Testing lipase activity on nitrophenol pNPP.

Asked:
I'm doing this experiment and bought different nitrophenol substrates from C2-C16, but to my bewilderment, substrates below C10 are easily hydrolyzed spontaneously in the buffer, which interferes with enzyme biometrics. Substrates larger than C10 are difficult to dissolve. Very confused! May I ask you have done this experiment prawns to give me some help, not very grateful!
:
30 mg of nitrobenzene (p-NPP) can be dissolved in 10 ml of isopropyl alcohol, a slight water bath
heating
can be completely dissolved. I am based on (p-NPP), solution A:30 mg of nitrobenzene (p-NPP) palmitic acid can be dissolved in 10 ml of isopropyl alcohol. Solution B: Phosphoric acid buffer solution containing 1% triton. Solution A is mixed with solution B1:9 (now accessories).
I now encountered in the experiment is: the enzyme living substrate solution is colorless, but a fermentation solution immediately turned yellow, the measured absorption of light value is particularly large, but the fermentation solution boiled for 20 minutes for the control substrate solution unchanged color, with alkaline measurement of fatase vitality is not high. There is no such phenomenon with standard lipase. The experiment was very depressing! I don't know what causes the substrate to change.
, please ask you to have done this experiment prawns to give me some help, not very grateful!
:
I think it's very sensitive to measure the vitality of lipase with nitrophenol fatty acid substrates, I don't quite understand the confusion on the second floor, from your description, your fermentation liquid should have enzyme activity. The fatase vitality was not high by alkali titration. Normal ah, because the PNPP method is more sensitive, so even if you use PNPP method found that the color reaction is very yellow, its use of alkali titration may also be relatively low enzyme activity. This is my experimental conclusion, do not know whether it is in line with everyone's experience.
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