For the p-NPB substrate system, 0.1 mL of crude lipase extract was added to 3.3 mL of substrate
solution and the reaction rate after incubating at 35 C for 5 min ° was measured by spectrophotometer at 410
nm.
For the p-NPL substrate system, 0.1 mL of crude lipase extract was added to 3.2 mL of the corresponding
substrate solution and the mixture was incubated in shaking water-bath at 35°C for 20 min.
The reaction was terminated by boiling for 5 min following centrifugation 6000 rpm for 10 min the absorbance of the clear
supernatant was measured at 410 nm.
In both cases, a sample without enzyme taken the same treatment was
used as a blank. One unit (U) of enzyme activity was defined as the amount of enzyme required for the
liberation of 1 μmol p-NP per minute under the assay conditions.