Ochrobactrum intermedium strain SCUEC4 is a newly isolated nicotine-degrading bacterium and can use the nicotine as the sole carbon source via a series of enzymatic catalytic processes. Mechanisms underlying nicotine degradation in this bacterium and the corresponding functional genes remain unclear. Here, we analyzed the function and biological properties of the ocnE gene involved in nicotine degradation pathways in strain SCUEC4. The ocnE gene was cloned by PCR with total DNA of strain SCUEC4, and used to construct the recombinant plasmid pET28a-ocnE. The overexpression of OcnE protein was detected by SDS-PAGE analysis, and functional insight of this protein was spectrophotometrically carried out with monitoring the changes of 2, 5-dihydroxypyridine. Moreover, effects of temperature, pH and metal ions on the biological activities of OcnE protein were analyzed. The results demonstrated that optimal conditions for the biological activities of OcnE, approximate 37.6 kDa, was determined as 25°C, pH 7.0 and 25 μM Fe2+, and the suitable storage conditions for OcnE protein was determined as 0°C and pH 7.0. Conclusively, the ocnE gene owns the ability of 2, 5-dihydroxypyridine dioxygenase. These findings will be beneficial in clarifying the mechanisms of nicotine degradation in Ochrobactrum intermedium strain SCUEC4.

Agrobacterium tumefaciens strain SCUEC1 is a nicotine-degrading bacterium, which has been recently isolated from the tobacco waste-contaminated field soil. However, the mechanism for nicotine degradation in this strain remains unclear. Here, we analyze the function and biological properties of the agnH gene in the strain SCUEC1. The overexpression of the AgnH protein was detected by SDS-PAGE analysis, and functional insight of the AgnH protein was carried out with monitoring the changes of maleic acid into fumaric acid by high performance liquid chromatography (HPLC). Moreover, the effects of temperature, pH and metal ions on the enzymatic activities of the AgnH protein were also analyzed. The results demonstrated that the agnH gene was successfully ligated to the plasmid pET28a. The optimal condition for the enzymatic activities for the AgnH, approximately 28.0 kDa, was determined as 37 °C, pH 8.0 and 25 µM Mg2+. Conclusively, the agnH gene fulfils an important role in the conversion of maleic acid into fumaric acid involved in nicotine-degradation pathways in Agrobacterium tumefaciens strain SCUEC1.