Real-time and simultaneous assay of monophenolase and diphenolase activity in tyrosinase catalyzed cascade reactions by combination of three-way calibration and excitation-emission matrix fluorescence.

A real-time assay for multiple enzyme activities in cascade reactions is required for research on metabolism and bioengineering. Tyrosinase has the bifunctional activity of monophenolase and diphenolase. A combined strategy of three-way calibration with excitation-emission matrix (EEM) fluorescence was developed for real-time and simultaneous determination of monophenolase and diphenolase activity with tyrosine as a substrate. Mathematical separation and second-order advantage were utilized to solve spectral overlapping and uncalibrated interferents during complex dynamic enzymatic processes. Kinetic evolution profiles of EEM were monitored to stack a fusion three-way data array together with static samples. Using a parallel factor analysis (PARAFAC) algorithm, pseudo-univariate calibration curves with limits of detection (LODs) of 3.00 µM and 0.85 µM were established to simultaneously and real-time measure tyrosine and DOPA. Progress curves for tyrosine consumption by monophenolase and DOPA consumption by diphenolase were obtained using the law of mass conservation to calculate the initial velocity. The LODs for monophenolase and diphenolase were 0.0232 U⋅mL-1 and 0.0316 U⋅mL-1. The method achieved real-time and simultaneous assays of multiple enzyme activities in cascade reactions. It showed potential application in the metabolic pathway and biochemical industry.