Archives of Microbiology

, Volume 183, Issue 1, pp 1–8

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

DOI: 10.1007/s00203-004-0738-5

Cite this article as:
Wu, J., Sun, C., Jiang, C. et al. Arch Microbiol (2005) 183: 1. doi:10.1007/s00203-004-0738-5

Abstract

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.

Keywords

Comamonas2-Aminophenol 16-DioxygenaseChloronitrobenzene degradation

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