Mass spectrometric mapping of the enzymes involved in the phenol degradation of an indigenous soil pseudomonad
Mass spectrometric mapping of the enzymes involved in the phenol degradation of an indigenous soil pseudomonad
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DOI：
10.1016/j.bbapap.2004.04.003
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发表时间：
2004-07-01
影响因子：
3.2
通讯作者：
Tsiotis, G
中科院分区：
文献类型：
--
作者：
Tsirogianni, I;Aivaliotis, M;Tsiotis, G
研究了一种新的土壤细菌假单胞菌phDV1降解苯酚的酶。利用蔗糖密度离心和阴离子交换层析分离得到了催化苯酚降解中间裂解途径第二步的关键酶——儿茶酚2,3-双加氧酶（C23O）。用吸收光谱法和肽图对纯化的C23O进行了检测和鉴定。此外，在葡萄糖或苯酚作为唯一碳源和能量源的不同生长底物条件下，对假单胞菌菌株phDV1蛋白质组进行了监测。采用蔗糖密度离心法收集和浓缩具有C23O活性的细胞部分，降低总蛋白混合物的复杂性。采用1-DE Tricine PAGE电泳分离结合MALDI-TOF质谱法鉴定参与代谢途径的蛋白。我们发现19种蛋白质的不同表达取决于生长底物（苯酚或葡萄糖），其中10种被确定为参与苯酚降解的酶。(C) 2004 Elsevier B.V.版权所有
The enzymes involved in the degradation of phenol by a new soil bacterium referred as Pseudomonas sp. strain phDV1 were characterized. The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol 2,3-dioxygenase (C23O), was isolated using sucrose density centrifugation and anion exchange chromatography. The purified C23O was detected and identified by absorption spectroscopy and peptide mapping. Further, the Pseudomonas sp. strain phDV1 proteome was monitored under different growth substrate conditions, using glucose or phenol as sole carbon and energy source. Sucrose density centrifugation was used to collect and concentrate the cell fraction exhibiting C23O activity and to reduce the complexity of the total protein mixture. 1-DE Tricine PAGE electrophoresis separation in combination with MALDI-TOF MS was attempted for the identification of the proteins involved in the metabolic pathway. We found a different expression of 19 proteins depending on the growth substrate (phenol or glucose) and 10 were identified as enzymes involved in the phenol degradation. (C) 2004 Elsevier B.V. All rights reserved.