Monophenolase assay using excitation-emission matrix fluorescence and ELMAN neural network assisted by whale...

Tyrosinase plays a vital role for melanogenesis and inherently involves both monophenolase activity and diphenolase activity. Monophenolase catalyzes hydroxylation of tyrosine to l-DOPA (L-3,4-dihydroxyphenylalanine). Real-time monophenolase assay method is of outstanding interest for both scientific research and industrial application. A combined strategy of three-dimensional excitation-emission matrix (EEM) fluorescence spectra and artificial neural network was developed to determine monophenolase activity. A quantitation system for tyrosine in presence of l-DOPA was designed based on ELMAN neural network. Principal component analysis (PCA) was conducted to reduce the dimensionality of fluorescence spectra. Four principal components was used as input variables. Whale optimization algorithm (WOA) was implemented to optimize the initial weights and threshold network. Real-time concentration of tyrosine in monophenolase reaction was monitored to calculate the initial velocity for tyrosine consumption. The exclusive monophenolase activity without interference from diphenolase reaction was determined. Limit of detection (LOD) for monophenolase assay is 0.0113 U mL^-1. Using the proposed method, enzyme kinetics for monophenolase was investigate. K_m was calculated as 14.16 μM. Inhibitor for monophenolase was screened by using model molecule kojic acid with IC₅₀ of 3.49 μM. The assay method exhibited a promising prospect to characterize the kinetics and inhibitor of monophenolase.