Biocatalysis of tyrosinase in organic solvent media using phenolic substrate models.

摘要

The biocatalysis of tyrosinase was investigated in selected organic solvent media, using catechin as substrate. The results showed that the optimal enzymatic activity was obtained at pH 6.2, 6.6, 6.0 and 6.2 in heptane, toluene, dichloromethane and dichloroethane media, respectively. The kinetic studies indicated that the Km values were 5.38, 1.03, 2.52 and 4.03 mM, for the enzymatic reaction in heptane, toluene, dichloromethane and dichloroethane media, respectively, whereas the Vmax values were 12.2 x 10--4, 3.3 x 10--4, 14.7 x 10--4 and 12.0 x 10--4 deltaA mug protein--1 sec--1 , respectively. The results showed that the change in acetone concentration, used as co-solvent for the tyrosinase biocatalysis, from 5 to 30% (v/v) in the heptane medium resulted in a decrease of 4.3 to 96.7% in enzymatic activity. However, the presence of 12.5, 22.0 and 22.0% of acetone in the media of dichloromethane, dichloroethane and toluene resulted in a maximal increase in enzymatic activity of 42.6, 71.8 and 92.1%, respectively. Moreover, the biocatalysis of tyrosinase in dichloromethane and heptane reaction media, using model phenolic substrates was also investigated. The Km values for the tyrosinase biocatalysis in dichloromethane medium, using 4-methyl catechol, catechol and catechin as substrates, were 2.21, 2.36 and 2.52 mM, respectively, whereas the Vmax values were 5.1 x 10--4 , 6.0 x 10--4 and 14.7 x 10 --4 deltaA mug protein--1 sec --1, respectively. In addition, the Km values for tyrosinase biocatalysis in the heptane medium, using p-cresol, catechol and catechin as substrates, were 1.07, 4.32 and 5.38 mM, respectively, whereas the Vmax values were 0.8 x 10--4, 1.0 x 10 --4 and 12.2 x 10--3 deltaA mug protein--1 sec--1, respectively. The characterization of the end products resulting from the tyrosinase biocatalysis, using selected substrates, was carried out by spectrophotometeric scanning, differential scanning calorimetry and pyrolysis/gas chromatography coupled to mass spectrometry. The results showed that the change in reaction media resulted in the formation of end products that differed with respect to their maxima absorbance, thermal parameters and wide range of pyrolysis residuals.