Optimization of growth and induction conditions for the production of recombinant whole cell cyclohexanone monooxygenase in Escherichia coli

Optimizing biocatalyst production conditions is essential for enhancing productivities and yields in biotransformation applications. This study focused on investigating the impact of the volumetric oxygen mass transfer coefficient (k_La) on the specific growth rate of recombinant E. coli cells and optimizing induction conditions for whole-cell cyclohexanone monooxygenase (CHMO) production. The results demonstrated that elevated k_La improved microbial growth rates, with optimal conditions achieved at k_La = 31 h⁻¹, where aerobic growth is no longer limited by dissolved oxygen. Additionally, the induction of CHMO during the exponential growth phase led to the highest specific biocatalyst activity, when used as resting cells. Further optimization of induction parameters, including the isopropyl-β-D-thiogalactopyranoside (IPTG) concentration and induction duration, significantly increased CHMO activity. The specific activity reached 54.4 U/g, representing an improvement of over 130%. Specifically, optimized conditions included a 5-hour cultivation period at k_La = 31 h⁻¹, resulting in a biocatalyst concentration of approximately 1 g/L, followed by a 20-minute induction with 0.16 mmol/L of IPTG. Bioreactor strategies for a biocatalytic Baeyer-Villiger oxidation process were evaluated, revealing that repeated batch experiments with cell washing between cycles maintained CHMO activity at 53 U/g over multiple cycles, making this the most favorable method for sustained CHMO activity and technology application. This study underscores the importance of induction optimization in maximizing biocatalyst activity for potential pilot-scale applications. These findings provide valuable insights into the optimization of biocatalytic processes, paving the way for enhanced efficiency and productivity in Baeyer-Villiger monooxygenase (BVMO)-driven processes.