut from the roll, hydrated by immersion in water for several minutes, and
clamped or knotted at one end to form a sealed ―dialysis bag‖. The protein is introduced into
this bag and the open end is sealed by clamping or knotting. The dialysis bag is immersed in a
large volume of distilled water or buffer for several hours at 4°C to effect exchange of the
permeable ions and molecules, the dialysis solution being changed at intervals (every few
hours). During dialysis, water enters the dialysis bag due to the osmotic pressure of the protein
solution. For this reason, a dialysis bag must not be filled, but a potential space must be left to
accommodate the increasing volume of the protein solution. Note that if the dialysis bag is
sealed with knots, the knot should be tightened by pulling only on the outside, not on the bag
side of the knot, to avoid stretching the bag and thus distorting the pores.

Ultrafiltration
Ultrafiltration is a technique related to dialysis, and can also be used to desalt protein solutions,
effect buffer exchange, or concentrate protein solutions. It is more expensive than dialysis,
however, as special equipment and membranes are required. In this technique, pressure is
applied to the solution to cause a bulk flow of water and dissolved low molecular weight
solutes, through the membrane, while high molecular weight solutes are retained.
Fig 6 An ultrafiltration cell.

Chromatographic separation of the enzyme proteins

The word ―chromatography‖ means ―writing with colour‖ and refers to the early observations
on the separation of dyes by paper chromatography. All chromatographic separations depend
upon the differential partition of solutes between two phases, a mobile phase and a stationary
phase. Such partition between two phases is described by the so-called partition coefficient or
distribution coefficient.
(i) Ion exchange chromatography

(ii) Adsorption chromatography

(iii) Gel filtration chromatography and

(iv) Affinity chromatography.

In general, the procedure of carrying the work is same in all types of chromatography. First,
the enzyme protein sample to be purified is applied onto the pre-equilibrated column and
thereafter, the sample from the column is eluted with buffer with a series of steps of different
solute concentrations, with a gradient of solute or with a specific ligand for the desired enzyme
protein. The effluent eluted out from the column is collected as a series of fractions using a
fraction collector, tested for enzyme activity and protein.
Ion exchange chromatography
The basic principle involved in ion exchange chromatography is binding of charged proteins
on the ion exchanger by electrostatic attraction (ionic bonds) between charged groups on the
proteins and opposite charges on the exchanger.

Unbound proteins are removed from the column by washing with the same medium used for
pre-equilibrium. Bound proteins are eluted by passing buffer of higher ionic strength (using
salts like sodium or potassium chloride) or by using buffer of different pH.

Two types of ion exchangers are in common use for separation of enzymes: Anion
exchangers and cation exchangers.

The most commonly used anion exchanger is diethyl amino ethyl cellulose (DEAE
cellulose). Some other are amino ethyl cellulose (AE cellulose), triethyl amino ethyl cellulose
(TEAE cellulose) and guanido ethyl cellulose (GE cellulose).
The most commonly used cation exchanger is carboxy methyl cellulose (CM cellulose). The
other examples of cation exchangers are phospho cellulose (P cellulose) and sulfo ethyl
cellulose (SE cellulose). These exchangers have cellulose matrix, which is considered to be
inert. The other matrices used in exchangers are Sephadex and Sepharose.
1. Equilibration of the ion exchanger in a buffer in such a way that the molecule(s) of interest
will bind in a desirable way.

2. a) Application of the sample. Solute molecules carrying the appropriate charge are bound
reversibly to the gel.
b) Unbound substances are washed out with the starting buffer.
3. Elution with a gradient of e.g. NaCl. This gradually increases the ionic strength and the
molecules are eluted. The solute molecules are released from the column in the order of the
strengths of their binding i.e. the weakly bound molecules elute first.

4. Substances that are very tightly bound are washed out with a concentrated salt solution and
the column is regenerated to the starting conditions.

Adsorption chromatography
The basic principle in this type of chromatography is binding of the proteins on the matrix by
physical adsorption on the surface of insoluble matrix (through weaker bonds like hydrogen,
van der Waals bonds). Afterwards, proteins are eluted from the column matrix by using a
suitable elution buffer either having change in ionic concentration or pH. The commonly used
matrices in adsorption chromatography are: (i) calcium phosphate gel; (ii) alumina gel and (iii)
hydroxylapatite gel.
In this type of chromatography, gel to protein ratio is important for physical adsorption.
It is preferable to carry a trial experiment in centrifuge tubes. A constant amount of the gel is
put in each tube and different amounts of protein sample are added in each tube so that ratio of
0.1 to 2.0 in different tubes be obtained. After addition of sample, it is mixed with the gel and
allowed to bind for few minutes. Afterwards, tubes are centrifuged and enzyme activity is
determined in different tubes supernatants. If enzyme activity is present in a supernatant, it
means binding of the enzyme protein (of interest) on the gel did not occur. From this trial
experiment, one can determine, what will be the optimum gel to protein ratio so that enzyme
protein of interest gets adsorped on the gel surface. Afterwards, accordingly, size of the packed
gel in the column be decided. For elution, generally either buffer of high ionic strength or buffer
with salt like NaCl or KCl is used. The gels used in adsorption chromatography are
commercially available. The gels may also be prepared in the laboratory. It is found that older
gels are more effective in separation compared to newly prepared gel. In the laboratory, calcium
phosphate gel is prepared by addition of sodium tri phosphate to a diluted solution of calcium
chloride and pH is adjusted to 7.4. A precipitate of calcium phosphate formed is washed to
remove excess ions. Alumina gel is prepared by the addition of a hot solution of aluminum
ammonium sulfate to a solution containing ammonium sulfate, ammonia and water at 60 C.
The solution is cooled, the precipitate of alumina formed is washed with water to remove excess
ions. Hydroxylapatite gel is prepared by addition of calcium chloride and di sodium hydrogen
phosphate to a solution of one molar sodium chloride. The precipitate of hydroxylapatite
formed is treated with alkali and heated to boiling for about 40 to 50 minutes. Afterwards, it is
cooled and washed with water to remove excess ions.
Gel filtration (Molecular sieve) chromatography
The basic principle is based on the size and shape of the proteins. Here, gel particles have
sponge like porous matrix as a structure with controlled dimension. The gel particles are
swollen and equilibrated with appropriate medium and afterwards is packed in the
chromatography column. Gel particles are spherical in shape. The molecules (proteins) to be
separated enter in the porous matrix of the gel particles and too large molecules are not entered
in the porous matrix and are eluted out from the column. Every gel is characterized by exclusion
limit that means the proteins of more than that molecular weight will not enter in the matrix
and eluted out as such (without separation). Void volume is considered as the space between
the gel particles in the packed column. It is determined by passing blue dextran, which has very
high molecular weight. Molecules with masses below the exclusion limit of the gel are eluted
from the column in order of their molecular mass (weight) with the largest eluting first. Larger
molecules have lesser of the interior volume of the gel available to them than the smaller
molecules.
The commonly used gel filtration gels are of dextran, agarose, polyacrylamide. These gels are
having registered trade names of the manufacturers. For example, dextran gels having
registered trade name ‘Sephadex‘ are in much common use. Gel filtration chromatography
(with matrix having much lesser exclusion limit such as Sephadex G- 25) is also used for
desalting purpose. Since in Gel filtration chromatography, separation is based on molecular
weight (if shape of all the molecules is same), this chromatography has been commonly used
for determination of molecular weight of proteins.
Advantages of Gel Filtration
• Can handle biomolecules that are sensitive to changes in pH
• Separations can be performed in the presence of essential ions, detergents, urea
guanidine hydrochloride at high or low ionic strength.
Common terms in size exclusion chromatography
1. The total volume (Vt): the sum of the volume of the gel matrix, the volume inside the gel
matrix, and the volume outside the matrix. The total volume is also, in most cases, equal to the
amount of the eluent required to elute a substance through the column, when the substance is
small enough to completely penetrate the pores of the gel.
2. Inner volume (Vi): the volume of the eluent inside the gel matrix. The volume inside the
beads.

3. Void volume (Vo): the volume of eluent outside the gel matrix. This is the volume required
to elute a substance so large that it cannot penetrate the pores at all. Such a substance is said to
be completely excluded, such as dextran blue 2000.

4. Elution volume (Ve): the volume of eluent required to elute any given substance.

Affinity chromatography
The basic principle involves bio-specific interaction of the enzyme protein of interest with an
immobilized ligand, which may be substrate, analogue of the substrate, inhibitor, activator.
Inert materials like agarose, polyacrylamide, glass beads, cellulose etc have been used as
supporting medium (matrix). The ligand is attached so that its enzyme interaction function is
not impaired. Subsequently, elution is done by treatment resulting in dissociation of the desired
enzyme ligand complex. Nowadays, affinity matrices (ligand immobilized with the matrix) are
commercially available. Immuno-affinity chromatography is also an affinity chromatography
where antibody of the protein is used as ligand. The basic principle of antigen antibody
interaction in this chromatography is applied. Although it is a good technique for purification
of a protein, it is not in common use for enzymes since generally enzyme gets inactivated after
binding with the antibody. \
The recovery and purification (briefly described below) steps will be the same for both
intracellular and extracellular enzymes, once the cells are disrupted and intracellular enzymes
are released. The most important consideration is to minimise the loss of desired enzyme
activity.

Drying and packing:

The concentrated form of the enzyme can be obtained by drying. This can be done by film
evaporators or freeze dryers (lyophilizers). The dried enzyme can be packed and marketed. For
certain enzymes, stability can be achieved by keeping them in ammonium sulfate suspensions.

All the enzymes used in foods or medical treatments must be of high grade purity, and must
meet the required specifications by the regulatory bodies. These enzymes should be totally free
from toxic materials, harmful microorganisms and should not cause allergic reactions.

ENZYME CHARACTERIZATION AND QUALITY ASSESSMENT

After purification, enzyme characterization is essential to ensure enzyme identity, stability, and
functionality. Techniques such as SDS-PAGE, isoelectric focusing, and mass
spectrometry confirm enzyme purity and molecular weight.
SDS PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique
used for the separation of proteins based on their molecular weight. It is a technique widely
used in forensics, genetics, biotechnology and molecular biology to separate the protein
molecules based on their electrophoretic mobility.

Principle of SDS-PAGE
The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the
opposite sign when placed in an electric field. The separation of the charged molecules depends
upon the relative mobility of charged species.
The smaller molecules migrate faster due to less resistance during electrophoresis. The
structure and the charge of the proteins also influence the rate of migration. Sodium dodecyl
sulphate and polyacrylamide eliminate the influence of structure and charge of the proteins,
and the proteins are separated based on the length of the polypeptide chain.
Role of SDS in SDS-PAGE
SDS is known as sodium lauryl sulfate sodium dodecyl sulfate. SDS is a detergent present
in the SDS-PAGE sample buffer. SDS along with some reducing agents function to break the
disulphide bonds of proteins disrupting the tertiary structure of proteins. The system is
primarily made with one of these previously mentioned components and gel that is
known as a polyacrylamide. The gel is of prime importance as it takes away basic
characteristics of the protein molecules so that the mass of the protein molecules do not
get influenced by the structure and charge. Then the protein molecules are separated
depending on the polypeptide chain length.

Materials Required

• Power Supplies: It is used to convert the AC current to DC current.

• Gels: These are either prepared in the laboratory or precast gels are purchased from the
market.

• Electrophoresis Chambers: The chambers that can fit the SDS-PAGE gels should be
used.

• Protein Samples: The protein is diluted using SDS-PAGE sample buffer and boiled
for 10 minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also
added to reduce the disulfide linkages to prevent any tertiary protein folding.

• Running Buffer: The protein samples loaded on the gel are run in SDS-PAGE running
buffer.

• Staining and Destaining Buffer: The gel is stained with Coomassie Stain Solution.
The gel is then destained with the destaining solution. Protein bands are then visible
under naked eyes.

• Protein Ladder: A reference protein ladder is used to determine the location of the
protein of interest, based on the molecular size.

Protocol of SDS-PAGE
Preparation of the Gel
• All the reagents are combined, except TEMED, for the preparation of gel.

• When the gel is ready to be poured, add TEMED.

 • The separating gel is poured in the casting chamber.

• Add butanol before polymerization to remove the unwanted air bubbles present.

• The comb is inserted in the spaces between the glass plate.

• The polymerized gel is known as the “gel cassette”.

Sample Preparation
• Boil some water in a beaker.
• Add 2-mercaptoethanol to the sample buffer.

• Place the buffer solution in microcentrifuge tubes and add protein sample to it.

• Take MW markers in separate tubes.

• Boil the samples for less than 5 minutes to completely denature the proteins.

Electrophoresis
• The gel cassette is removed from the casting stand and placed in the electrode assembly.

• The electrode assembly is fixed in the clamp stand.

 • 1x electrophoresis buffer is poured in the opening of the casting frame to fill the wells
of the gel.

• Pipette 30ml of the denatured sample in the well.

• The tank is then covered with a lid and the unit is connected to a power supply.

• The sample is allowed to run at 30mA for about 1 hour.

• The bands are then seen under UV light.

Applications of SDS-PAGE
1. It is used to measure the molecular weight of the molecules.
2. It is used to estimate the size of the protein.
3. Used in peptide mapping
4. It is used to compare the polypeptide composition of different structures.

5. It is used to estimate the purity of the proteins.

6. It is used in separation of protein associated with disease.

7. Analysing the size and number of polypeptide subunits.

8. To analyze post-translational modifications.

Isoelectric Focusing
Isoelectric focusing refers to the separation of proteins electrophoretically on the basis of
their pI that is on the basis of their relative acidic and basic residues. The isoelectric point
(pl) of a protein is the pH at which its net charge remains zero. Therefore, at this pH
(isoelectric point), the electrophoretic mobility of the protein will also be zero since the
proteins will not be able to migrate in any electric field. When a mixture of proteins is
subjected to gel electrophoresis across a pH gradient, each protein will tend to move in the
gel until it reaches a position where the pH becomes equal to the pI of the protein. At this
position, the migration of the protein will stop. Figure 15 outlines the method of isoelectric
focusing for separation of proteins.
Two-dimensional electrophoresis is an advanced method of protein separation where the
separation of proteins on a gel is conducted in two dimensions. It involves the combination of
isoelectric focusing with SDSPAGE to obtain protein separations with a very high resolution.
The sample to be purified is first subjected to isoelectric focusing as described in the previous
section (1 dimension). For the second dimension, the gel is then subjected to SDS PAGE
electrophoresis. The proteins now move through polyacrylamide gel vertically based on how
far they migrated during isoelectric focusing. Post the electrophoresis, the gel is further stained
by Coomassie or silver staining to yield visible two-dimensional pattern of spots. Thus, in 2D
gel electrophoresis, the proteins are separated horizontally on the basis of isoelectric point and
vertically on the basis of their mass. The proteins separated by 2D electrophoresis can further
be identified by coupling with mass spectrometric techniques.

Mass Spectrometry
The separated protein using SDS-PAGE or isoelectric focusing strip can be removed and
analysed in mass spectrometry to identify the protein or the amino acid sequence. Mass
spectrometry is an analytical technique to evaluate known materials and determine unknown
compounds. It also helps to determine the structure and chemical properties of several
molecules. The food we eat, the water we drink, medicine we consume when we fall ill all are
first tested to check the presence of any harmful elements or any contamination with the help
of mass spectrometry. Mass spectrometry is also used in isotope determination, carbon dating,
identification of protein, etc.
In mass spectrometry, the sample compound is first converted to gaseous ions with or without
the fragmentation method, which is further identified by their mass-to-charge ratio and relative
abundances (intensity).
Principle of Mass Spectrometry
The principle involved in mass spectrometry is the formation of several ions from the sample.
Further, these ions are separated according to their mass to charge ratio, which is also expressed
as m/z and then taking a record of the relative abundance of each ion.
In the first step, the sample compound is converted to ions in the gas phase by the electron
ionisation method. After that, the molecular ions undergo fragmentation. Each ion is separated
from the other in a mass spectrometer depending on their mass-to-charge ratio and identified
according to their relative abundance. A mass spectrum is then formed, which shows the
spectrum of ion abundance versus mass-to-charge ratio.
The ions present give information about the structure and properties of the compound. In the
spectrum, the molecule ion of the pure compound has the highest value of mass to charge ratio,
followed by ions of heavier isotopes. By this, the molecular weight of the compound is
determined.
Instrumentation of Mass Spectrometry
There are three major components present in mass spectrometry which are discussed below.
• Ion Source: It produces gaseous ions from the given sample.

• Analyzer: It is used to analyse and separate the ions into their characteristic mass
according to their mass-to-charge ratio.

• Detector System: Detectors in mass spectrometry detect the ions and maintain their
relative abundance.

Apart from these, a sample introduction system is required to add the sample to the ion source.
A high vacuum is maintained (10-5-10-8 torr), and a computer system is needed to control the
instrument, store the data and compare the spectrum with the references.
 Instrumentation of Mass Spectrometry
Working of Mass Spectrometry
In the ion source, the sample molecules are mostly bombarded by electrons from a heated
filament. The volatile liquid samples and gases come into the ion source from the reservoir,
and the non-volatile solids and liquids are added directly. The cations are pushed away by the
charged repeller plate and moved towards other electrodes, and anions are attracted to the plate.
The plate has a slit from where the ions pass as a beam.
The perpendicular magnetic field deflects the ion beam into an arc. The lighter ions are
deflected higher than, the heavier ions. By analysing the strength of the magnetic field, the
ions having different masses are detected by the detector. According to the mass spectrum
formed by the charged ions, one can determine the molecule or atom compared with the
known molecular masses.
Mass Spectrum
A mass spectrum is a vertical bar graph of mass to charge ratio versus the relative abundance
(intensity) of the ions where each bar represents the ion of specific mass to charge ratio and the
length of the bar is the relative abundance of ion. The ion with the most intensity has an
intensity of 100 and is called the base peak. The mass-to-charge ratio is equal to the mass of
the ion as the ion has a single charge.

High-Resolution Mass Spectrometry

High-resolution mass spectrometry is used to analyse complex chemical compounds,

determine isotopes, etc. It has increased resolution, which helps in differentiating the isotopes,
generating fragments pattern, and library matching for compound verification. HRMS is not
the replacement for low-resolution mass spectrometry, but it is an advanced technique used to
determine unknown compounds and also generate their molecular formula. High-resolution
mass spectrometry is quite expensive as compared to low resolution.
Advantages and Limitations of Mass Spectrometry

Advantages:

1. Works with a small sample size

2. Fast
3. Can differentiate isotopes
Limitations:

1. Does not give direct structural information

 2. The requirement of pure samples
3. Not ideal for non-volatile compounds
Application of Mass Spectrometry
• Environmental Analysis: It is used in water testing, soil contamination, analysis of trace
elements, carbon content and pollution analysis.

• Pharmaceutical Analysis: It is used in producing new drugs, preclinical development,

protein identification etc.

• Clinical Application: It is used in identifying infectious agents, drug therapy

monitoring, clinical tests, screening of diseases, etc.

• Forensic Analysis: It helps in confirming drug abuse, identifying explosives, arson

investigation, etc.
Mass spectrometry is a spectroscopic technique used to determine the nature and structure of
unknown inorganic and organic compounds based on their mass-to-charge ratio and relative
abundance. This technique is widely used in different industries because of its advanced
technology and application. It is used in forensic analysis, analysing different environmental
issues, clinical trials, etc. High-resolution mass spectrometry is used to analyse complex
chemical compounds which a low resolution is not able to determine accurately.

DETECTION OF ENZYME ASSAY

Enzyme assay
• It is a laboratory method for measuring enzymatic activity. They are vital for the study of
enzyme kinetics and enzyme inhibition.
• The assay is the act of measuring how fast a given (unknown) amount of enzyme will
convert substrate to product (the act of measuring a velocity).
• Enzyme assays measure either the disappearance of substrate over time or the appearance
of product over time.
• An assay requires to determine the concentration of a product or substrate at a given time
after starting the reaction.
• Enzymes are usually present in very small quantities in biological fluids • Therefore,
Enzymes are not directly measured
• They are commonly measured in terms of their catalytic activity
• We don’t measure the molecule but we measure how much “work” it performs (catalytic
activity) That means the rate at which it catalyzes the conversion of substrate to product
 • The enzymatic activity is a reflection of its concentration
• Activity is proportional to concentration
Features of a good Enzyme Assay
• 1. Simple and Specific
• 2. Rapid (one doesn’t need to wait for hrs or weeks for the results to appear)
• 3. Sensitive (little sample)
• 4. Easy to use
• 5. Economical
Types of Enzyme assay
Enzyme assays can be split into two groups according to their sampling method:
Continuous assays, where the assay gives a continuous reading of activity, – multiple
measurements, usually of absorbance change, are made during the reaction, – either at specific
time intervals (usually every 30 or 60 seconds) – or continuously by a continuous-recording
spectrophotometer. – These assays are advantageous over fixed-time methods because the linearity
of the reaction may be more adequately verified.
Discontinuous assays, where samples are taken for a fixed time and the reaction is stopped (usually
by inactivating the enzyme with a weak acid) then the concentration of substrates/products
determined.
Continuous Assay
Spectrophotometer
• The spectrophotometric assay is the most common method of detection in enzyme assays.
• It uses a spectrophotometer, a machine used to measure the amount of light a substance
absorbs, to combine kinetic measurements and Beer's law by calculating the appearance of
product or disappearance of substrate concentrations.
• If this light is in the visible region, we can actually see a change in the color of the assay.
UV light is often used, since the common coenzymes NADH and NADPH absorb UV light
in their reduced forms, but do not in their oxidized forms.
• The conversion of one to another is followed by change in absorption and by measuring
the change the progress of the reaction can be quantified.
• The enzyme is allowed to react with substrate and the decrease in substrate and increase in
product will be followed spectrophotometrically.
Coupled Reaction
• In many reactions, changes in substrates or products are not observable by
spectrophotometric methods because they do not absorb light.
• Even when such enzyme reaction does not result in a change in the absorbance of light, it
can still be possible to use a spectrophotometric assay for the enzyme by using a coupled
assay.
• These reactions can be measured by coupling them to enzymes that can be detected by a
spectrophotometer.
 • The product of one reaction is used as the substrate of another, easily-detectable reaction.
• This help to follow the first enzymatic reaction.

Fluorometric method
• Fluorescence is when a molecule emits light of one wavelength after absorbing light of a
different wavelength.
• Uses a Fluorometer
• Fluorometric assays use a difference in the fluorescence of substrate from product to
measure the enzyme reaction.
• FLAVIN COMPOUNDS: fluorescence in reduced form and loose their fluorescence in
oxidised form
• Example of these assays is again the use of the nucleotide coenzymes NADH and NADPH.
• Here, the reduced forms are fluorescent and the oxidised forms non- fluorescent.
• Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction
reactions by an increase.
• More sensitive than spectrophotometric assays, but can suffer from interference caused by
impurities and the instability of many fluorescent compounds when exposed to light.
• Detection in small quantities
• Non dangerous
Colorimetric
• The measurement of the heat released or absorbed by chemical reactions.
• These assays are very general, since many reactions involve some change in heat and with
use of a micro-calorimeter, not much enzyme or substrate is required.
• These assays can be used to measure reactions that are impossible to assay in any other
way.
Chemiluminescent
• The emission of light by a chemical reaction.
• Some enzyme reactions produce light and this can be measured to detect product formation.
• These types of assay can be extremely sensitive, since the light produced can be captured
by photographic film over days or weeks,
• A spontaneous chemical reaction between nitric oxide and ozone (an unstable molecule
formed of three oxygen atoms: O3) is known to produce chemiluminescence:
• NO + O3 → NO2 + O2 + light
• Although this process of generating light is quite inefficient (only a small fraction of
the NO2 molecules formed by this reaction will emit light), it is predictable enough to
be used as a quantitative measurement method for nitric oxide gas. Ozone gas is very
easy to produce on demand, by exposing air or oxygen to a high-voltage electric
discharge.
• In order to use chemiluminescence to measure all oxides of nitrogen, we must
chemically convert the other oxides into nitric oxide (NO) before the sample enters the
reaction chamber. This is done in a special module of the analyzer called a converter
• As with many optical analyzers, a photomultiplier tube serves as the light-detecting
sensor, generating an electrical signal in proportion to the amount of light observed
inside the reaction chamber. The higher the concentration of NO molecules in the
sample gas stream, the more light will be emitted inside the reaction chamber, resulting
in a stronger electrical signal produced by the photomultiplier tube.
 • The main drawback is can be hard to quantify, because not all the light released by a
reaction will be detected.
Electrode Method
In the glass-electrode method, the known pH of a reference solution is determined by using
two electrodes, a glass electrode and a reference electrode, and measuring the voltage
(difference in potential) generated between the two electrodes. The difference in pH
between solutions inside and outside the thin glass membrane creates electromotive force
in proportion to this difference in pH. This thin membrane is called the electrode
membrane. Normally, when the temperature of the solution is 30 ℃, if the pH i nside is
different from that of outside by 1, it will create approximately 60 mV of electromotive
force.
In other words, a glass electrode is devised to generate accurate electromotive force due to
the difference in pH. The reference electrode is devised not to cause electromotive force
due to a difference in pH.
Polarimetric Method
It is used for isomerases that convert one isomer to another
It converts optically active to inactive or vice versa
It can be used both in substrate and product are optically active but different in specific rotation
D Glucose L Glucose
Discontinuous assay
Discontinuous assay are when samples are taken from an enzyme reaction at intervals and the
amount of product production or substrate consumption is measured in these samples by different
chemical methods.
Radiometric:
• Radiometric assays measure the incorporation of radioactivity into substrates or its release
from substrates.
• The radioactive isotopes most frequently used in these assays are 14C, 32P, 35S and 125I.
• Since radioactive isotopes can allow the specific labelling of a single atom of a substrate,
these assays are both extremely sensitive and specific.
• They are frequently used in biochemistry and are often the only way of measuring a specific
reaction in crude extracts (the complex mixtures of enzymes produced when you lyse
cells). • Radioactivity is usually measured in these procedures using a scintillation counter.,
which measures the ionizing radiation.
• Very sensitive but hazardous
Chromatographic assay
• It measure product formation by separating the reaction mixture into its components by
chromatography.
• This is usually done by high-performance liquid chromatography (HPLC), but can also use
the simpler technique of thin layer chromatography.
• Enzymes can be assayed by HPLC by calculating the amount of substrate(s) left over, or
product formed, through the peak area ratios with a suitable internal standard. However,
sometimes the substrates used are contaminated with small amounts of products and this
can lead to errors in the determination of the enzyme activity.
• A method for a HPLC test of such enzymes, which prevents eventual errors, uses the ratio
substrate/product at time zero as internal standard and the kinetics can be followed with
the aid of a simple mathematical equation. This approach was applied to the
determination of the activities of papain, urokinase, NAD glycohydrolase, and pyruvate
 kinase samples and it was compared with the data obtained by the internal standard
method, giving reproducible results in all cases.
• Although this approach can need a lot of material, its sensitivity can be increased by
labelling the substrates/products with a radioactive or fluorescent tag.

UNIT V
Recent developments
Asymmetric aldol reactions catalyzed by the aldo–ketoreductase enzyme The promiscuous
aldo–ketoreductase (AKR) enzyme is used as a sustainable biocatalyst for the first time to
catalyze asymmetric aldol reactions in aqueous medium. In the absence of enzyme at ph 5.5
No reaction took place While with enzyme reaction takes place with higher yield and
enantioselectivity AKR catalyzed the aldol addition of cyclohexanone with aromatic aldehydes
to give the desired products in reasonable yields (up to 65%), enantioselectivities (up to 60%
ee), and moderate to excellent diastereoselectivities (up to 96:4, anti:syn).
Enzyme-catalyzed asymmetric Mannich reaction using acylase from Aspergillus melleus
Conventional mannich reaction Yield= up to 83% Less enantioselective not diastereoselective
Enzyme catalysed mannich reaction Yield= up to 85% Enantioselectivities= up to 89%
Diastereoselectivities=u p to 90:10 Yield = 92% Enantioselectivity = 75% When MeCN used
as solvent
Reduction of C=O to CHOH Using Enzymes and Microorganisms • Ketoreductases (KREDs)
can be used to generate chiral alcohols with good yields and excellent selectivity (often >99%
ee). • Most KREDs use either NADH or NADPH cofactors and catalyse the reduction of
carbonyl groups or the oxidation of alcohols. The reaction starts with the binding of the
NAD(P)H cofactor to the enzyme. Next, the ketone substrate is bound to the enzyme. Substrate
binding is followed by hydride transfer from the cofactor to the ketone to produce an alcohol.
The enzyme then releases the product alcohol. Whole cell Isolated enzyme Low yield and
enantioselectivity Yield >90% and ee>98%
Advances in synthesis of biodiesel via enzyme catalysis: Novel and sustainable approaches •
Lipases can effectively convert triglycerides to FAAE, thus attracting interest in the biodiesel
field. • Feedstock oil and short chain alcohols acting as acyl acceptors react in the presence of
lipases. Lipases effectively convert triglycerides as well as FFA to FAAE.
Lipase Feed stock (oil) Acyl acceptor Yield (%) Candida sp. Glycerol Methanol 80.6
Novozyme435 Sunflower Ethyl acetate 92.7 Novozyme435 palm Isobutanol 100 Candida
rugosa Soybean Methanol 87 Pseudomonas cepacia Soybean Methanol 90 Burkholderia
cepacia Palm Methanol 100 Candida antarctica Cotton seed Methanol 97 Geotrichum sp. Waste
cooking oil Methanol 85 Different Lipase with different feed stock and acyl acceptor
Conclusion
that use of enzymes in above mentioned techniques provides efficient organic synthesis in
terms of selectivity, yield and conversion. At the same time overcomes drawbacks of
conventional methods involving enzymes.

ARTIFICIAL ENZYMES
The term artificial enzyme loosely describes a molecule that attempts to imitate, recreate or
modify naturally occurring biochemical reactions. Molecules are designed and modified to
display enzyme-like characteristics. For instance, artificial enzymes may mimic the catalytic
ability and specificity of a well-characterized enzyme or catalyze non-naturally occurring
chemical reactions using known mechanisms of enzymatic catalysis.
Enzymes Are Highly Effective Natural Catalysts
Catalysts facilitate non-spontaneous chemical reactions by lowering the activation energy and
stabilizing the transition state. In catalyzed reactions, catalysts spontaneously react with
molecules of the reactants to initiate the transformation of reactants into products After the
reactants have transformed into products, they become free to interact with the excess reactant
molecules, starting another round of catalysis. Living cells in any biological system
use enzymes as biocatalysts to accelerate the rate of naturally occurring chemical reactions.

They are proteins that interact only with specific reactants, referred to as substrates, and other
regulatory molecules such as cofactors and effectors. These molecules must complement the
three-dimensional structure and electrical charges of the enzyme catalytic and regulatory sites.
Enzymes are active only in narrow temperatures and pH ranges generally favorable to the
corresponding organisms. Unlike inorganic catalysts, it is possible to control and change the
rate and direction of enzyme-catalyzed reactions by manipulating the reaction setting and the
abundance of the enzyme and substrate in the system.

Enzymes also have a high turnover rate even though typical enzymatic reactions occur in mild,
aqueous conditions without extra heat or pressure added to the system.

Artificial Enzymes Are Modeled on Enzymes or Enzymatic Reactions

The effectiveness and environmental friendliness of enzymes have inspired scientists to create
catalytic molecules that display similar characteristics.Artificial enzymes are structurally more
complex than inorganic catalysts, although some are less sensitive to temperature and pH
changes than natural enzymes. However, they may not necessarily be derived from chains of
amino acids like enzymes, though they possess the substrate selectivity and specificity that
amino acids provide.[4]

Types of Artificial Enzymes

These enzymes first emerged with the term biomimetic chemistry in the early eighties. Ronald
Breslow first used them to describe attempts to emulate the catalytic activity and functionality
of known enzymes. However, the term now has a broader meaning, covering the modification
of enzyme functionality and application of enzymatic mechanisms.

1. Enzyme Mimics
Enzyme mimics is a group of artificial enzymes that fit the definition of biomimetic
chemistry, which display functional abilities resembling known natural enzymes.
The construction of enzyme mimics revolves around creating catalytic and other necessary
binding sites on a suitable host molecule.

How Do Host Molecules Contribute to Artificial Enzyme-Substrate Selectivity?

Host molecules are soluble in water, and their three-dimensional structure typically contains a
confined inner space. The host’s confined inner space accommodates the substrate, performing
much the same role as the catalytic site of a natural enzyme.
In the absence of the substrate, water molecules fill in the inner space of the host molecules.
When the substrate is present, substrate molecules act as the so-called guest molecules and
replace the occupying water molecules.
Only substrates that are complementary to the shape of the inner space and electrically
compatible with the attached functional groups can access and occupy the inner space of the
host.
Like natural enzymes, an enzyme mimic’s host molecule shields the bound substrates from
other molecules, preventing them from interacting with other non-targeted molecules while
substrate-to-product transformation occurs.

2. Designer Enzymes
Designer enzymes are those enzymes derived from de novo enzyme design. In other words,
designer enzymes do not mimic the catalytic activity of natural enzymes. Instead, they are
modified and created based on structural and functional information of several naturally
occurring enzymes and insights into their catalytic mechanisms.

Biological method
The earliest host molecule used to construct artificial enzymes is cyclodextrin, a water-soluble
polymer that forms a supramolecular structure. It is made of cyclic glucose rings and shaped
like a donut that has a hydrophobic inner space. The size of cyclodextrins varies, depending on
the number of glucose molecules in the glucose ring. Thus, cyclodextrin-derived enzymes can
accommodate hydrophobic substrates that fit the size of the cyclodextrin inner space. Other
than supramolecular structures, complex molecules such as porphyrin cage and polypeptide
chains serve as host molecules of artificial enzymes.
Directed Evolution and Computational Design
Also referred to as in vivo evolution, the designer enzyme is randomly mutagenized and
screened for new variations with the most promising potential. Both processes are repeated
until the variation displaying the most desirable catalytic activity can be identified.
De novo enzyme design is a popular approach to modify enzyme catalytic sites and catalyze
non-naturally occurring chemical reactions.

One of the earliest examples of a designer enzyme is Merck-Codexis, used for the catalysis of
Diel-Alder reaction, a final step in the large-scale production of sitagliptin, an active ingredient
in the type 2 diabetes drug Januvia. At the time of development, no natural enzyme existed to
catalyse such reactions

Traditionally, the last step in sitagliptin production involves an expensive rhodium-catalyzed

reaction that requires additional purification steps to remove the heavy metal – rhodium.
To create designer enzymes that allow skipping that process, scientists selected a known natural
enzyme that displays a desirable catalytic activity but is too small to accommodate the
substrate. Using computational analysis, the amino acids that influence the size of the active
site were mutagenized.

The initial designer enzyme went through several random mutation events using directed
evolution before acquiring the designer enzyme with the designated functional activity.

As it turned out, the artificial enzyme-catalyzed route could avoid using rhodium as catalysts,
reduce the produced waste and increase the overall product yield

3. Enzymes From Artificial Building Blocks

This type of enzyme is the most recently developed approach. This approach is based on the
idea that alterations in the characteristics of the enzyme’s building blocks will lead to changes
in the enzyme’s inherent characteristics.

Examples of artificial building blocks are:

3.1. Synthetic Nucleic Acids (XNAs)
Nucleic acids are not building blocks for proteins, but they serve as a template for RNA
transcription and subsequent protein translation. Changes in the DNA sequence could lead to
changes in the amino acid sequence.
Synthetic nucleic acids (XNAs) with unnatural backbones have reportedly been used as
templates to express known enzymes. Reportedly, XNA-derived enzymes did not possess the
exact amino acid sequence as that from natural nucleic acids.
Nonetheless, XNA-derived enzymes displayed comparable catalytic activity despite the
predicted conformational change.

3.2. Non-Canonical Amino Acids (ncAAs)

Amino acids are the enzyme’s most basic unit. The sequence of amino acids determines the
three-dimensional structure, which affords enzymes with their selectivity and specificity.
Incorporating non-canonical amino acids (ncAAs) into a protein will, therefore, affect the
selectivity, specificity, and possibly, the enzyme’s catalytic ability.
To date, two methods are used to create ncAAs: Selective Pressure Incorporation (SPI) and
Stop Codon Suppression (SCS).
In SPI, manipulated transfer RNAs (tRNAs) carry ncAAs, which are structurally similar to
normal amino acids. In an alternative method, SCS, tRNA with the non-preferring stop codons,
and the associated translation mechanism are altered to carry the desired ncAAs.
Reportedly, ncAAs created from both methods can expand the enzymes’ functionality,
including the addition of certain unnatural characteristics.

3.3. β-Amino Acids

Similar to the idea of ncAAs, artificial enzymes have been created using β-amino acids instead
of the natural ɑ-amino acid. The main difference between the two amino acid species is the
placement and distance between their functional groups.
Reportedly, the enzymes made from β-amino acid chains can catalyze an aldol reaction despite
protein folding differences. The findings suggested the potential of β-amino acid-enzymes to
serve as a substitution when natural enzymes do not function.[11]

Benefits of Artificial Enzymes

Naturally occurring enzymes apply to the large-scale production of consumer products,
especially in the food and pharmaceutical industries. Nevertheless, limitations in natural
enzymes exist, and artificial enzymes show great potential to fill such gaps.
 • Attempts to create artificial enzymes, especially enzyme mimics, have provided
scientists with insights into the mechanisms of catalysis in several natural enzymes and
the behavior of water as a reaction solvent.
• Many artificial enzymes display enhanced catalytic activity or novel catalytic activity
to reactions that natural enzymes cannot catalyze, presenting new opportunities and
solutions to many industries.
• Artificial enzyme-catalytic reactions occur in mild, aqueous conditions. As a result,
they are more cost-effective, environmentally friendly, and more sustainable when
compared to inorganic catalysts and other solvents.

Conclusion
The effectiveness of natural enzymes has sparked the idea to emulate their characteristics and
functionality. Early artificial enzymes are mostly enzyme mimics created based on the known
natural enzymes. The advancement of science and the understanding of catalytic mechanisms
have made it possible to design artificial enzymes or synthesize ones from unnatural building
blocks, expanding the existing catalytic capacity and catalyzing novel chemical reactions not
previously catalyzed by natural enzymes. All in all, efforts to create these enzymes can
overcome the limitation of natural enzymes in their industrial applicability, foster our
understanding of biological catalysis and promote sustainable consumption and production.

CATALYTIC ANTIBODIES
‘Catalytic antibodies’ (antibody specificity + enzyme’s catalytic power) (CatAbs),are immuno-
modulators which can increase certain metabolic, physiological and chemical reactions in the
body. This is made possible by binding to a chemical group.CatAbs are produced by combining
an antibody to a hapten molecule for improving the immunogenicity. The carrier haptens are
usually so designed that they resemble the transition state of metabolic reactions, which also
evoke a specific response in the organism.Normally larger molecules can effectively elicit
antibodies via immunization and hence prior to the actual immunization, small-molecule
haptens are mostly attached to a larger protein molecule, called carrier proteins. Antibody
molecules normally are known for its specificity for binding to its antigen through
paratopeepitope interaction; normally they bind simply and are not involved in catalysis of any
biochemical reactions. However, when animals are immunized with carrier molecules, CatAbs
are produced. The carrier molecules are specially designed to elicit antibodies that have binding
pockets which are capable of catalyzing chemical reactions; endorsing an enzymatic nature by
lowering a reaction's activation energy barrier. This is only possible when we design antibodies
having a site of binding which is complementary to the transition state, both in terms of charge
distribution and 3D structure. This complementarity encourages the substrate to adopt a
transition-state-like geometry and charge distribution, increase the reaction selectivity, thus
leading to catalysis. On contrary to enzymes, one of the most interesting features of CatAbs is
that the desired reaction selectivity can be engineered into the antibody using a carrier hapten,
which has been appropriately designed. CatAbs demonstrate a high degree of substrate
selectivity as well as specificity. Furthermore, CatAbs that have region selectivity

.Characteristics of catalytic antibodies: CatAbscan either be present naturally (innate, in case

of autoimmune diseases) or be elicited by vaccination or immunization. Catalytic antibodies utilize
– antibody’s specificity & enzyme’s catalytic power
The mode of action include nucleophilic catalysis, coordination with metal ions, induction of
conformational strain and stabilization of transition states Haptens: The term ‘hapten’ derived
from a Greek word ‘Haptein’ means to fasten. Small molecules that stimulates the production of
antibodies are often conjugated to larger molecule, called a carrier molecule or haptens.

Transition state analogs:Enzyme does catalysis by maintaining the middle state; known as the
transition state and certainchemical compounds resembles the transition state of a substrate by its
chemical structure and are known as transition state analogs.In general, for CatAbs compounds
are synthesized with closer similarity towards transition state other than the substrate itself and
hence will be highly specific and potent inhibitors of enzymes.

Construction & generation of catalytic antibodies

An artificial catalytic antibody designed as per need is known as ‘Abzyme’. It’s usually a
monoclonal antibody raised in lab, against specific synthetic haptens, where they specificially bind
and catalyze a reaction. The major components for Abzyme generation are:

• A transition state analogue which is able to mimic the transition state of desired reaction is
synthesized. These are called as haptens
• Haptens are attached to carrier molecule which is capable of eliciting an antibody response.
These are called as antigens
• Specific antigen designed is injected to invivo models like mouse or rabbit
• Monoclonal antibodies are isolated and purified
• These monoclonal antibodies are further tested for catalytic activity by cat ELISA, HPLC
etc
The major stages associated with the production of catalytic antibodies include designing haptens,
generating monoclonal antibodies by hybridoma technology and further purification and screening
for catalysis.
Applications:
The various applications of abzymes include : Catalytic antibodies play immense role in drug
and pharmaceutical industries. Abzymes have been developed for detoxification of drugs like
cocaine. To eliminate the toxic effect of the drug, CatAbs have been generated which cleaves the
cocaine molecule at specific bonds.
Abyzme technology is also used for destructing hyper-proliferating cancer cells using the
characteristic determinants on their surface, called TCA-tumor cell antigens. Cancerous cells can
be targeted and destroyed using antibodies specific to these TCAs, known as magic bullets with
two distinct paratopes (Figure 11): which is bivalent and binds to TCA as well as a cytotoxic
prodrug or toxin. The antibody is administered to patients, and it binds specifically to TCA,
followed by prodrug through intravenous route of administration. Later the prodrug is introduced
into the bloodstream, but only becomes activated in the vicinity of the targeted antibody.
38C2 is the world's first commercially available catalytic antibody. This was used to or
intramolecular aldol reactions have been identified. catalyze aldol addition of a wide variety of
aliphatic open chain/cyclic ketones to various aromatic and aliphatic aldehydes. More than 100
different substrate combinations of cross aldol Novel catalytic antibodies were developed for
synthetic carbohydrate synthesis, insect pheromones production, and for producing derivatives of
the epothilone (anti-cancer drugs). Catalytic antibodies against allergy, viral and bacterial
infections were also developed. Conclusion: Catalytic antibodies or abzymes are tailor-made
enzyme which has been exploited to mimic enzyme-like rates. Various strategies of hapten design
including transition state analog, strain induced, bait-and-switch and reactive immunization based
hapten design have led to numerous antibody catalysed reactions. The primary mode of action in
both enzymes and catalytic antibodies is stabilizing a high energy transition state. However,
enzymes have a superior evolutionary advantage over catalytic antibodies. Moreover, additional
mechanisms that assist enzyme-based catalysis play a larger role. CatAbs on the other hand possess
numerous applications in medical & pharmaceutical research.