Expression and cloning of catA encoding a catechol 1,2-dioxygenase from the 2,4-D-degrading strain Cupriavidus campinensis BJ71

Abstract

Catechol 1,2-dioxygenases catalyze catechol ring-opening, a critical step in the degradation of aromatic compounds. Cupriavidus campinensis BJ71, an efficient 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, was previously isolated from an environment contaminated with 2,4-D. In this study, catA encoding a catechol 1,2-dioxygenase was cloned from the BJ71 strain. The gene was 939 bp long and encoded a polypeptide of 312 amino acids with a molecular weight of 34 kDa. To investigate its enzymatic characteristics, CatA was heterologously expressed in Escherichia coli. Optimal reaction conditions for the pure enzyme were 35 °C and pH 8.0. The enzyme remained stable within a range of 25 °C–45 °C and pH 6.0–9.0, thus indicating that CatA has wide temperature and pH adaptability. After incubation at 45 °C, the enzyme activity of CatA decreased to 37.12%, but its activity was not affected by incubation at pH 9.0. The pure enzyme was able to use catechol, 4-methyl-catechol and 4-chlorocatechol as substrates. Enzyme kinetic parameters K_m and V_max were 39.97 µM and 10.68 U/mg, respectively. This is the first report of the cloning of a gene encoding a catechol 1,2-dioxygenase from a 2,4-D-degrading bacterial strain.

Keywords:

• catA
• catechol 1,2-dioxygenase
• cloning
• Cupriavidus campinensis BJ71
• expression

Acknowledgments

We thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Additional information

Funding

This work was supported by the Research Fund for Introducing Talents of Guizhou University under Grant [[2015]57].