Materials
General chemical reagents were purchased from Sigma-Aldrich, Sangon Biotech Co., Ltd. (Shanghai, China). High-performance liquid chromatography (HPLC) was performed on an Agilent 1260 Infinity apparatus with a diode array detector (DAD). HPLC-MS experiments were performed on Agilent 1290 HPLC system coupled with a Thermo Electron LTQ-Orbitrap XL mass spectrometer. All chemicals and solvents were of analytical or chromatographic grade. The resuspended cells were lysed by an ultrasonic processors VCX750 (Sonics and Materials Inc, PA, USA). Common biochemicals and chemicals were purchased from standard sources for laboratory use.
Bacterial strains, plasmids, and culture conditions
All strains and plasmids used in this study are listed in Supplemental Table S2. Escherichia coli DH5α was used for general cloning. Escherichia coli BL21 (DE3) was used as a host for protein expression. E. coli strains were routinely cultured in Luria–Bertani (LB) liquid medium at 37 °C, 220 rpm, or LB agar plate at 37 °C. When appropriate, kanamycin (Kan; 100 μg mL-1 for E. coli) was added to the medium.
DNA isolation and manipulation
DNA manipulations were performed using standard procedures or the manufacturer’s protocols for E. coli. Plasmid extraction, DNA purification and gel extraction were carried out using commercial kits (Omega Biotech Co., Ltd., Beijing, China). Oligonucleotide synthesis and DNA sequencing were performed at Tsingke Biotech Co., Ltd. (Beijing, China). PCR reactions were carried out using Pfu DNA polymerase (TIANGEN Biotech Co., Ltd., Beijing, China).
Site-directed mutagenesis of P450S18
The reverse complementary primers with mutation sites were designed and listed in Table S2. The pET28a plasmids carrying P450S18 gene with mutation sites was linearized by reverse PCR using 2 × Phanta® Flash Master Mix DNA polymerase. The linear plasmids were ligated to circular molecules by seamless cloning using ClonExpress® Ultra One Step Cloning Kit (Nanjing, China). The introduced mutations were verified by DNA sequencing.
Expression of P450S18 variants
Single colonies of E. coil BL21 (DE3) strain containing protein expression vectors were inoculated were inoculated in 100 μL of LB supplemented with 100 μg/mL kanamycin in 96-well plates and grown at 37 °C for 12 h. Then, 2% of the E. coli culture volume was transferred to 500 μL of LB supplemented with 100 μg/mL kanamycin in a 1.5 mL microtube and grown to an optical density at OD600 of 0.5–0.6 at 37 °C and 220 rpm. Next, protein expression was induced overnight by the addition of 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and culturing at 16 °C and 220 rpm. After centrifugation of the cultures at 13,000 rpm and 4 °C for 5 min, the resulting cell pellets were resuspended in 100 μL of bacterial protein extraction reagent (Cell Biolabs Inc., San Diego, CA, USA), incubated for 15 min at room temperature, and centrifuged at 12,000×g for 10 min. The resulting supernatants were used as the crude enzyme.
In vitro assays
A typical reaction (50 μL) consists of OPD (1 mM), H2O2 (20 mM), P450S18 in Tris-HCl buffer (50 mM, pH 7.5). The reaction mixtures were incubated at 30 °C for 2 h, and were quenched by the addition of ACN (50 μL), and the denatured protein was removed by centrifugation. The assays were monitored by HPLC analysis, using a C18, YMC pack ODS-AQ column (5 μm, 150 × 4.6 mm) with UV detection at 260 nm with a gradient program (0-5 min, 10% B; 5-20 min, 10 % to 25 % B; 20 - 35 min, 100% B; 1 mL min-1).