Full Papers
doi.org/10.1002/cctc.202002011
ChemCatChem
pET28a/CaeEnR1 plasmid was transformed into E. coli BL21 Gold
DE3). E. coli cells were cultivated in Terrific Broth medium (TB)
Kinetic parameters
(
Steady-state kinetic parameters were calculated by incubating
purified CaeEnR1 with different concentrations according to the
used substrates ranging from 7.5–300 μM and 0.2 mM NADPH.
Resulting data was fitted with the Michaelis-Menten equation in
OriginPro 2018 software.
containing 12 g tryptone, 24 g yeast extract and 5 g glycerol,
supplemented with an equivalent of 1x TB-salts from a 10x stock
À 1
solution (0.17 M KH PO4 and 0.72 M K HPO ) and 50 mg·L
2
2
4
kanamycin as selection marker at 37°C until an OD of 0.4–0.6 was
600
reached. Expression was induced by adding isopropyl-β-d-thioga-
lactopyranoside to a final concentration of 0.1 mM. Cultivation was
continued for another 24 h at 24°C. Cells were harvested by
centrifugation (4,000 g, 30 min, 4°C) and stored at À 20°C until
further use or directly processed.
Product analysis
The reaction products were extracted with 400 μL ethyl acetate,
followed by mixing on a vortexer and centrifugation for 2 min at
1
3000 g. Analysis of the reaction products in the organic layer was
Protein purification
carried out on a Agilent Technologies 7890 A GC-MS-system (Santa
Clara, USA), equipped with a VFWax column (30 m×0.25 mm×
The cell pellet was thawed on ice and resuspended in lysis-buffer
0.25 μm film thickness, Santa Clara, USA) operated in splitless mode
(50 mM phosphate, 300 mM NaCl, 20 mM imidazole, pH 7.5).
under the following parameters: carrier gas, Helium with a constant
Disruption of cells was carried out by sonification (3 cycles for 60 s
on, 60 s rest) on ice using a sonifier (Bandelin Sonopuls, Berlin,
Germany). After complete disruption, cell debris was removed by
centrifugation (11,000 g, 45 min, 4°C). The resulting supernatant
was further processed, by either using Ni-NTA spin columns,
following the instruction of the manufacturer (Qiagen, Venlo,
Netherlands) or by using a 5 mL HisTrap column (GE-Healthcare).
The HisTrap columns were equilibrated with 5 column volumes (CV)
lysis-buffer and then loaded with the supernatant, washed twice
with 5 CV lysis buffer and eluted with an increasing imidazole
concentration from 20 mM imidazole (lysis-buffer) to 500 mM
imidazole (elution-buffer, 50 mM phosphate, 300 mM NaCl, 500 mM
imidazole, pH 7.5). Fractions with purified CaeEnR1 were analyzed
À 1
flow of 1.2 mL·min . Oven temperature was at 40°C (3 min),
À 1
1
0°C·min to 240°C and hold for 7 min. The mass spectrometer
operated in electron impact mode with an electron energy of 70 eV
and scanned in a range of m/z 33–300. Conversion rates were
calculated by using the peak areas. Retention times and mass
spectrum was compared with authentic standards, the NIST data-
base or by the characteristic fragmentation patter and molecule
ion.
In silico analysis of CaeEnR1
Phylogenetic analysis and amino acid alignments: Sequences for
phylogenetic analysis and sequence alignments were obtained via
the National Center of Biotechnology Information (NCBI, https://
www.ncbi.nlm.nih.gov/). Amino acid alignments and tree building
were performed with the Clustal Omega web tool (https://
www.ebi.ac.uk/Tools/msa/clustalo/). Visualization of the phyloge-
netic tree was carried out via the online tools, interactive tree of life
®
via SDS-PAGE, concentrated and rebuffered with Amicon Ultra
4
mL centrifugal filters (Merck KGaA, Darmstadt, Germany). Protein
concentration was photometerically determined by using the
2
4
60/280 ratio and the specific extinction coefficient (E
0340 M ·cm ), calculated with the ExPASy ProtParam tool.
=
280
À 1
À 1
[29]
(iTOL, https://itol.embl.de/) and http://www.phylogeny.fr.
Enzyme assay
UV method: CaeEnR1 activity was determined by recording the
À 1
À 1
oxidation of NADH/NADPH at 340 nm (E340 =6620 M ·cm ) on a
Nanophotometer (Implen, Munich, Germany). The typical reaction Acknowledgements
mixture contained 0.2 mM NADPH, 0.2 mM substrate and an
appropriate amount of enzyme in a total volume of 1 mL. For pH
We gratefully acknowledge the financial support by the Deutsche
dependency, the mixture was incubated in either 50 mM acetate
buffer (pH 4.5–6.0), 50 mM phosphate buffer (pH 6.5–8.0) or 50 mM
borate buffer (pH 8.5–10.0) to determine the pH-optimum. Effects
of the temperature were measured by incubating the reaction
mixture at different temperatures, ranging from 4°C–50°C. One
unit of enzyme activity was defined as the conversion of μmol
substrate per minute.
Forschungsgemeinschaft (DFG, German Research Foundation)-
Funder Id: https://doi.org/10.13039/501100001659, Grant Number:
RU 2137/1-1. Open access funding enabled and organized by
Projekt DEAL.
GC-MS method: A typical reaction mixture of 1 mL contained Conflict of Interest
.2 mM NADPH, 0.1 mM substrate, an appropriate amount of
0
enzyme in 50 mM phosphate buffer, pH 7.5. The mixture was
The authors declare no conflict of interest.
incubated at 24°C for 3 h.
Keywords: ene-reductase
· biocatalysis · regioselectivity ·
Preparative scale biotransformation
double bond reductase · alkynes
Biotransformation in a preparative scale contained 25 mM of
substrate 1 (solved in ethanol, 10% final solvent concentration),
+
0
.1 mM NADP , 100 mM glucose, 100 U glucose dehydrogenase
[
[
from Pseudomonas sp. (Sigma Aldrich) and an appropriate amount
enzyme in 50 mM phosphate buffer, pH 7.5 with a final volume of
2] M. C. Bryan, P. J. Dunn, D. Entwistle, F. Gallou, S. G. Koenig, J. D. Hayler,
1
00 mL. The mixture was incubated for 16 h at 24°C. After defined
times (1 h, 3 h, 6 h and 16 h) an aliquot was taken to monitor the
reaction progress.
[
ChemCatChem 2021, 13, 2191–2199
2198
© 2021 The Authors. ChemCatChem published by Wiley-VCH GmbH