Abstract

Although the bioproduction of complex, functionalised N-heterocycles has been reported, bioproduction of unsubstituted platform N-heterocycles has not yet been achieved. Therefore, the suitability of putrescine oxidase from Rhodococcus erythropolis (PuO_RH) for bioproduction of Δ¹-piperideine was studied. PuO_RH catalyses the oxidation of cadaverine to 5-aminopentanal.  Although this product is known to cyclise spontaneously into Δ¹-piperideine, direct formation of this product catalysed by PuO_RH has not been demonstrated, except by using o-aminobenzaldehyde as a reagent to trap Δ¹-piperideine and shift the equilibrium for cyclisation. The PuO_RH gene was cloned and expressed in E. coli BL21 (DE3) using the pET20b vector, and the His-tagged enzyme was purified. Steady state kinetics of PuO_RH were determined by monitoring oxygen consumption; the K_M and k_cat values were 0.24 ± 0.05 mM and 26.6 ± 0.08 s^-1 for cadaverine, and 0.17 ± 0.03 mM and 147.4 ± 0.4 s^-1 for putrescine, respectively. Whereas the k_cat/K_M values are lower than those reported using peroxidase-coupled assays, this result should represent the true kinetics of PuO_RH. Using the purified enzyme, the conversion of cadaverine to Δ¹-piperideine was demonstrated qualitatively using LC-ESI-MS and ¹H NMR; 5-aminopentanal could not be detected. Δ¹-Piperideine also formed the corresponding dimer and trimers, a known spontaneous reaction, and the product ratio could be adjusted by varying the pH. This preliminary study indicates that PuO_RH is suitable for bioproduction of Δ¹-piperideine.  The next steps are to optimise the reaction conditions, quantify, extract and purify the products, and develop whole cell bioproduction of N-heterocycles from renewable feedstocks.