I am working on laccase production from fungi and bacteria. During plate assays, I observe a reddish-brown color forming around the colonies, indicating that guaiacol (used as a substrate) is being oxidized by laccase present in the medium. However, when I scale up production and perform the laccase enzyme assay using guaiacol as the substrate, I do not observe any color change in the test solution.
What could be the reason for this discrepancy between the plate assay and the enzyme assay? Could it be related to enzyme activity, substrate concentration, or assay conditions?
Same condition with my bacteria sample.
For laccase assay, I use 2,5mL acetate buffer (pH 5, 10 mM), 0.1 mL guaiacol (2mM), and 0.4 mL supernatant as crude enzyme. The absorbance is measured at 450nm using spectro in 5 minutes. The absorbance is increased but not stable, and the value is low (under 0.1) for all sample.
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