Beilstein J. Org. Chem. 2018, 14, 697–703.
Production of PeAAOx
PeAAOx were pooled, concentrated and desalted using HiTrap
E. coli cultivation
desalting columns (GE Healthcare) and 10 mM sodium phos-
slightly modified literature protocol was used [28]. Pre-cultures lated based on the absorbance using the molar extinction coeffi-
of LB media containing 100 μg mL−1 of ampicillin were cient of ε463 11,050 M−1 cm−1.
inoculated with E. coli W3110 containing pFLAG1-AAO and
incubated overnight at 37 °C and 180 rpm. Overexpression Activity assay
was carried out in 5 L flasks with 1 L of TB medium supple- The activity of PeAAOx was determined by UV–vis spectros-
mented with 100 μg mL−1 of ampicillin. The medium was copy, using an Agilent Cary 60 UV–vis spectrophotometer,
inoculated with the pre-culture to an OD of 0.05 and grown following the oxidation of ABTS (ε405 = 36,800 M−1 cm−1) by
at 37 °C and 180 rpm. At an OD600 of 0.8, 1 mM isopropyl horseradish peroxidase (POD) at the expense of hydrogen
β-D-thiogalactopyranoside (IPTG) was added and the cultures peroxide. In general, 0.044 µM PeAAOx was used to convert
were incubated for additional 4 h at 37 °C and 180 rpm. The 3 mM of trans-2-hex-2-enol. The hydrogen peroxide formed in
bacterial pellets, obtained after harvesting the cells, were this reaction was subsequently used to convert 2 mM of ABTS
re-suspended in a total volume of 40 mL 50 mM Tris/HCl to ABTS·+ by an excess of POD (500 U mL−1). The reactions
buffer, pH 8.0, containing 10 mM EDTA and 5 mM dithio- were performed at 30 °C in oxygen-saturated 50 mM KPi buffer
threitol (DTT).
at pH 7.0.
Refolding
Flow reactor experiments
The re-suspended cells were disrupted by incubation with PFA microreactor coils (750 μm ID) with a volume of 3 and
2
mg mL−1 lysozyme for 1 h at 4 °C. Afterwards, 0.1 mg mL−1 6 mL were constructed. The reaction mixture was introduced
DNase, 1 mM MgCl2 and 0.1 mM PMSF were added followed via a syringe pump (Fusion 200, Chemyx), while the pure
washed three times with 20 mL 20 mM Tris/HCl buffer, pH 8.0, Information File 1, Figure S8). Residence times were taken as
containing 10 mM EDTA and 5 mM DTT using a potter the time between the solution entering and exiting the coil and
homogenizing device. The pellets obtained after centrifugation were varied by altering the flow, keeping the ratio of oxygen to
(
15 min at 15,000 rpm and 4 °C) were solubilized in a total liquid at three to one. Samples were collected on ice and as
volume of 30 mL 20 mM Tris/HCl buffer, pH 8.0, containing soon as enough volume was collected, extracted with ethyl
mM EDTA, 50 mM DTT and 8 M urea. After incubation on acetate and analysed by GC (vide infra).
ice for 30 min, the solution was cleared by centrifugation
15 min at 15,000 rpm and 4 °C). The obtained supernatant was GC analysis
2
(
used as stock solution for the in vitro refolding.
The collected reaction mixtures were extracted into an equal
volume of ethyl acetate, dried with magnesium sulphate and
The PeAAOx was solubilized using 150 µg mL−1 protein in analysed on a CP-wax 52 CB GC column (50 m × 0.53 m ×
2
1
4
0 mM Tris/HCl buffer, pH 9.0, containing 2.5 mM GSSG, 2 µm) (GC method: 60 °C for 3 min; 30 °C/min to 105 °C;
mM DTT, 0.02 mM FAD, 34% glycerol and 0.6 M urea at 105 °C for 7 min; 30 °C/min to 250 °C; 250 °C for 1 minute).
°C for 80 h. After the incubation for PeAAOx activation/ Dodecane (5 mM) was added as standard.
refolding, the refolding mixture was concentrated to 100 mL
and the buffer exchanged against 10 mM sodium phosphate Work-up semi-preparative scale
buffer, pH 5.5 by diafiltration (DV 20) and subsequently The reaction mixture was directly collected in deuterated
(
and 4 °C), the soluble fraction was further purified using anion- porting Information File 1). The organic mixture was diluted
exchange chromatography.
and introduced into a separation funnel and washed with brine.
The aqueous phase was backwashed once with DCM. The
collected organic phase was dried over MgSO4, filtered and
Purification
The concentrated PeAAOx solution was purified using a 58 mL concentrated under reduced pressure. Purification of the isolat-
Q Sepharose column (GE Healthcare). PeAAOx was eluted ed mixture was performed by flash chromatography on silica
with a linear NaCl gradient (0–0.6 M over 6 CV) using 10 mM (pure DCM). The final product was obtained as colourless oil
sodium phosphate buffer, pH 5.5. Fractions containing (200 mg).
701