Fig. 5

Cyclase activity. a LC–MS analysis and mass configuration of ε-caprolactam produced by CF3HBD from 6-ACA (blue peak) and 6-ACAwithout CF3HBD as a control (green peak). Analysis of the reaction mixture by LC–MS. The reaction was carried out using 0.5 mg/ml purified CF3HBD and 1 mM 6-ACA as the substrate at 35 °C with 50 mM HEPES buffer (pH 7.5) b NMR analysis of the reaction mixture for 3HBD in the presence of 6-ACA. The reaction was carried out using 0.5 mg/ml purified CF3HBD and 1 mM 6-ACA as the substrate at 35 °C with 50 mM HEPES buffer (pH 7.5). c Schematic representation of the ACA-GESS system consisting of CL-GESS and cyclase in E. coli. Intracellular ε-caprolactam compounds were generated by 3HBD from ω-fatty acids, such as 5-AVA and 6-ACA, and were visualized by sfGFP, whose expression was induced by the ε-caprolactam–NitR complex