A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli
A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli
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DOI：
10.1007/s00203-004-0738-5
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发表时间：
2004
影响因子：
2.8
通讯作者：
Jian-feng Wu;Cui-wei Sun;C. Jiang;Zhipei Liu;Shuangjiang Liu
中科院分区：
文献类型：
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作者：
Jian-feng Wu;Cui-wei Sun;C. Jiang;Zhipei Liu;Shuangjiang Liu
从处理对氯硝基苯生产废水的生物反应器中分离到一株丛毛单胞菌CNB-1。菌株CNB-1以对氯硝基苯为唯一碳源、氮源和能量源。从CNB-1菌株的细胞中纯化了一种2-氨基苯酚1，6-双加氧酶。纯化的2-氨基苯酚1，6-双加氧酶具有130 kDa的天然分子量，并由α和β亚基组成，分别为33和38 kDa。该酶与目前已知的2-氨基苯酚1，6-双加氧酶的不同之处在于：（a）对2-氨基-5-氯苯酚（Km=0.77 μM）的亲和力高于对2-氨基苯酚（Km=0.89 μM）的亲和力;（B）利用原儿茶酸作为底物。这些结果表明，2-氨基-5-氯苯酚，对氯硝基苯降解的中间体，是该酶的天然底物。N端氨基酸序列分析表明，α亚基和β亚基的N端氨基酸序列分别为T-V-V-S-A-F-L-V和M-Q-G-E-I-I-A-E。从CNB-1菌株的总DNA中构建了粘粒文库，获得了3个具有2-氨基酚1，6-双加氧酶活性的克隆（BG-1、BG-2和CG-13）。克隆BG-2的DNA测序显示一个15 kb的片段，包含两个ORF，ORF 9和ORF 10，其N-末端氨基酸序列分别与纯化的2-氨基酚1，6-双加氧酶的β和α亚基的氨基酸序列相同。当编码ORF 9和ORF 10的基因被克隆到大肠杆菌中时，该酶被活跃地合成。
Comamonasstrain CNB-1 was isolated from a biological reactor treating wastewater from ap-chloronitrobenzene production factory. Strain CNB-1 usedp-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of α- and β-subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (Km=0.77 μM) than for 2-aminophenol (Km=0.89 μM) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate duringp-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the α- and β-subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the β- and α-subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned intoEscherichia coli.