I have a crude extract of enzymes produced by Solid State Fermentation, but the microorganism produces a black/ brown dye. I already tried to different methods to remove dye from extracts, like filtration, ultrafiltration and carbon active without good results ( active carbon removes dye but in the filtered we can't found protein neither activity). So, I saw a closely association between dye/total protein/Enzymatic activity, in all experiments, when the color it's removed, the activity and the protein are also removed. I used Spectrophotometric techniques to determinate the amount of enzymatic activity and total protein, and the presence of the dye causes interferences because the amount of absorbance are really higher with great rates of standard error.
I'm sure that the amount of protein (Bradford method) and the Enzymatic activity are not product of the error caused for the dye, because I made some dilutions ( when the dye is diluted ) with linear results of activity and protein.
Any suggestion?
I think you can try precipitate the proteins out the crude extract using trichloroacetic acid( TCA ) at low temperature i.e. 4 C ,so the dye will be in the supernatant while the proteins including your enzyme will be in the pellet, which you can dissolve in any buffer suitable for your assay
It will be better to dialysis your sample using larger amount of buffer with a dialysis bag having smaller cutoff than the size of your enzyme.
Dear Angel, you described your experiments in a very wise and detailed way I like very much! TCA will kill every enzyme activity thus please do not destroy your enzyme. I agree with Martin Mahro idea of size exclusion, but rather do not suppose it is covalently bound since you are able to separate the "color" from your protein. What could be the explanation of your experiments is that you have an "colored" ligand is essential for your enzyme activity. In order to get the Nobel;) for this ligand discovery I would separate your crude by for example size-exclusion, then try the color fraction mixed with the rest of the clear fractions. If addition of the color to any of them restores your activity, Nature will accept it!
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