| ρ-Hydroxyphenylpyruvate dioxygenase (HPPD) is an enzyme which is needed in the tyrosine catabolic pathway. It catalyzes the formation of homogentisate fromρ-hydroxyphenylpyruvate. HGA is an important precursor substance of plastoquinone and tocopherol synthesis in plant, HPPD is a key enzyme of this biosynthesis pathway. HPPD became a new target for herbicide in 1990s.The recombinant CjHPPD catalyzes the formation of homogentisate(HGA) fromρ-hydroxyphenylpyruvate(HPP). The HGA auto-oxidizes and polymerizes to a brown pigment, called pyomelanin.In this paper, some characterizations and the yield optimum conditions of pigment were studied. The temperature, the concentration of tyrosine and oxygen, and other physical or chemical factors had a great influence on the production of pigment. The results of experiment showed it was more in favor of the generation of pigment when the initial pH was 7.0 and the culture temperature was 30℃.In the mean time, when the medium was supplemented with tyrosine concentration of 2 mg/mL, the production of pigment reached to the maximum. With the UV-visible spectrum scanning, it was no characteristic absorption peak in all the band, and the absorbance changed little in the visible band, but increased rapidly in the ultraviolet band. It had stability against high temperature, low temperature, hydrogen peroxide, sodium sulfite, and light intensity. It was free from sucrose, glucose, but the absorption degree change greatly after added citric acid. It also showed protective effect for bacterium under ultraviolet irradiation.In the same time, the study about the susceptibility of recombinant CjHPPD to the pyrazole herbicides DTP, triketones herbicide Callisto, isoxazole herbicides Isoxaflutole were developped. And the IC50 value for the three herbicides was 19.67μmol/L, 2.17μg/mL, 7.27 ppm respectively through the direct measurement of the pigment. |
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