It is difficult to say without knowing more about your reaction conditions. I have seen other colors (e.g. purple) with other enzymes but not colorless.

Are you sure you have laccase activity? A positive control, which you can titrate, would be useful just to make sure all is well with your system and dilution of enzyme. [HRP is found in many labs (as antibody conjugates), which could also be used just to check your ABTS substrate if you have no laccase control].

If you do have laccase activity and yet get no green color, it suggests some further reaction of product is happening with a component in your reaction mix to prevent formation of the green form of the dye.

Dear Zeba,

Respect to Nick's opinion, I suggest you to use guaiacole to be sure about the laccase activity. The color change of guaiacol is so clear if there is a few laccase in the medium due to its sensivity. Then after, repeat your test to maesure the enzyme acctivity.

I think t your reaction mixture contains other components like salts or antioxidants substances. Native ABTS in a saline solution is colorless. Also, ABTS is used for antioxidant capacity assay depending on the reduction of ABTS+ from blue-green to colorless.

Nick Gee Thank you very much for your suggestions.

I did not use any positive control instead i used a negative control with water in place of enzyme in supernatant and even Negative control has the original ABTS Colour ( light green). but in presence of enzyme solution it shows colourless.

I used 100mM Citrate-phosphate buffer of PH-4 and 3mM ABTS to make ABTS screening assay solution. I followed this paper attached here for the assay. See highlighted text

Mina Karimi-Avargani . Thank you! sure i will try

Nashwa Fetyan . so what is the solution to avoid these?

As your paper mentioned, It is likely that the presence of certain metabolites released by yeast cells could negatively affect laccase activity/stability at this stage of fermentation

Good luck

Nashwa Fetyan . okay thank you

Dear Mina Karimi-Avargani , If i use guaiacole as a laccase substrate instead of ABTS and as Nashwa Fetyan mentioned that certain metabolites could affect the laccase activity by using ABTS. I wonder if it may also act as same as ABTS in presence of certain metabolites?

Dear Zeba,

The mechanisim of laccase with guaiacol is so different in comparision to ABTS.

I have attached three articles about using guaiacol. I hope they would be useful.

In this case, I think you must have positive control, and the adjustment of the reaction pH must be very accurate to prevent overlapping of other enzymes ( peroxidases).

good luck

I expressed fungal Laccase in Yeast for lignin degradation purpose and its RNA and proteins are successfully transcribed and translated. I confirmed it by q RT PCR and Western blot respectively.

Now i want to test their enzymatic activity by using ABTS or Guaiacol . Since i already done with ABTS and found no color and now i am thinking to go for Guaiacol as Mina Karimi-Avargani mentioned. Before going for Guaiacol, i want to make sure that if i need to culture yeast with Lignin also? Because so far i just cultured the laccase containing yeast in media and collected the cultured supernatant to do the enzymatic assays.

Any leads would be appreciated. Thanks

i hope u carried out the reaction in acidic ph. Abts turns colorless to green in acidic ph upon reduction by laccase

Please check the pH of the test media, it need pH3.