isolation, purification and characterisation of ...isolation, purification and characterisation of...

ISOLATION, PURIFICATION AND

CHARACTERISATION OF ENZYMES

Steps involved in isolation of a protein or enzyme:

1) Cell disruption (which can done via a number of number of

different processes of choice e.g Detergents lysis, Osmolysis, freeze-

thaw cycles, enzymatic lysis, ultrasonication, Homogenisation)

2) Centrifugation (at a specific speed depending on the organ,

tissue, organelle or fluid).

3) Removal of supernatant (Decantation to obtain supernatant)3) Removal of supernatant (Decantation to obtain supernatant)

A HOMOGENIZER

A CENTRIFUGE

Steps involved in purification of a protein or enzyme:

1) Salt precipitation using (NH4)2SO4

2) Dialysis

3) Size exclusion chromatography (also called Gel filtration

or Molecular sieving)

4) Ion exchange chromatography

5) Affinity chromatography

ENZYME ACTIVITY, SPECIFIC ACTIVITY,

PURIFICATION FOLD & YIELD

CALCULATIONS USING TABLES CONTAINING ENZYME ACTIVITIES

ENZYME CHARACTERISATIONEnzyme characterisation simply refers to the determination of the various chemical

and physical properties (characteristics) of an enzyme. It involves the use of a series

of laboratory procedures. Examples of assays for commonly characterised enzyme

properties in biochemical researches include:

1) Determination of the effect of changes in temperature on enzyme’s activity

and optimum temperature.

2) Determination of enzyme’s thermal stability.

3) Determination of the effect of changes in pH on enzyme’s activity and

optimum pH.

4) Determination of pH stability.

5) Determination of the effect of changes in substrate concentration on 5) Determination of the effect of changes in substrate concentration on

enzyme’s activity and kinetic constants e.g Vmax, Km, Kcat, Km/Kcat etc.

6) Determination of substrate specificity

7) Determination of molecular weight (Mw) of enzyme

8) Determination of the effect of metal ions, chelating agent or denaturating

agents.

9) Determination of enzyme’s isoelectric point (pI)

10) Determination of the effect of duration of incubation.

11) Determination of active site fractional saturation (V/Vmax) at a particular

substrate concentration.

12) Determination of Enzyme’s turnover number (Vmax/ET)

13) Determination of activation energy (Ea)

14) Determination of salt tolerance