| Autor: |
Marina Schramm, Stephanie Friedrich, Kai-Uwe Schmidtke, Jan Kiebist, Paul Panzer, Harald Kellner, René Ullrich, Martin Hofrichter, Katrin Scheibner |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
|
| Zdroj: |
Antioxidants, Vol 11, Iss 2, p 284 (2022) |
| Druh dokumentu: |
article |
| ISSN: |
2076-3921 |
| DOI: |
10.3390/antiox11020284 |
| Popis: |
Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal biocatalysts that have attracted considerable interest for application in chemical syntheses due to their ability to selectively incorporate peroxide-oxygen into non-activated hydrocarbons. However, the number of available and characterized UPOs is limited, as it is difficult to produce these enzymes in homologous or hetero-logous expression systems. In the present study, we introduce a third approach for the expression of UPOs: cell-free protein synthesis using lysates from filamentous fungi. Biomass of Neurospora crassa and Aspergillus niger, respectively, was lysed by French press and tested for translational activity with a luciferase reporter enzyme. The upo1 gene from Cyclocybe (Agrocybe) aegerita (encoding the main peroxygenase, AaeUPO) was cell-free expressed with both lysates, reaching activities of up to 105 U L−1 within 24 h (measured with veratryl alcohol as substrate). The cell-free expressed enzyme (cfAaeUPO) was successfully tested in a substrate screening that included prototypical UPO substrates, as well as several pharmaceuticals. The determined activities and catalytic performance were comparable to that of the wild-type enzyme (wtAaeUPO). The results presented here suggest that cell-free expression could become a valuable tool to gain easier access to the immense pool of putative UPO genes and to expand the spectrum of these sought-after biocatalysts. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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