Isolation and purification of

enzymes
Dr Pooja Rana
Why isolate enzymes?​

•It is important to study enzymes in a simple system (only with small

ions, buffer molecules, cofactors, etc.) for understanding its structure,
kinetics, mechanisms, regulations, and role in a complex system​
•Also isolating pure enzyme is important to use it for medical and
industrial purposes​
Objectives of enzyme purification

Objectives :
• maximum possible yield
• maximum catalytic activity
• maximum possible purity​
• Enzymes that are used for biocatalysis are typically purified from
microbial cells or from culture media after or during growth of
microorganisms (in case of excreted proteins).
• The enzyme purification generally starts with a cleared cell
extract in which the enzyme is present in a soluble form.
• If the enzyme to be purified is excreted into the culture medium,
it is usually sufficient to remove the cells from the medium by
centrifugation (for small-scale purifications) or by filtration (for
large-scale industrial purifications).
• In the case of an intracellular enzyme, cells should be broken
first to release the protein into solution. Depending on the type of
cells, different techniques are employed
• The microbial cells are first harvested from the culture medium
by centrifugation and re-suspended in a small amount of buffer.
• The cells can be broken using a variety of techniques, e.g. by
treatment with enzymes that digest cell walls (e.g. lysozyme),
followed by osmotic shock, by using lysis buffers containing
detergents, by exposure to ultrasound using sonicators, by
pushing cells under high pressure through a small orifice using
a pressure cell system, or by grinding frozen cells in liquid
nitrogen.
• Extracts thus obtained are cleared from unbroken cells and
large, insoluble particles by centrifugation or filtration.
• To prevent enzyme inactivation during these treatments, and
also in the following purification steps, the temperature of the
enzyme solution is usually kept around 4 °C.
• Proteolytic degradation of the enzyme to be purified can be
precluded by adding a protease inhibitor cocktail during
breaking of the cells.
• Once a cell-free extract has been obtained, several methods
can be employed for further purification of the desired
enzyme.
Isolation of Enzymes
• Generally enzymes are isolated in the cold condition (at 0 to
4o C). For the purpose, homogenizing medium as well as
container should be in the chilled condition.
• Pestle and mortar-
• Pestle and mortar is a moderate technique for tissue
homogenization. Mechanical breakdown occurs during the process.
Sometimes, grinding is done in the presence of purified sand or
glass beads for aberration.
• Blenders –
• Waring blender (commonly called as mixie) is comparatively harsh
technique of grinding the tissue compared to pestle and mortar and is
mostly used for homogenizing the harder tissues (generally the plant
tissues).
• Vir-Tis homogenizer
• This is considered to be a mild technique and generally used for
homogenization of soft tissues such as animal tissues.
• Here a motorized pestle with teeth like aberrations is used. With Vir-
Tis homogenizer, generally no rupturing of cell organelles occurs
during grinding provided isotonic medium with no detergent is used.
Vir-Tis homogenizer Potter Elvejm homogenizer Ultra- Sonicator
• Potter Elvejm homogenizer
• This is also a mild technique and is used for homogenization of soft animal tissues.
• Potter Elvejm Homogenizer is a simple equipment having a pestle like glass rod with
teeth like aberrations on its tip. There are down aberrations in the tube too on which
teeth of the rod are fitted during up and down process of the rod. Up and down
process of the pestle is done manually by hand or by mechanical device.
• Razor blade
• It is comparatively very mild technique.
• It is generally used only for isolation of intact cell organelles for the purpose of
studying the intracellular localization of the enzyme proteins.
• In the technique, razor blade is used for chopping the tissue in the presence of
isolating medium. Although the technique is good for the isolation of intact
organelles, but it is unable to rupture all the cells. Therefore, there is low recovery of
the enzyme due to left out of unruptured or partially ruptured cells. These un-
ruptured or partially ruptured cells are removed as cell debris after centrifugation.
• Ultra- Sonicator
• This technique of rupturing the cells is generally used for microbial/
bacterial cells. Ultrasonicator generates low as well as high
wavelength ultrasonic waves.
• For the purpose, a suitable probe depending on the volume of the
homogenizing medium is selected and connected with the ultra-
sonicator.
• The container having cells and homogenizing (isolating) medium is
put in chilled condition by covering the container with ice.
• There is much generation of heat during ultra-sonication, therefore,
ultrasonic waves are thrown in the sample after few seconds interval,
every 10 to 15 seconds ultrasonication.
• Extrusion method-This method relies on the principle that forcing a
cell suspension at high pressure through a narrow orifice will provide
a rapid pressure drop. This is a powerful mean of disrupting cells
especially from bacteria.
• Lytic enzymes -Cell wall and cell membrane lytic enzymes like
cellulase, pectinase, xylanase, pectin methyl esterase, lysozyme etc.
can be used for rupturing the cells.
• Enzymes being costly are not commonly used for making cell free
preparation for isolation of enzymes. In plant tissue culture, lytic
enzymes are used to prepare protoplast.
• Freeze-Thaw- With certain susceptible microbes and eukaryotic cells,
repeated freezing and thawing results in extensive membrane lesions
with release of periplasmic and intracellular proteins.
• Acetone powder- Drying with acetone is a good method for rupturing
the cell membrane. Using acetone, powder of the tissue may be
prepared which may be stored in a Deep freezer for a long time. It
forms a convenient starting material from which the enzyme may be
extracted with the isolating medium, whenever required. However,
one has to take much precautions of low temperature (generally –
20oC), otherwise, acetone may denature the enzyme protein.
• Isolation of enzymes from sub-cellular organelles requires rupturing of
the organelle. Generally for the purpose, organelle is isolated in intact
form thus removing the contaminating proteins of the cytoplasm and
other cell organelles.
• Afterwards, cell organelle is ruptured in the presence of a suitable
detergent like tween, teepol, digitonin etc.
Methods of enzyme purification
• The purification of a particular enzyme involves removal of other
substances (proteins as well as non-proteins) present in the preparation.
• Purification of an enzyme protein is generally a multi-step process
exploiting a range of biophysical and biochemical characteristics such
as its relative concentration in the source, solubility, charge, size
(molecular weight), hydrophobicity/ hydrophilicity of the target
protein.
• In general, design of the purification technique/ protocol should be
focused on: (i) high recovery, (ii) highly purified enzyme protein, (iii)
reproducibility of the methods, (iv) economical use of the chemicals
(reagents) and (v) shorter time for complete purification.
• Proteins are relatively labile and get denatured at high temperatures
and variation in pH.
• Each protein has its own physico-chemical characteristics. The
techniques selected for enzyme purification should be moderate and
native conformation of the enzyme protein should not change as a
result of purification.
• During purification, degree of purity and percent of recovery should
be checked after each step of purification. It is not always necessary
to purify the enzyme protein to homogeneity since enzyme
purification is a costly affair and also the time consuming. Therefore,
it is necessary to have an idea of the degree of purity necessary for the
intended use of the targeted enzyme.
These separation methods can be roughly
divided into the following categories:
(i) selective precipitation
(ii) separation based on charge
(iii) separation based on molecular size
(iv) separation based on bio-affinity, and
(v) separation based on adsorption principles
• Except for the first category, all these methods generally make use of
column chromatography
• A good purification results in the recovery of most of the enzyme
activity (i.e. a high yield) and in removal of many “contaminating”
proteins and other types of (bio)molecules (i.e. a strong increase in
specific activity).
• The purity of the final enzyme preparation can be tested in several
ways. The most common methods used are sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) analytical gel
filtration, and mass spectrometry.
• The techniques selected for enzyme purification should be moderate
and native conformation of the enzyme protein should not change as a
result of purification
• Traditional enzyme purification procedures many times start with an
ammonium sulfate fractionation. This type of fractionation makes use of
the fact that individual proteins precipitate at different concentration
ranges of ammonium sulfate.
• To make an estimation of the fractionation range, a small-scale pilot
experiment can be performed.
Ammonium sulfate precipitation

• The solubility of proteins varies according to the ionic strength of the

solution, and hence according to the salt concentration.
• Two distinct effects are observed: at low salt concentrations, the
solubility of the protein increases with increasing salt concentration
(i.e. increasing ionic strength), an effect termed salting in.
• As the salt concentration (ionic strength) is increased further, the
solubility of the protein begins to decrease. At sufficiently high ionic
strength, the protein will be almost completely precipitated from the
solution (salting out).
• Since proteins differ markedly in their solubilities at high ionic
strength, salting-out is a very useful procedure to assist in the
purification of a given protein.
• The commonly used salt is ammonium sulfate, as it is very water
soluble and has no adverse effects upon enzyme activity. It is generally
used as a saturated aqueous solution which is diluted to the required
concentration, expressed as a percentage concentration of the saturated
solution (a 100% solution).
• In the preliminary test, the ammonium sulfate concentration is increased
stepwise, and the precipitated protein is recovered at each stage.
• This is usually done by adding solid ammonium sulfate, but calculating
how much ammonium sulfate to add to a solution at one concentration to
achieve a desired higher concentration is tricky, since addition of
ammonium sulfate significantly increases the volume of the solution.
• The amount to add can be determined either from published nomograms or
by using an on line calculator.
• Each protein precipitate is dissolved individually in fresh buffer and
assayed for total protein content and amount of desired protein. The aim is
to find the ammonium sulfate concentration which will precipitate the
maximum proportion of undesired protein, whilst leaving most of the
desired protein still in solution.
• The precipitated protein is then removed by centrifugation and then the
ammonium sulfate concentration is increased to a value that will
precipitate most of the protein of interest whilst leaving the maximum
amount of protein contaminants still in solution.
• The precipitated protein of interest is recovered by centrifugation and
dissolved in fresh buffer for the next stage of purification.
• This technique is useful to quickly remove large amounts of contaminant
proteins, as a first step in many purification schemes.
• It is also often employed during the later stages of purification to
concentrate protein from dilute solution following procedures such as gel
filtration
 Ion-exchange chromatography
✔ IEX chromatography separates ions and charged molecules based on their
affinity towards the appositively charged matrix (ion exchanger).
✔ It works on almost any kind of charged molecule—including large proteins,
small nucleotides, and amino acids.
✔ Mobile phases consist an aqueous buffer system into which the mixture to be
resolved.
✔ The stationary phase usually made from inert organic matrix chemically
derivative with ionizable functional groups.
✔ Ions which exist in a state of equilibrium between the mobile phase and
stationary phases giving rise to two possible formats, anion and cation exchange
are referred to as counter ion.
✔ Ions which exist in a state of equilibrium between the mobile phase and stationary phases
giving rise to two possible formats, anion and cation exchange are referred to as counter ion.
The interaction between matrix and analyte is determined by net charge, ionic
strength and pH of the buffer. For example, when analytes loaded onto a
negatively charged matrix, the neutral or negatively charged analyte will not
bind to the matrix whereas positively charged analyte will bind as per their
relative charge and needed higher concentration of counter ion to elute from
matrix.
a- Cation exchange chromatography
✔ Analyte is positively charged and matrix has a negatively charged functional group.

✔ The matrix have negative functional groups such as carboxyl (- COO-), carboxy methyl (-
CH2COO-) and sulpho (- SO3-), and sulpho methyle (- CH2SO3-).
b. Anion Exchange chromatography-
✔ Analyte is negatively charged and matrix has a positively charged functional group. The
negatively charged analyte replaces the reversible bound anion and binds to the matrix

✔ Matrix such as amino

ethyl (- CH2CH2 NH3+)
and trimethyl amino
ethyl.
• The stationary phase (matrix) in IEC carries charged functional groups
fixed by chemical bonds.
• The fixed groups are associated with exchangeable counter ions.
• In anion exchange chromatography, the fixed groups have positive
charges.
• In cation exchange chromatography, these groups are negatively
charged.
• Proteins bind to an anion exchanger at pH values above their
isoelectric point (pI) and to a cation exchanger at pH values below the
pI
• IEC is a very powerful (preparative) purification method because
• (i) the high binding capacity of ion exchange columns allows elution
of proteins in a very concentrated form and
• (ii) a proper choice of elution conditions results in separation of the
bound proteins at high resolution.
• IEC technique appeared to be crucial for the purification of a wide
range of oxidoreductases, including monooxygenases, oxidases,
dioxygenases, peroxidases, reductases, and dehydrogenases
Gel Filtration Chromatography
✔ The column is packed with the beads containing pores to allow entry
of molecules based on their sizes.
✔ Smallest size in the inner part of pore followed by gradual increasing
size and largest molecule excluded from entering into the gel.
✔ The separation occur due to the time they travel to come out from the
pores.
✔ The small molecules present in the inner part of the gel takes longer
flow of liquid (or time) and travel longer path to come out where as
larger molecules travel less distance to come out.
✔ As a result, the large molecule and small molecule get separated from
small sizes molecules.
The most important parameters in SEC are
(i) the diameter of the pores allowing access to the internal volume of
the beads,
(ii) the total internal volume of the beads,
(iii) the hydrodynamic diameter of the sample molecules,
(iv) the flow rate of the liquid phase, and
(v) the operation temperature and viscosity of the buffer used.
 Gel Filtration

Porous beads

Large molecules are “excluded” from the pores and

Column matrix travel through the column fastest
Small molecules are “included” – can diffuse into
the pores and elute later
Theory
Elution Profile

Ve Ve Ve

Ve = Elution volume (volume of solvent between injection

and elution). Dictated by proportion of porous matrix
available to molecules (Kd).
 Applications of gel filtration
• Purification of enzymes and other proteins.
• Estimation of molecular weight mainly for globular proteins:
 Choice of Source​
• Classical approach involves choosing a source containing large quantity of enzyme
• Acetyl CoA carboxylase (mammary gland)​
• Alkaline phosphatase (kidney)​
• Modern approach with DNA recombinant technology​
• 3-phosphoshikimate-1-carboxyvinyl transferase in E. coli (1984)​
• 100-fold increase in productivity​
• Prokaryotes as host organisms (E. coli and​
• Bacillus)​
• Rapid growth and simple medium components​
• Disadvantages: lack of post-
translational modification (glycosylation) and forming inclusion bodies​
 Choice of Source​
• Yeasts as enzyme source​
• Saccharomyces cerevisiae rarely forms inclusion bodies, but grow slowly and ma
ke hyperglycosylation​
• Kluyveromyces lactis and Pichia pastoris are also being developed​
• Insect cell with baculovirus vector​
• It can employ many of the
protein modification, processing, and transport system in higher eukaryotic cells​
• ‘Fusion Protein’​
• Glutathione-S-transferase, maltose binding protein, or His-
tag are popularly used​
• They greatly enhance the power of purification and sometimes solubility of prot
ein​
Choice of Source​
• Production occurred in the strain known to make the enzyme of inter
est​
• alkaline protease or -amylase -> Bacillus licheniformis,​
• glucoamylase -> Aspergillus,​
• acid cellulase -> Trichoderma,​
• glucose/xylose isomerase -> Streptomyces​
Methods of homogenization​
• Mechanical methods​
• High pressure homogenizer* (55 MPa) : cooling is important​
• Wet grinding by mills or glass balls​
• Non-mechanical methods​
• Drying​
• Lysis by osmotic shock, detergents, or enzymes​
• Ultrasound*​
• Cooling and protease inhibition are important to recover the enzyme​
 Methods of homogenization​
• Animal cells (organs)​
• It is easy to homogenize due to the lack of cell wall​
• Fat and connective tissue must be removed before homogenization​
• Bacteria and Fungi​
• Cell wall must be digested by enzymes (Protoplasts can be made by treating
lysozyme or chitinase/3-glucanase)​
• Plant​
• Disruption of vacuole can damage enzymes​
• Membrane proteins​
• Usually detergent (anionic, cationic, or neutral) is added​
• Detergent must be chosen by considering the choice of purification method, especially col
umn chromatography​
Methods of separation​
1. Size and mass​

• Ultracentrifugation (300,000g)​
• Mr is the major factor for separation​
• Not very efficient to separate a enzyme from enzyme pool : Usually used to remove impurities​
• Gel filteration (Mr ~ hundreds of thousands)​
• Sephadex, Bio-Gel P, Sephacryl, and Sepharose –​
• expensive and time-consuming​
• Usually in later stage of purification​
• Dialysis (Mr ~ tens of thousands)​
• Usually used for removing salts, organic solvents, etc..​
• Ultrafilteration​
• Small molecules are filtered out by pressure​
• Used for concentrating proteins​
• Alternatively, centrifugation with dialysis membrane​
Methods of separation​
2. Polarity​

• Ion-exchange chromatography​
• Electrostatic property​
• Flow through in low salt and at appropriate pH​
• Desorption by changing salt conc’ and pH​
• Enzymes can be separated by gradient condition​
• Large scale is possible​
• Usually 10-fold increase
Methods of separation​
2. Polarity​

• Electrophoresis​
• Separation by movement of charged molecules​
• Capillary electrophoresis (cross section less than 100m)​
• Isoelectric focusing​
Methods of separation​
2. Polarity​

• Hydrophobic interaction chromatography​

• Depending on the nonpolar amino acid on the surface of enzyme​
• Octyl- or phenyl-Sepharose with high ionic strength​
• Desorption by lowering ionic strength or adding organic solvents (or d
etergents)​
Methods of separation​
3. Solubility​

• Change in pH​
• Enzymes are least soluble at pI because there is no repulsive force between enzymes​
• Enzyme must not be inactivated in a range of pH​
• Change in ionic strength​
• Large charged molecules are only slightly soluble in pure water; Addition of ion promotes sol
ubility (Salting in)​
• Beyond a certain ionic strength, the charged molecules are quickly precipitated (Salting out)​
• Ammonium sulfate is popularly used​
• 10-fold increase in purity​
• Fructose-
bisphosphate aldolase from rabbit muscle can be purified in high purity by ammonium sulfat
e​
Methods of separation​
3. Solubility​

• Decrease in dielectric constant​

• Addition of water-miscible organic solvent (ethanol or acetone)​
• Decrease dielectric constant​
• Sometimes deactivate the enzyme​
• Work at low temperature​
• PEG (poly ethylene glycol) ~ Mr 4000 to 6000 is commonly used​
Methods of separation​
4. Specific binding sites​

• Affinity chromatography​
•​
•​
•​
•​
•​
•​
• Substrate or inhibitor is linked to a matrix​
• Desorbed by a pulse of substrate or changed pH, ionic strength​
Methods of separation​
4.1 Affinity chromoatography​

• Problems​
• Attaching a suitable substrate or inhibitor to the matrix can be difficult​
• Linking b/n substrate and matrix itself may inhibit the binding b/
n enzyme and substrate: Spacer arm (diaminehexane) may be needed​
• Binding affinity b/n enzyme and substrate must be in a proper range​
• Special attention is necessary to separate the enzymes using same substrate or using mo
re than one substrate​
• Fusing proteins to solve the problems​
• Glutathione-S-transferase : glutathione​
• Maltose binding protein : maltose​
• Hexahisitidine : Ni2+ (Elution by imidazole or thrombine cleavage site is added after the t
ag)​
Methods of separation​
4. Other chromoatographies​

• Affinity elution​
• Affinity occur at desorption step​
• Can solve some problems of affinity chromatography and easy to scale up​
• Dye-ligand chromatography​
• Cibacron Blue F3G-A can bind to a number of dehydrogenases and kinases​
• Procion Red HE-3B binds well with NADP+-dependent dehydrogenase​
• Immunoadsorption chromatography​
• Immobilize the antibody to CNBr treated Sepharose​
• Achieve much higher purity​
 Methods of separation​
4. Other chromoatographies​
​

• Covalent chromatography​
• Separation of cysteine containing protein using thiol-Sepharose 4B​
Methods of separation​
5. Choice of method​

• Time/Large scale -
> Precipitation by ethanol or ammonium sulfate or purification based
on solubility​
• Small scale/high purity -> Column chromatography or electrophoresis​
• FPLC or HPLC -> Fast and high purity, expensive​
How to know the success of purification​
• Tests for catalytic activity​
• By enzyme assay​
• Check cofactors and inhibitors​
• Stabilizing factors​
• Neutral pH, storage in 50% glycerol may help​
• 2-mercaptoethanol or DTT(Dithiothreitol)*​
• Protease inhibitor PMSF (Phenylmethylsulfonyl flouride)​
• Active site titrations​
• Checking the proportion of active enzyme in the purified enzyme
Affinity chromatography
• The affinity chromatography works on the principle of mutual
interaction between a ligand (L) and receptor (R) forms ligand-
receptor complex (RL) with a dissociation constant Kd, which is
expressed as follows
✔ Dissociation constant Kd is specific to the receptor-ligand pair and number of interaction between them.

✔ when a crude mixture is passed through an affinity column, the receptor present on the matrix reacts
with the ligand present on different molecules.
•
✔ The affinity between receptor on matrix and ligands and consequently the best choice bind to the
receptor whereas all other molecules do not bind and appear in flow through.
•
✔ A wash step removes remaining weakly bound molecules on matrix.

✔ Subsequently, a counter ligand is used to elute the bound molecule through a competition between the
matrix bound molecule and counter ligand.
•
Advantages of Affinity chromatography-

• . Specificity: Affinity chromatography is specific to the analyte in comparison to other

purification technique which are utilizing molecular size, charge, hydrophobic patches or
isoelectric point etc.
•
• 2. Purification Yield: Compared to other purification method, affinity purification gives very
high level of purification fold with high yield. In a typical affinity purification more than
90%recovery is possible.

• 3. Reproducible: Affinity purification is reproducible and gives consistent results from one
purification to other as long as it is independent to the presence of contaminating species.

• 4. Easy to perform: Affinity purification is very robust and it depends on force governing
ligand-receptor complex formation. Compared to other techniques