Crude Extract: Characterization and Analytical Application.

In the present work crude extract (ABE) has been prepared and characterized by its tyrosinase activity, protein composition and substrate specificity. The presence of mushroom tyrosinase (PPO3) in ABE has been confirmed using two-dimensional electrophoresis, followed by MALDI TOF/TOF MS-based analysis. GH27 alpha-glucosidases, GH47 alpha-mannosidases, GH20 hexosaminidases, and alkaline phosphatases have been also detected in ABE. ABE substrate specificity has been studied using 19 phenolic compounds: polyphenols (catechol, gallic, caffeic, chlorogenic, and ferulic acids, quercetin, rutin, dihydroquercetin, l-dihydroxyphenylalanine, resorcinol, propyl gallate) and monophenols (l-tyrosine, phenol, -nitrophenol, -nitrophenol, guaiacol, -cresol, -cresol, -cresol). The comparison of ABE substrate specificity and affinity to the corresponding parameters of purified tyrosinase has revealed no major differences. The conditions for spectrophotometric determination have been chosen and the analytical procedures for determination of 1.4 × 10-1.0 × 10 M l-tyrosine, 3.1 × 10-1.0 × 10 M phenol, 5.4 × 10-1.0 × 10 M catechol, 8.5 × 10-1.0 × 10 M caffeic acid, 1.5 × 10-7.5 × 10 M chlorogenic acid, 6.8 × 10-1.0 × 10 M l-DOPA have been proposed. The procedures have been applied for the determination of l-tyrosine in food supplements, l-DOPA in synthetic serum, and phenol in waste water from the food manufacturing plant. Thus, we have demonstrated the possibility of using ABE as a substitute for tyrosinase in such analytical applications, as food supplements, medical and environmental analysis.