@misc{GroebeUllrichPecynaetal., author = {Gr{\"o}be, Glenn and Ullrich, Ren{\´e} and Pecyna, Marek J. and Kapturska, Danuta and Friedrich, Stephanie and Hofrichter, Martin and Scheibner, Katrin}, title = {High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula}, series = {AMB Express}, journal = {AMB Express}, issn = {2191-0855}, abstract = {An extracellular peroxygenase from Marasmius rotula was produced in liquid culture, chromatographically purified and partially characterized. This is the third aromatic peroxygenase (APO) that has been characterized in detail and the first one that can be produced in high yields. The highest enzyme levels of about 41,000 U l-1 (corresponding to appr. 445 mg l-1 APO protein) exceeded the hitherto reported levels more than 40-fold and were detected in carbon- and nitrogen-rich complex media. The enzyme was purified by FPLC to apparent homogeneity (SDS-PAGE) with a molecular mass of 32 kDa (27 kDa after deglycosylation) and isoelectric points between 4.97 and 5.27. The UV-visible spectrum of the native enzyme showed a characteristic maximum (Soret band) at 418 nm that shifted after reduction with sodium dithionite and flushing with carbon monoxide to 443 nm. The pH optimum of the M. rotula enzyme was found to vary between pH 5 and 6 for most reactions studied. The apparent Km-values for 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, naphthalene and H2O2 were 0.133, 0.118, 0.279, 0.791 and 3.14 mM, respectively. M. rotula APO was found to be highly stable in a pH range from 5 to 10 as well as in the presence of organic solvents (50\% vol/vol) such as methanol, acetonitrile and N,N-dimethylformamide. Unlike other APOs, the peroxygenase of M. rotula showed neither brominating nor chlorinating activities.}, language = {en} } @misc{FriedrichGroebeKlugeetal., author = {Friedrich, Stephanie and Gr{\"o}be, Glenn and Kluge, Martin and Brinkmann, Tobias and Hofrichter, Martin and Scheibner, Katrin}, title = {Optimization of a biocatalytic process to gain (R)-1-phenylethanol by applying the software tool Sabento for ecological assessment during the early stages of development}, series = {Journal of Molecular Catalysis : B, Enzymatic}, journal = {Journal of Molecular Catalysis : B, Enzymatic}, number = {103}, issn = {1381-1177}, doi = {10.1016/j.molcatb.2013.10.002}, pages = {36 -- 40}, abstract = {Ecological assessment using the software tool Sabento was conducted to compare different processes to gain the fine chemical (R)-1-phenylethanol from ethylbenzene. The software was applied during the biocatalytic process development using the unspecific peroxygenase (EC .11.2.1) of the fungus Agrocybe aegerita. The process could be systematically improved with respect to the ecological performance during process development. Compared to a modern chemical process and a further biotechnological process, it now reaches the best environmental key indicator. The software tool Sabento proved to be well suited to work out the most important factors determining the ecological burdens in the early stages of process development.}, language = {en} } @misc{KiebistKonczFriedrichetal., author = {Kiebist, Jan and Koncz, Tino and Friedrich, Stephanie and Scheibner, Katrin}, title = {Oxidative biocatalysts to design new oxyfunctionalization tools for drugs and added value bio-based products}, series = {International Biotech Innovation Days 2020 (IBID), 28th - 29th October 2020}, journal = {International Biotech Innovation Days 2020 (IBID), 28th - 29th October 2020}, address = {Senftenberg}, language = {en} } @misc{SchrammFriedrichSchmidtkeetal., author = {Schramm, Marina and Friedrich, Stephanie and Schmidtke, Kai-Uwe and Panzer, Paul and Kellner, Harald and Ullrich, Ren{\´e} and Hofrichter, Martin and Scheibner, Katrin}, title = {Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases}, series = {Antioxidants}, volume = {11}, journal = {Antioxidants}, number = {2}, issn = {2076-3921}, doi = {10.3390/antiox11020284}, pages = {1 -- 15}, abstract = {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.}, language = {en} } @misc{KellnerFriedrichSchmidtkeetal., author = {Kellner, Harald and Friedrich, Stephanie and Schmidtke, Kai-Uwe and Ullrich, Ren{\´e} and Kiebist, Jan and Z{\"a}nder, Daniel and Hofrichter, Martin and Scheibner, Katrin}, title = {Draft genome sequence of Truncatella angustata (Anamorph) S358}, series = {Microbiology resource announcement}, volume = {11}, journal = {Microbiology resource announcement}, number = {7}, issn = {2169-8287}, doi = {10.1128/mra.00052-22}, abstract = {The ascomycete Truncatella angustata has a worldwide distribution. Commonly, it is associated with plants as an endophyte, pathogen, or saprotroph. The genome assembly comprises 44.9 Mbp, a G+C content of 49.2\%, and 12,353 predicted genes, among them 12 unspecific peroxygenases (EC 1.11.2.1).}, language = {en} } @misc{FriedrichSchrammKiebistetal., author = {Friedrich, Stephanie and Schramm, Marina and Kiebist, Jan and Schmidtke, Kai-Uwe and Scheibner, Katrin}, title = {Development of translationally active cell lysates from different filamentous fungi for application in cell-free protein synthesis}, series = {Enzyme and microbial technology}, volume = {185}, journal = {Enzyme and microbial technology}, publisher = {Elsevier BV}, address = {Amsterdam}, doi = {10.1016/j.enzmictec.2025.110588}, pages = {1 -- 10}, abstract = {There is an enormous potential for cell-free protein synthesis (CFPS) systems based on filamentous fungi in view of their simple, fast and mostly inexpensive cultivation with high biomass space-time yields and in view of their catalytic capacity. In 12 of the 22 different filamentous fungi examined, in vitro translation of at least one of the two reporter proteins GFP and firefly luciferase was detected. The lysates showing translation of a reporter protein usually were able to synthesize a functional cell-free expressed unspecific peroxygenase (UPO) from the basidiomycete Cyclocybe (Agrocybe) aegerita. For the most promising candidate Neurospora crassa, the influence of different conditions of cultivation and lysate preparation on in vitro translation of the reporter proteins was investigated and optimized. In general, the greatest improvements in the translational activity were achieved by the choice of the growth medium, the addition of organic nitrogen being most beneficial. Optimizing the culture and preparation conditions of the N. crassa platform improved protein yield of the original lysate by a factor of 25 for firefly luciferase and 17 for GFP, respectively. In addition to the reporter proteins, the aforementioned UPO as well as a functional UPO from Aspergillus niger were cell-free expressed using the different lysates from N. crassa. CFPS with fungal lysates opens the door to expressing UPOs in high throughput and in parallel, for example to optimize synthesis conditions or adapt catalyst properties. The presented method proves the general potential of fungal lysates for application in cell-free syntheses.}, language = {en} }