CHEMBIOCHEM
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overnight at 378C. Induced cells were covered with an agar solu-
tion in potassium phosphate buffer (pH 7.4, 0.1m), Triton X-100
(0.2%) and EDTA (10 mm) at a maximum temperature of 708C.
Once the agar had cooled down to room temperature, cells were
permeabilised with potassium phosphate buffer (pH 7.4, 0.1m,
5 mL), Triton X-100 (0.1%) and EDTA (200 mm). This step was done
three times with gentle stirring for about 15 min. The plates were
washed three times with potassium phosphate buffer (pH 7.4,
0.1m) before addition of the substrate solution [3.5 mL, formate
(1.25m), NAD+ (5 mm) in potassium phosphate buffer (pH 7.4,
0.1m)]. The reaction was allowed to proceed for 5 min with stir-
ring. NADH formation was revealed by the addition of PES (60 mm,
120 mL) and NBT (6 mm, 200 mL). The reaction was allowed to take
place in the dark for 20–30 min. Cells were washed with water, and
positive clones appeared as dark spots.
FDH purification: Wild-type and mutant FDHs were purified by
modifications of protocols by Ansorge-Schumacher et al.,[29] who
incorporated a PEG-based ATPS and by Schꢃtte et al.,[31] who used
hydroxyapatite gel chromatography and Cibracon blue Sepharose
affinity chromatography. Bacterial pellets were resuspended in po-
tassium phosphate buffer (1 mL, 0.1m, pH 7.6). PEG 400 (30%, v/v)
was added to the suspension, which was homogenised. The mix-
ture was heated for 2 h at 378C and then allowed to cool to room
temperature (5–10 min). Dipotassium phosphate salt (K2HPO4, 5%,
w/w) and water (21%, w/w) were added. After K2HPO4 dissolution,
PEG 1550 (7% w/w) and sodium chloride (6%, w/w) were added.
The solution was mixed until homogenised, and separation into
two phases was achieved after 2 h. The upper phase was recov-
ered and mixed with PEG 6000 (20%, w/w) and water (10%, w/w).
The enzyme precipitated from this solution after 2 to 3 h at room
temperature and was recovered by centrifugation at 10000g for
10 min before dissolution in potassium phosphate buffer (60 mm,
pH 7.6). This solution was concentrated three times in potassium
phosphate buffer (60 mm, pH 7.6) with centrifugal filters (Ultracel
YM-30, Millipore, Molsheim, France) before purification by Cibracon
blue Sepharose affinity chromatography. After washing with potas-
sium phosphate buffer (60 mm, pH 7.6), the enzyme was eluted by
addition of NADH (1 mm) and concentrated three times in potassi-
um phosphate buffer (5 mm, pH 7.6) by the same protocol as
before. The enzyme was then purified by hydroxyapatite chroma-
tography. After washing with potassium phosphate buffer (5 mm,
pH 7.4), the enzyme was eluted by use of a step gradient in potas-
sium phosphate buffer (20–100 mm, pH 7.6). The FDH was recov-
ered in potassium phosphate (60 mm). Once the protocol was opti-
mised for wild-type FDH, the enzymes were purified five to 24
times with overall yields of 20–70%, depending of the mutation.
Construction of single mutants: The single mutant N187S was
constructed by digesting the pET-28b expression vectors (2 mg)
containing the wild-type FDH gene or the double mutant M60
gene with with Pac I and Xho I [2 units each, 20 UmLꢁ1, New Eng-
land Biolabs, in Bis·Tris·propane·HCl buffer (1 mm, pH 7), MgCl2
(1 mm) and DTT (0.1 mm)] in a final volume of 100 mL, with agita-
tion for 2 h at 378C. The insert coding for the FDHwt and the
vector coding for the double mutant M60 containing only the
N187S mutation were isolated by agarose gel electrophoresis and
use of the QIAquick gel extraction kit (Qiagen, Courtaboeuf,
France). The insert and the vector were desalted (QIAquick kit,
Qiagen) and incubated together in ratios of 1:1 (85 ng/18 ng) or
1:3 (85 ng/54 ng; vector/insert) in the presence of T4 DNA ligase
(1 Unit, Invitrogen, Cergy-Pontoise, France) in Tris·HCl buffer
(40 mm, pH 7.6), MgCl2 (10 mm), ATP (0.5 mm) and DTT (10 mm) for
a final volume of 20 mL for 1 hour at 378C. Ligation medium (1 mL)
was used to transform competent E. coli DH10B cells by electric
shock (2.5 kV during 5.7 ms). Cells transformed by the pET-28b-
N187S were selected on LB/agar/kanamycin (100 mgmLꢁ1). The cor-
rect orientation of the insert was verified by PCR screening of the
plasmid DNA of 12 clones (Nucleospin kit, Macherey–Nagel), and
the nucleotide sequence was determined by Eurofins MWG
Operon (Courtaboeuf, France). The single mutant T321S was con-
structed in a similar manner by digesting the plasmid DNAs coding
for the FDHwt and the double mutant M60 with BamHI (2 Units,
10 Umlꢁ1, New England Biolabs) and Pac I (2 Units, 10 Umlꢁ1, New
England Biolabs) and subsequently using the same protocol as
above to isolate the single mutation.
Enzyme activity assays: Enzyme activity was measured from the
NADH absorbance at 340 nm (e3M40 nm =6.22·103 mꢁ1 cmꢁ1
) with
a Tecan Infinite M200 (Salzburg, Austria) microtitre plate reader at
308C. A single well contained enzyme solution (10 mL, 0.4 UmLꢁ1),
NAD+ solution (10 mL, 50 mm), sodium formate (10 mL, 3m) and
[MMIm][Me2PO4] (0–70%, v/v) in potassium phosphate buffer
(20 mm, pH 7.6). Optical pathways were determined by measuring
the absorbance of water at 975 nm in each individual well (eM975 nm
=
3.05 10ꢁ3 mꢁ1 cmꢁ1). Activities are average values of at least three
measurements and are expressed in mmolminꢁ1 mgꢁ1. The kinetic
parameters (Km, kcat and kcat/Km) of the different enzymes were de-
termined with a final concentration of formate at 300 mm and final
concentrations of NAD+ ranging from 25 mm to 5 mm. The param-
eters were extracted from the Hanes–Woolf plot.
Colorimetric screening assay: Cells presenting FDH activity on the
agar plate were cultured overnight in LB/kanamycin (1 mL,
100 mgmLꢁ1) from the mother plate in a 96-well culture plate. Pro-
tein expression was induced with IPTG (1 mm) for 5 h. Cells were
harvested by centrifugation at 2000g for 15 min at 48C and resus-
pended in PBS buffer (500 mL, 0.1m, pH 7.4, potassium salt), Triton
X-100 (0.1%) and EDTA (200 mm, sodium salt) and centrifuged
again at 2000g (15 min, 48C). The supernatant was used for the
screening assay as follows: the reaction medium was composed of
supernatant (10 mL), substrate solution [10 mL, NAD+ (25 mm), for-
mate (1.5m), PES and NBT solution (10 mL, 140 mm and 1 mm, re-
spectively)] and either 0 or 60% (v/v) of [MMIm][Me2PO4]. The final
volume was adjusted to 100 mL with PBS buffer (pH 7.2). The for-
mation of NBT diformazan blue precipitate was detected after
5 min by measuring the absorbance at 560 nm. The activity was
normalised by determining the protein content in each superna-
tant by a Bradford assay by the standard procedure. The colorimet-
ric assay was preferred in the screening due to the high absorb-
ance of the IL in the UV region.
Effect of temperature and of [MMIm][Me2PO4] on the stabilities
of wild-type FDH and the double mutant: Thermal stabilities
were determined by incubating the enzymes (0.04 mgmLꢁ1) at 40
and 608C in sodium phosphate buffer (20 mm, pH 7.6). The con-
centrations of NAD+ and sodium formate were 5 mm and 300 mm,
respectively. Aliquots (160 mL) in 1.5 mL Eppendorf tubes were
placed in a water bath (40 or 608C). Every 15 min, a sample (90 mL)
was removed, and its residual activity was assayed spectrophoto-
metrically as described above. Enzyme stability in the presence of
[MMIm][Me2PO4] was assayed with a 96-well plate. The enzyme
was incubated (0.04 mgmLꢁ1) in sodium phosphate buffer (pH 7.6,
20 mm) containing [MMIm][Me2PO4] (62.5%, v/v). Every 12 h, the
residual enzyme activity was determined spectrophotometrically.
The concentrations of NAD+ and sodium formate were 5 and
300 mm, respectively.
Fluorescence: Protein unfolding was evaluated by quenching of
the protein fluorescence with acrylamide as previously reported.[36]
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Carter, Julie L.L.
