- I am doing the lipase activity assay using 4-nitrophenyl dodecanoate as lipase substrate and lipase from Aspergillus oryzae(L4277). 4-nitrophenyl dodecanoate was dissolved in 100mL 5mM Sodium acetate solution(1% Triton X-100).
- Positive control: 100μL lipase+100μL 0.1mg/ml orlistat+300μL substrate
- Negative control：100μL lipase+ 100μL deionized water+300μL substrate
- The reaction was processed in 40℃ and more than 6h, but there was no any different between positive control and negative control.
- Could somebody tell me why? or help me optimize my experiment process.
Engelbert Buxbaum
You should also have a control reaction without enzyme. What is the time course of lipase inactivation by Orlistat? Perhaps, enzyme and inactivator (please, do not say inhibitor, inhibitors are in association-dissociation equilibrium with the enzyme, while Orlistat performs a permanent chemical modification) need to be incubated together first, without substrate.
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Michael J. Benedik
Did both reactions go to completion or was there no activity in either reaction? That makes a big difference, but I agree with Engelbert Buxbaum that you need a no enzyme control.
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Junying Li
Stjepan Krešimir Kračun
Exactly, the substrate is unstable. I store the substrate at the refrigerator and Ishould use the hot water bath to dissolve the substrate solution again. I felt the color of substrate solution also will be a little bit yellow without the enzyme. Is it right to add 1ml Triton X-100 to 100ml substrate solution, or could you tell me a better method to dissolve the substrate? Thank you!
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Junying Li
Stjepan Krešimir Kračun
Thank you, professor! You help me a lot. But I still have some questions. First, someone told me i should use Na2CO3 solution to stop the reaction of lipase and substrate, and at the same time, he also said the absorbance of p-nitrophenol at 405nm will be more reliable when the solution is alkaline. And you just told me that this substrate will chemically hydrolyze if the solution is too alkaline or acidic. So, is there a better way to stop the reaction? Can I use boiling water？Another question is that the lipase i used is from Aspergillus oryzae. It is recommended to be used in 40℃ and pH 8.0. If I use the buffer to make the lipase react at pH 7.0. Does it will make the lipase lose its activity?
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