Protein engineering of thioether monooxygenase to improve its thermostability for enzymatic synthesis of chiral sulfoxide

Abstract

• Consensus analysis and random mutagenesis were conducted to engineer a cyclohexanone monooxygenase from the Acinetobacter calcoaceticus (AcCHMO-M6) for improving its thermostability as well as specific activity. • The half-lives of the resultant mutants at 40 °C were increased up to 8.5 h as well as with up to 4-fold enhance in specific activity. • By employing the best mutant AcCHMO-M10, complete conversion (>99% conversion, >99% ee) asymmetric Kagan oxidation of omeprazole sulfide was achieved in 16 h, while the AcCHMO-M6 only afford 39% conversion. Esomeprazole, the S -enantiomer of omeprazole, is the best-selling proton pump inhibitor. In our previous work, a mutant of cyclohexanone monooxygenase from the Acinetobacter calcoaceticus (named Ac CHMO-M6) was successfully obtained through protein engineering which could catalyze the oxidation of omeprazole sulfide. However, its practical application is still hindered by the poor thermostability, especially in the up-scaled reaction process. In this work, site mutagenesis based on consensus analysis and directed evolution were used to engineer this enzyme in order to improve the stability of Ac CHMO-M6. The half-lives of the resultant mutants Ac CHMO-M9 (F29L/R444E) and Ac CHMO-M10 (F29L/R444E/A145S/G430T) at 40 °C were increased from 2.2 h to 8.5 h and 5.9 h respectively, while the corresponding T m values were increased by 7 °C and 5.3 °C in comparison to Ac CHMO-M6. The specific activity of Ac CHMO-M9 was comparable to that of Ac CHMO-M6, and the specific activity of Ac CHMO-M10 was about 4-fold that of Ac CHMO-M6. The Ac CHMO-M10 catalyzed sulfide oxidation reaction reached 100% conversion after 16 hours at 30 °C, in contrast to 39.4% conversion in the case of Ac CHMO-M6. These results show that the potential of this thioether monooxygenase can be significantly improved by protein engineering.