Methods of enzyme isolation and purification
- 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.