| Tyrosinase(tyrosinase,EC1.14.18.1, TYR) is a polyphenol oxidase, sometimes also called phenol oxidase, is a multi-subunit complex structure of copper-oxide reductase, widely present in microorganisms, plants, animals and humans. Both dioxygenase and dual function oxidase, can add single phenolic oxygen into two phenol, phenol and other two and can be oxidized to quinones polyphenol. Not only is the organism, such as a key enzyme in the synthesis of melanin pigmentation produced, and participate in the formation of polyphenols. Plants tyrosinase mainly phenolic metabolism, plant disease related. It can induce anti-nutritive plant defense response, reduce the nutritional effects on herbivores, but also inhibit the bacteria and prevent the spread of bacteria. Currently, tyrosinase is used in medicine, environment, food, chemical and other fields widely, such as in the food and fruit industry.on the one hand, due to the oxidation of phenols tyrosinase melanin reducing its nutritional value and flavor, thereby inhibiting tyrosinase has important significance. On. the other hand, in order to increase the tone, interested in joining the tyrosinase, as in the production of black tea, raisins, The drying system ofjujube, beer production, in order to increase the color of wine, often need to add the right amount of tyrosinase. So to get a wide range of low-cost source of tyrosinase and study some properties of important theoretical and economic significance.Ipomoea Aquatica Forsk, also called water spinach,known as bindweed Ipomoea annual or perennial vegetables, high yield, strong resistance, fewer pests and diseases, And etc, is blocking the lack of choice of vegetables fill light. This paper is separated from the purified water spinach leaves TYR, and some properties of studied, not only can provide a reference for seeking Ipomoea tyrosinase inhibitor, can alst provide a cheap and readily available source of tyrosinase to improve its economic valuThe results of our study were follows:1. Purification of TYR from Ipomoea Aquatica Forsk leavesFresh Ipomoea Aquatica Forsk leaves after homogenization buffer extraction, ammonium sulfate fractionation, DEAE-Sepharose ion-exchange chror atography, Superdex-200 gel filtration chromatography, electrophoresis to obtain pure yrosinase. The enzyme specific activity reached 114.53U/mg, activity recovery was 11.33%, purification factor of 99.59.2. Enzymatic characteristics of TYR from Ipomoea Aquatica Forsk leavesThe results showed that water spinach leaves tyrosinase full molecurar mass of 77.60kD, subunit molecular mass of 38.70kD, presumably enzyme is compesed of two identical subunits; The optimum temperature is 45 ℃, the optimum pH value of 7.5, the enzyme in the range of 25-55℃ and pH6.0-8.0 has better stability; under optimal conditions with L-DOPA as substrate Km values measured obtaining 10.05mmol/L.3-The effect of some chemical reagent to TYR from Ipomoea Acmatica Forsk leavesMethanol, ethanol, isopropanol, and ascorbic acid, citric acid, Ca2+ and Pb2+ inhibited the enzyme. Mn2+, Zn2+, Co2+ and Cu2+ to the low concentration of a certain enzyme activation. Urea, SDS, Li+, K-, Fe3+almost no effect on the activity, EDTA at 0 40mmol/L range significantly inhibited enzyme activity, there is no effect on the activity when concentration of more than 40mmol/L.4. Functional group of TYR from Ipomoea Aquatica Forsk leavesThe results show that:Imidazolyl disulfide and histidine may be Ipomoea Aquatica Forsk leaves the active site of tyrosinase a desired group, a lysine residue, arginine guanidino group, a phenolic hydroxyl of tyrosine and tryptophan indole group may not be necessary to constitute the active site of the enzyme group. The chloramine-T has a certain enzyme activation, indicating that the enzyme methionine thioether group catalytic role in promoting a certain function. For chloromercuribenzoate (pCMB), phenylmethylsulfonyl fluoride (PMSF) enzyme has a certain extent, be described cysteine sulfhydryl and serine residues may not be necessary to constitute the active site... |
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