Enzymes are important biological molecules found in living systems. Early attempts at purifying enzymes were made in the 1920s. Methods are needed to isolate enzymes for further study and applications. Common methods to isolate enzymes include breaking open cell walls through grinding, freezing and thawing, using hydrolytic enzymes, blending, altering pH or ionic strength, and using organic solvents. Further purification techniques include centrifugation, gel filtration chromatography, affinity chromatography, and changing solubility through pH or salt concentration. Specific methods have also been developed for isolating individual enzymes like urease and pepsin.

2. Introduction
• Enzymes are found distributed widely in the
living systems and are a major factor in the
growth, maintenance and propagation of living
system.
• Willstatter and colleagues in 1920 carried out
the early attempts of purification of enzymes.
• The First Enzyme to be crystallized was Urease in
1926 by Sumner.

3. Need of Isolation of Enzyme
• Required for study about the various
aspects of that enzyme.
• Required for researches and as diagnostic
components.
• They employed in forensics, food
processing and Industrial applications,
fermentation.
• Required for leaching of ores.

4. Methods of Isolation
1) Grinding with Abrasives :
The shear and strain causes the disruption of the cell wall
leading to the collection of cell sap as crude homogenate.

5. Methods of Isolation
• Alternate Freezing
and Thawing :
Freezing water
molecules form ice
which expand in size
and cause a strain on
the cell leading to
disruption.
• Use of Appropriate
Hydrolytic Enzymes :
Proteases and
lipases are used to
cause localized removal
of membrane particles
leading to formation of
pores or holes.

6. Methods of Isolation
Long periods of Blending :
Cells broken due to
mechanical shear…
Use of Hypotonic
Solutions :
• It lead to absorption of
water by cells and their
swelling leading to a
turgor pressure that is
great enough to cause
the cells to burst.

7. Methods of Isolation
Alteration of pH of The
Solution :
• The cell membrane
proteins to coagulate
leading to formation of
pores through which the
cell sap oozes out as
homogenate.
Organic Solvents :
• Organic solvents such as
acetone, ether .. are
employed to dissolve the
lipid constituents leading
to formation of pores.

8. Methods of Purification of Enzymes
• Centrifugation : Depends upon size, higher the molecular
weight, the greater the rate of sedimentation.

9. Gel Filteration / Gel permeation
chromatography
• Column of porous gel beads with solvent is used. Small molecules
are retarded and large molecules move down with little resistance.

10. Affinity Chromatography
• It exploits the capacity for binding of enzyme with ligands by
substrate analog.

11. Methods Based on Change in
Solubility
Change in pH :
• An enzyme is least
soluble at its isoelectric
point since at this pH the
repulsive forces within
enzyme cease to exist
and tend to
precipitate……
Change in Ionic
Strength :
• Enzymes often
precipitate on addition
of high amount of salts.
At high salt
concentrations, the conc.
Of water is greatly
reduced.
• Ammonium Sulfate if
widely used salt due to
many advantages..

12. Some other methods of Enzyme
Purification..
• Sumner’s Method For Isolation of Urease.
• Northrop’s Procedure For Isolation of
Pepsin.
• Fractional Precipitation By Heat.
• Electrophoresis.
• Homogenization by Potter-Elvehjem
Homogenizer or High-Speed Blender.