Fluorometric assay of laccase in mushroom extracts and comparisons with absorption spectrophotometry

Abstract

Laccase is a lignin-degrading enzyme that is expected to move industrial applications to a greener form of biotechnology. Here, we used 2,2'-azinobis(3-ethylbenzthiazolin-6-sulfonic acid) (ABTS) as a mediator and N-benzoyl leucomethylene blue (BLMB) as a substrate to develop a fluorometric assay that we used to measure laccase activity in mushroom extracts. We then compared this novel approach to conventional absorption spectrophotometry. With this novel approach, laccase oxidizes ABTS to produce ABTS radicals that show an absorption maximum at 415 nm. The ABTS radicals oxidize BLMB to generate fluorescent methylene blue that is measured by fluorometry while absorption spectrophotometry directly measures the absorbance of the ABTS radicals at 415 nm. Under the optimal conditions, the fluorometric assay showed a linear calibration curve with limits of detection and quantification of 1.0 × 10-2 mg mL-1 and 3.2 × 10-2 mg mL-1, respectively, and those values are 1.4-fold lower than the results using conventional absorption spectrophotometry to measure ABTS radicals. Laccase activity of extracts from species of mushrooms that include eryngii and shiitake were successfully determined via both fluorometry and absorption spectrophotometry. The eryngii extract showed the highest level of activity, which was followed by the shiitake extract, but laccase activity was not observed in the shimeji extract.