To assess the S oxidation activity, the aromatic thioethers methyl-p-tolyl sulfide sulfide (Cat. # 275956-5G, Sigma-Aldrich) and the bulky benzyl phenyl sulfide (Cat. # 8415660025, Sigma-Aldrich) were tested52 (link),53 (link). For the N oxidation activity, benzydamine (Cat. # B5524-5G, Sigma-Aldrich) and tamoxifen (Cat. # T5648-1G, Sigma-Aldrich) were selected54 (link),55 (link). To follow the BV activity the aliphatic hepta-2-one (Cat. # 537683-100 ML, Sigma-Aldrich), the alicyclic cyclohexanone (Cat. # 29140-100 ML, Sigma-Aldrich) and the aromatic phenylacetone (Cat. # 135380-100G, Sigma-Aldrich) were selected56 ,57 .
Substrate conversions were done in duplicates, using 1.0–5.0 mM substrate (1% methanol), 0.10 mM NADPH, 2.0 µM enzyme, 5.0 µM phosphite dehydrogenase (PTDH, produced in-house58 ), and 20 mM sodium phosphite. The last two components were used as a regeneration system for NADPH and the control did not contain any tAncFMO. All compounds were prepared in storage buffer (50 mM KPi, 250 mM NaCl, 0.05% TritonTm X-100 reduced, pH 7.5) the final reaction volume was adjusted to 1.0 ml and put into 4 ml vials before being incubated at 30 °C, with shaking, for 16 h. Conversions of phenylacetone, heptan-2-one, cyclohexanone, benzyl phenyl sulfide and methyl-p-tolyl sulfide were analyzed by GC–MS while benzydamine and tamoxifen conversions were monitored by HPLC. Due to their poor solubility, tamoxifen, benzydamine, and benzyl phenyl sulfide conversions were done using 1.0 mM substrate while the remaining substrates were tested at 5.0 mM.
Substrate conversions were done in duplicates, using 1.0–5.0 mM substrate (1% methanol), 0.10 mM NADPH, 2.0 µM enzyme, 5.0 µM phosphite dehydrogenase (PTDH, produced in-house58 ), and 20 mM sodium phosphite. The last two components were used as a regeneration system for NADPH and the control did not contain any tAncFMO. All compounds were prepared in storage buffer (50 mM KPi, 250 mM NaCl, 0.05% TritonTm X-100 reduced, pH 7.5) the final reaction volume was adjusted to 1.0 ml and put into 4 ml vials before being incubated at 30 °C, with shaking, for 16 h. Conversions of phenylacetone, heptan-2-one, cyclohexanone, benzyl phenyl sulfide and methyl-p-tolyl sulfide were analyzed by GC–MS while benzydamine and tamoxifen conversions were monitored by HPLC. Due to their poor solubility, tamoxifen, benzydamine, and benzyl phenyl sulfide conversions were done using 1.0 mM substrate while the remaining substrates were tested at 5.0 mM.
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