Purification and characterization of tyrosinase enzyme from pleurotus ostreatus mushroom by affinity chromatography and investigation the effects of organophosphate pesticides on tyrosinase enzyme

Tyrosinase (E.C. 1.14.18.1) is a copper-containing enzyme in the active site, a member of oxidoreductases, catalysing the rate-limiting step in melanin synthesis. Melanin is an indolic heteropolymer that provides protection from the harmful effects of ultraviolet rays from the sun and is primarily responsible for hair, eye and skin pigmentation in animals. Tyrosinase, besides its importance in melanin synthesis, is an important enzyme with various industrial applications such as cosmetics, textiles, environment, food and medicine. Within the scope of the thesis study, tyrosinase enzyme was purified from Pleurotus ostreatus, a wild and edible mushroom, by following the steps of homogenisation, precipitation, dialysis and finally Sepharose-4B-L-tyrosine-p-aminobenzoic acid affinity gel with a yield of 15,14%. Enzyme purity was controlled by SDS and native gel electrophoresis. The molecular mass of tyrosinase enzyme was determined to be approximately 97 kDa. Tyrosinase enzyme, the optimum pH and stable pH values were pH 5,5 and pH 5,25 and the optimum temperature value was determined as 20°C, respectively. The in vitro inhibition effect of glyphosate pesticide on the activity of the enzyme was also investigated. Activity (%)-[I] and Lineweaver-Burk graphs were plotted. The type of inhibition was determined uncompetitive with the graphs drawn; IC50 value, which is the inhibitor concentration that halves the enzyme activity from the curve equation, was calculated as 0,807 mM and Ki value, which shows the interest of the inhibitor to the enzyme from the Lineweaver-Burk graph, was calculated as 2,97 µM.