Automatic flow methodology for kinetic and inhibition studies of reactions with poorly water-soluble substrates in...

In the present work an automatic generic tool, based on sequential injection analysis (SIA) for kinetic and inhibition studies of reactions with poorly water-soluble compounds in ionic liquid (IL)-containing systems, is described. The oxidation of the poorly water-soluble phenolic compound, caffeic acid, catalyzed by the mushroom tyrosinase, in different 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)])/buffer mixtures as reaction media, was investigated. This determination was based on measuring depletion rate of the substrate caffeic acid at its maximum wavelength (λ(max) 311 nm). The influence of several parameters such as substrate and enzyme concentration, temperature, pH, delay times and measurement periods on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. The obtained results showed that tyrosinase was active in oxidising caffeic acid in this water-miscible IL and the presence of an impaired tyrosinase activity with increase in [bmim][BF(4)] concentration as an increase in the apparent Michaelis-Menten constant (K(M)(app)) was observed while the maximum reaction rate (V(max)(app)) remained fairly constant. The results were compared to those obtained when the assay was performed in water/methanol mixtures under the same conditions to substantiate [bmim][BF(4)] as an alternative to conventional organic solvents. Additionally, it was shown that tyrosinase is effectively inhibited by the substrate analogues tested (trans-cinnamic acid and 3,4-dihydroxybenzoic acid) in the IL-containing aqueous system used.