A simple preparative method was developed for purification of TYROSINASE from edible mushroom (Agaricus bispora). A homogenized extract of mushroom was first saturated by ammonium sulfate. The desired precipitate was mixed thoroughly with DEAE-Cellulose (DE-52) and washed out to produce melanin free precipitate. The obtained protein solution was dialyzed against running water for 4 hrs, then, concentrated and chromatographed on a DE-52 column. On the basis of the activities assay, the eluted fractions by 150 mM salt solution were selected for further purification. The collected fractions were pooled and chromatographed on a Sephadex G-200 column. Polyacrylamide gel electrophoresis (PAGE) of the purified TYROSINASE produced a single band right beside the commercial sample obtained from Sigma Company at 128 kDa. The lyophilized form of the purified TYROSINASE had a purification degree of 104 and showed strong cresolase and catecholase activities when compared to a commerically available TYROSINASE.

In recent years, enzymatic activity of TYROSINASE has been the focus of investigation due to its potential applications in medicine, agriculture and cosmetics. TYROSINASE, entitled poly-phenol oxidase, is a key enzyme that catalyzes synthesis of melanin in plants, microorganisms and mammalian cells. Presence of some antioxidants can delay or inhibit the activity of this enzyme as well. In this survey, molecular docking calculation method using Autodock 4.0 software for prediction of binding energy of the protein with some antioxidant ligands was executed. The pose with the lowest energy of binding or inhibition constant was extracted at 298.15 K for kojic acid. Number of conformations in the cluster of rank was 13. The first and second boxes free energy and the inhibition constant were as follows: -5.60 kcalmol-1, 78.99 mM and -3.32 kcalmol-1, 3.66 mM, respectively. Since the first box presented a lower value of free energy, it was considered as the best mode of structure of kojic acid and the protein docking for further analysis. Thus, our present study could contribute to development and discernment of TYROSINASE inhibitors in order to prevent hyper pigmentation.

The inhibition effects seed and peel of melon on diphenolase activity of mashroom TYROSINASE were investigated. The IC50 values and Ki values of seed and peel of melon were evaluated. increased the Km value and decreased the Vm value so it show mixed type inhibition on mushroom TYROSINASE when L-DOPA was used as a substrate.

Mushroom TYROSINASE (MT) is a copper-containing enzyme, which is widely distributed in microorganisms, animals and plants. It is also a key enzyme in melanin biosynthesis, which plays a crucial role in determining the color of mammalian skin and hair. Nowadays melanoma is the one of the most terrified and lethal cancers. In this work, the modification of TYROSINASE by Woodward’s reagent k has been done and its thermodynamic stability was investigated. For the study of stability, thermodynamic parameters obtained from thermal and chemical denaturation of the native and modified enzyme. Tm values in thermal denaturation showed thermal instability for modified enzyme. Tm values for the native and modified enzyme with different concentrations of the modifier (0.5, 1, 5 and 10 mM) were determined 61.2, 60.1, 58.3, 53.9 and 45.5 (0C) respectively. In chemical denaturation 8 M Guanidium Hydrochloride was used. The Cm (half of modifier’s concentration) and DGH2O (free energy) values for the native and modified enzyme were obtained. The values of ΔGH2O for the native and modified enzymes were 17.22, 16.75, 15.0, 13.7 and 11.5 KJ/mol and the values of Cm are 8.0, 7.5, 6.7, 10.0 and 8.0 (M) respectively. Thus, decreasing in values of DGH2O for the modified enzyme in comparison with its native form indicate the protein instability.

Background: Mushroom TYROSINASE (MT) a potent candidate in clinical studies known as polyphenol oxidase, is a metaloenzyme from the oxidase superfamily widely distributed from lower to higher life forms. It plays a crucial role in sclerotization of exoskeleton in insects, also responsible for skin pigmentation in mammalians. Objective: In this study, after reconstitution of MT by some metal ions, the activity and structure of native, apo and reconstituted enzymes were investigated. Materials and Methods: Kinetic of reconstituted TYROSINASE carried out in catecholase reaction by depletion of caffeic acid. Tertiary and secondary structure of apo, native and metal reconstituted TYROSINASE obtained with fluorescence and circular dichroism techniques respectively. Reconstitution confirmed by Atomic Absorption Spectroscopy. Results: Kinetic assessment showed higher activity of MT reconstituted by Cu2+ and Ni2+ in comparison with native, Zn2+ and Co2+ reconstituted enzyme. The tertiary structure of enzyme by fluorescence technique indicated more stability of Ni2+ reconstituted MT and the apo form showed the lowest tertiary structure. Circular dichroism study showed that Ni2+ reconstituted MT form has more regular secondary structure and it caused higher stability of the enzyme. The molar ratio values from atomic absorption indicate that Ni2+ and Cu2+ have got the most binding to the apoenzyme. Conclusions: It has been shown that Ni2+, Zn2+ and Co2+ can replace Cu2+ in TYROSINASE, indicating that the histidines at the active site of the TYROSINASE family enzymes can be reconstituted with this metals, but, the most stabilization and well-structured enzyme was observed in the apoTYROSINASE reconstituted by Ni2+.