Presently I am working on bacterial laccase mediated delignification of lignocellulosic biomass. In regards to that, the isolated bacteria was grown in Luria Broth and the laccase assay was carried out by monitoring the absorbance at 420 nm up to 7 days post-inoculation. The reaction mixture contained- 510ul buffer (citrate phosphate buffer, pH 5), 300 ul of enzyme and 90ul of ABTS (0.5mM).For the assay the enzyme was diluted 5 times. The absorbance was found in the range of 0.2-0.9 (1st Day to 7th Day). However, the light green color of the ABTS disappeared once added to the reaction mixture (diluted enzyme and the buffer). As per literature, the color of the reaction mixture should change to green once ABTS is added. The color of the supernatant was also reddish brown on 7th Day. So the day wise absorbance might be the reflection of the supernatant. Also I am planning to check for the intracellular laccase production. Please suggest the required changes so that it works out. Thanks in advance.
N.B. - The bacterial colonies was found to be reddish brown after a certain incubation period on guaiacol plates and so was considered laccase positive.
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