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In this article we will discuss about the tests for estimation of lipase in plants.
Principle:
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.
Materials:
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a. Substrate:
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Take 2 ml of any clear vegetable oil, neutralize to pH 7.0, if necessary, and stir well with 25 ml of water in the presence of 100 mg bile salts (sodium taurocholate) till an emulsion is formed. Addition of 2 g gum Arabic hastens emulsification.
b. 0. 1 N NaOH
c. Enzyme Source
Grind a known quantity of sample with a mortar and pestle. Homogenise 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 1 g of the powder in 20 ml ice-cold water or a suitable buffer. Centrifuge at 15,000 rpm for 10 min and use the supernatant as enzyme source.
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50 mM phosphate buffer (pH 7.0).
Test Procedure:
1. 20 ml of substrate is taken in a 500 ml beaker. Add 5 ml of phosphate buffer (pH 7.0).
2. The beaker is set 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.
3. Enzyme extract (0.5 mL) is added; immediately record the pH and set the timer on. Let it be pH at zero time.
4. At frequent intervals (say 10 min) or as the pH drops by about 0.2 unit add 0.1 N NaOH to bring pH to the initial value. Continue the titration for 30-60 min period.
5. The volume of alkali consumed is noted.
Calculation:
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 expressed as milliequivalents/min/mg protein:
Activity meq/min/g sample = Volume of alkali consumed-Strength of alkali/Weight of sample in g × Time in min
