Exploring the promiscuity of native enzymes presents a promising strategy for expanding their synthetic applications, particularly for catalyzing challenging reactions in non-native contexts. In this study, we explore the promiscuous potential of old yellow enzymes (OYEs) to facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction reaction. Using mass spectrometry and spectroscopic techniques, we confirm promiscuity of Gk OYE in both MBH and reduction reactions. By blocking H - and H + transfer pathways, we engineer Gk OYE.8, which loses its reduction ability but enhances its MBH activity. The structural basis of MBH reaction catalyzed by Gk OYE.8 is obtained through mutation studies and kinetic simulations. Furthermore, enantiocomplementary mutants Gk OYE.11 and Gk OYE.13 are obtained by directed evolution, exhibiting the ability to accept various aromatic aldehydes and alkenes as substrates. This study demonstrates the potential of leveraging substrate similarities to unlock enzyme functionalities, enabling the catalysis of new-to-nature reactions.
Asymmetric reduction of the conjugated C C bonds by the old yellow enzymes (OYEs) presents a promising field in the synthesis of chiral chemicals. Nevertheless, few natural OYEs have been applied in large-scale applications due to the requirement of costly NADPH and low operational stability. Herein, a stable and efficient fusion of YqjM from Bacillus subtilis and glucose dehydrogenase (GDH) from Bacillus megaterium was constructed to stereoselectively reduce the conjugated C C bonds in a self-sufficient continuous process. The effects of the enzyme order and different linkers on the fusions were investigated by structural analysis and all-atom molecular dynamics simulation. The best fusion YqjM_G_GDH gave 98% conversion of 100 mmol/L 2-methylcyclopentenone with an excellent ee value (>99%) in 3 h, while the mixture of individual enzymes only obtained 68% conversion after more than 8 h. The improved substrate conversion of YqjM_G_GDH fusion was probably attributed to the increased flexibility of each fused enzyme and the shortening of the diffusion distance of NADPH regenerated. A one-pot process was designed to purify and immobilize the fusion on the Ni 2+ -nitrilotriacetic acid functionalized magnetic mesoporous silica nanoflowers. The resulting immobilized biocatalyst not only catalyzed the asymmetric reduction of various α,β -unsaturated ketones (20 mmol/L) continuously with only 50 μmol/L NADP + to initiate the whole process, but also retained more than 82% of the initial activity after seven cycles, serving as a good candidate for the industrial applications.
危险品化学品经营许可证（带存储）