Detection and quantification of 4-substituted phenols: a comparison of mushroom tyrosinase and cell extracts of Pseudomonas putida F6

A comparison of the spectrophotometric detection and quantification of a number of 4-substituted phenols by two sources of the enzyme tyrosinase (Agaricus bisporus (mushroom) versus Pseudomonas putida) is described. Incubation of either source of tyrosinase with selected 4-substituted phenols results in the formation of coloured products that absorb light maximally within a narrow wavelength range (400-423 nm). The inclusion of the nucleophile 3-methyl-2-benzothiazolinone (MBTH) in the tyrosinase assay results in more intensely coloured products that also absorb light within a narrow wavelength range (440-475 nm). The molar extinction coefficient of the reaction products in the tyrosinase and tyrosinase-MBTH assay differed dramatically with values between 714-1580 and 14213-26563 M−1 cm−1, respectively. The addition of MBTH improved the sensitivity of the reaction between 1.3- and 100-fold, depending on the substrate and source of the enzyme. The limit of detection of 4-substituted phenols also varied according to substrate and the source of enzyme used in the assay. The lowest detectable concentration of 4-substituted phenol was 2.5 μM 4-hydroxyphenoxy acetic acid in the presence of mushroom tyrosinase and MBTH and 2.5 μM 2-(4-hydroxyphenyl) ethanol in the presence of cell extract of P. putida F6 and MBTH.