ChemBioChem
10.1002/cbic.201800814
COMMUNICATION
be the best organic solvents in the micro-aqueous media for the
fused protein FMO-E–HLADH. Overall, the here presented
cascade reaction catalyzed by fused oxidoreductase enzymes in
predominantly organic media shows the high potential for these
fragile enzymes to be employed in non-conventional conditions.
group of Prof. Dr. Marion Ansorge-Schumacher (Chair of
Molecular Biotechnology, TU Dresden).
Keywords: Fusion Enzyme • Baeyer-Villiger Monooxygenase •
Alcohol Dehydrogenase • Convergent Cascade Reaction •
Biocatalysis in Organic Media
Experimental Section
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1
Chemicals, reagents, enzymes and strains: Chemicals, media
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USA), Carl Roth (Karlsruhe, Germany), Merck (Darmstadt, Germany),
Fluka (Buchs, Switzerland) or Acros Organics (Geel, Belgium) and used
without further purification. Nickel-NTA affinity resin was ordered from
Expedeon (Cambridgeshire, UK) and BCA protein quantification kit
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®
purchased from Agilent Technologies (Santa Clara, USA). E. coli NEB
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media: Lyophilized CFE of fusion enzyme (from 2 mL CFE) was re-
dissolved in 500 µL ddH O. Cyclobutanone (5.95 µL) at final concentration
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of 20 mM and 1,4-butanediol (3.6 µL) at final concentration of 10 mM were
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9
2
50 µL substrate stocks. Therefore, the starting concentrations were
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o
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(
50 µL) from the organic phases were taken at definite time intervals and
mixed with 250 µL of ethyl acetate, followed by vigorously mixing and
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4
If phase separation occurred, the ethyl acetate phase was taken for GC
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Acknowledgements
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S.K. thanks the Fonds der Chemischen Industrie (Frankfurt,
Germany) for the financial support (Grant No: SK 196/20). L.H.
thanks China Scholarship Council (CSC) for the financial support.
F.S. Aalbers and M.W. Fraaije received funding from the
European Union (EU) project ROBOX (grant agreement
n°635734), EU’s Horizon 2020 Program Research and Innovation
actions H2020-LEIT BIO-2014-1. The views and opinions
expressed in this article are only those of the authors and do not
necessarily reflect those of the European Union Research Agency.
The European Union is not liable for any use that may be made
of the information contained herein. The authors thank Dr.
Diederik Johannes Opperman (University of the Free State, South
Africa) for the recombinant plasmid containing HLADH gene. R.
Röllig contributed to some parts of the experimental work and
interpretation of the data as an intern student from the research
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