Article Improved method for qualitative screening of lipolytic bacte...

http://ijpsr.com/bft-article/extracellular-lipase-from-bacillus-subtilis-kubt4-isolation-identification-and-characterization/?view=fulltext

Pare nitrophenylpalmitate is highly sensitive to the pH of the buffer and works well in a pH range of 7.0 to 8.5, and at a strongly alkaline pH it auto degrades and at an acidic pH say pH 4.0 it doesn't degrade much and you need to use para nitrophenylacetate. Firstly, be specific whether you need to isolate alkaline lipase producers or esterase

Hello Dr. Shamsher S. Kanwar , thanks a lot for taking the time to answer me.

I've searched information about para nitrophenylacetate.

Isn't it an esterase substrate?

https://www.sigmaaldrich.com/catalog/product/sigma/n8130?lang=en®ion=TW

I did the qualitative estimation on agar plates by following this article ( https://scialert.net/fulltextmobile/?doi=ijbc.2011.116.126 ).

"The present method could also successfully differentiate between esterase and lipase by using tributyrin and oil, respectively, as substrates. Esterase gave positive results (yellow on pink background) only on tributyrin plates and lipase was positive for both the substrates."

Yes, it is a substrate to check esterase activity of an enzyme. The true lipases efficiently hydrolyze p-nitrophenyl palmitate.

Hi, Mark Stor

I'm planning to do the stability test of enzyme activity ──

【 Prepare the same concentration of commercially purified lipase at different pH value (4~9), test different ratio of the sample and substrate buffer, to check which ratio have similar optical density (OD). That means if the ability of the buffer is sufficient, the OD of sample should be consistent at different pH values. Then reconfirm the lipase activity of crude extract from different strains.】

Please give me some suggestions or recommended literature about methods of lipase activity.

Mark Stor

The method I described above is based on someone's thesis, which is the procedure I have found that can improve the stability of enzyme measurement.

I think the purpose of this method is to confirm the buffer's ability to achieve similar results after reacting with different pH samples.

I have also thought about the method you mentioned, so I am confused now.

And I can tell you, at this stage, what I want to improve is to let the enzymes react well in the substrate buffer, and let the product (p-nitrophenol, p-NP) be stable.