Methodology for Enzymes

(Triacylglycerol acylhydrolase EC

Lipase hydrolyses triglycerides to release free fatty acids and glycerol.

Triglycerides + H2O

Glycerol + Fatty Acids
During germination of oilseeds, lipases play an important role in hydrolysing the stored oils so that the required energy for growth and carbon skeleton for synthesis of new compounds are produced. The germinating seeds of castor bean, sunflower, groundnut are good sources of lipases.

The quantity of fatty acid released in unit time is measured by the quantity of NaOH required to maintain pH constant. The milliequivalent of alkali consumed is taken as a measure of the activity of the enzyme.

» Substrate
   Take 2mL of any clear vegetable oil, neutralize to pH 7.0, if necessary, and stir well with 25mL of water in the presence of 100mg bile salts (sodium taurocholate) till an emulsion is formed. Addition of 2g gum arabic has­tens emulsification.
» 0.1N NaOH
» Enzyme Source
    Grind a known quantity of sample with a mortar and pestle. Homogenize the tissue with twice the volume of ice-cold acetone. Filter and wash the powder successively with acetone, acetone:ether (1:1) and ether. Air dry the powder. This acetone powder, can be stored in a refrigerator. Extract 1g of the powder in 20mL ice-cold water or a suitable buffer. Centrifuge at 15,000rpm for 10min and use the supernatant as enzyme source.
» 50mM phosphate buffer (pH 7.0)

Take 20mL of substrate in a 500mL beaker. Add 5mL of phosphate buffer (pH 7.0).
Set the beaker on top of a magnetic stirrer cum hot plate and stir the contents slowly. Maintain the temperature at 35°C. Dip the electrodes of a pH meter in the reaction mixture. Note the pH and adjust it to 7.0.
Add enzyme extract (0.5mL), immediately record the pH and set the timer on. Let it be pH at zero time.
At frequent intervals (say 10min) or as the pH drops by about 0.2 unit add 0.1N NaOH to bring pH to the initial value. Continue the titration for 30 - 60 min period.
Note the volume of alkali consumed.

The enzyme activity is defined as the amount of enzyme which releases one milliequivalent of free fatty acid per minute per g sample. Specific activity is expressed as milliequivalents/min/mg protein.
Activity meq/min/g sample
                                                   =   Volume of alkali consumed x Strength of alkali
                                                                Wt. of sample in g x Time in min

1.  Jayaraman, J (1981) In: Laboratory Manual in Biochemistry Wiley Eastern Limited New Delhi p 133.