Expression and characterization of the key enzymes involved in 2-benzoxazolinone degradation by Pigmentiphaga sp. DL-8

In this study, the key enzymes involved in 2-benzoxazolinone (BOA) degradation by Pigmentiphaga sp. DL-8 were further verified and characterized in Escherichia coli. By codon optimization and co-expression of molecular chaperones in a combined strategy, recombinant BOA amidohydrolase (rCbaA) and 2-aminophenol (2-AP) 1,2-dioxygenase (rCnbC_αC_β) were expressed and purified with the highest activity of 1934.6 U·mg protein⁻¹ and 32.80 U·mg protein⁻¹, respectively. BOA could be hydrolyzed to 2AP by rCbaA, which was further transformed to picolinic acid by rCnbC_αC_β based on identified catalytic product. The optimal pH and temperature for rCbaA are 9.0 and 55 °C with excellent stability for catalytic environments, and the residual activity was >50% after incubation at temperatures <45 °C or at pH between 6.0 and 10.0 for 24 h. On the contrary, rCnbC_αC_β composed of α-subunit (33 kDa) and β-subunit (38 kDa) showed poor stability against environmental factors, including temperature, pH, metal ions and chemicals.