芳香烃龙胆酸降解途径蛋白质组学的研究

Proteome Study of Degradation of Aromatic Hydrocarbons Via Gentisate Pathway

芳香烃是一类重要的环境污染物,微生物降解是其主要的处理方法。研究显示降解过程中产生保守型和诱导型的各一组同工酶。目前,仅有保守型的龙胆酸加双氧酶(GDOI)及其下游片段被克隆。产碱假单胞菌NC IB9867(P25X)的突变株--SNZ28 GDO I被打断,在龙胆酸诱导的情况下,该突变株仍能检测到龙胆酸加双氧酶活性。采用二维蛋白电泳分析突变株SNZ28在有和没有龙胆酸诱导条件下的蛋白质表达差异。电泳结果显示了两者存在有15个蛋白点的差异。通过MALD I-TOF和Q-TOF分析,其中的12个蛋白质点与数据库中已知多肽片段有同源性。其中,P4点与青枯菌(Ralstonia species)龙胆酸1,2加双氧酶同源。该结果在蛋白质组学上证实了GDO II的存在。

Aromatic hydrocarbons are capital environmental contamination. Pseudomonas alcaligenes NCIB 9867 （P25X） is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalysed reactions. One set of the enzymes is constitutive and the other is strictly inducible. To date, only the genes for the constitutively-expressed gentisate dioxygenase and the downstream enzymes have been cloned. A mutant strain of P25X, designated SNZ28, which had the constitutive copy of the gentisate 1, 2-dioxygenase gene interrupted by a streptomycin/ spectinomycin resistance gene cassette, was found to still express gentisate dioxygenase when induced by gentisate. The proteome profiles of P25X and mutant SNZ28, grown in the presence and absence of the aromatic inducer gentisate, were compared after 2D-PAGE. Fifteen distinctive protein spots which were observed only in induced cells of P25X and mutant SNZ28 but absent in non-induced cells of both were further analyzed by MALDITOF and Q-TOF. Of the 15 proteins, 12 showed significant sequence similarity to proteins with assigned function in other microorganisms. The identification of protein P4 which showed positive identification to a gentisate dioxygenase from Ralstonia species indicated the putative role of this protein to encode gentisate 1, 2-dioxygenase in P. alcaligenes.