BIOCATALYSIS BY TYROSINASE IN ORGANIC-SOLVENT MEDIA - A MODEL SYSTEM USING CATECHIN AND VANILLIN AS SUBSTRATES

The oxidative activity of mushroom tyrosinase was optimized in chlorof orm, using catechin and vanillin as substrates, The presence of 1.75 a nd 5.00% (% v/v) methanol, respectively, showed an activating effect ( 92.3%) with catechin, but an inhibitory one (87.1%) with vanillin. The addition of acetone to the reaction medium, 30% (% v/v) for catechin and 25% for vanillin, reduced the enzymatic activity by 92.1 and 93.5% , respectively. The V-max values of tyrosinase in chloroform were 0.14 15 and 0.0250 dA/mu g protein/s for catechin end vanillin, respectivel y; in addition, K-m values were 0.7940 and 0.0825 mu mol, respectively for the two substrates. The use of 0.25 mu mol catechol activated the tyrosinase activity by 56.2% when catechin was used as substrate; how ever, no effect was found with vanillin. In addition, the use of 0.85 and 0.45 mu mol of ethylenediaminetetraacetic acid, with catechin and vanillin as substrates, inhibited the tyrosinase activity by 44.3 and 84.7%, respectively. FT-IR analyses suggested that native mushroom tyr osinase is predominately of alpha-helical conformation; while that in chloroform the enzyme is mainly composed of beta-sheet structure.