Molecular Engineering and Utilization of Cyclohexanone Monooxygenase in the Cascade Biocatalytic Synthesis of ε-Caprolactone

The utilization of cyclohexanone monooxygenase (CHMO) as an oxidizing agent to synthesize the biodegradable plastic precursor ε-caprolactone offers a sustainable alternative to traditional chemical synthesis. However, the unsatisfactory catalytic efficiency of CHMO presents a major challenge. Herein, several CHMO mutants with enhanced catalytic performance were constructed by using a semi-rational design strategy in enzyme molecular engineering. The G14A mutation significantly improved operational stability, while the L143A mutation remarkably increased enzyme activity. The combination mutant CM9 (G14A+L143A) performed the best, with a 98% increase in specific activity, a 67% increase in kcat/Km, and a prolongation of t1/2 (30 °C) from 7 to 18 h. Subsequently, CM9 was employed in multiple batches of whole-cell catalysis to produce ε-caprolactone efficiently. Only 0.475 g of wet cells achieved the catalytic process of converting 12.7 mmol (1.27 g) of cyclohexanol into 9.9 mmol (1.13 g) of ε-caprolactone, achieving a total yield of 78%. Moreover, the space-time yield reached 50.6 mg gwcw–1 h–1, marking the highest rate reported for ε-caprolactone biocatalytic synthesis to date. The evolved CHMO mutants enhance the competitiveness of biocatalytic synthesis of ε-caprolactone, promoting the green production and sustainable development of the biodegradable plastics.