PRODUCTION OF ENZYMES

Enzyme technology broadly involves production, isolation, purification and use of enzyme for the
ultimate benefit of humankind. The first enzyme produced industrial was takadiastase (a fungal
analyse) in 1896, in united states. It was as a pharamaceutical agent to cure digestive disorders.
Commercial enzymes can be produced from a wide range of biological sources. At
present, a great majority (80%) of them are 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%
Enzymes from animal and plant sources
In the early days, animal and plat sources largely contributed to enzymes. Even now for
certain enzymes they are the major sources.
A selected list of plant ( Table 1) and animal (Table 2) enzymes with their sources aqnd
applications are given.
Table 1. Commercially produced enzymes from plant sources their applications.

Enzyme Source(s) Application(s)

β -Amylase Barely, soy bean Baking, preparation of maltose syrup.
Bromelain Pineapple Baking
Esterase Wheat Ester hydrolysis
Ficin Fig Meat tebneriser
Papin Ppaya Meat tenderizer, tanning, baking
Peroxidase Horse radish Diagnostic
Urease Jack bean Diagnostic

Table 2
Commercially produced enzymes from animal sources and their applications
Enzymes Sources Applications
Amylase, esterase Lamb,calf Digestive aids
Pepsin, trypsin Bovine Preparation of cheese
Lipase, rennin Poreine
(chymosin),phospholipase, phytase
Lysozme Hen eggs Cell wall breakage in bacteria
Human urine Urokinase For dissolution of blood clots
Animal organs and tissues are very good sources foe enzymes such as lipases, esterase
and proteases. The enzyme lysozyme is mostly obtained from hen eggs. Some plants excellent
sources for certain enzymes-papain (papaya), bromelain (pincapple).
Limitation
There are several dracobacks associated with the manufacture of enzymes fraom 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 enzume and the cost
factor. For these reasons microbial production of enzymes is preferred.

4.1.1 ENZYMES FROM MICROBIAL SOURCES

 Microorganisms are the most signicant and convenient sources of commercial enzyme.
They can be made to produce abundant quantitic of enzymes under suitable growth conditions.
Microorganisms can be cultivateo by using inexoansive 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
process are easy with microbial enzymes than that with animal or plant sources.
In fact, most enzymes of industrial applications have been successfully produced by
microorganisms. Various fungi, bacteria and yeasts are employed for this purpose. A selected list
of enzymes, microbial sources and the applications are given in Table-3.

4.1.2 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 sailent 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.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 oraganism is selected, strain improvement for
optimizing the enzyme production can be done by appropriate methods (mutagens, OV rays).
From the organism chosen, inoculum can be prepared in a liquid.
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. 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 fields 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, celluloses, proteases.
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
minimized by adding antifoan agents.
The production of enzymes is mostly carried ouet 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 of several other metabolitesare also produced. The
enzyme(s) have to be recovered and purified.
Purification of Enzymes
 After fermentation, the cells are separated from the growth medium by filtration or by
centrifugation. Depending on nature of enzyme namely, intracellular or extracellular either the
cells or the fermentation broth is further processed to separate and purity the enzyme.
Table.3. A selected list of industry (microblally) produced enzymes, their sources and
APPLICATIONS
Enzyme Source(s) Application(s)
α -Amylase Aspergillus oryzae Production of beer and alcohol,
Aspergillus niger Preparation of glucose syrups,
Bacillus subbtilus As a digestive aid
Bacillus licheniforns Removal of starch sizes
Amyloglucosidase Aspergillus niger Stacrh hydrolysis
Rhizopus nivous
Cellulose Aspergillus niger Alcohol and glucose production
Tricoderma koningi
Glucoamylase Aspergillus niger Production of beer and alcohol
Bacillus Starch hydrolysis
amyloliquefaciens
Glucose isomerase Arthrobacter sp Bacillus Manufacture of high fructose syrups
sp
Glucose oxidase Aspergillus niger Antioxidant in prepared foods

Invertase Saccharomyces cerevisiae Surcose inversion

Prepartion of artificial honey,
Confectionaries
Keratinase Streptomyces fradiae Removal of hair from hide
Lactase Kluyveromyus sp Lactose Hydrolysis
Saccharomyces fragilis Removal of lactose from whey
Lipase Candida lipolytica Preparation of cheese
Aspergillus niger Flavour production
Pectinase Aspergillus sp Clarification of fruit juices and wines
Sclerotina libertina Alcohol production, coffee concentration
Penicillin acylase Escherichia coli Production of 6-aminopenicillanic acid
Penicillanase Bacillus subtills Removal of Penicillin
Protease, acid Aspergillus niger Digestive aid,Substitute for calf rennet
Protease, neutral Bacillus Fish and meat tenderizer
amyloliquefaciens
Protease, alkaline Aspergillus oryzae Meat tenderizer
Streptomyces griseus Detergent additive
Bacillus sp Beer stabilizer
Pollulanase Klebsiella aerogens Hydrolysis of starch
Takadiastase Aspergillus oryzae Suplement to bread,Digestive aid

Extra cellular enzyme purification is easy from the broth. The recovery of intracellular enzyme is
more complicated and involves the disruption of cells and removal of cell debris and nucleic
acids. In some cases, enzyme may be both intracellular and extracellular, which requires
processing of both broth and cells. Intracellular enzymes may be released to medium by
increasing the permeability of cell membranes.

4.2 RECOVERY AND PURIFICATION OF ENZYMES

 The desired enzyme produced may be excreted into the culture medium (extracellular
enzymes) or my be present within the cells (intracellular enzymes). Depending on the
requirement, the commercial enzyme may be crude or highly purified. Further, it may be in the
solid or liquid form. The steps involved in downstream processing i.e recovery and purification
steps employed will depend on the nature of the enzyme and the degree of purity desired.
The extraction and purification of a biotechnological product from fermentation is
referred to as downstream processing (DSP) or product recovery.
In fig.1 an outline of the major steps in downstream processing is given, and they are described in
some detail as follows
The four main stages of down stream processing is,
1. Solid-liquid separation
2. Concentration
3. Purification
4. Formulation
4.2.1 Release of Intracellular Products
In general, recovery of an extra cellular enzyme which is present in the broth is relatively
simpler compared to an intracellular enzyme. For the release of intracellular enzymes, special
techniques are needed for cell disruption. The method of disruption varies with the type of cells
and the nature of intracellular products. Since there is a wide veriation in the property of cell
disruption or breakage for instance, Gram-negative bacteria and filamentous fungi can be more
easily broken compound to Gram positive bacteria and yeasts.

4.2.2 CELL DISRUPTION

The cell disruption methods can be broadly classified into mechanical and non
mechanical methods
Mechanical methods
Mechanical methods can be applied to a liquid or solid medium. First consider some
methods applied to a liquid medium.
Ultrasonication:
Ultrasonication vibrators (sonicators) are used to disrupt the cell wall and membrane of
bacterial cells. Wave density is usually around 20 kHz/S. Rods are broken more readily than coai
and gram-negative cells more easily than gram-positive cells.
An electronic generator is used to generate ultrasonic waves and a transducer converts
these waves into mechanical oscillations by a titanium probe immersed in cell suspension.
Intracellular enzymes are released into the broth upon cell disruption.
High pressure Homogenization
This technique involves forcing of cell suspension at high pressure through a very narrow
orifice to come out to atmospheric pressure. The sudden release of high pressure creates a liquid
shear that can break the cells.

SOLID SHEAR
Grinding with glass beads
The cells mixed with glass beads are subjected to a very high speed in a reaction vessel.
The cells break as they are forced against the wall of the vessel by the beads. Several factors
influence the cell breakage size and quantity of the glass beads. Concentration and age of cells,
temperature and age of cells, temperature and agitator speed. Under optical conditions, one can
expect a maximal breakage of about 80% of the cells.
A diagrammatic representation of a cell disrupter employing glass bead is shown in figure 3.
It contains a cylindrical body with an inlet, outlet and central motor – driven shaft. To
this shaft are fitted radial agitators. The cylinders are fitted with glass beads. The cells
suspension is added through the inlet and the disrupted cells come out through the outlet. The
body of the cell disrupter is kept cool while the operation is on. Eg. Dyno – mill, Ball mill,
French press, etc.

NON – MECHANICAL – PHYSICAL METHODS:

Osmotic Shock:
Osmotic shock and rupture with ice crystals are commonly used methods. By slowly
freezing and then thawing a cell paste, the cell wall and membrane may be broken, releasing
enzymes into the media. Changes in osmotic pressure of the medium may result in the release of
certain enzymes, particularly periplasmic proteins in gram – negative cells.

Heat Shock:
Breakage of cells by subjecting them to heat is relatively easy and cheap. But this
technique can be used only for a very few heat – stable intracellular products.

CHEMICAL METHODS:
Treatment with alkalies, organic solvents and detergents can lyse the cells to release the
contents.

ALKALIES:
Alkalie treatment has been used for the extraction of some bacterial proteins. However,
the alkali stability of the desired product is very crucial for the success of this method, e.g.
recombinant growth hormone can be efficiently released from E.Coli by treatment with sodium
hydroxide at pH 11.

ORGANIC SOLVENTS:
Several water miscible organic solvents can be used to disrupt the cells. Eg. Methanol,
ethanol, isopropanol, butanol. The organic solvent toluene is frequently used. It is believed that
toluene dissolves membrane phospholipids and creates membrane pores for release of
intracellular contents.
Detergents:
Detergents that are ionic in nature, cationic – cetyl trimethyl ammonium bromide or
aninonic – sodium lauryl sulfate can denature membrane proteins and lyse the cells.
Non – ionic detergents Triton X – low or Tween are also used some extent.

ENZYMATIC METHODS OF CELL DISRUPTION:

Cell disruption by enzymatic methods has ceratin advantages i.e. lysis of cells occurs
under mild conditions in a selective manner. This is quire advantageous for product recovery.
Lysozyme is the most frequently used enzyme and is commercially available (produced from hen
egg white). It hydrolyses β - 1, 4 – glycosidic bonds of the mucopeptide inbacterial cell
walls. The Gram – positive bacteria (with high content of cell wall mucopeptides) are more
susceptible for the action of lysozyme.
For Gram – negative bacteria, lysozyme in association with EDTA can break the cells. As
the cell wall gets digested by lysozyme, the osmotic effects break the periplasmic membrane to
release the intracellular contents.
Certain other enzymes are also used, although less frequently for cell disruption. For the
lysis of yeast cell walls, glucanase and mannanase in combination with proteases are used.

SOLID – LIQUID SEPARATION:

 The first step in product recovery is the separation of whole cells (cell biomass) and other
insoluble ingredients from the culture broth. Several methods are in use for solid – liquid
separation. These include filtration and centrifugation.
Filtration:
Filtration is the most commonly used technique for separation the biomass and culture
filtrate.
The efficiency of filtration depends on many factors – the size of the organism, presence
of other organisms, viscosity of the medium and temperature. Several filters such as depth filters,
absolute filters, rotary drum vacuum filters and membrane filters are in use.
Depth filters:
They are composed of a filament matrix such as glass wool, asbestos or filter paper. The
particles are trapped within the matrix and the fluid passes out. Filamentous fungi can be
removed by using depth filters.
Absolute filters:
These filters are with specific pore sizes that are smaller than the particles to be removed.
Bacteria from culture medium can be removed by absolute filters.
Rotary Drum Vacuum filters:
These filters are frequently used for separation of broth containing 10 – 40% solids (by
volume) and particles in the size of 0.5 – 10 µ m. Rotary drum vacuum filters have been
successfully used for filtration of yeast cells and filamentous fungi.
The equipment is simple with low power consumption and is easy to operate (Fig.4).
The filtration unit consists of a rotating drum partially immersed in tank of broth. As the
drum rotates, it picks up the biomass which gets deposited as a cake on the drum surface. This
filter cake can be easily removed.
Membrane Filters:
In this type of filtration, membranes with specific pore sizes can be used. However,
clogging of filters is a major limitation. These are two types of membrane filtrations – static and
cross – flow filtration (Fig. 5).
In cross – flow filtration, the culture broth is pumped in a crosswise fashion across the
membrane. This reduces the clogging process and hence better than the static filtration.

CENTRIFUGATION:
The technique of centrifugation is based on the principle of density differences between
the particles to be separated and the medium. Thus centrifugation is mostly used for separating
solid particles from liquid phase.
The different types of centrifuges are depicted in fig.6. and briefly described Lereunder.
Tubular bowl centrifuge: (Fig 6.a.)
This is a simple and a small centrifuge, commonly used in pilot plants. Tubular bowl
centrifuge can be operated at a high centrifugal speed and can be run in both batch or continuous
mode. The solids are removed manually.
Disc Centrifuge: (Fig. 6.b.)
It consists of several discs that separate the bowl into settling zones. The feed slurry is
fed through a central tube, the clarified fluid moves upwards while the solids settle at the lower
surface.
Multi chamber Centrifuge:
This is basically a modification of tubular bowl type of centrifuge. It consists of several
chambers connected in such a way that the feed flows in a zig – zag fashion. There is a variation
in the centrifugal fore in different chambers. The force is much higher in the periphery chambers,
as a results smallest particles settle down in the outer most chamber.

Concentration:
 The filtrate that is free from suspended paticles (Cells, cell debris, etc) usually contains
80 – 98% of water. The desired product is a very minor constituent. The water has to be removed
to achieve the product concentration. The commonly used techniques for concentrating biological
products are evaporation, liquid – liquid extraction, membrane filtration, precipitation and
adsorption. The actual product adopted on the nature of desired product (quality and quantity to
be retained as far as possible) and the cost factor.
Evaporation:
Water in the broth filtrate can be removed by a simple evaporation process. The
evaporators in general have a heating device for supply of steam and unit for the separation of
concentrated product and vapour, a condenser for condensing vapour, accessories and control
equipment. The capacity of the equipment is variable that may range from small laboratory scale
to industrial scale. Some of the important types of evaporators in common uses are as follows:
Eg. Plate evaporators, falling film evaporators, forced film evaporators, centrifugal forced film
evaporators.
LIQUID – LIQUID EXTRACTION:
The concentration of biological products can be achieved by transferring the desired
product (solute) from one liquid phase to another liquid phase, a phenomenon referred to liquid –
liquid extraction. Besides concentration, this technique is also useful for partial purification of a
product. The efficiency of extraction is dependent on the partition coefficient i.e. the relative
distribution of a substance between the two liquid phases.
The process of liquid – liquid extraction may be broadly categorized as ‘extraction of low
molecular – weight products’ and extraction of ‘high molecular weight products’.
EXTRACTION OF LOW MOLECULAR WEIGHT PRODUCTS:
By using organic solvents, the lipophilic compounds can be conveniently extracted.
EXTRACTION OF HIGH MOLECULAR WEIGHT PRODUCTS:
Proteins are the most predominant high molecular weight products produced in
fermentations industries. Organic solvents cannot be used for protein extraction, as they lose
their biological activities. They are extracted by using an aqueous two – phase systems.
Aqueous two – phase systems (ATPs):
They can be prepared by mixing a polymer (e.g. polyethyle glycol) and a salt solution
(ammonium sulphate) or two different polymers. Water is the main component in ATPs, but the
two phases are not miscible.
Cells and other solids remain in one phase while the proteins are transferred to other
phase. The distribution of the desired product is based on its surface and ionic character and the
nature of phases. The separation takes much longer time by ATPs.

PRECIPITATION:
Precipitation is the most commonly used technique in industry for the concentration of
macromolecules such as proteins and polysaccharides. Further, precipitation technique can also
be employed for the removal of creation unwanted by products, e.g. nuclei acids, pigments,
neutral salts, organic solvents, high molecular weight polymers (ionic or non – ionic), besides
alteration in temperature and pH are used in precipitation.
Isoelectric point:
Enzymes and other proteins are highly charged molecules and can be precipitated with
appropriate change neutralizing chemicals. Once their changes are broken they form aggregates
and settle down as precipitate. When an acid or base is added, the enzyme protein can be brought
to its isoelectric pH. At this pH, there is no net charge on enzyme molecules and electrostatic
repulsion between them is low so that they tend to aggregate. Therefore adjusting the pH to the
isoelectric point of a protein causes its precipitation.
Salting Out:
 ‘Salting Out’ of proteins is achieved by increasing the ionic strength of a protein
containing solution by adding salts such as (NH4)2SO4 or Na2SO4. The added ions interact with
water more strongly, causing protein molecules to precipitate. The solubility of proteins in a
solution as a function of the ionic strength of the solution is given by,
log (S/S0) = – K'S (I)
Where, S – Solubility of protein in solution (g/l)
S0 – Solubility of protein when Z = 0, Z – ionic strength of solution
K'S – Salting – out constant, which is a function of temperature and pH.
Organic solvents:
Ethanol, acetone and propanol are the commonly used organic solvents for protein
precipitation.
Organic solvents addition at low temperature (T< - 5°C) cause the precipitation of
proteins by the reducing the dielectric constant of the solution. Thesolubility of protein as a
function of the dielectric constant of a solution is given by
log (S/S0) = – K' /DS2
Where, DS is the dielectric constant of a solution results in stronger electrostatic forces between
the protein molecules and facilitates protein precipitation. The addition of solvents reduces
protein – water molecule interactions and therefore decreases protein solubility.
NON – IONIC POLYMERS: Polyethylene glycol (PEG) is a high molecular weight non – ionic
polymer that can precipitate proteins. It reduces the quantity of water available for protein
salvation and precipitates proteins.
IONIC POLYMERS: The charged polymers such as polyacrylic acid and polyethyleneimine are
used. They form complexes with oppositely charged protein molecules that causes charge
neutralization and precipitation.
Increases in temperature:
The heat sensitive proteins can be precipitated by increasing the temperatures.
PRECIPITATION BY LIGANDS:
Ligands with specific binding sites for proteins have been successfully used for selective
precipitation.
FURTHER PURIFICATION PROCEDURE:
Further purification of enzymes after extracting the crude enzyme extract by the above
methods, involves Dialysis, Chromotography and electrophoresis.
Dialysis:
Dialysis is the process that is used to remove small molecules from enzyme. For this
enzyme precipitate obtained in previous step is dissolved in a small quantity of buffer solution in
which the enzyme was originally extracted.
The solution can be taken in a dialysis bag (may be a semi permeable membrane such as
a cellulose membrane with pores). The bag is suspened in either distilled water of a buffer of
known molarity and ionic composition. Some other salts or chemical smay have to be added
sometimes in the outer solution to prevent the loss of enzyme activity during dialysis (Fig. 7.).

Molecules having dimensions significantly greater than the pore diameter are retained
inside the dialysis bag, whereas smaller molecules and ions traverse the pores of such a
membrane and emerge in the dialysate outside the bag.

CHROMATOGRAPHY:
Chromatographic separation of proteins is the most common method of enzyme
purification.
It is basically an analytical technique dealing with the separation of closely related
compounds from a mixture. Chromatography usually consists of a ‘stationary phase’ and ‘mobile
phase’. The stationary phase is the porous solid matrix packed in a column onto which the
mixture of compounds to be separated is loaded? The compounds are elated by a mobile phase.
The eluate from the column can be monitored continuously (E.g. protein elution can be monitored
by ultraviolet adsorption at 280 nm and collected in fractions of definite volumes.
The different types of chromatography techniques used for separation (mainly proteins)
along with, the principles are given in Table.1.
Table 1: Chromatographic techniques along with the principles for separation of proteins

Chromatography Principle
Gel – filtration (size exclusion) Size and Shape
Ion – exchange Net charge
Affinity Biological affinity and molecular recognition

A large number of matrices are commercially available for purification of proteins e.g.
agarose, cellulose, polyacrylaminde, porous silica,m cross – linked dextan polystryrene. Some of
the important features of selected Cheomalographic techniques are briefly described.

GEL – FILTRATION CHROMATOGRAPHY:

In this chromatography, various proteins are separated on the basis of differences in their
molecular sizes. This type of chromatography is also known as molecular exclusion
chromatography or molecular sieve chromatography (Fiog. 8)
A column made up of glass or steel is taken and packed with a gel (E.g. Sephadex,
Sepharose and Biogel) or Porous bead made of an insoluble but highly hydrated polymer such as
dextran or agarose or polyaccylamind. (The beads are typically 100µ m (0.1 mm) in diameter).
A solution mixture containing molecules of different sizes (e.g. different proteins) is applied to
the top of the column and eluted. The smaller molecules enter the gel beads through their pores
and get trapped. On the other hand, the larger molecules cannot pass through the pores and
therefore come out first with the mobile liquid.
The elution volume is logarithmically proportional to the molecular weights.

ION EXCHANGE CHROMATOGRPHY:

It involves the separation of molecules based on their surface charges. Ion exchangers are
of two types:
• Cation exchangers – which have negatively charged groups like carboxymethyl and
sulfonate, phosphocellulose.
• Anion exchangers – With positively charged groups like diethyl amino ethyl (DEAE),
quaternary ammonium, groups such as triethyl amino ethyl (TEAE) and QAE.
The matrix material for the column is formed from beads of some inactive material, often
a carbohydrate such as cellulose or dextrans.
In ion – exchange chromatography, the pH of the medium is very crucial, since the net
charge caries with pH. In other words, the pH determines the effective charge on hboth the target
molecules and the ion – exchanger. (Fig. 9)
If an protein has a net positive charge at pH 7, it will usually bind two a column of beads
containing carboxylate groups, whereas a negatively charged protein will not.
A positively charged protein bound to such a column ear than be eluted (released) by
increasing the concentration of sodium chloride or another salt in the elulting buffer because
sodium ions compete with positively charged groups on the protein for finding to the column.
Proteins that have a low density of net positive charge will emerge first, followed by those having
a higher charge density.
Positively charged proteins (Cationic proteins) can be separated on negatively charged
carboxymethyl – cellulose (CM – Cellulose) columns. Conversely, negatively charged proteins
(anionic proteins) can be separated by chromatography on positively charged diethylamine ethyl
– cellulose (DEAE – Cellulose) columns.

AFFINITY CHROMATOGRAPHY:
In this method, enzymes are purified according to their speficicity for a particular
substrate or cofactor.Affinity chromatography is based on the highly specific interaction between
solute molecules and ligands attached on polymeric or ceramic beads in a packed column.
The concept of affinity chromatography is described in Fig.10.
The matrix is usually agarrose. However pollyaisylamide, hydroxyethyl methaerylate,
cellulose and porous glass can also be used as the matrix bead. Spacer arms between the matrix
and ligand are usually llnear aliphatic hydrocarbons. The use of space arms between the matrix
and ligand may reduce the steric hindrance generated by the matrix.
Coupling between the matrix and ligand depends on the functional groups present on the matrix
and ligand. Chemically reactive groups on the support matrix usually are – OH, - NH2 or –
COOH groups. If the reactive group on the matrix is an – OH group (polysaccharides, glass
hydrooxyalkyl methacrylate), then cyanogens bromide (CnBr) is used as a coupling agent. The
cyanogens bromide – activated agarose reacts with primary amine groups present in proteins that
act as ligands.
After the desired solutes are fbound to theligand, elution is achieved by changing the pH or ionic
strength in the column. Ligand – solute molecule interaction in affinity chromatography are very
specifi. That is an enzyme inhibitor or substare may be used as a lighand in separating specific
enzyme from a mixture.

ELECTROPHORESIS:
Electroporesi is a technique in which enzyme molecules are separated by difference in
their net charge in the presence of an externally applied electric field. This technique is routinely
used in enzyme purification and isozyme separation in the laboratories, although it has found only
limited application at large scale. Since the technique is time consuming and is a bit expensive.
Various types of instrumental approaches have been used to separate and purity charged
molecules using electrophoresis. However, the most common method for purifying enzymes is
though electrophoresis on polyacrylamide gel. Polycrylamide is a polymer of acrylamide and
methylene bisacarylamide and when prepared as a gel it is transparent, thermostable, non – ionic
and extremely regular in structure.
The gel may be taken either in the form of a column or a slab, although the later is
preferred over the former (F.g 11). The protein mixture is loaded in the gel and the components
are separated under a direct current of constant voltage. The migration rate of various components
of the mixture is dependent upon their charge and molecular weight.
Sodium doderyl sulphate polyacrylamide gel electrophoresis (ADS – PAGE):

This form of polyacrylamide gel electrophoresis is the most widely used method for
analyzing protein mixtures qualitatively. It is particularly useful for monitoring protein
purification and because the method is based on the separation of proteins according to size, the
method can also be used to determine the relative molecular mass of proteins.
SDS, (CH3 – (CH2)10 – CH2OSO3– Na+) is an anionic detergent, samples to be run on SDS
– PAGE are firstly boiled for 5 min in sample buffer containing β - mereaptoethanol and SDS.
The mercaptoethanol reduces any disulphide bridges present that are holding together the protein
tertiary structure and the SDS binds strongly to and denatures the protein. Each protein in the
mixture is therefore fully denatured by this treatment and opens up into a red – shaped structure
with x series of negatively charged as molecules along the polypeptide chain.
Once the samples are loaded a current is passed through the gel. The negatively charted
protein – SDS complexes now continue to move towards the anode and because they have the
same charge per unit length, they travel into the separating gel under the applied electric field
with the same mobility. However, as they pass through the separating gel the proteins separate,
owing to the molecular siwving properties of the gel. Quite simply, the smaller the protein the
more easily it can pass through the pores of the gel, whereas large proteins are successively
retarded by frictional resistance due to the sieving effect of the gels.
The sample buffer also contains an ionisable tracting due, usually bromophenol blue, that
allows the electrophoretic run to be monitored. When the dyue reaches to bottom of the gel, the
current is turned off and the gel is removed from between the glass plates andn shjaken in an
appropriate stain solution (usually Coomassie Brilliant Blue) for a few hours and then washed in
destain solution overnight. The destain solution removes unbound background due from the gel
leaving stained proteins visible as blue bands on a clear background.

DRYING AND PACKING:

The concentrations form of the enzyme can be obtained by dying. This can be done by
film evaporator or freezer dryers (lyophilizers).

FREEZE DRYING:
Freeze drying or lylophilization is the most preferred method for dying and formulation
of an enzyme. This is mainly because freeze – drying usually does not cause loss of biological
activity of the enzyme.
Lyophilization is based on the principle of sublimation of a liquid from a frozen state. In
the actual techniques the liquid containing the product in frozen and then dried in a freeze – dryer
under vacuum. The vacuum can now be released and the product containing vials can be sealed.
The dried enzyme can be packed and marketed. For certain enzymes, stability can be
achieved by keeping them in ammonium sulphate 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 micro organisms and should not cause allergic reactions.

MONITORING OF PURIFICATIONS OF AN ENZYME:

Successful enzyme purification is recognized by high specific activity of the final

purified reaction, may fold purification and high yield.

Yield (in fraction ) =

Units of enzyme in fraction
Units of enzyme in original preparation
The amount of enzyme present in a particular fraction is usually expressed in terms of
units, which are based upon the rate of the reaction that the enzyme catalyses. One International
unit of an enzyme that will convert 1µ mole of substarate to product in 1 min under defined
conditions (such as temperature at 30°C and the optimum pH).
A good indicator of enzyme purification is the fold purification. It is determined by
dividing the specific activity of enzyme in a particular fraction by the original specific activity of
enzyme in a particular fraction by the original specific activity of enzyme in a particular fraction
by the original specific activity (of crude homogenate).
Specific activity of fraction
Fold purification =
Original specific activity
The specific activity relates to the total activity in a fraction to the toal amount of protein
present in that fraction.
Total Units of enzyme in fraction
Specific activity =
Total amount of protein in fraction
 A steady increase in the specific activity of fractions from the crude homogenate
indicates the successful approach in enzyme purification.
CENTRIFUGATION
Centrifugation is used to separate particles of size between 100 and 0.1µ m from liquid
by centrifugal forces. The sub-cellular location of many enzymes may be revealed by
microscopy, provided suitable fixation and staining procedures are followed.
The sedimentation characteristics of the various sub-cellular organelles are different, so it is
possible to separate them by centrifugation of a tissue homogenate, and then to investigate which
enzymes are associated with each cell fraction.
There are two types of centrifugation, they are as follows,
A. Differential centrifugation
B. Density-gradient centrifugation

A) Differential Centrifugation
The simplest and most cuidely used method for separating the various sub-cellular organelles
from each other is different centrifugation.
A tissue homogenate is prepared in a medium of low density (e.g. 0.25 M sucrose) and
centrifuged in a series of stages, the centrifugal field of each step being higher than for the
previous one. At the end of each stage the sedimented pellet, consisting of particles of similar
sedimentation characteristics is removed. (fig .1)
A simplified scheme for the fractionation a rat liver homogenate is shown in fig. 2

Liver homogenate

Centrifuge 1000 g, 10 minutes

Supernatant

Pellet Centrifuge 10,000 g, 10 minutes

Nuclei unbroken cells

Supernatant
Pellet
miltochondrialysosomes Centrifuge 100,000 g, 90
minutes

Pellet supernatant
Microsomes Free ribosomes
enzymes of cytosol
Fig 2. Simplified scheme for the fractionation by differential centrifugation of a 10% rat liver
homogenate in 0.25M sucrose at 0° C. Note that microsomes are not organelles but particles
produced during homogenization. Largely from endoplasmic reticulam, and consisting a specific
sedimentation fraction.

B) DENSITY-GRADIENT CENTRIFUGATION
Centrifugation techniques where the density of the suspending medium is not uniform
throughout. There are two methods of density gradient centrifugation, the rate zonal technique
and the isopycnic (isodensity or equal density) technique, and both can be used when the
quantitative seperation ofall the components of a mixture of particle is required.
RATE ZONAL CENTRIFUGATION
Particle seperation by the rate zonal technique is based upon differences in size, shape
and density of the particles, the density and viscosity of the medium and the applied centrifugal
field. (fig.3)
Seperation of similar types of similar types of particles by the rate, zonal technique is
based mainly upon differences in their size. Subcellular organelles, therefore, such as
mitochondria, lysosomes and peroxisomes, whoch have different densities but are similar in size,
do not separate efficiently using this method.
The technique involves carefully layering a sample solution on top of a preformed liquid
density gradient, the highest density of which does not exceed that of the densest particles to be
separated.
The sample is then centrifuged, until the desired degree of seperation is effected, i.e. for
sufficient time for the particles to travel through the gradient to form discrete zones or bonds
(fig.3) which are spaced according to the relativevelocities of the particles.
ISOPYNIC CENTRIFUGATION
Isopynic centrifugation depends solely upon the density of the particle and not its shape
or size and is independent of time, the size of the particle affecting only the rate at which it
reaches it s isopycnic position in the gradient.
The technique is used to separate particles of similar size but of differing density.
The subcellular organelles such as Gol;gi apparatus, mitochondria, and peroxisomes can be
effectively separated.The sample is initially mixed with the gradient medium to give a solution of
uniform density, the gradient self-forming, by sedimentation equilibrium, during centrifugation.
(fig.4).

CHARACTERIZATION OF ENZYMES
Molecular Weight Determination of an Enzymes
The information regarding the complete three-dimensional structure of an enzyme at
atomic or near atomic resolution provides a basis for understanding the properties of the enzyme,
especially its catalystic activity. The determination of this detailed strucure is a daunting task for
even the smallest enzyme and has been found to require several years of work. Assuming that a
supply of purified enzyme is available, the primary stage of the work is the determination of
relative molecule lass Mr.
The term relative molecular mass (Mr) is now used in place of molecular weight. Mr is a
dimensionless number and is the ratio of the molecular mass of a molecule to 1/12 the mass of
one atom of 12C. The latter value is known as a Dalton. Molecular masses are often quoted in
Daltons or kilodaltons.
The determination of Mr.
Enzymes are macromolecules with Mr values ranging from about 10,000 to several
million. Therefore methods for determining Mr such as mass spectrometry or freezing point
depression which are applicable to small molecules are not suitable for enzymes. The majority of
present-day determinations of Mr values of enzymes are performed by use of one or more of the
following techniques,

1. Gel filteration
2. Ultracentrifugation
3. Sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE).
4. GEL FILTERATION

Which separate molecules on the basis of size provides one way of doing this. A packed column
is calibrated by applying proteins of known molecular weight to the top of the column and
determining the volume of buffer required for the elution of each protein; as each protein leaves
the column there should be an absorbance peak at 280nm in the column eluate, thus providing a
simple way of monitoring the elution.
From the data obtained, a graph may be drawn of elution volume against molecular
weight. The protein of unknown molecular weight is then passed through the column and its
elution volume determined exactly as for the marker proteins. Hence, by reference to the
calibration graph, the molecular weight of the protein may be estimated.

ULTRA CENTRIFUGATION
This technique is also widely used for the determination of molecular weights of proteins.
In this, the ultracentrifuge is operated at high speeds to generate centrifugal forces that are
sufficiently intense to sediment the macromolecules. The sedimentation of an enzyme can be
monitored by suitable optical means, and from the measurements the sedimentation coefficient
‘S’ can be calculated.
The sedimentation coefficient cannot by itself be used to calculate the Mr of the enzyme,
since the rate of sedimentation will depend on other factors such as the shpe of the
macromolecule. However, if we have other information, such as the value of the diffusion
coefficient (D) of the macromolecule, its partial specific volume (v) and the density of the
solution (ρ), the Mr can be calculated from the formula,
This is known as the Svedberg equation.
Where, R – gas constant, T – absolute temperature and D – diffusion constant of the molecule.

RTS
Mr =
D(1 – ύρ)

SDS – Polyacrylamide gel electrophoresis:

The mobility of a charged molecule in an electric field is a function of various factors

such as the size and shape of the molecule and the charge it carries and it would therefore be
expected that electrophoresis would not normally give any reliable estimates of Mr.
Hedrick and Smith have shown that the Mr of a protein can be estimated by measuring its
mobility as a function of acryl amide concentration. However, this method is only reliable if the
standard proteins for calibration have the same shape, degree of hydration and partial specific
volume as the unknown protein.
In the case of proteins we use the detergent sodium dodechyl sulphate, SDS which has
the structure CH3(CH2)11OSO3– Na+. A reducing agen6t such as 2 – merraptoethanol is also added
to break disulphide bonds. Addition of the detergent has two principal effects.
 1. Nearly all proteins bind SDS in a more or less constant ratio, 1.4 g SDS per gram of
protein. Since the negative charge carried by the SDS overwhelms any charge carried by
the protein, the protein – SDS complex has a constant charge/ mass ratio.
2. The three dimensional structure of the protein is Dost and the protein – SDS complex is
rod – shaped with a length proportional to the Mr of the protein.

Since the charge and hydrodynamie properties of the protein – SDS complex are both simple
functions of the Mr, the mobility of electrophoresis is a function of Mr alone. The larger
molecules have lower mobilities means that the hydrodynamic effects (i.e., sieving) predominate
over the charge effects.
Relative Mobility →

1.0 –

0.5 –

A graph of the logarithm of the unknown protein can be determined by reference to the
standard line. Different ranges of Mr can be examined by the use of gels of different
polyacrylamide concentration or by the use of gradienet gels.
0.0 – | | |
4.0 4.5 5.0
Uses of Mr information: log (Mr) →
The Mr of an enzyme is a fundamental piece of information because it cane be used in a
variety of ways such as in consideration of composition, catalytic activity and ligand binding.
Measurements of Mr made in the absence of presence of denaturing agents will show whether or
not the enzyme is composed of subunits and may indicate the number of such subunits.

IMMONOCHEMICAL METHODS OF ENZYMATIC ASSAY

These are currently two main types of enzyme – Immunoassay (EIA), they are

• Enzyme – Linked Immunosorbent assay (ELISA)

• Enzyme Multiplied Immnoassay Technique (EMIT)

Enzyme – Linked Immunosorbent Assay (ELISA):

‘ELISA’ is an immunological technique used for the quantitative determination of the

concentration of certain antigens or antibodies.
The ‘ELSIA’ technique was first introduced in 1970 by Engvall and Perlmann.
An antibody (AB0 reacts with the concerned Antigen (Ag) in a highly specific manner
(i.e. an antibody reacts only with that determinant or region of an antigen for which it is specific)
to produce an Ag – Ab complex.
ELISA PROCEDURE:
A generalized procedure and the basic principle of ELISA is as follows:

Primary Reaction:
• The antigen (Ab) of interest is immobilized on the surface of a test tube, petriplate or
micro titter well.
• Now, Antibody (Ag) specific to the Ag (Ab) is added and allowed to react with the
absorbed antigen. Unreacted molecules of the Ab(Ag) are washed away, leaving only the
Ag- Ab complex.

Secondary Reaction:
In the secondary reaction on anti – immunoglobulin (anti – Ig: an antibody that reacts
with the antibody0 is added into the vessel and allowed to react with the Ag – Ab complex
already formed; the anti – Ig binds to the antibody component of the Ag – Ab complex.
The anti – Ig is linked to an appropriate enzyme molecule (i.e. labeled with an enxyme
molecule) in such a way that its anti – Ig activity is not impaired (eg. Alkaline phosphatase,
horseradish peroxidase and β - galactosidase).
An enzyme conjugated with an antibody reacted with a colourless substrate to generate a
coloured reaction product. This substrate are known as chromogenic substrates.
The unreacted anti – Ig is washed away and finally substrate of the enzyme is added
alongwith the necessary reagents to develop colour due to the enzyme activity.
The intensity of colour is proportional to the enzyme concentration; therefore colour
intensity is used to determine the quantity of antigen or antibody or simply to detect their
presence. The sensitivity of ELISA isin the range of nanograms (10-4g)/ml.
For an easy and rapid assay a computerized ELISA reader may be used.

VARIOUS TYPES OF ELISA (Fig. 1):

• Direct ELISA (Sandwich ELISA)
• Indirect ELISA

DIRECT ELISA:
Antigen can be detected or quantititated by a sandwich or direct ELSIA.
In this technique, the primary antibody (Ab1) is immobilized on a microtiter well.
A sample containing antigen is added and allowed to react with the bound antibody. After
any free Ag is washed away, the presence of antigen bound to the antibody is detected by added
an enzyme – conjugated antibody specific for a different epitope on the antigen is added and
allowed to react with the bound antigen.
Any free Ab2 then is washed away and a substrate for the enzyme is added. The coloured
reaction product that foams is measured by specialized spectrophotometric plate readers, which
can measure the absorbance of a 90 – Well plate in less than a minute.

INDIRECT ELISA:
Antibody can be detected or quanititated with an indirect ELISA.
Serum or some other sample containing primary antibody (Ab1) is added to an antigen
coated microtiter well and allowed to react with the antigen attached to the well. After any free
Ab1 is washed away the presence of antibody bound to the antigen is detected by added an
enzyme – conjugated secondary anti – isotype antibody (Ab2), which binds to the primary
antibody. Afy free Ab2 is washed away and a substrate for the enzyme is added. The amount of
colored reaction product that forms is measured.
ENZYME MULTIPLIED IMUNOASSAY TECHNIQUE (EMIT):
This is a homogeneous procedure, since no separation of components is required. As with
some types of ELISA, an enzyme is attached to a specimen of antigen to act as a label. However,
in contrast to ELISA, the subsequent binding of the enzyme – labeled antigen to the antibody
results in a significant change in activity of the enzyme in most types of EMIT procedure,
enzyme activity is lost completely on binding to the antibody either as a result of steric lindranace
or conformational changes. In such an assay, specified amounts of antibody and enzyme – labeled
antigen are mixed with the sample. The antigen in the sample (Ag) then competes with the
enzyme – labeled antigen (E. Ag) for the available antibody (Ab) according to the following
reactions:
Ag + Ab Ag. Ab
E.Ag + Ab E'.Ag.Ab
The enzyme – antigen – antibody complex formed (E'.Ag.ab) has no catalytic activity, so
the total activity present is contributed by E.Ag. Hence the antigen content of the sample may be
calculated from the total enzyme activity at equilibrium; the more antigen there is in the sample,
the less E.Ag is able to bind to the antibody, so the greater will be the total enzyme activity.
EMIT has so far been used principally for the determination of relatively small (io.e. non
– protein) molecules, e.g. barbiturates. The enzymes which have been involved include lysozyme
and malate dehyrogenase.

RADIO IMMUNO ASSAY (RIA):

One of the most sensitive techniques for detecting antigen or antibody is radio immuno
assay (RIA).
The technique was first developed by two endocrinologists, S.A. Berson and Rosalyn
yalow in 1960. It combines the specificity of the immune reaction with the sensitivity of radio
isotope techniques.

PRINCIPLE:
The principle of RIA involves competitive binding of radio labeled antigen and
unlabelled antigen to a high affinity antibody.
The antigen is generally labeled with a gamma – emitting isotope such as 125I. The
labeled antigen is mixed with antibody at a concentration just saturates the antigen – binding sites
of the antibody molecule and then increasing amounts of unlabelled antigen of unknown
concentration are added.
The antibody does not distinguish labeled from unlabelled antigen and of the two kinds of
antigen compete for available binding sites on the antibody.
With increasing concentration of unlabelled antigen, more labeled antigenwill be
displaced form the binding sites.

4Ag* + 4 Ab 4Ag*Ab
4Ag + 4Ag* + 4 Ab 2Ag*Ab + 2Ag Ab + 2Ag* + 2 Ag
12Ag + 4 Ag* + 4Ab Ag*Ab + 3 Ag Ab + 3 Ag*+ 9 Ag

where, Ag* - Labeled antigen

Ag - Unlabeled antigen
Ab - Antibody

To determine the amount of labeled antigen bound, the Ag – Ab complex is precipitated

to separate it from free Ag. (Ag not bound to Ab) and the radio activity in the precipitate is
measured.
 It will be realsed that the labeled antigen competes with the antigen in thesample for the
available binding sites on the antibody, so the higher the concentation of antigen in the sample,
the less radioactive antigen will be able to bind to the antibody and the greater will be the radio
active content of the free antigen fraction. In this way, the concentration of antigen in the sample
can be estimated.

Total amount of Free-labeled antigen Amount of labeled

– =
labeled antigen remaining antigen bound
A typical radio immunoassay calibration curve:

A standard curve has been plotted between bound labeled antigen (%) and known concentrations
of unlabeled antigen added.
Once a standard curve had been plotted unknown concentrations of the unlabeled antigen
can be determined from the standard curve.
Antibodies for the RIA of enzymes may be prepared in the form of antisera by
immunizing rabbits with the required enzyme; for example, the blood of a rabbit immunized in
the foot pad with human pancreatic α - amylase contains sufficient antibodies within a few
weeks to be useable as an antiserum.
The part of the enzyme which binds to the antibody is likely to be quite distinct from the
active site, so RIA and catalytic assay of enzymes can give different information. Catalytically
inactive forms of enzymes (eg. Prienzymes) may be detected by RIA if they contain the structural
part which is recognized by the antibody. On the other hand, QRIA procedure is likely to be
specific for one particular isoenzyme (that used to produce the antibody). Hence, it can be seen
that catalytic assay and RIA procedures are not alternat6ives but can be used to complement each
other RIAs also have the advantage of being particularly sensitive (upto a thousand times more
sensitive than catalytic assays).
ENZYME ASSAYS
An assay is a measurement of a given enzyme of known characteristics in a sample. Ideally, this
means measuring a specific and characteristics biological property (or ability to catalyze a
chemical reaction) of the desired enzymes. Less ideally, we use a method, which does not in
principle, derive from the biological activity of the protein, but is a general method in which it
has a specific behaviour. Thus enzyme assay may be
(1). The former, catalytic assay and
(2). The later, stoichiometric assay
A catalytic assay is obviously more sensitive than a stoichiometric assay, which measures
amount of the enzyme by measuring a molar equivalent amount of something, such as bound
Coomassie Blue or silver stain. In a catalytic assay the amount of measured product, at least in
principle, increases indefinitely with time of incubation, yielding a greater sensitivity while a
stoichiometric assay only reaches equilibrium.

There are two general purposes for enzyme assay:

1. To measure how much of the enzyme is present in the sample. The enzyme is the variable
measured.
2. With a constant amount of the enzyme present, how does its activity vary with conditions
such as pH, temperature, variation of substrate concentration, effect of inhibitors, etc or
in prior incubation (stability to heat, chemical modification, etc)

Assay may be either

 1. Continuous: A continuous assay is one in which some change caused by the enzyme is
monitored continuously while it is happening such as absorbance of light, uptake of base
or acid in a pH – stat, or change in oxygen electrode. The quantity monitored usually
expressed as a line graph and usually one is interested in the slope of the line, the rate of
the reaction, which should be proportional to the amount of your protein present.
2. Discontinuous: In a discontinuous assay or stop – sample assay, on the other hand, the
reaction is stopped at some definite time, and do something to the reaction mixture, such
as adding acid or base or heating to develop a color, or separating radioactive products
from starting material, which makes possible the determination of amount of product
formed by that time (or amount of starting material remaining). Reactions are stopped by
adding a solution which makes further reaction impossible, such as strong acid or base or
EDTA for Mg++ dependent reaction, boiling.
3. Semi continuous: There is also semi – continuous assays, where you can make a number
of measurements on the same reaction mixture while the reaction is running, but each
measurement takes a finite amount of time – manommetric and viscometric assays, for
instance. In principle any stop – sample assay can be made semi – continuous, by taking
multiple samples from a single assay mixture, instead of setting sponutiple assays and
stopping them at different times.

Note: Enzyme activity is measured as the amount of substate lost (or product gained) per unit
time, and it should also be specimen used for assay. In 1961, the enzyme commission of the IUB
defined an ‘Enzyme Unit’ (U), later to be known as an International Unit (IT), as the amount of
enzyme causing loss of 1μmol substrate per minute under specified conditions. Later, in 1973, the
commission of Biochemical Nomenclature introduced the Katal (Kat) as the system International
(SI) units of enzyme activity; this is defined as the amount of enzyme causing loss of 1 mol
substrate per second under specified conditions. Both units are in current usage.
For a successful assay system, the reaction being catalyzed should be capable of being
accurately monitored. That is, during reaction there should be change in optical, electrical or
other properties that is directly proportional to the product formed or substrate utilized. The
product formed/substrate utilized can be directly monitored (either by stop and sample method or
by continuous method) or any of the following method. If product or substrate is not having any
detectable trait, it may be possible to study the course of reaction indirectly by coupling it to
another detectable reaction.
Mg2+
D-glucose + ATP ------------------→ D-glucose-6-phosphate + ADP
Hexxokinase
For example, the product in this reaction (D-Glu-6-P) is not easily assayable because
there is no spectrophometric absorbtion for this compound. But if this rection is coupled to the
production of D-glucono δ -lactone 6-phosphate by the reduction of NADP + to NADPH using
glucose-6phosphate dehydrogenase enzyme. The amount of NADPH can be monitored easilyas it
has an absorption spectrum at 340nm, whereas NADP+ is not having. Thus D-glucose 6-
Phosphate can be indirectly measured in a couple assay.
D-glucose + ATP D-glucose-6-phosphate + ADP

NADP+
NADPH+H+
D-glucono δ -lactone 6-phosphate
Another example of coupled assay is
D-alanine + O2 pyruvate + NH4+ + H2O2 (D-alumino acid oxidase)
H2O2 + chromogen colored product + H2O (peroxidase)
Cycled assays are also there which use a small amount of a compound as rate-limiting
intermediate in reactions going both ways. Strictly these are assays for the compound rather than
for an enzyme, but the amount of compound started with might be the product of an enzyme
reaction carried out on a very small scale, say one cell. An example,
Pyruvate + NADH +H+ L-lactate + NAD+ (lactate dehydrogenase)
Ppyruvate + H2O2 L-lactate +O2 (lactate oxidase)

IIn this case the amount of pyruvate is the rate limiting compound and increase in concentration
of pyruvate is directly pproportional to the rate of reaction and it can be monitored as the
absorption of NADH.
Some standard methods for enzyme assay methods which are fairly quick and easy are:
1. Colorimetry: Any substrate or product which absorb radiations in visible range can be
monitored by this method. The colorimetric assays can be extended by the use of artificial
substrate and by the production of colored derivates of the substrate or product. In some cases,
substrate or product containing certain functional group which which can also be converted to
colored derivative.
2. Spectrophotometry: This method depends on absorption of light at a specified wave length
(When the wave length range is only narrowed down by filters) by substrate or product.
Spectrophotometry typically measures compounds in the 10-3 – 10-6 M.
Two products are very commonly monitored spectrophotometrically –the coenzymes
NADH and NADPH, which have a molar extinction coefficient of 6200 L/mole.cm at 340nm.,
and p-nitrophenol, which has an extinction coefficient of 18,300 L/mole.cm. Mny hydrolytic
enzymes are assayed using p-nitrophenyl esters and glycosides, which are artificial substrates.
Many reactions are coupled to production of NADH or NADPH, or their disappearance, because
they are so convenient to observe.
If a product absorbs uniquely and the spectrophotometer is attached to a recorder this can be a

continuous assay; also, modern spectrophotometers allow us to determine the rate directly

through the instruments software.

3. Fluorimerty: Some compounds when excited by higher-energy light, UV or visible, re-emit

light lower-energy of a longer wave length. This assay is very sensitive, down to 10-9 M.
Disadvantage is fewer compounds fluoresce, and the instrumenty is more expancive. It can be
more specific, since the wave length of both the exciting and emiotted light can be selected.
NADH and NADPH can fluoresce, and can be measured moer sensitively. Methylumbelliferol
and methylumbelliferylamine are the fluorescent equivalents of p-nitrophenol; but parent
glycosides of these compounds, esters and amides donot give fluorescence, so assays for
glycosidases, esterases and amidases can use them as artificial substrates.
4. Luminometric assay: In this assay, light is produced by the chemical reaction without prior
irradiation. The best known in assay of ATP by the firely tail luciferin-luciferase reaction, for
which ATP is a cofactor, but now of some importance is measurement of Ca ++ by the jellyfish
protein, which undergoes a light-producing oxidative reaction when it binds Ca++.
Luciferin +ATP +O2 Oxyluciferin + AMP + PPi + CO2 + Light
5. Titration: This assay is with an instrument called a pH-stat, which is essentially a pH meter
controlling an automatic buret, so that if the pH drops below a set value base is added to restore
it, and the amount of base is added is recorded. The same can be done for acid added to counter a
rising pH. Thus reactions producing acid, such as the hydrolysis of acetyltyrosine ethyl ester, a
chymotrypsin substrate, can be followed continuously, by plotting a chart recording of base
addition vs time, recording the total amount added over the period of linearity of the rate. On the
other hand, reactions producing acid or base can be measured spectrophotometrically if an
indicator is present.
6. Manometric assays: This is applicable if either product or substrate is a gas. The assay
measures uptake or release of a gas in a closed system. Enzymes which can be measured
manometrically include glutamate carboxylase, catalase, monoamine oxidase. These enzymatic
reaction results in the production of CO2 or O2. This can be monitored using Warburg manometer
or respirometers.
2H2O2 2H2O + O2 (gas)
7. Oxygen electrode and ion-selective electrode: The development of ion-selective electrode
such as those for ammonium ion, and the oxygen electrodes measuring O2 concentration in
solution are attractive methods for enzyme assays. This method is very sensitive, reproducible
can us very volume of sample.
8. Viscometric assays measuring change usually decrease in the viscosity of a solution as a
polymer is broken down. This can be useful for glycosides assay which releases reducing sugar
from a polysaccharide and to know whether it is an endoglycosidase (breaking the polymer in the
middle) or an exoglyucosidase, (chewing on the ends). The endoglycosidase will reduce the
viscosity of a solution of the polymer, the exoglycosidse won’t. The same could apply to
aminopeptidase vs a true protease (endo cleaving).
9. Radioisotopic assay: A radioactive substrate is acted on by an enzyme, then the product is to
separated from the substrate very carefully and measured by tis radioactivity. This must therefore
be a ‘stop and sample’ assay. Radioactive assays are best if the separation is quick and complete.
Although very sensitive, the use of radio isotope in an assay is restricited to applications where it
is possible to separate easily the radio labeled form of substrate or product. Exceptionally this
assay is having application if one of the product is a gas, which can be radio labeled.
HOOC CH2 CH2 CH (NH2)14 COOH → 14CO2 + HOOC CH2.CH2.NH2.14COOH
Glutamic acid Amino butyric acid
10. Immunochemical methods: Antibodies raised against a particular enzyme can be used for
highly specific assays. Enzyme Linked Immuno Sorbent Assary (ELISA) is an example. This
assay can distringuish between isoenzymes, which is of imortace in clinical diagnosis. A
monoclonal assay is available for serum prostatic acid phosphatase, which diagnose carcinoma of
prostrate.
11. Micro calorimetric methods: Most enzymatic reactions are accompanied by a minute
change in heat (enthalpy) that gives rise to a temperature change of the order of 10–2 to 10–4 °C.
Measurment of such small changes is possible using thermistors which are temperature sensitive
metal oxides. The technique requires stringent insulation of the reaction vessel. This assay may
be coupled to a secondary reaction where heat formed can be amplified to an easily detectable
level.
12. Gas chromatography and liquid chromatography: reactant and product differ in the time
they take coming through a column, whether as a volatile molecule in a flow or carrier gas or as a
solute in a flowing fluid. Gas chromatographs measure material passing thr detector by flame
ionization or by capture of an electron from a radioactive source is they contain nitrogen or
phosphorus; liquid chromatographs can detect spectrophometrically flurometrically by other
methods. The recorder can integrate the area under each peak to quantitate it. The power and
versatility is great such methods are particularly useful when substrate and product are very
similar chemically, as for instance cellobiose and glucose. Since being a continuous assay, the
drawback is the time each measurement takes.
13. Capillary electrophoresis and gel electrophoresis as for assay of a restriction endonuclease.
But the separation takes the more you’d like to find another way.
14. Biological assay which measure a more specific biological action of the protein rather than a
chemical action. Biological assays are particularly important when the chemical event carried out
is unremarkable, the importance being in its specificity – such as the many proteases which cause
specific biological effects by cleavage of one or a few bonds in specific protein substrates.
Another example, assay of growth hormone by thickness of the knees of hypophysectomized rats.
Another is the blood clotting system, the result of a cascading series of proteolytic events, the
product of each reaction being the enzyme for the next, culminating in the cleavage of fibrinogen
to fibrin and the formation of the lot. The clotting assay is timing how long it takes a clot to form;
it may be sued as an assay for any of the factors involved if a serum deficient in that factor so that
the sample added supplies what is needed to bring about clotting.

ISOELECTRIC FOCUSING (IEF) GELS:

This method is ideal for the separation of amphoteric substances such as proteins because
it is based on the separation of molecules according to their different isoelectric points. The
method has high resolution, being able to separate proteins that differ in their isoelectric pints by
as little as 0.01 of a pH unit. The most widely used system for IEF utilizes horizontal gels on
glass plates or plastic sheets. Separation is achieved by applying a potential difference across a
gel that contains a pH gradient. The pH gradient is formed by the introduction into the gel of
compounds known as ampholytes, which are complex mixtures of synthetic polyamino – poly
carboxylic acids.

– CH2 – N – (CH2)n – N – CH2 – where R = H or – (CH2)n – COOH , n = 2 or 3.

| |
(CH2)n (CH2)n
| |
NR2 COOH
‘The general formula for ampholytes’
Commercial available ampholytes include Bio – lyte and Pharmalyte. To prepare a thin layer
IEF gel, carrier ampholytes covering a suitable pH range amd riboflavin are mixed with the
acrylamide solution and the mixture is then poured over a glass ploate (typically 25 xm x 10
cm), which contain the space. The second glass plate is then placed on the top of the first to
form the gel cassette and the gel polymerized by photopolymerisation by placing the gel in
front of a bright light. The photo decompostion of the riboflavin generates a free radiacal
which initiates polymerization. This takes 2 – 3 hour.
Once the gel has set, the glass plates are prized apart to to reveal the gel stuck to one of the glass
sheets. Electrode wicks, which are thick (3 mm) strips of welted filter paper (the anode is
phosphoric acid, the cathode sodium hydroxide) are laid along the long length of each side of the
gel and a potential difference applied. Under the effect of this potential difference, the
ampholytes form a pH gradient between the anode and cathode.
The power is then turned off and samples applied by laying on the gel small squares of filter
paper soaked in the sample. A voltage is again applied for about 30 min to allow the sample to
electrophores off the paper and into the gel at which time the paper square can be removed from
the gel. Depending on which point on the pH gradient the sample has been loaded, proteins that
are initially at a pH region below their isoelectric point will be positively charged and will
initially migrate towards the cathode. As they proceed, however the surrounding pH will be
steadily increasing and therefore the positive charge on the protein will decrease correspondingly
until eventually the protein arrives at a point where the pH is equal to its isoelectric point. The
protein will now be in the Zwitterion form with no net charge, so further movement will cease.
Likewise, substances that are initially at pH regions above their isoelectric points will be
negatively charged and will migrate towards the anode until they reach their isoelectric pints and
become stationary.
Following electrophoresis, the gel must be stained to detect the proteins. The gel’s stained with
coomassie Brilliant Blue and then destained. The method is particularly useful for separationg
isoenzymes, which are different forms of the same enzyme often differing by only one or two
amino acid residues. Since the proteins are in their native form, enzymes can be detected in the
gel either by washing the unfixed and unstained gel in an appropriate substrate or by over
layering with agarose containing the substrate.

(A). Low pH (+) High pH (–)

(B). Low pH (+) Low pH (–)

A pH gradient is established in a gel before loading the sample.

(A). The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric
– pH, the location at which they have no net charge.
(B). The proteins form bands that can be excised and used for further experimentation.

Further Purification Procedure:

Although a considerable degree of purification is achieved by fractional precipitation,
much other soluble protein will still be present because of overlap of solubility ranges. Precipitate
is collected by centrifugation and dissolved in a suitable volume of extractant for further
purification. Before further purification by instrumental methods generally chromatography,
extractant is adsorbed onto a suitable adsorbent. All adsorption and chromatographic methods
depend on distribution of the material being purified (here enzyme) between a stationary (usually
solid, sometimes adsorbed liquid) phase and a moving (usually liquid) phase. Distribution
coefficient [adsorbed material] /[total material].
Batch adsorption:
This is a simple technique intermediate between precipitation and chromatographic
methods. The most commonly used adsorbent is calcium phosphate. Originally in an gel form but
now usually in crystalline forms, brushite and hydroxylapatite, which can be used in columns.
Adsorbent is added to the crude extractant of enzyme and allowed enzymes to adsorb on to its
surface. After adsorption, the solid phase is collected by filtration or centrifugation like
precipitation process. Effective adsorbents, except affinity adsorbents, are likely to adsorb many
other proteins and thus require that a substantial amount be used. And since separation is only
between two phases, adsorbed and suppressant resolution is generally much less than in
chromatography. Zinc hydroxide has been used to remove pigments from enzyme preparations.
Ion exchange chromatography:
Proteins differ from one another in the proportions of the charged amino acid (asparic and
glutamic acids, lysine arginine and histidine) than can contain. Hence proteins will differ in net
charge at a particular pH. This difference is exploited in ion exchange chromatography to
separate enzymes . In this method, enzyme of interest is bound on a solid support material
(ionexchange resin) bearing charged group of the opposite sign. Most of the enzyme purification
is done on anion exchange columns because most enzymes are negatively charged at
physiological pH. Elution of the bound enzyme is by exchanging the charged enzyme by a
corresponding cation/anion. Based on the charge exchanged, it may be a cation exchange
chromatography or anion exchange chromatography. Ion exchanger consists of a water insoluble
matrix namely dextra or cellulose to which charged groups have been charged group have been
covalently bound. Cation exchangers have acidic group with a net negative charge on the matrix
and positively charged exchangeable counter ions. Example is diethyl amino ethyl cellulose
(DEAE.)
(C2H5)2 NH CH2 CH2 – O Cellulose

Quarternary ammonium groups, such as triethylaminoethyl (TEAE) and QARE are also
used as anion exchangers. The most commonly used cation exchange group is carboxymethl
(CM). Phosphocellulose is another example of cation exchanger.
Ion exchange adsorbents are usually eluted by means of a gradient or steps of increasing KCl or
NaCl concentrations, in presence of a constant concentration of buffer. The charged form of the
buffer should be of the same charge as the ion exchange material, i.e. use Tris or another amine
with DEAE, phosphate or another acid with CM.

Chromatofocusing:
A variation of ion exchange technique, it chromatofocusing in which a linear pH gradient
is generated in the column with 2 or 4 pH units lower at the top as the column is eluted using the
acid form of an ampholyte at low ionic strength. When a protein is added to this pH gradient with
a buffer whose pH is similar to that prevailing at the top of the column, it will migrate down the
column as cation, encountering an increasing pH, until it reaches a pH corresponding to this
isoelectric ponit. Just beyond this pint it will become an anion and will be able to bind to the
positive groups of the exchanger (in this example column is anionexchanger). This process is
repeated in the column by changing the pH of the buffer and at the end protein is eluted at a pH
slightly above is isoelectric point. Proteins are claimed to to emerge in sharp, highly resolved
peaks. This technique has high capacity. Chromatofocusing gives a good resolution of quire
complex mixture of proteins, provided that there are discrete differences in their isoelectric point.
Proteins possessing very similar isoelectric points tend to be poorly resolved.

Hydrophobic chromatography:
Enzymes canstick to hydrophobic material byhydrophobic interation with nonpolar
regions of their surface (by val, phe, etc). The hydrophobic groups used in the columninclude
alkyl (octyl – Sepharose), phenyl (phenyl – Sepharose) andalkyl amino achains. The capacity is
high. Adsorption is strongest at high salt concentration, so a sample may be applied immediately
on redissolution after (NH4)2SO4 precipitation. Proteins are eluted by decreasing the salt
concentration. Resolution is not as good as in ion exchange chromatography.
Affinity Chromatography:
Affinity chromatography is a bio – specific process which exploits the formation of
specific and reversible complexes between a pair of biomolecules. One of the pair is called ligand
and is usually immobiliex on to a stationary phase while the other called counter ligand, is
adsorbed from the extract that is passing through the chromatographic column containing the
immobilized ligand. This technique enables separate closely related proteins from a mixture.
Method depend on a specific interaction the enzyme of interest and specific ligands, which may
be substrate analogue binding to its respective enzyme (affinity chromatorgraphy), a synthetic
dyes which can bind to specific protein (dye – lignad chromatography) a lectin, chinch can bind
to glycoproteins (lectin – affinity chromatography) or an antibodies binding to specific enzymes
antigen (immunoardsorbent chromatography).
Ligands and counter- lignds in affinity chromatography:
Lignad Counter Ligand Chromatography
Substrate, subnstrate,
1. Enzyme Affinity chromatography
analogue, cofactor, inhibitor
2. Glyco protein enzyme Lectin Lectin – affinity chromatography
3. Enzyme (an antigen) Antibody Immunoabsorbent chromatography
4. Enzyme Dye Dye- ligand chromatography
5. Metalloenzyme Metal ions Metal – chelate chromatography
The matrix or support is usually agarose or a cross – linked derivative, because it is very
poprous and admits large proteins to the pores, but has good strength and stability and is
reasonably derivatizable. In general any matrix useful for ion exchange or gel filtration
chromatography is also good for affinity chromatography. The attachment of ligand usually
proceeds by treating the matrix with a reactive compound like cyanogens bromide and
glutaraldehyde, which either leaves reactive groups to which ligands can be attached. The ligand
is usually attached with a spacer arm between it and the matrix to assure that theligand will be
fully accessible to the desiredprotein. An example of non-specific space is 1.6 – diaminohezane.
The protein mixture is applied to the column and the relevant enzyme is trapped by the
immobilized ligands while all other proteins pass through and are discarded. The enzyme is the
liberated from the column either by eluting with a deforming buffer at a pH which changes the
characteristics of the enzyme and nolonger allows it to bind to immobilized lignad. Another
method is using a competitive counter ligand, which displaces the immobilized ligand on the
enzyme.
Advantages of affinity chromatography included:
1. High selectivity compared to other purification techniques.
2. Extremely good purification upto several thousand folds in a single step and recoveries
greater than 90% can be expected provided conditions are carefully selected.
3. Affinity chromatography had a high concentration effect, especially when the enzyme of
interest is a minor component of a complex mixture.
4. Affinity methods can also be used to remove unwanted materials from a mixture.\

Immunoadsorbent chromatography:
Here immobilized ligand is antibodies to the desired enzyme. In principle, this technique
is the last word in specificity and tight binding, but of course there are drawbacks. First of all, in
order to prepare the antibodies it is required to purify the protein first and the antibody
preparation procedure is cumbersome. Another problem is elution of the bound enzyme antibody
will be difficult.
Dye ligand chromatography
Some reactive triazine-dyes (about 40 dyes) Cibacron F3GA, Procion Red H-E3B, etc.,
WW have very affinity for protein and can there for use in enzyme purification. These dyes will
easily attach to agarose or other matrices and thus can be used in an easy way. Elution is as with
‘true affinity columns, either with specific ligands which compete with the dye for the protein
binding site, or with high salt concetration or high pH.
Metal-binding chromatography
This can be a general approach for proteins with exposed histidines, cysteines or carboxyl groups
near each other, but in practice it is mainly for cloned proteins. To cloned protein, a short
sequence is added which facilities purification by binding to specific metals. The commonly used
method is to add a sequence of six or so histidinesd at the C-terminus. These bind well to divalent
cations of transition metals such as nickel. A column is prepared by attaching nitrilotriacetic acid
to a solid support. This binds nickel ions tightly; the resin is washed with 5mM imidazole to
remove unbond nickel. The fusion protein, perhaps denatured in 6M urea to ensure that the hexa-
His sequence is exposed, is applied to the column. The column is washed with dilute imidazole,
then more concentrated imidazole to elute the desired protein.Some variations are: mercuric ions
bound tightly to immobilized sulfhydryl groups, which can bind proteins by their exposed
sulfhydryl groups – elution is with excess free SH compound such as mercaptoethanol; and Fe+++
bound to iminodiacetic acid, which binds phosphoproteins by the phosphate groups.
Gel filtration (size-exclusion or molecular sieve chromatography)
 The gel filtration material is porous, with pores the size of protein molecules. Large
molecules, too large to enter any of the pores, pass down the column through the space between
the gel particles. Very small molecules enter all the pores, and therefore spend much of their time
not moving and elute out only solely. Intermediate size molecules enter some of the pores, and
are eluted somewhere in between. Eluting buffer should be of high ionic strength to counteract
the few changes which may be present on the gel. Gel filtration is also used to separate proteins
from salts such as ammonium sulfate, using a small-pored gel such as Sephadex G-25 or BioGel
P10 which excludes all proteins; it is much faster than dialysis. Gel filtration is widely used to
separate protein detergent miscelles from excess of detergent, during purification of membrane
bound enzymes.
The gel filtration materials are the Sephadexes (cross-linked dextrans), ‘sephacry’ (cross-
linked acrylamide) and biogel (agarose).
High performance chromatographic techniques
HPLC stands for “high performance liquid chromatography” (through HP could also be
said to stand for “high pressure”). The stainless steel columns and robust packing material of
104m or less which can with stand high pressure are used and it enables the resolution in minutes.
These columns yield good separation and high resolution in short period. One advantage of fast
operation is that they can be run at room temperature without denaturing the protein, because the
protein comes off in 5 to 60 min. However, only small volumes can be purified. For protein
chromatography it was necessary to develop materials both strong enough like silica, to stand the
pressure and porous enough to have a high surface area for adsorption, or for gel filtration. HPLC
is a high resolution, but low capacity method. High performance Size Exclusion
Chromatography (HPSEC) utilizes rigid beads of porous silica with bonded hydrophilic polar
groups. High Performance Ion Exchange Chromatography (HIPEC) utilizes amines as anion
exchanges and sulphonic or carboxylic acids as cation exchanges, each bonded to a rigid support
as silica. High Performance Liquid Affinity Chromatography utilizes ligands bounds to supports
such as epoxy-silica. Proteins can also be separated by reverse phase HPLC (RP-HPLC) on
alkylsilica columns, the eluting solvents being buffered aqueous and organic mixtures.
Electrophorectic Tecniques
Electrophoresis is mainly an analytical procedure as it is suited for small amount of metal
it has also been used for purification of enzymes. The rate and direction of migration of a protein
in an electric field depends on its net charge and size of the molecule. In zone electrophoresis, the
separation occurs in a solid matrix commonly agarose gel or polycrylanide gel. This may be
vertical ‘disc gel electrophoresis’ and horizontal ‘thin slab gel electrophoresis’. Electrophoresis in
the absence of any support material is free solution electrophoresis or moving boundary
electrophoresis. The different analytical gel electrophoresis are
1. Simple or native gel electrophoresis
2. SDS-PAGE
3. Urea gel electrophoresis.

1. Native gel electrophoresis: The enzyme mixture to be separated is mixed with a buffer at a pH
where the proteins remain stable and in their native conformation. The pH range is usually 8-9
where most of the proteins carry negative charges and move towards the anode placed at the other
end. Any basic contaminant protein will remain in the cathodic buffer. In negative PAGE, the gel
is prepared by polymerzing acrylamide and a cross linker N,N-Methyl bisacrylamide (30:1)
together with ammonium persulphate as initiator of polymerization and TEMED (N,N,N,N-
Tetraethylendiamine) as catalyst. The pore size of the gel can be tailored to suit the molecular
weight of the sample proteins by altering the concentration of either the monomer acrylamide or
cross-linker. Increase in concentration of either of the two decreases the pore size and vice versa.
2. SDS-PAGE: This method involves the electrophorectic seperation of denatured protein on
polyacrylamide gel. The proteins in the solution are completely denatured by treatment with the
detergent sodium dodecyl sulphate (SDS) and β -mercaptoethnol and boiling the mixture for a
few minutes. Addition of mercaptoethnol disrupts disulphide linkage in protein. Each SDS
contains a negative charge and approximately one SDS bind to two amino acid residues giving
the polypeptide a large negative charge. Consequently the charge/size ratio is almost the same for
all polypeptide chains and separation of the polypeptides can occur only due to the molecular
size. The electrophoresis is carried out in vertical slab polyacrylamide gel using buffers such as
tris HCl-glycine and tris-tricine buffer for small molecular weight proteins.

The method has a high resolution and gives sharp zones. The molecular weight of sample
polypeptide chains can be determined by comparing their mobility with standard poly peptides
whose molecular weight is known.

3. Urea gel electrophoresis: This method is used particularly for protein that is insoluble at low
ionic strength. The proteins are solubilised by denaturing them completely with urea in the
presence of meracaptoethanol to disrupt any disulphide linkages. Urea containing starch gels are
easier to handle compared to polyacrylamide.

IMMUNOELECTROPHORESIS
Immunoelectrophoresis is an identification and quantofication by separating components
I mixtures by electrophoresis in an ager gels followed by immunoprecipition reaction one the
same gel. Imunoprecipitin reaction is a specific reaction between an antigen and its corresponding
antibody and it is observable as white precipitate in the pH range of 7 – 9.

CAPILLARY ELECTROPHORESIS:
Capillary electrophoresis is an analytical technique requiring only micro – to nagogram
samples. The components of the sample are allowed to seprate in a high voltage inside a capillary
tube filled with a suitable buffer. Column is usually made of stainless steel with a diameter of 100
micrometer and about 30 cm long.

ISOELECTRIC FOCUSING:
Isoelectric focusing is an example of moving boundary electrophoresis, in which a pH
gradient is set up between electrodes by allowing an acid (eg. Phosphoric acid) to diffuse from
anode and a base (eg. Ethanolamine) from cathode. The stabilization of this pH gradient is
achieved by using buffers called ampholytes. Ampholytes are synthetic aliphatic polyamino –
polycarboxylic acid and they have large number of positive and negatively charged functional
groups with closely spaced isoelectric points. During isoelectric focusing the ampholytes
migrates to their respective isoelectric pH and stabilizes the pH zones, usually a pH gradient is set
up with a difference of 0.02 pH units. The protein mixture when introduced and electrophotesis
gel, the components will be moving thl it its reach isoelectric pH and get precipitated there. The
zones containing each protein are very sharp as a result of this focusing and proteins whose
issoelecctric points differ by as little as 0.02 pH units can be distinguishable by this method.
Isotachophoresis:
Isotachophoresis is also moving boundary electrophoresis where migration of different
ionic species of the same signs all having the same counter ion in an applied electric field. In
principle, the mixture containing two ionic species A – and B – are separated on the basis of their
mobility in an electric field. For enzyme isolation, at mildly alkaline pH, when most would be
anions, a leading anion (eg. Phosphbbate) is added which has a faster mobility towards the anode
than any of the sample anion. A trailing or terminating anion with a slower mobility than any in
the sample is also added. The system is buffered by a counter cation, Tris. The leading and
trailing ions are applied at different sides of sample, leading near the anode. When high voltage is
applied all components will migrate toward the anode in discrete zones at the same
velocity(‘isotacho’ GK. ‘Same speed’). The sample will arranged in the order of mobilities. The
ions with higher effective mobility will move fast and those with lower effective mobility will
follow in decreasing order of their effective mobilities. The polarity of electric field is such that
with a homogeneous current density all the ions move with same speed at equilibrium and get
separated into a number of consecutive zones in immediate contact with each other and arranged
in order of their effective mobilities. Isotachophoresis is used in preparatory level.
Final Concentrating steps:
When further purification, it is considered that the relevant enzyme has been seprated
completely from other protein, mineral salt and other small molecules are removed by dialysis.

Dialysis: Dialysis is a membrane separation used for removal of low MW solutes such as
organic acids (< 500 MW) and inorganic ions (< 100 MW) from a solution. In enzyme
purification, dialysis is commonly used after salting out, to separation salt ions before further
purification steps. Cellophane or cellulose acetate membranes of different porosity are used for
dialysis. Complete removal of ions can be achieved but concentration of enzyme mixture is not
possible.
The purified enzyme preparation is likely to be quite dilute, so it might require
concentrating. Concentration of the enzyme preparation is achieved by lyephilisation or
ultrafiltration.

Lyophilisation is freeze-drying technique. The sample is frozen first and to it vacuum is applied
to sublimate all the ice crystals to vapour state. The powder obtained can be resuspended in a
required volume of buffer.

Ultrafiltration: This is the filtration of a protein solution through a membrane with pores small
enough to retain the protein of interest. It is a two – phase method – what is retained and what
passes through the filter. To hasten the process, it required either gas pressure on the solution
above the filter (for large volumes) or increase of gravitational force by centrifugation (for small
volumes). It is usually used just to concentrate a dilute protein solution, such as a crude culture
brotn and sometimes after dialysis for concentrating again until practically all small molecules
have been flushed through the membrane. It is sometimes used as a purification method with
retain the protein of interest usually large molecules, but pass through smaller ones. The bigget
problem is clogging of the pores by protein accumulating on the membrane surface. The enzymes
are purified and concentrated by this process.
The ideal way to complete purification is to crystallize the enzyme. Crystallization is achieved by
adding enough quantity of ammonium sulphate to cause precipitation of the enzyme and leaving
this in cold room for several days.
Membrane bound enzymes may be inactive after purification unless reintroduced into
phospholpids environment.

Some general comments:

A good industrial protein purification processes should follow the five rules:

“Rule 1: Choose separation processes based on different physical, chemical or biochemical

properties”. Repeating the same process doesn’t gain much, though sometimes it makes later step
more efficient.
“Rule 2: Separate the most plentiful impurities first”. This means especially non – protein
impurities such as cell debris and small molecules.
“Rule 3: Choose those processes that will exploit the differences in the physico chemical
properties of the product and impurities in the most efficient manner”. This is done when you
know the properties of the purified protein and are designing a large-scale process, which you
want to be as efficient as possible. For purification of a recombinant protein it is also useful to
know properties of the commonest proteins of the host cell and how to remove them efficiently.
“Rule 4: Use a high – resolution step as soon as possible”. This is less obvious but eliminates as
many impurities as possible at an early stage. They mean affinity chromatography where
possible, otherwise probably ion exchange chromatography.
“Rule 5: Do the most arduous step last”. This means removal of the last few percent impurities.
High-resolution gel filtration is often the best step here.
MONITORINIG OF PURIFICATION STEPS
To monitor the efficacy of purification or to follow the progress of process, an assay of
the desired enzyme and it gives the measure of biological activity (total activity). It must be also
convenient and easy to perform because at times many fractions obtained after purification steps
are to be assayed for the desired enzyme and the fractions positive for the enzyme are pooled
together before going to next step. A detailed description of assay methods to find out biological
activity is given in the notes “enzyme assay”. The biological activity is measured as IU or units
(SI unit is katal). See the notes attached “unit of enzyme activity” for definitions. The important
criterion of purity is specific activity. Specific activity is biological activity divided by total
protein in the sample, This will show, irrespective of preconceived ideas, precisely which fraction
contain the important enzyme and will enable the degree of purification to be calculated.
Total protein in the sample can be estimated by any of the following method.
1. Kjeldahl analysis: The nitrogen content of most protein is 16% by weight. Total nitrogrn in
the biological sample is determined by kjeldahl titration method and the total protein is calculated
from that.
2. Ultraviolet absorption method: The method is relatively sensitive and applicable only to
partially or highly purified enzyme preparations. The aromatic amino acids, tyrosine and
tryptophane give an absorption maximum at a wavelength of 230. The major advantage of this
protein assay is that it is non-destructive and protein concentration of effluents from
chromatographic column can be measured continuously.
3. Biuret method: This is suitable method for measuring protein concentration of crude
homogenate and in subcellular fractions. Biuret reagent is a mixture of alkaline copper sulphate
and sodium potassium tartarate. The cupric ions form a coordination complex with four -NH
groups present in peptide bonds giving an absorption maximum at 540-560 nm wavelength.
4. Lowry (Folin-Ciocalteau) method: This is the most widely used protein assay method, which
measures protein concntration as low as 10mg cm-3 the principle is comparable to biuret method.
Protein complexes with copper in alkaline solution. This complex then reduces a
phosphomolybdate-phosphotugstic reagent to yield an intense blue colour. The intensity of the
blue-purple colour is measured at 660nm.
5. Turbidimetric method: Certain organic acids such as trichloroacetic acid and sulphosalistic
acid precipitate protein. The amount of precipitate formed can be measured by its light scattering
intensity. Scattering is measured in colorimeter or spectrophotometer.
Specific activity is used to calculate yield and purification factor. Purification factor gives
degree of purification and it is also called fold purification. Purification factor will be high for
efficient separation steps.
Specific activity after a particular step
Purification factor =
Specific activity of previous step
 Yield can be directly calculated in each step as

Biological activity after a particular step

Yield =
Biological activity of initial step
If the fractions containing desired enzyme are combined, the total activity of the enzyme
in that fraction should be the same as that started with, whereas total amount of protein in this
pooled sample will be less than that originally present. The specific activity of the enzyme in the
combined fraction should therefore be greater than that in the preparation before purification and
increase in specificity will be a measure of the purification achieved. With each successive
purification step, the specific activity of the sample containing the enzyme should be greater than
before until complete purification is achieved and specific activity reaches a limiting value.
However, the finding of the same specific activity value before and after a purification
step does not necessarily mean that the enzyme preparation is completely pure, it could be simply
mean that the contaminating proteins have passed through the procedure in the same fraction as
the enzyme. Similarly crystallization cannot be taken as proof that only one protein is present, for
many mixed protein crystals have been found. Hence other criteria of purity have to be
considered.
Protocal for purification of Adenylate kinase

Minced Muscle
Step 1 Extract with 0.01 Mol dm-3 KCl
strain trough cheesecloth.

Extract
Step 2 Incubate at pH 3.5 then at pH 7.0;
Centrifuge

Supernatant
Step 3 Load on to phospho cellulose column. Elute
with pulse of AMP (5 mmol dm-3 )
Pooled fraction
containing activity
Concentrate by (NH4)2 SO4 Gel filtration –
Step 4
Scphadex G. 15
Pooled fraction
containing activity
Crystallization at 62% saturation of
Step 5
(NH4)2SO4

Crystalline enzyme
Purification table for adenylate cyclase from 6Kg of pig muscle
S.No Step Total Total Total Specific Yield Purification
 Volume protein activity activity
% factor
Cm3 mg Katal Katal/Kg
I Extraction 16600 435000 0.0413 0.095 100 1.0
II Precipitation 15700 112000 0.0365 0.325 88.3 3.42
Phosphocellulose
III 1380 1716 0.0223 13.02 54.0 40.0
column
IV Gel filtration 211 462 0.0200 43.17 48.4 3.32
V Crystallization - 344 0.016 46.5 38.7 1.08
ENZYME ASSAYS
As assay is a meaurement of a given enzy,e of known characteristics in a sample. Ideally
this means measuring a specific and characteristic biological property (or ability to catalyze a
chemical reaction) of the desired enzyme. An assay must be specific for the enzyme being
purified and highly sensitive to its presence. Further more, the assay must be convenient to use
because it is to be done repeatedly especially during enzyme purification processes.

There are two general purposes for enzyme assay:

1. To measure how much of the enzyme is present in the sample. The enzyme is the variable
measured.
2. With a constant amount of the enzyme present, how does its activity vary with conditions
such as pH, temperature, variation of substrate concentration, effect of inhibitors, etc or
in prior incubation (stability to heat, chemical modification, etc). These studies help to
characterize the enzyme.

Another example of coupled assay is

D – alanine + O2 -----------------→ Pyruvate + NH4+ + H2O2 (D – amino acid odidase)
H2O2 + Chromogen ------------ → Coloured product + H2O (Peroxidase)

Cycle assays are also there which use a small amount of a compound as related liming
intermediate in reactions going both ways. Strictly these are assays for the compound rather than
or an enzyme, but the amount of compound started with might be the product of an enzyme
reaction carried out on a very small scale say one cell. An example,

Pyruvate + NADH + H+ ↔ L – lactate + NAD+ (Lactate dehydrogenase)

Pyruvate + H2O2 ↔ L – lactate + O2 (lactate oxidase)

In this case the amount of pyruvate is the rate-limiting compound and increase in
concentration of pyruvate is directly proportional to the rate of reaction and it can be monitored
as the absorption of NADH.

Enzyme concentration in the sample can be directly measured by the following analytical
methods.

Method Comments
Molecular weight determination.
Ultra filtration
Impurities determination < 5% level
Elecctrophoresis Enzymes with non – identical subunits
SDS – PAGE Mr determinator, excellent in impurity determination
Capillary electrophoresis Excellent analytical technique for Mr determination
Isoelectric focusing Sensitive method
Sub unit determination drawback – determination of enzymes
N – Terminal analysis
with blocked N – Terminus or more than one poly peptide
Specialized technique to detect primary structure and post
Mass Spectroscopy
translation modification.