10.1002/cbic.201900599
ChemBioChem
FULL PAPER
extracted by using n-pentane and a subsequent concentration step by
applying reduced pressure to obtain an enriched product fraction.
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The same procedure was repeated at higher scale. 200 mL reaction
volume in a 250 mL round-bottom flask was prepared as described above
in phosphate buffer: 15 vol% acetone, 25 mM (751 mg) (R)-carvone, 25
mM BNAH (initial + 7.5 mM hourly), and 1.5 µM FOYE-1. The reaction was
started by adding the enzyme (1.5 µM FMN loaded FOYE-1) and
incubated at 30 °C (gently shaken) until the conversion stopped after about
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Product identification. In case of small scale biotransformations, the
products and purity were identified by GC-FID or HPLC-UV/vis analyses
as described previously.[14] Otherwise, substrate and product
concentration were determined by GC-FID with a Shimadzu 2010 GC
system containing
a Hydrodex β-6TBDM column (Macherey-Nagel,
Germany). The samples were extracted with 1:1 volume of ethyl acetate
containing 1-octanol as internal standard. After extraction, the organic
phase was dried with anhydrous magnesium sulfate and the supernatant
placed in an analytic vial for further measurement. The column
temperature was kept constant at 120 °C and the measurement took place
for 10 minutes. Retention times were determined by means of authentic
standards as follows: 1-octanol 3.5 min, dihydrocarvone (R/S-
enantiomers) 5.2/5.7 min, and (R)-carvone 7.7 min.
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(R)-Carvone: 1H NMR (200 MHz, DMSO-d6) δ 7.00–6.67 (m, 1H), 4.88–
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2H), 2.47–2.13 (m, 4H), 2.13–1.95 (m, 1H), 1.92–1.51 (m, 5H), 1.40–1.15
(m, 1H), 0.99–0.77 (m, 3H).
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Acknowledgements
This work was supported by the Saxon Ministry of Science and
Fine Arts and the European Union (EU) in the framework of the
European Social Fund (ESF; project numbers 100101363 and
100236458). DT, AGB and CM where supported by the Federal
Ministry for Innovation, Science and Research of North Rhine -
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van der Geest, L.G. Otten, U. Hanefeld, Appl. Microbiol. Biotechnol. 2018,
102, 5545–5556; b) D.J. Mansell, H.S. Toogood, J. Waller, J.M. Hughes,
C.W. Levy, J.M. Gardiner, N.S. Scrutton, ACS Catal. 2013, 3, 370–379;
c) G. Steinkellner, C.C. Gruber, T. Pavkov-Keller, A. Binter, K. Steiner,
C. Winkler, A.j Łyskowski, O. Schwamberger, M. Oberer, H. Schwab, K.
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N.S. Scrutton, J. Am. Chem. Soc. 2016, 138, 1033–1039.
Westphalia (PtJ‐TRI/1411ng006) - ChemBioCat.
Keywords: biotransformations • oxidoreductases • old yellow
enzyme • solvent stability • biocatalysis • cofactor mimics
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36,1329–1333; b) Y. Fu, K. Hoelsch, D. Weuster-Botz, Process Biochem.
2012, 47, 1988–1997.
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