Preparation of Crude Enzymes
Centrifugation
The enzyme extract, prepared as above, is centrifuged to remove cell debris, cell organelles and sometimes other molecular aggregates, leading to partial purification of enzymes. It also helps in characterization of an enzyme, since, depending upon its mass and shape, the enzyme will move through a solution at a definite speed and occupy a characteristic position in the centrifuge tube.

Cell fractions by different centrifugation protocols

Acids and bases, however, often inactivate the enzyme, so that their use for precipitation is not recommended in most cases. Instead ammonium sulphate and other salts are used for precipitation in a process called salting out. Salts can change the structure of the solvent, which can lead to large changes in protein conformation by altering the electrostatic interaction between charged groups on the protein surface. The salt also competes with the protein for solvent molecules and thereby lowers its solvation. In large scale enzyme precipitation, use of many other neutral salts is preferred over ammonium sulphate, which is corrosive and releases NH₃ at higher temperatures.

Some organic solvents like acetone, methanol and ehtanol are also used for enzyme precipitation, since water miscible solvents decrease the solubility of proteins, leading to precipitation. They are cooled upto 40-60°C before their use, and precipitation is carried out at 0°C, because precipitation at room temperature causes denaturation of the enzyme, in most cases. Organic solvents are added drop' by drop to avoid local concentration.