Normally for active molecules PMSF can be used to stop enzyme activity. However, in my case I am going to test the viability/release of encapsulated bacteria in simulated in vitro gastrointestinal fluids. When I sample from media over the time, I need to stop enzyme activity in the sample I withdraw. I cannot use PMSF since it is soluble in methanol. I don´t know if water soluble version 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, AEBFS, can be used or not...if it has any negative effect on bacteria or not..
Also, can I keep withdrawn samples in freezer after adding enzyme inhibitor and snap freezing into liquid nitrogen in order to enumerate later?(considering many samples and impossibility in plating them all freshly)
If you are familiar with the topic and give your suggestion, I appreciate.
Thank you
Best
Cigdem
enzyme activity inhibition depends on type of enzymes,
1- lypophlized sample (freeze frying)
2- freezing -18 C
3- by chemical solution
4- coagulate of protein enzyme (by heating) you need to study the physical properties of your enzyme
It would be useful to know which enzymes were added to the simulated gastrointestinal fluid. There may be selective, water-soluble inhibitors that could be added instead of general serine protease inhibitors like PMSF. For example, you could add bovine pancreatic trypsin inhibitor protein instead of PMSF to inhibit trypsin.
Dear Adam,
Thank you for your input. I have pepsin in gastric fluid and bile salts+pancreatin in intestinal fluid.
Pepsin can be potently inhibited with pepstatin. Pancreatin contains trypsin, which can be inhibited by bovine pancreatic trypsin inhibitor. Pancreatin also contains amylase and lipase. Do you need to inhibit those enzymes as well?
Snap freezing the samples is a good way to store them for future use.
Thank you for your answer. yes I need to stop all enzyme activity since I will test the resistance of encapsulation and release of bacteria from encapsulation during in vitro gastrointestinal passage. That means I am going to plate the samples to count bacteria survived and/or released.
Additionally you can try to play with the pH, but again, if you need to plate the samples AFTER stopping the reaction this might influence your survival rate. I m not sure whether the samples you are collecting already have the bacteria you are plating later or not.