I use para nitrophenyl palmitate substrate with tris buffer ph 8... i read many temperature for Yellow color of para nitro phenol production.. 37 and 50 and 40 at water bath ... I confused as i use 37 degree the reaction produce yellow color after 1 h and many times no color produced although enzyme is produced at plate assay
Dear Heba Kamal Issa
Activity : assay reaction is precisely explained in the attached protocol
Generally, lipase activity is measured at 37 deg celsius. Your lipase may be getting deactivated at 50 deg. You should be sure that your p-nitrophenolate has dissolved in the buffer giving a clear solution. It may be precipitating out when you add it to the buffer. In that case you will not see any yellow colour. You should not need more than 10 min to see the colour. If you have wait for an hour, it means the lipase activity is nil (you may see the protein band but it has lost enzyme activity during your work-up) and what you see is the self hydrolysis of the substrate.
Please look at the following below links which may help you in your analysis:
https://www.journalofdairyscience.org/article/S0022-0302(56)94883-4/pdf
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/1/mak047bul.pdf
http://www.scielo.br/pdf/qn/v35n6/a20v35n6.pdf
Thanks
Nitin Fadnavis but when i use 50 degree, yellow color appear after 30 min comparing with blank there is no color. no color at 37 degree....... what is the reason in your opinion?
Your lipase may be thermophilic which works well at high temperatures. Still I think 30 min is very long time. If you increase enzyme concentration, does the reaction becomes faster? It would be an indication of actual enzymatic reaction.
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