methods ofenzymeisolationandpurification
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The document discusses various methods of enzyme isolation and purification, highlighting the historical context and the significance of enzymes in living systems. It details techniques such as grinding, freezing and thawing, use of hydrolytic enzymes, and purification methods like centrifugation and chromatography. Additionally, the document emphasizes the importance of enzyme isolation for research, diagnostics, and industrial applications.
methods ofenzymeisolationandpurification
- 1. 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. Thank you…..