Flavin‐dependent halogenase (FDH) is highly prized in pharmaceutical and chemical industries for its exceptional capacity to produce halogenated aromatic compounds with precise regioselectivity. This study has devised a multi‐enzyme self‐assembly strategy to construct an effective and reliable in vitro coenzyme cycling system tailored for FDHs. Initially, tri‐enzyme self‐assembling nanoclusters (TESNCs) were developed, comprising glucose dehydrogenase (GDH), flavin reductase (FR) and FDH. The TESNCs exhibited enhanced thermal stability and conversion efficiency compared to free triple enzyme mixtures during the conversion of L‐Trp to 6‐Cl‐L‐Trp, resulting in a 2.1‐fold increase in yield. Subsequently, an ordered co‐immobilization of GDH, FR, and FDH was established, further amplifying the stability and catalytic efficiency of the FDH coenzyme cycle system. Compared to the free TESNCs, the immobilized TESNCs demonstrated a 4.2‐fold increase in catalytic efficiency in a 5 mL reaction system. This research provides an effective strategy for developing a robust and efficient coenzyme recycling system for FDHs.

中文翻译：

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蛋白质支架介导的多酶自组装和黄素依赖性卤素酶辅酶循环系统的有序共固定化，用于 6-Cl-L-Trp 的高效生物合成


黄素依赖性卤素酶 （FDH） 因其生产具有精确区域选择性的卤代芳香族化合物的特殊能力而在制药和化学工业中备受推崇。本研究设计了一种多酶自组装策略，以构建为 FDH 量身定制的有效且可靠的体外辅酶循环系统。最初，开发了三酶自组装纳米簇 （TESNC），包括葡萄糖脱氢酶 （GDH）、黄素还原酶 （FR） 和 FDH。在将 L-Trp 转化为 6-Cl-L-Trp 的过程中，与游离的三酶混合物相比，TESNC 表现出增强的热稳定性和转化效率，导致产量增加 2.1 倍。随后，建立了 GDH 、 FR 和 FDH 的有序共固定化，进一步放大了 FDH 辅酶循环系统的稳定性和催化效率。与游离 TESNC 相比，固定化 TESNC 在 5 mL 反应系统中的催化效率提高了 4.2 倍。这项研究为开发稳健高效的 FDH 辅酶回收系统提供了一种有效的策略。