pH stat methods have been exhaustive and inaccurate in measuring enzyme activity.
The enzyme in question is lipase in a crude form (fermentation broth). I was hoping for an improvement on this method. Please help.
Hi Revathi, are you measuring PLA2 acivity?
There are chromogenic and fluorogenic substrates commercially available for measuring PLA2 activity, without doing the pH stat (I always hated doing that).
There may be a more convenient enzyme-coupled assay available, but I would need to know what you are using for your substrate and what is the product in order to think about it some more.
Steingrimur Stefansson, I am measuring CALB activity. I am stressing the use of pH stat because what I am performing is a validation study. And I need to reproduce the results.
Adam B Shapiro, Yes I agree, there are many more accurate methods but I am helpless due the current scenario. The substrate I use is Tributyrin which when acted upon by Lipase, releases F.A.s and the resultant mixture is titrated to know the activity.
Hi Revathi, unfortunately I am not familiar with lipase b.
But tributeryn is a simple triester of glycerol and butyric acid. (I bet that the assay smells really bad).
Anyway, lipase b it is an esterase with a catalytic triad similar to serine proteinases, it should be able to cleave synthetic esterase substrates.
Can you try out any of these synthetic substrates?
http://www.sigmaaldrich.com/catalog/search?interface=All&term=lipase%20b%20substrate&N=0&focus=product&lang=en®ion=US
I forgot to add that many lipases have to adhere to organized lipid micelles in order to cleave triglyceride ester bonds of long chain FAs.
I may be wrong, but the butyric acid alkyl tail in tributeryn may be too short to form organized micellar structures in solution.
Maybe lipase b can act on small ester substrates.
Steingrimur Stefansso- Of course, I'll try out with other substrates if that's what it takes. We have been using a emulsified Tributyrin solution in order to circumvent the problem that you've pointed out.
The smell...not so much when you have a media which smells a gazillion times worse as a sample.
Steingrimur Stefansson- Thank you ;),we scientists need it. Crunching up numbers and sitting for hours at a stretch with little to show can suck the life outta People. A little humor goes a long way at keeping our sanity intact.
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