Enzymes

1. By:
Dr.Farid Shehta.
Enzymes Purification

2. Enzymes are generally globular proteins, acting alone or in larger
complexes. The sequence of the amino acids specifies the structure
which in turn determines the catalytic activity of the enzyme
Enzymes

3. Methods of enzyme purification
Purification of an enzyme protein is generally a multi-step
process exploiting a range of biophysical and biochemical
characteristics such as its relative concentration in the source,
solubility, charge, size (molecular weight), hydrophobicity/
hydrophilicity of the target protein. In general, design of the
purification technique/ protocol should be focused on:
 High recovery.
 Highly purified enzyme protein.
 Reproducibility of the methods.
 Economical use of the chemicals (reagents).
 Shorter time for complete purification.

4. Salting in & out
Generally, salting out of proteins using ammonium sulfate is
used as the first step in the enzyme purification.

5. Enzyme Stabilization
• In many cases, the enzyme protein to be purified is fairly stable at
temperature like 55oC or 60oC. At this temperature, many unwanted
proteins get denatured and precipitated out with little or no loss of
enzyme activity.
• In few cases of allosteric enzymes, the enzyme is stable at high
temperature in the presence of an effector molecule.

6. Alcohol Disrupts Hydrogen Bonding
Organic Solvents
• New hydrogen bonds are formed instead
between the new alcohol molecule and
the protein side chains.
ethanol & acetone) change in dielectric
constant andhydration of ionic groups
(disinfectant action and precipitation
ofprotein.)
(e.g. ethanol & acetone) change in dielectric
constant andhydration of ionic groups(disinfectant
action and precipitation ofprotein.)

7. solubility of the protein decreases with increase in the ionic strength.
The solubility of the protein depends on the following factors:
(i) pH.
(ii) temperature and.
(iii) type of the salt used.
log s

8. Chromatographic separation of
the enzyme proteins
Chromatography is a separation technique based on partitioning
of the proteins between moving phase and a stationary phase.
For enzyme purification, commonly used chromatography
techniques are:
 Ion exchange chromatography.
 Adsorption chromatography.
 Gel filtration chromatography.
 Affinity chromatography.

9. Ion Exchange Chromatography
- Ion exchange resins contain charged groups.
- Separates molecules based on charge.
- Mobile phase
- Generally liquid
- Stationary phase
Electrostatically charged ions bound to insoluble, chemically inert
matrix.
Elution of protein Add salt to compete with binding of sample to
stationary phase.
- Change pH (alters charge of protein).

10. Ion Exchange Chromatography
Anion Exchanger
-weak exchanger – diethyl
aminoethyl (DEAE)
-strong exchanger
quatenaryaminoethyl (QAE)
-This type of resin is positively
charged
-The resin binds negative proteins
-Proteins are eluted by NaCl or
altering pH - how does this work?
weak exchanger -
carboxymethy (CM)
weak exchanger -
sulfipropyl (SP)
-This type of resin is negatively
charged
-The resin binds positive proteins
Proteins are eluted by NaCl or
altering pH - how does this work?
Cation exchanger

11. Ion Exchange Chromatography

12. Adsorption chromatography
The basic principle in this type of chromatography is
binding of the proteins on the matrix by physical
adsorption on the surface of insoluble matrix (through
weaker bonds like hydrogen, van der Waals bonds).
The commonly used matrices in adsorption
chromatography are:
1. Calcium phosphate gel.
2. Alumina gel.
3. Hydroxylapatite gel.

13. is performed to analyse samples or to follow
reactions as they proceed, and column
chromatography is used to separate and purify
compounds after a reaction. The most common
stationary phases used in the lab are silica (SiO2)
and alumina (Al2O3), in very fine and pure powder
form.
TLC (thin layer chromatography)

14. HPLC
- high pressure to drive the solutes through the column
faster.
- diffusion is limited and the resolution is improved.
- The most common form is "reversed phase" hplc,
where the column material is hydrophobic.
- The proteins are eluted by a gradient of increasing
amounts of an organic solvent, such as acetonitrile. The
proteins elute according to their hydrophobicity. After
purification by HPLC the protein is in a solution that
only contains volatile compounds, and can easily
be
lyophilized

16. Gel-Filtration” of proteins
Size-Exclusion Chromatography
-Gel filtration separates proteins on the basis of their
molecular weights.
-Larger molecular weight proteins move more rapidly.
Hence, large MW proteins elute from the column before the
smaller ones
-Small pores in the gel allow smaller molecules to become
trapped in the matrix, slowing their flow through the
column.

18. Affinity chromatography
purification based on a natural interactions for a protein and a
substrate or chemical group (ligand)
 only proteins which recognize the molecule on the stationary
phase will bind
 Elute by competition with the bound ligand
 generally a good method but it doesn’t always work - Some
non-specific interactions can occur
 Spacer arm may be needed to make the
compound available to the protein

19. Affinity Chromatography” of proteins:
(binding specificity)
- Separates proteins by binding specificities.
- The proteins retained on the column are
those that bind specifically to a ligand cross-
linked to the beads.
- After proteins that do not bind to the ligand
are washed through the column, the bound
protein of particular interestis eluted by a
solution containing free ligand

20. Affinity Chromatography

21. Electrophoresis techniques
-Paper Electrophoresis
-Gel Electrophoresis
-SDS-PAGE
- Isoelectric Focusing
-Capillary Electrophoresis
Now-a-days, electrophoretic techniques are not in
common use for enzyme purification. Electrophoretic
techniques are more commonly used for analytical
purpose for checking the purity of the enzyme protein.

22. Paper Electrophoresis

24. Gel electrophoresis
is a method for separation and analysis
of macromolecules (DNA, RNA and proteins)
and their fragments, based on their size and
charge. It is used in clinical chemistry to
separate proteins by charge and/or size (IEF
agarose, essentially size independent) and
in biochemistry and molecular biology to
separate a mixed population of DNA and RNA
fragments by length, to estimate the size of
DNA and RNA fragments or to separate
proteins by charge.

25. SDS-PAGE
Here, the proteins (Enzyme) are separated
based on size (in SDS-PAGE) and size/ charge
(Native PAGE).

26. Isoelectric focusing (IEF), also known as
electrofocusing, is a technique for separating
different molecules by differences in their
isoelectric point (pI).[1][2] It is a type of
zone electrophoresis, usually performed on
proteins in a gel, that takes advantage of the
fact that overall charge on the molecule of
interest is a function of the pH of its
surroundings.
Isoelectric Focusing

27. The heart of capillary electrophoresis (CE) is electroosmotic flow
(EOF). This is the mobile phase “pump” in CE. Unlike gas
chromatography (GC), there is no pressurized gas acting as the mobile
phase in CE. Unlike high performance liquid chromatography (HPLC)
these is no (high pressure) pumped mobile phase. And unlike paper
chromatography, there is no capillary action that pulls the solvent
through the stationary phase.
Instead, the electrical potential maintained across the CE’s capillary
tube by the electrical circuit of the
1) Capillary.
2) Buffer.
3) Reservoirs.
4) Electrodes.
5) Power supply sets up some pretty interesting conditions that makes
the buffer solution flow from one buffer reservoir to the other, just as if
it were being pumped.
Capillary Electrophoresis