Characterization of lipase
The lipase activity was measured in
triplicate by the method modified from Markweg
et al. (1995) using 0.01 M para–
nitrophenylpalmitate (pNNP) dissolved in iso–
propanol as the substrate. The substrate (100 μl)
was incubated with crude enzyme extract (50 μl)
and buffer solution (800 μl) at various pH levels:
0.1 M glycine–HCl pH 2; 0.1 M citrate buffer pH
3–5; 0.1 M phosphate buffer pH 6–8; 0.05 M
carbonate buffer pH 9–10; 0.05 M Na2HPO4
buffer pH 11; and 0.1 M KCl - NaOH buffer pH
12 – 13; for 60 min at 30°C. Then, 250 μL of 0.1
M Na2CO3 was added to stop the reaction and
centrifugation was carried out at 10,000 g for 15
min. The absorbance of the supernatant was
measured at 410 nm against a blank similarly
prepared, but without the crude extract sample.
Para–nitrophenol (pNP) at a concentration of 100–