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.

中文翻译：

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肌红蛋白基卡宾转移生物催化剂的有机溶剂稳定性和长期储存。

近年来，酶在化学合成和制造中的应用迅速增长，包括使用多酶工艺在工业规模合成药物。从操作的角度看，这些生物过程通常需要能够耐受高浓度有机溶剂并具有长保存期限稳定性的坚固的生物催化剂。在这项工作中，我们研究了在有机溶剂存在下和冻干后基于肌红蛋白（Mb）的卡宾转移生物催化剂的活性和稳定性。我们的研究表明，基于Mb的环丙烷酶具有出色的有机溶剂稳定性，可以在浓度高达30％–50％（v / v）的各种有机溶剂（包括乙醇，甲醇，N，N-二甲基甲酰胺，乙腈和二甲基亚砜。此外，它们耐受冻干形式的长期储存，既作为纯化的蛋白质，也作为完整细胞，而不会显着降低活性和立体选择性。这些稳定性是由基于Mb的卡宾转移酶共享的，针对其他类型的不对称卡宾转移反应进行了优化。最后，我们报告了用于整个细胞系统的催化剂回收的简单方案，并避免了严格的厌氧条件来进行这些转化。这些发现证明了基于Mb的卡宾转移酶在操作相关条件下的稳健性，并应有助于指导这些生物催化剂在合成应用中的应用。