Organic solvent stability and long-term storage of myoglobin-based carbene transfer biocatalysts

Recent years have witnessed a rapid increase in the application of enzymes for chemical synthesis and manufacturing, including the industrial-scale synthesis of pharmaceuticals using multienzyme processes. From an operational standpoint, these bioprocesses often require robust biocatalysts capable of tolerating high concentrations of organic solvents and possessing long shelflife stability. In this work, we investigated the activity and stability of myoglobin (Mb)-based carbene transfer biocatalysts in the presence of organic solvents and after lyophilization. Our studies demonstrate that Mb-based cyclopropanases possess remarkable organic solvent stability, maintaining high levels of activity and stereoselectivity in the presence of up to 30%-50% (v/v) concentrations of various organic solvents, including ethanol, methanol, N,N-dimethylformamide, acetonitrile, and dimethyl sulfoxide. Furthermore, they tolerate long-term storage in lyophilized form, both as purified protein and as whole cells, without significant loss in activity and stereoselectivity. These stability properties are shared by Mb-based carbene transferases optimized for other type of asymmetric carbene transfer reactions. Finally, we report on simple protocols for catalyst recycling as whole-cell system and for obviating the need for strictly anaerobic conditions to perform these transformations. These findings demonstrate the robustness of Mb-based carbene transferases under operationally relevant conditions and should help guide the application of these biocatalysts for synthetic applications.