Kinetic characterization of diphenolase activity from Streptomyces antibioticus tyrosinase in the presence and absence of cyclodextrins

Streptomyces antibioticus tyrosinase was kinetically characterized after purification by PEG-8000/phosphate phase partitioning and ammonium sulfate fractionation using tert-butylcathechol (TBC) and dopamine. The enzyme showed an optimal pH at 6.5 and a KM of 1.2 mM and 8.4 mM, respectively. The effect of several modulators was studied on this Gram-positive bacterium tyrosinase. In addition, previously undescribed characterization of apparent inhibition and activation of a bacterial tyrosinase using different kinds of cyclodextrins was carried out. When a hydrophobic substrate of S. antibioticus tyrosinase, in this case, tert-butylcatechol was used, a marked substrate sequestrant effect was observed in the presence of hydroxypropyl-β-cyclodextrins (OH-β-CDs) and gamma cyclodextrins (γ-CDs). This sequestrant effect was due to the complexation of TBC into the CD cavity. Moreover, the effect of some hydrophobic inhibitors in the presence of OH-β-CDs and γ-CDs was studied using dopamine, a hydrophilic substrate of S. antibioticus tyrosinase. Increasing concentrations of CDs in the presence of inhibitors like hexestrol or hinokitiol, were able to reactivate the inhibited enzyme to reach the non-inhibited level, as a result of the complexation of these inhibitory compounds in the hydrophobic core of the CDs. This dual effect of CDs as apparent inhibitor and activator has never before described being observed in bacteria.