Effects of the immobilization supports on the catalytic properties of immobilized mushroom tyrosinase: A comparative study using several substrates

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, d-sorbitol, d-manitol, 1,2-O-isopropylidene-α-d-glucofuranose, d-glucuronic acid, d-gulonic acid, sucrose, d-glucosone, d-arabinose, d-fructose, d-glucose, ethyl-d-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-d-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-α-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and dl-dopa, were studied. When immobilized tyrosinase acted on 4-tert  -butylcatechol, the values of Kmapp were lower than these obtained for tyrosinase in solution while, when dopamine and dl-dopa were used, the Kmapp were higher. The order of the substrates as regards their ionizable groups, dl-dopa (two ionizable groups) > dopamine (one ionizable group) > 4-tert  -butylcatechol (no ionizable group) coincided with the order of the Kmapp values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The Kmapp values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.