I am studying the effect of different organic solvents on lipase stability. To compare the wild-type enzyme with it's mutants in terms of stability, should I work at very drastic conditions like incubation in 100 % organic solvents then perform the assay or work around what is reported in literature around 30 %?
Hi there,
There are 2 different approaches: study the effect of the presence of the solvent during the assay (ie. characterizing the immediate effect of the solvent on enzyme activity) or study the effect of the solvent during enzyme storage (ie. long term effect on protein stability). If the experiment is already documented, set your own using published protocol and compare with the published data. You may somehow extend the range of solvent concentration tested.
I agree with Dominique in that you should measure the effect of organic solvent in the assay itself or incubate the protein with organic solvent and then dilute into assay.
For the second part, you can vary the concentration of organic solvent, as well as the time incubated with such- temperature is another factor. I would not be surprised if the solvent at high concentration would unravel the protein and you may not be able to refold it when added into the assay and find out you have zero activity. That is why the dose/response is necessary.
Keep in mind that when you assay for enzymatic activity, you should have the optimal pH for the enzyme and other factors (chemicals) to optimize its activity. Keep these constant for all doses.
Good luck.
Dear Prof.Gustavo,
I am interested in the second part. Actually, my study is both theoretical and experimental. During the theoretical part I performed MD simulations in which the protein is immersed in 100% organic solvent. But, when we come to wet lab as you said this would kill the protein. So, I think I will follow what is in literature as Prof. Liger stated in his answer.
Thanks !
concentration of the solvent in biocatalytic reactions depends on the enzyme activity in particular solvent
most of the enzymes are not stable in solvents more particularly in the polar solvents
only lipases and enzymes obtained from thermophilic environment are stable in organic solvents and in higher temperatures of 50oC one has to follow the work of Alexander M. Klibanov
some people say that the enzymes are stable at all solvents at 100 oC one has to take the statement with pinch of salt.
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