Laccase: addressing the ambivalence associated with the calculation of enzyme activity

Laccase (benzenediol: oxygen oxidoreductase) a unique multi-copper oxidase enzyme has been studied rigorously since its identification. However, there is ambivalence associated with various aspects of laccase, e.g., assay conditions and calculations. Our aim was to minimize its ambivalence, thus, total of five formulas (F1-F5) were used to determine laccase activity of white and blue laccase. In case of enzymatic profiling of blue laccase, its activity ranged from 0.04 to 464.3 U L^-1 whereas in case of white laccase it ranged from 0.05 to 1404.7 U L^-1. The affinity of laccase at various enzyme concentration (0.3-0.9 mg mL^-1), time (5 and 10 min) along with various substrates, i.e., 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol (GCL), 2,6-dimethoxyphenol (DMP) and syringaldazine (SYZ), and its concentration (ABTS 0.5-1.5 mM, GCL 20-30 mM, DMP 1-5 mM, SYZ 10-30 mM) were inferred. The optimal substrate concentrations were 1.5 and 0.5 mM ABTS for blue and white laccase, respectively, with 30 mM GCL and 2 mM DMP being the common parameter. The optimal substrate concentrations were 0.5 mM ABTS, 20 mM GCL, 1 mM DMP and 30 mM SYZ for commercial laccase. It was observed that the optimal protein load and reaction time was 0.3 mg mL^-1 and 5 min in all the cases, however, in case of white laccase it was 0.6 mg mL^-1 at 10 min for DMP and in case of commercial laccase it was 0.9 mg mL^-1 and 5 min for SYZ. In the present study, F1 was most appropriate among the five formula used as it incorporates all the significant factors and use of single formula will help reduce future ambiguity.