Y. Fu et al. / Process Biochemistry 47 (2012) 1988–1997
1989
2. Materials and methods
and 50 mM Tris–HCl applied in a pH range of pH 5.5–9.5. The effect of organic sol-
vents was studied using ethanol, 1-propanol, iso-propanol and dimethylformamide
(DMF). For the determination of kinetic parameters, 10 mM maleimide were kept
constant, whereas the concentration of NADPH was varied between 0.01 mM and
0.6 mM. Kinetic parameters were estimated according to the Michaelis–Menten
equation [36] and data were analyzed using non-linear regression analysis (Sigma
Plot 8.0, SPSS, Chicago, USA). Investigation of the substrate spectrum was conducted
with 0.5 mM NADPH using substrates at a concentration of 10 mM (added as an
ethanol solution, <5% (v/v) final ethanol concentration). Appropriate controls were
included and experiments were conducted at least in triplicates. One unit of enzyme
activity was defined as the oxidation of 1 mol NADPH per minute. Background
oxidase activity was determined and subtracted from the specific activity.
2.1. Chemicals and enzymes
Chemicals and solvents were of analytical grade from commercial sources. Sub-
strates and reference materials were obtained from Sigma–Aldrich (Schnelldorf,
Germany). 2-Methylmaleimide and rac-2-methylsuccinimide were synthesized as
reported previously [26,34]. NMR spectra were in accordance with those reported
in the literature [26]. NADH and NADPH were purchased from Carl Roth (Karlsruhe,
Germany). Enzymes used for DNA manipulations were bought from New England
Biolabs (Frankfurt, Germany). Primers were synthesized by Metabion (Martinsried,
Germany).
2.2. Cloning
2.6. Bioreduction on a mL-scale
The DNA sequence of the ene-reductase from Synechoccocus sp. PCC 7942 was
obtained from the NCBI database (RefSeq ID: NC 007604). Genomic DNA from
Synechoccocus sp. PCC 7942 (Pasteur Culture Collection of cyanobacteria, Paris,
France) was used as template for the amplification of the ER gene. The PCR
was performed using Phusion® HF DNA polymerase (forward primer, pET21a(+):
5ꢀ-AGAGATCATATGTCCGAATCGCTCAAACTGCTGACG-3ꢀ; reverse primer, pET21a(+):
5ꢀ-AGAGATGCGGCCGCGACAGATGCTGCTTCCAAACTGGGATAG-3ꢀ; forward primer,
pETM-41: 5ꢀ-AGAGATCCATGGACATGTCCGAATCGCTCAAACTGCTGACG-3ꢀ; reverse
primer, pETM-41: 5ꢀ-AGAGATGCGGCCGCTTAGACAGATGCTGCTTCCAAACTGGGA-
3ꢀ). The ER gene was integrated into pET21a(+) (Novagen, San Diego, USA) using
the restriction sites NdeI and NotI for a construct with a C-terminal His6-tag
(Syn7942ER-His6), as well as into pETM-41 (EMBL, Heidelberg, Germany) using
NcoI and NotI. The vector pETM41 contained an N-terminal His6-maltose-binding-
protein (His6-MBP) tag (His6-MBP-Syn7942ER), which was removed using the
tobacco etch virus (TEV) protease cleavage site to obtain the enzyme without
any tags (Syn7942ER). The constructed vectors were transformed into competent
Escherichia coli (E. coli) DH5␣ (Invitrogen, Carlsbad, CA, USA). After confirming the
accuracy of the constructed vector by sequencing (GATC, Konstanz, Germany), plas-
mids were transformed into E. coli BL21(DE3) (Novagen, San Diego, USA) for protein
expression.
The bioreduction of alkenes was carried out in 1 mL sodium phosphate
buffer (100 mM, pH 7.0) containing 5 mM substrates (added as
a DMF solu-
tion, <2% (v/v) final DMF concentration), 15 mM NADH and 85 g mL−1 (2.1 M)
Syn7942ER. The reactions were agitated at 30 ◦C and 300 rpm (Thermomixer com-
fort, Eppendorf, Hamburg, Germany) for 24 h. The reduction of ketoisophorone
with 5–20% (v/v) ethanol, iso-propanol or DMF was performed on a 1 mL-scale
with 57 g mL−1 (1.2 M) NADP+-dependent MycFDH C145S/D221Q/C255V [37],
250 mM sodium formate, 0.5 mM NADP+, 10 mM ketoisophorone and 25 g mL−1
(0.6 M) Syn7942ER for 6 h at 30 ◦C and 150 rpm (WiseCube, Witeg Labortechnik,
Wertheim, Germany). Reactions were stopped by extraction with ethyl acetate (1:1)
containing 36 mM (R)-limonene as internal standard.
2.7. Analytical procedures
reduction products as described previously [4,17,25]. Ketoisophorone and (R)-
Levodione were determined on a CP-Chirasil-DEX CB column (25 m, 0.32 mm,
Agilent Technologies, Böblingen, Germany) according to [4]. The absolute config-
uration of products was identified by comparison with reference materials on chiral
GC according to the literature [4,17,25].
2.3. Recombinant expression and purification
A 5 mL Terrific Broth (TB) preculture supplemented with the respective antibi-
otics (50 mg L−1 ampicillin for pET21a(+) and 34 mg L−1 kanamycin for pETM-41)
was inoculated with a single colony from an agar plate, incubated over night at
37 ◦C and then subcultured into 200 mL TB in 1000 mL shake flasks without baffles.
As the cell density reached an OD600 of 0.6–0.8, the protein expression was induced
by the addition of 1 mM isopropyl -d-1-thiogalactopyranoside (IPTG). Afterwards,
cells were incubated overnight (18–20 h) at 20 ◦C and 160 rpm. After harvesting
cells by centrifugation, pellets were stored at −20 ◦C or used directly for protein
purification.
3. Results
3.1. Sequence analysis
The ene-reductase from the cyanobacterium Synechococcus sp.
PCC 7942 (RefSeq ID: YP 399492) was identified using the basic
local alignment search tool (BLAST) of the National Center for
Biotechnology Information (NCBI) databank [38]. The amino acid
sequence exhibited a high similarity to OPR1 from Oryza sativa (51%
similarity). In contrast, the similarity to OYE1 from Saccharomyces
only moderate (40–42% identity, 56–57% similarity). A sequence
alignment with other OYE homologues (Fig. 1) demonstrated that
sidered to be a monomeric protein based on the P-[LM]-T-R-X-R
pattern in the loop 1 region and the G-[FYW]-X(3)-P-G-[ILV]-
[FHYW] pattern in the loop 2 region according to Oberdorfer et al.
[39].
TEV protease, His6-MBP-Syn7942ER fusion protein and Syn7942ER-His6 were
purified according to a modified protocol described by Hölsch et al. [30]. Cell pel-
lets were suspended in binding buffer (20 mM sodium phosphate, 500 mM sodium
chloride, 40 mM imidazole, pH 7.4) and disrupted using 50% (v/v) glass beads
(0.25–0.5 mm; Carl Roth, Karlsruhe, Germany) for 3 min at 30 Hz in a mixer mill
(Retsch, Haan, Germany). After centrifugation (47,808 × g, 4 ◦C, 30 min), the super-
natant was filtered (0.45 m, Minisart HF, Sartorius Stedim Biotech, Göttingen,
Germany) and applied to a 1 mL HisTrap FF crude column (GE Healthcare, Uppsala,
Sweden). Buffer exchange and concentration of purified proteins were performed
using a Vivaspin ultrafiltration device (molecular weight cutoff: 5 kDa; Sartorius
Stedim Biotech, Göttingen, Germany).
2.4. Protein analysis
Protein concentration was determined using the BCA Protein Assay (Thermo
Fisher Scientific, Rockford, USA). SDS-PAGE was performed using 3% and 12.5%
Bis–Tris gels in Tris–glycine running buffer with Roti®-Mark Standard (Carl Roth,
Karlsruhe, Germany) for the estimation of the molecular mass and protein purity.
Gels were stained according to Fairbanks et al. [35].
2.5. Enzyme assay
3.2. Cloning, protein expression and purification
The enzyme activity was determined by a photometric assay monitoring the
oxidation of NADPH concentration at 340 nm using a molar absorption coeffi-
cient of 6.22 mM−1 cm−1. In case of ketoisophorone and 3-phenyl-2-methylpropenal
Cloning of the ER gene enabled the expression of a protein
protein without an affinity tag (Syn7942ER). Typical yields after
protein purification were about 5.6 mg protein per gram dry cell
the assay was performed at 365 nm using
a
molar absorption coefficient of
200 L scale in sodium
3.51 mM−1 cm−1
.
All reactions were performed on
a
phosphate buffer (100 mM, pH 7.0) at 30 ◦C or 25 ◦C using microplate spectrom-
eters (EL808, BioTek Instruments, Winooski, USA/infinite M200, Tecan, Crailsheim,
Germany).
−1
weight (mg gDCW−1) for Syn7942ER-His6 and 0.5 mg gDCW
for
Syn7942ER. A SDS-PAGE analysis of purified Syn7942ER is shown
in Fig. 2. Purified enzymes were used for the characterization of
the substrate spectrum, cofactor preference and stereoselectivity,
All assays contained 10 mM maleimide and 0.5 mM NADPH, unless stated oth-
erwise. Buffers for the investigation of the pH profile were 50 mM sodium citrate,
50 mM sodium phosphate, 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS)