期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 166, 期 -, 页码 -出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2019.105508
关键词
Fluorinase; Actinopolyspora mzabensis; Inclusion bodies; Protein refolding; C-F bond; Biocatalyst
类别
资金
- National Research Foundation of South Africa [111329]
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 degrees C. At these conditions, the enzyme exhibited an activity of 0.44 +/- 0.03 mu M min(-1) mg(-1). Furthermore, the Am-fluorinase showed exceptional stability at 25 degrees C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.
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