Biomanufacturing of chemicals using biocatalysts is an attractive strategy for the production of valuable pharmaceuticals since it is usually more economical and has a much-reduced environmental impact. However, there are often challenges such as their thermal instability that should be overcome before a newly discovered enzyme is eventually translated into industrial processes. In this work, we describe a roadmap for the development of a robust catalyst for industrial resolution of Vince lactam, a key intermediate for the synthesis of carbocyclic-nucleoside-related pharmaceuticals. By a genome mining strategy, a new (+)-γ-lactamase (MiteL) from Microbacterium testaceum was successfully discovered and biochemically characterized. In vitro studies showed that the enzyme exhibited high activity but poor enantioselectivity (E = 6.3 ± 0.2) toward racemic Vince lactam, and thus, it is not suitable for industrial applications. Based on structural modeling and docking studies, a semi-rational engineering strategy combined with an efficient screening method was then applied to improve the enantioselectivity of MiteL. Several mutants with significant shifting stereoselectivity toward (-)-γ-lactam were obtained by site-saturation mutagenesis. Synergy effects led to the final mutant F14D/Q114R/M117L, which enabled efficient acquisition of (-)-γ-lactam with a high E value (> 200). The mutant was biochemically characterized, and the docking studies suggested a plausible mechanism for its improved selectivity. Finally, a sunflower-like nanoreactor was successfully constructed to improve the mutant's robustness via protein supramolecular self-assembly. Thus, the synergism between semi-rational protein engineering and self-assembling immobilization enabled construction of a nanoreactor with superior properties, which can be used for resolution of Vince lactam in large scale.

中文翻译：

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整合半合理蛋白质工程和纳米反应器设计的基因组采矿：Vince内酰胺工业拆分的稳健生物催化剂路线图。

使用生物催化剂对化学药品进行生物制造是生产有价值的药物的一种有吸引力的策略，因为它通常更经济并且对环境的影响大大减少。然而，在将新发现的酶最终转化为工业过程之前，通常应克服诸如热不稳定性之类的挑战。在这项工作中，我们描述了开发用于工业拆分文斯内酰胺的稳健催化剂的路线图，文斯内酰胺是合成碳环核苷相关药物的关键中间体。通过基因组挖掘策略，成功地发现了来自睾丸微细菌的新的（+）-γ-内酰胺酶（MiteL），并对其进行了生化表征。体外研究表明该酶表现出高活性但对映选择性差（E = 6.3±0。2）朝向外消旋文斯内酰胺，因此，它不适合工业应用。基于结构建模和对接研究，然后应用半理性工程策略结合有效的筛选方法来提高MiteL的对映选择性。通过位点饱和诱变获得具有向（-）-γ-内酰胺明显的立体选择性偏移的几个突变体。协同效应导致了最终的突变体F14D / Q114R / M117L，该突变体使得能够以高E值（> 200）有效地获得（-）-γ-内酰胺。该突变体已进行了生化鉴定，对接研究表明其选择性提高的合理机制。最后，成功构建了向日葵样纳米反应器，以通过蛋白质超分子自组装提高突变体的稳健性。从而，