8.2.4.1.3 Extraction of Enzymes

8.2.4.1.3 Extraction of Enzymes

Fresh tissue is crushed into a paste with an extraction medium (often a buffer) in a mortar and pestle, or in a tissue homogenizer, or in a blender  or by  ultrasonic vibrations (sonication).

The molarity and pH of the buffer is suitably adjusted (which may vary for different enzymes) to achieve maximum solubility and activity of the enzyme.

EDTA (ethylene diamine tetra acetic acid) is often included in the extraction medium to remove heavy metals (which otherwise inhibit enzyme activity), and for disrupting the membranes of cells and cell organelles. Detergents such as Triton-X are also used sometimes to solubilise the membranes. 

Many enzyme proteins contain disulfide (S-S) bonds due to the presence of cysteine residues, which are easily broken during enzyme extraction leading to loss of enzyme activity. To overcome this problem are added, thiols such as mercaptoethanol whose sulfhydryl (-SH) group is able to maintain the S-S linkage in enzymes. 

If the extract is not homogeneous, the homogenate (extract) is filtered to remove cell derbis, fibres etc., otherwise filtration may be avoided. All operations of extraction and purification are generally carried out in cold (O-4°C), since most of the enzymes get inactivated at higher temperatures.

Last modified: Saturday, 12 November 2011, 6:46 AM