I am working on a novel bacterial laccase (recombinant), but I am not sure how to begin studying best conditions for enzyme activity. Studying optimal pH, optimal temperature, thermostability...e.t.c
Also what is the minimal required enzyme concentration to show activity? and what is the required substrate concentration too? Different papers use different concentrations.
I tried several protocols for enzyme activity from past research reports and none of them showed activity. I am not sure if the conditions are wrong, or if my procedure is wrong.
In this form, your question is unanswerable. However, you should approach this problem systematically. You have a recombinat laccase, then what is the source? How is the natural enzyme detected, what are the optimal reaction conditions?
Use the natural enzyme to develop your assay and as positive control, the same conditions should also work for the recombinant enzyme.
Of course, it is possible that the recombinant enzyme is inactive because of incorrect folding, missing or excessive S-S bridge formation, missing cofactors, damage during purification... Then you'd have to optimise your production.
Thank you for your reply, @Engelbert Buxbaum
Let me clarify, I cloned a potential laccase gene, expressed the enzyme at different temperatures and, based on the thickness of the SDS band, chose the best temperature. Then I purified the enzyme using the His-tag available in the plasmid.
When trying activity assay for crude and purified fractions, I don't get any results.
So your suggestion is that my enzyme could be inactive?
@Mina Karimi-Avargani
Thank you for your reply. I will try using these papers. But does procedure differ based on the source of laccase, wether fungal or bacterial?
If the enzyme from the natural source is active under your assay conditions, but the recombinant enzyme is not, then yes.
As I said, you need to do this systematically:
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