Hello everyone,
Does it make sense to think of re-using enzyme substrates? Just been curious. Given the fact that I know there is plenty of enzyme substrate left once the rxn is over.
Thanks.
Hello Misbah,
I haven't re-used the substrate, as I had no means to quantify how much was left after the enzyme-controlled reaction finished. If you used excess substrate and are able to quantify the remaining substrate I don't see why you couldn't re-use it. Is your product inhibiting the enzyme? In this case it needs to be removed entirely. How would you quantify the enzyme concentration in the mixture after the first run?
The enzyme could easily be removed by passing the reaction mixture through a centrifugal ultrafiltration device with a molecular weight cutoff smaller than the enzyme's molecular weight. The substrate would pass through. Removal of the products would have to be achieved by some other means if there is any product inhibition, as Mihaela mentioned, or if the presence of the products interferes with monitoring the reaction.
Hello, @Mihaela, Thanks for your reply. Yes, I know the amount of left-over substrates, since my enzyme works very slowly and I have got an idea based upon my quantifications that I have almost 50% of substrate and starter-COA left in my reaction mixtures, as everything is controlled and I know about every nano and picomole utilized and remaining. I do extract the products by doing partition into organic and aqueous layer and I am pretty sure that I extract all of the enzyme product this way. I am sorry I did not get the idea of quantification of enzyme concentration in reaction mixture after first run, since I do overnight reaction. I let it go until it could go so I guess that after overnight, enzyme would have degraded or most of it would have degraded.
@Shapiro, very nice idea, I had not even thought of it. What I am careful about is, I work with [14C-malonyl COA] and I cannot bring this radioactivity anywhere.
Thanks a lot to both of you.
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