Chryseobacterium gleum DSM 16776 was obtained from the German collection of microorganisms (DSMZ). E. coli strain JM109 [genotype endA1 recA1, gyrA96, thi, hsdR17,(rK-, mK+), relA1, supE44, λ-, Δ(lac-proAB), (F', traD36, proAB, lacIqZΔM15)] and the pQE-30 expression vector were purchased from Promega (Madison, USA) and Qiagen (Valencia, USA), respectively. The origin of replication in pQE-30 is ColE1 (pBR322) and transcription of the inserted gene is controlled by the bacteriophage T5 promoter (recognized by the E. coli housekeeping RNA polymerase) and two lac operator sequences (conferring inducibility by IPTG). For efficient repression the host strain JM109 which over-expresses the LacI repressor was used. JM109 was transformed with the plasmid pRARE2, which contains the tRNA genes argU, argW, ileX, glyT, leuW, proL, metT, thrT, tyrU, thrU and argX. The usage of the rare codons AGG, AGA, AUA, CUA, CCC, GGA and CGG is thereby supplemented. The plasmid was isolated from Rosetta2 (DE3) (Merck Chemicals, UK) (F-ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2) cells. The resulting chloramphenicol-resistant strain JM109-pRARE2 was the expression host.
Cloning of choA from C. gleum
The putative cholesterol oxidase gene choA of C. gleum was identified by Protein blast (NCBI website) using the cholesterol oxidase sequence of Streptomyces sp. (UniProt accession no. P12676) as search template. The cholesterol oxidase gene of C. gleum (accession no. ACKQ02000004) was PCR amplified from genomic DNA with forward primer 5’ GCG GCA TGC GAC AGA AAA AAA TTC ATC AGG ACA AGT GC 3’ (introducing a SphI site around the native start codon) and reverse primer 5’ CCG AAG CTT TTA ACC CAG GTT AAA TTC ATT TTG CCG G 3’ (introducing a HindIII site after the native stop codon). PCR was performed with high fidelity Phusion polymerase (New England Biolabs, Ipswich, USA) and a diluted solution of genomic DNA of C. gleum DSM 16776 as template source. Genomic DNA was isolated using the GenElute Bacterial genomic DNA kit (Sigma-Aldrich, CH). Plasmid DNA and PCR products were purified using the Gene Jet Plasmid Miniprep Kit (Fermentas) and the GenElute PCR clean-up kit (Sigma-Aldrich). DNA from agarose gels was recovered using the GenElute Gel extraction kit (Sigma-Aldrich, CH). The 1596 bp PCR product was cloned into the pQE-30 expression vector in frame with a sequence coding for an N-terminal hexa-histidine tag to allow purification by immobilized metal affinity chromatography. The in frame cloning of the choA gene from C. gleum DSM 16776 in the final expression plasmid pCgChoA was confirmed by DNA sequencing (GATC, Germany).
Cell cultivation and protein purification
C. gleum DSM 16776 was grown overnight at 30°C at 180 rpm in trypticase soy yeast extract medium (trypticase soy broth 30 g/L, yeast extract 3 g/L). E. coli JM109-pRARE2 was transformed with pCgChoA. Expression of the recombinant protein was performed in medium containing 1× M9 salts, 20 g/L N-Z-amine, 20 g/L glycerol, 1 mM MgSO4, 1 mM MgCl2, 100 μM CaCl2, 100 μM thiamine, 0.025% glucose and trace metal mixture . A 100 mL overnight culture was grown from a single colony (LB agar) and used to inoculate 700 mL of medium (dilution 1:50). The culture was grown at 37°C with shaking at 180 rpm. At an OD600 = 0.8, protein production was induced at 0.1 mM isopropyl thio-β-D-galactoside (IPTG). At the same time, the temperature and shaking were reduced to 16°C and 120 rpm for 16–18 hours. For plasmid selection 100 μg/mL ampicillin and 20 μg/mL chloramphenicol were added to plates and liquid media. For protein purification cells were harvested by centrifugation at 4°C for 30 min at 4,495 × g, washed in 0.1 M sodium phosphate buffer pH 7, centrifuged again and subsequently stored at -20°C. Frozen cells were thawed on ice and resuspended in 0.1 M sodium phosphate buffer pH 7 with 20 mM imidazole and 0.5 M sodium chloride (buffer A) containing 1 mg/mL lysozyme and protease inhibitor mix (Roche Complete Protease Inhibitor Mix, EDTA-free) and re-frozen at -80°C. Cells were thawed, Benzonase Nuclease (Roche) was added and the suspension incubated for 1 h at 37°C at 120 rpm. The suspension was subjected to twelve 10 s rounds of sonication with a Branson sonicator equipped with a microtip at a setting of 80%. Cellular debris was removed by centrifugation at 4°C for 40 min, 47,000 × g. Purification was performed on an Äkta purifier FPLC system (GE-Healthcare). The sample was loaded onto a 1 mL HisTrap FF chromatography column (GE-Healthcare), previously equilibrated with buffer A. Proteins were eluted with a imidazole gradient from 0 to 1 M. Fractions displaying cholesterol activity were pooled and concentrated by ultrafiltration using a 30 kDa cut-off. The sample was loaded onto a Superdex 200 column (GE-Healthcare), previously equilibrated with 20 mM MOPS buffer pH 6.75 containing 0.1 M NaCl. Fractions with cholesterol oxidase activity were pooled and concentrated by ultrafiltration. The purity of the sample was analyzed by SDS-PAGE using a 10% polyacrylamide gel. The gel filtration kit (GE-Healthcare) was used to calibrate a Superdex 200 column with high and low molecular weight standards, previously equilibrated with 20 mM MOPS buffer (pH 6.75) containing 0.1 M NaCl.
Activity assay and protein determination
A 27.2 mM stock solution/dispersion of cholesterol was prepared and diluted in water in the presence or absence of 5% (v/v) Triton X-100, 2.9% (w/v) of taurocholic acid sodium salt (Sigma Aldrich), and a combinations thereof. Cholesterol oxidase activity was assayed by quantifying H2O2 formation from the coupling reaction with HRP. The activity assay mixture contained 40 μL of cholesterol at the selected concentration, 10 μL of HRP (concentration 1 mg/mL, in ddH2O), 10 μL of ABTS (concentration 10 mM, in ddH2O), 110 μL of 0.011 M MOPS buffer pre-heated to 37°C, and 30 μL of the purified enzyme preparation in a total volume of 200 μL. The spectrophotometric cholesterol activity assay was carried out in a 96-well plate using a BioTek Synergy Mx spectrophotometer. ABTS (0.6 mM), pyrogallol red (0.15 mM) and o-dianisidine (0.5 mM) were used as substrates for the HRP coupled assay using 0.011 M MOPS buffer pH 6.75 at 37°C. The reaction was started by adding cholesterol oxidase and followed for oxidation of ABTS at 420 nm (ε = 36 000 M-1 cm-1), of pyrogallol red at 550 nm (ε = 30 900 M-1 cm-1) and of o-dianisidine at 440 nm (ε = 13 000 M-1 cm-1). Kinetic parameters of cholesterol oxidase samples were determined between 0.17 μM – 5.5 mM cholesterol at 35°C, and results were analyzed with the Enzyme Kinetics Module of the software SigmaPlot (Systat Software Inc., CA, USA).
Cholesterol activity as a function of the pH was recorded via the HRP coupled assay with 0.5 mM ABTS and 0.55 mM cholesterol using Teorell-Stenhagen buffer (pH 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, and 8.5), 0.1 M sodium phosphate buffer (pH 6.0, 6.4, 7.0, 7.5, and 7.8), 0.11 M MOPS pH 6.75, 0.1 M potassium phosphate buffer (pH 6.0, 6.5, 7.0, 7.5, and 7.8), and McIlvaine buffer (pH 4.0, 5.0, 6.0, 7.0, 7.5, and 8.0). Further 0.55 M, 0.275 M, 0.11 M 0.055 M, 0.0275 M and 0.011 M MOPS buffers (pH 6.75, 7, 7.25, 7.5, 7.75, and 8.0) were tested. The temperature optimum was recorded between 24 and 48°C following ABTS oxidation with 0.55 mM cholesterol, in an assay volume of 3 mL using a magnetically stirred, temperature-controlled cuvette device using a Varian Cary 50 Bio spectrophotometer. Total protein concentration was determined by the method of Bradford, with bovine serum albumin as standard.
The substrate cholesterol was added from a stock solution, which was made up as described above (containing Triton X-100 and taurocholate), to a final concentration of 1 mM in 0.011 M MOPS buffer pH 6.75. The reaction was adjusted to 600 μL and 0.04 mg of purified cholesterol oxidase from C. gleum was added (0.67 U/mL). For the blank reaction water was used instead of enzyme solution. All reactions were prepared in triplicate. The reaction mixture was left shaking at 250 rpm at 30°C for 42 hours in 3 mL screw cap glass vials.
Analysis of cholesterol and cholest-4-en-3-one by HPLC-MS
The total reaction was extracted on 1 mL chloroform. After evaporation of the solvent at room temperature, the product was dissolved in the solvent, which was the same as the mobile phase used for HPLC. 10 μL of the analyte sample were injected into a Phenomenex Gemini® 5 μ C18 110 Å column (250 × 4.6 mm, 5 micron), and chromatography under isocratic conditions was performed using methanol:water 100:2 (v/v) at a flow rate of 0.8 mL/min at room temperature. Cholesterol and cholest-4-en-3-one were purchased from Sigma-Aldrich and used as reference. Product formation was monitored at 200 and 250 nm, whereas cholesterol was detected at 200 nm. The Agilent HPLC 1100 system equipped with a DAD was coupled to an esquireHCT ion trap mass spectrometer (Bruker. Germany), and an atmospheric pressure chemical ionization (APCI) source was operated in the positive ion mode. Conditions were as follows: scan range, m/z 50–600; dry gas flow of 11 L/min, nebulizer pressure 35 psi, drying gas temperature 320°C and the APCI heater temperature was 350°C. The extracted ion current (EIC) signals were deduced based on the exact masses for protonated cholesterol after water elimination (m/z 369.34) as well as for the protonated oxidation product cholest-4-en-3-one (m/z 385.34).