Gene Mining of Cyclohexanone Monooxygenase and Efficient Catalytic Synthesis of Omeprazole

Abstract:

[Objective] The aim of the study is to synthesize omeprazole efficiently. [Method] With CHMO_{NCIMB 9871} from Acinetobacter johnsonii （A. johnsonii）and P450 BM3 from Priestia megaterium （P. megaterium） as templates, the monooxygenase with the highest transformation rate was screened out and the optimal conditions for inducing protein expression were determined. Single factor experiments and response surface methodology were employed to optimize the conditions of this recombinant enzyme for producing omeprazole. [Result] The cyclohexanone monooxygenase CHMO_KT2440 from Pseudomonas sp. synthesized omeprazole with the highest transformation rate. The optimal inducing conditions of CHMO_KT2440 expression were as follows: OD₆₀₀ value of 0.6, induction temperature of 17 ℃, and final IPTG concentration of 0.15 mmol/L. The single factor experiment results showed that the reaction with 0.05 mmol/L FAD, 3.0 g/L substrate mass concentration at 33 ℃ and pH 9.0 for 10 h produced omeprazole with a yield of 78.9%. The CHMO_KT2440-catalyzed synthesis of omeprazole was fitted by the regression equation: y=60.78+9.56A-19.74B+9.55C+1.80AB+5.13AC+1.48BC-18.04A²-0.79B²-9.41C². Based on the single factor experimental results, the transformation conditions were finally optimized as follows: the substrate mass concentration of 3.0 g/L, 34 ℃, pH 9.0, 0.05 mmol/L FAD, and reaction time of 10 h. Under these conditions, the yield of omeprazole reached 79.9%, which increased by 90.23% compared with that before optimization. [Conclusion] This study provides an elite cyclohexanone monooxygenase candidate CHMO_KT2440 for omeprazole synthesis.