GDO Ⅰ酶阻断对芳烃降解途径的影响

产碱假单胞菌NCIB 9867株(P25X)能够通过龙胆酸途径降解芳香烃.研究显示P25X在龙胆酸途径可能产生一组同工酶-其中一种酶是保守型,另一种则为严格诱导型表达.龙胆酸降解途径主要的酶是龙胆酸加双氧酶(GDO),它促进芳香环的裂解,产生顺丁烯丙酮酸,并通过一系列的反应最终进入TCA循环.目前,仅有保守型的龙胆酸加双氧酶及其下游片段被克隆.P25X的衍生株SNZ28,它的龙胆酸加双氧酶的基因(GDO I)被链霉素/奇霉素抗性基因所打断.本研究运用二维蛋白电泳法分析降解菌株SNZ28的蛋白提取物,并用考马斯亮兰染色鉴别.经软件比对分析,由龙胆酸诱导与元龙胆酸诱导菌株的蛋白表达提取物存在明显的蛋白质表达差异,其中有15个蛋白质点被识别.这一结果为诱导型龙胆酸酶的进一步研究打下了基础.

Effect of GDO I interruption in degradation of aromatic compounds

Pseudomonas alcaligenes NCIB 9867 (strain P25X), a soil bacterium, is capable of degrading aromatic hydrocarbon via the gentisate pathway. It has been established that the gentisate pathway harbors isozymes for the gentisate metabolism - one set is constitutively expressed whereas the other set is strictly inducible. Gentisate 1,2-dioxygenase (GDO) is the key enzyme of the gentisate pathway. It catalyzes the fission of the gentisate aromatic ring to yield maleylpyruvate, which then yields TCA cycle intermediates. To date, the genes for the constitutively expressed gentisate 1,2-dioxygenase and its downstream enzymes have been cloned. We have obtained a derivative strain of P25X, designated as SNZ28, which had its constitutive copy of the gentisate 1,2-dioxygenase genes interrupted by a streptomycin/spectinomycin resistance gene cassette. In this study, the proteome profiles of SNZ28,grown in the minimal medium with or without the aromatic inducer, gentisate, were compared. Crude extracts of SNZ28 cells were subjected to two-dimensional polyacrylamaide gel electrophoresis (PAGE) and stained with Coomassie blue and silver staining. 9 de novo protein spots and 7 up - regulated protein spots were observed in the extracts obtained under gentisate induction condition. The results obtained provide us clues for further investigation of inducible GDO enzyme.