Separation, Purification And Characterization Of A Tyrosinase From Fruits Of Cinnamomum Camphora

Tyrosinase (TYR) is a copper-containing enzyme, as known as polyphenol oxidase, which is widely distributed in microorganisms, plants and animals and involved in some pigment biosynthesis. Tyrosinase has very important biochemical character and can be used in industry. The process of application research of tyrosinse in drug, environment, food and fine chemical industry was reviewed. As so far, the studies for tyrosinase including the separation, purification, characterization of tyrosinase have been paid attention to in the world.In this investigation, some kinds of plants were selected as candidate experiment materials and Cinnamomum camphora (L.) Presl was screened as experiment materials due to its high activity of TYR in crude extract. The testing conditions of crude enzyme, 20 mmol/L , pH 7.5 Tris-HCl buffer, 10 mmol/L L-DOPA was verified.Effect of treatment with different NAA concentrations on the isozymes and activities of TYR from leaf and fruit of Cinnamomum camphora were investigated. The result revealed that different concentrations of NAA had the different effect on activity of TYR in both leaves and fruits. High concentrations of NAA (10-4mol/L) stimulated not only the maturity of the fruit, but also increased the activity of TYR Cinnamomum camphora fruit; different osmotic potential had the different effect on activity of TYR in fruits; lower concentrations H2O2 (1mmol/L) treatment could also induce the activity of TYR in fruits.TYR from Fruits of Cinnamomum camphora was purified by the procedure: tissue homogenation, ammonium sulfate precipitation, Sephadex G-100 gel filtration, and DEAE-Sepharose chromatography. The optimual pH and temperature of the purified TYR was 7.5 and 50℃respectively. The molecular weight of its subunit was 43kDa. Its apparent Km of L-DOPA was 12.83 mmol/ L, the Vmax was 180.65 U/mg protein.The purified TYR from fruits of Cinnamomum camphora had high thermostablity. The residue activity remained more than 90% after incubation at 60℃for 1h. The TYR was stable in the media under the pH value from 4 to 7. Two kinds of peptides, e.g. Ala-Asp and Lys-Val, could improve the thermostablity of the purified TYR, when TYR was incubated at 80℃for 30 min.The activity of the purified TYR from Fruit of Cinnamomum camphora is strongly inhibited by 0.01 mmol/ L Al3+ and Cu2+, 1mmol/L Vc, 1mmol/Lβ-mercaptoethanol, and extractants of Armenica mume (Sieb.) and enhanced by 0.001mmol/L Zn2+, 250mmol/L EDTA and extractants of Ligustrum Lucidum (Ait.).