Cloning and characterization of four enzymes responsible for cyclohexylamine degradation from Paenarthrobacter sp. TYUT067.

Paenarthrobacter sp. TYUT067 is a soil bacterium that can degrade and use cyclohexylamine as the sole source of carbon and energy. However, the responsible enzymes involved in cyclohexylamine degradation by TYUT067 have not been cloned and characterized in detail yet. In this study, four possible cyclohexylamine degradation genes, one cyclohexylamine oxidase (Pachao), two cyclohexanone monooxygenases (Pachms) and one lactone hydrolase (Pamlh) were successfully cloned and heterologous expressed in Escherichia coli T7 host cells. The four enzymes were purified and characterized. The optimal pH and temperature of the purified enzymes toward their own substrates were 7.0 (PaCHAO), 8.0 (PaCHM1), 9.0 (PaCHM2 and PaMLH) and 30 °C (PaCHAO and PaMLH), 40 °C (PaCHM2) and 45 °C (PaCHM1), respectively, with KM of 1.1 mM (PaCHAO), 0.1 mM (PaCHM1), 0.1 mM (PaCHM2) and 0.8 mM (PaMLH), and yielding a catalytic efficiency kcat/KM of 16.1 mM-1 s-1 (PaCHAO), 1.0 mM-1 s-1 (PaCHM1), 5.0 mM-1 s-1 (PaCHM2) and 124.4 mM-1 s-1 (PaMLH). In vitro mimicking the cyclohexylamine degradation pathway was conducted by using the combined three cyclohexylamine degradation enzymes (PaCHAO, PaCHM2 and PaMLH) with 10-50 mM cyclohexylamine, 100% conversion of cyclohexylamine could be finished within 12 h without any detected intermediates. The current study confirmed the enzymes responsible for cyclohexylamine degradation in TYUT067 for the first time, provide basic information for further investigation and application of these specific enzymes in pollution control.