1) Explain three methods used in purifying enzymes
The first method of enzyme purification is using the precipitation technique based on salt concentration. Ammonium sulfate is a common solution used in this method. Usually the protein/enzyme solution is brought to a 50 percent saturation with a saturated ammonium sulfate solution. Due to the salt balance, the proteins will coagulate and precipitate to the bottom when centrifuged at a high speed for approximately 15 minutes.
Chromatography methods include ion exchange, bio-affinity and hydrophobicity. Ion exchange uses molecular charge, and bio-affinity uses biomolecular interaction. Initial preparation for such methods includes the lysing of cells and centrifugation for a pure supernatant. Supernatants contain the appropriate isolated enzymes and can be further purified by one of the above chromatography methods.
a. Adsorption chromatography
It is based on the principle of adsorption. Solute/ enzyme get adsorbed onto the particular sites of the adsorption chromatographic column depending on the effective distribution coefficient. These are then eluted using various solvents. The packing materials used include starch, diatomaceous earth etc.
Effective distribution coefficient is the ratio of distribution of solute across the different phases of chromatography. The process is widely used for initial recovery of extra cellular enzymes manufactured on a large scale.
b. Affinity chromatography
This technique makes use of enzyme substrate interactions. A matrix with a ligand is packed in the column. As the enzyme solution pass through the column, the solute/enzyme molecules get attached to the ligand. These are then eluted by suitable eluants/solvents. In effect the Matrix ligand enzyme complexes remain adsorbed to the column till it gets eluted.
Ion exchange chromatograph
Ion exchange chromatography separates compounds according to the nature and degree of their ionic...