In High throughput screening for cellulase using fluorogenic substrate, how can we ensure that we still can see activity from enzymes that have higher Km values? (accept increase molarity of the substrate)
Thank you very much Rouhollah Barahimipour for your answer,
Actually in our case, a library of thousands of recombinant bacteria has to be screened using fluorogenic substrates which makes the calculation of Km values for each too difficult, I found that a high molarity of the substrate can solve the problem but I want to economize the small quantity of the substrate that I have. For this reason I am trying to find an other solution.
Cordially!
You can compensate for the lower rate caused by a higher Km by increasing the enzyme concentration.
Thank you Dr Adam for answering
I am doing screening for the enzyme so how can I control the enzyme concentration? I don't even know if the enzyme is present or not, I only can control the bacterial growth and substrate concentration.
Thanks again
You can vary the amount of the bacterial extract you put into the assay. If you put in as much as you can and still don't see any activity, then either the enzyme is not present or its catalytic activity is too low to detect. If you don't have an independent method to see whether the protein is present (e.g. SDS-PAGE, Western blot) and you can't detect the activity, then you just have to set that particular strain aside as a negative, even if you don't know why.
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