Archives of Microbiology

, Volume 183, Issue 1, pp 1–8 | Cite as

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

  • Jian-Feng Wu
  • Cui-Wei Sun
  • Cheng-Ying Jiang
  • Zhi-Pei Liu
  • Shuang-Jiang Liu
Original Paper


Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-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 during p-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 into Escherichia coli.


Comamonas 2-Aminophenol 1 6-Dioxygenase Chloronitrobenzene degradation 



This work was supported by grants (NSFC30230010 and KSCX2-SW-113) from Chinese Academy of Sciences and National Natural Science Foundation of China. We are grateful to Harold L. Drake at the University of Bayreuth, Germany, for his assistance during the revision of this paper.


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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Jian-Feng Wu
    • 1
  • Cui-Wei Sun
    • 1
  • Cheng-Ying Jiang
    • 1
  • Zhi-Pei Liu
    • 1
  • Shuang-Jiang Liu
    • 1
  1. 1.Institute of MicrobiologyChinese Academy of SciencesBeijingChina

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