The incorporation of fluorine has been shown to improve the biophysical and bioactive properties of several organic compounds. However, sustainable strategies of fluorination are needed. Fluorinases have the unique ability to catalyse a C-F bond, hence, have vast potential to be applied as biocatalysts in the preparation of fine chemicals. But fluorinases are extremely rare in nature with only five representatives isolated thus far. Moreover, the heterologous expression of fluorinases is challenged by low yields of soluble protein. This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8 M urea, and successfully refolded into a biologically active form. Following hydrophobic interaction chromatography, >80 mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An additional gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temperature was 65 °C. At these conditions, the enzyme exhibited an activity of 0.44 ± 0.03 μM min-1 mg-1. Furthermore, the Am-fluorinase showed exceptional stability at 25 °C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.

中文翻译：

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鉴定和鉴定了来自Actinopolyspora mzabensis的氟化酶。

已表明掺入氟改善了几种有机化合物的生物物理和生物活性。但是，需要可持续的氟化策略。氟化酶具有独特的催化CF键的能力，因此，在制备精细化学品中作为生物催化剂具有巨大的潜力。但是氟化物在自然界中极为罕见，到目前为止仅分离出​​五个代表。此外，荧光素酶的异源表达受到可溶性蛋白产量低的挑战。这项研究描述了从Actinopolyspora mzabensis的荧光酶的鉴定。Am-氟化酶在大肠杆菌BL21（DE3）中的过表达导致包涵体（IBs）的形成。从IBs中回收了该酶，将其溶于8 M尿素中，并成功地重新折叠成生物活性形式。疏水相互作用色谱后，以适合于生物催化应用的纯度获得了> 80 mg的活性氟化酶。额外的凝胶过滤步骤可得到≥95％的纯Am-氟化酶。使用LC-MS / MS，活性的最佳pH为7.2，而最佳温度为65°C。在这些条件下，该酶的活性为0.44±0.03μMmin-1 mg-1。此外，Am-氟化酶在25°C下表现出出色的稳定性。初步结果表明，新发现的Am-氟化酶相对稳定。2，最佳温度为65°C。在这些条件下，该酶的活性为0.44±0.03μMmin-1 mg-1。此外，Am-氟化酶在25°C下表现出出色的稳定性。初步结果表明，新发现的Am-氟化酶是相对热稳定的。2，最佳温度为65°C。在这些条件下，该酶的活性为0.44±0.03μMmin-1 mg-1。此外，Am-氟化酶在25°C下表现出出色的稳定性。初步结果表明，新发现的Am-氟化酶相对稳定。