Cloning, expression and biochemical characterization of the cholesterol oxidase CgChoA from Chryseobacterium gleum
© Reiss et al.; licensee BioMed Central Ltd. 2014
Received: 7 June 2013
Accepted: 25 March 2014
Published: 21 May 2014
Cholesterol oxidases are important enzymes for applications such as the analysis of cholesterol in clinical samples, the synthesis of steroid derived drugs, and are considered as potential antibacterial drug targets.
The gene choA encoding a cholesterol oxidase from Chryseobacterium gleum DSM 16776 was cloned into the pQE-30 expression vector and heterologously expressed in Escherichia coli JM109 co-transformed with pRARE2. The N-terminally His-tagged cholesterol oxidase (CgChoA) was assigned to be a monomer in solution by size exclusion chromatography, showed a temperature optimum of 35°C, and a pH optimum at 6.75 using 0.011 M MOPS buffer under the tested conditions. The purified protein showed a maximum activity of 15.5 U/mg. CgChoA showed a Michaelis-Menten like kinetic behavior only when the substrate was dissolved in water and taurocholate (apparent Km = 0.5 mM). In addition, the conversion of cholesterol by CgChoA was studied via biocatalytic batches at analytical scale, and cholest-4-en-3-one was confirmed as product by HPLC-MS.
CgChoA is a true cholesterol oxidase which activity ranges among the high performing described cholesterol oxidases from other organisms. Thus, the enzyme broadens the available toolbox of cholesterol oxidases for e.g. synthetic and biosensing applications.
KeywordsChryseobacterium gleum Cholesterol oxidase Recombinant expression in Escherichia coli Biocatalysis Taurocholate
It has been reported that in biotransformation reactions whole cells of Chryseobacterium gleum were successfully used for the biotransformation of cholesterol to androsta-1,4-diene-3,17-dione, which is a precursor of antifertility drugs (e.g. estrogens), androgens and the diuretic drug spironolactone [14, 15]. This strain might therefore be an ideal candidate for strain engineering in order to optimize such biotransformation approaches. In this study, a novel cholesterol oxidase from Chryseobacterium gleum DSM 16776 (CgChoA) was cloned, expressed in E. coli, purified, and biochemically characterized. Moreover, we confirm that enzymatic reactions with purified CgChoA and cholesterol as substrate yields cholest-4-en-3-one as reaction product by HPLC-MS analysis. The isolated enzyme might thus be useful in fields focused on the biosensing of cholesterol.
In silicoamino acid analysis of ChoA variants
For the identification of a novel bacterial cholesterol oxidase, a Protein Blast search was performed using the cholesterol oxidase amino acid sequence from Streptomyces sp. (UniProt accession number P12676; PDB code 2GEW) as template. Protein sequences of ChoA were retrieved from public databases, aligned using the ClustalW algorithm of the MegAlign software (LASERGENE, Madison, USA), and analyzed in order to identify conserved residues possibly important for the catalytic activity. Out of numerous homologues, the gene choA encoding a hypothetical protein (CgChoA) annotated as cholesterol oxidase was found in the fully sequenced genome of Chryseobacterium gleum ATCC 35910 (DSM 16776; UniProt accession number D7VYA1). The gene was selected for cloning and recombinant expression in E. coli.
The cDNA sequence encoding CgChoA was cloned into the expression vector pQE-30 such that the final construct pCgChoA coded for an N-terminal His-tag MRGSHHHHHHGSAC fused to CgChoA. The wild-type CgChoA amino acid sequence (without the His-tag) of C. gleum DSM 16776 (UniProt D7VYA1, 528 aa) showed 46.1% identity to that from Streptomyces sp. (PDB code 2GEW, 540 aa), 42.8% identity to that from B. sterolicum (UniProt P22637), 16.1% to that from Mycobacterium tuberculosis (PDB code 2XKR, 398 aa) and 14.1% to that from Chromobacterium sp. (PDB code 3JS8, 587 aa). The CgChoA cholesterol oxidase with the N-terminal His-tag consists of 541 amino acids and has a hypothetical molecular mass of 60.4 kDa.
Expression of cholesterol oxidase from C. gleum choA in E. coli
The gene choA from C. gleum DSM 16776 contains 8% rare codons with respect to the codon usage of E. coli. Therefore, the expression host E. coli JM109 was additionally transformed with the pRARE2 plasmid, which encodes extra copies of genes coding for tRNAs recognizing the codons AGG, AGA, AUA, CUA, CCC, GGA and CGG. E. coli JM109 cells producing CgChoA in the absence of pRARE2 showed only low activity. In the presence of pRARE2, the choA gene was expressed at 30°C, but the protein was found in inclusion bodies. Activity could only be detected in the insoluble fractions. Only when the cultivation temperature was decreased to 16°C immediately after induction, soluble and active protein was present.
Protein purification and characterization
Enzymatic conversion of cholesterol to cholest-4-en-3-one
Searching for novel cholesterol oxidases is of great interest in fields such as biosensing and enzymatic synthesis. The oxidation of cholesterol to cholest-4-en-3-one has been reported for cholesterol oxidase from whole cells of C. gleum, Bacillus subtilis and Streptomyces sp. [13, 14, 19]. Especially those enzymes with considerable low amino acid homology to already described ones may have novel optimal working conditions and thus be suitable for innovative applications.
Comparison of different aspects concerning the production and selected properties of cholesterol oxidases
Source (UniProt ID)
Specific activity on cholesterol (U/mg)
Brevibacterium sterolicum (Q7SID9)
pET24b(+), E. coli BL21(DE3) pLysS, TB + 20 mL/L glycerol, 25/ 30°C
Potassium phosphate buffer pH 7.5 at 25°C (propan-2-ol and Thesit) monitoring H2O2 production using HRP and o-dianisidine
7 (crude extract)
Brevibacterium sterolicum nov. sp. ATCC21387
pUC19, E. coli MM294, LB, 30°C
Ammonium sulfate precipitation, DEAE-cellulose, Superose, hydroxyapatite
Sodium phosphate buffer pH 7.0, 37°C (Triton X-100), monitoring H2O2 production coupled to 4-aminoantipyrine and phenol via HRP
Brevibacterium sterolicum nov. sp. ATCC21387 (Q2I2N2)
pET28a(+), E. coli BL21(DE3), LB, 28°C
Affinity chromato-graphy, (riboflavin bound to, Sepharose 4B)
Quantifying H2O2 by coupling to HRP reaction with aminopyrine, 37°C
(3.7, with His tag)
Chromobacterium sp. DS-1 (B5MGF)
pET-21d(+), E. coli Rosetta, LB, 30°C
Heat purification at 70°C for 30 min, DEAE-cellulose DE52
Sodium potassium phosphate buffer pH7.0, 30°C (sodium cholate, Triton X-100), 4-aminoantipyrine, phenol, HRP
Chryseobacterium gleum (D7VYA1)
pQE30 (pRARE2), E. coli JM109, modified M9, 16°C
HisTrap FF (IMAC), SEC on Superdex 200 pg
HPLC assay and coupled enzyme assay MOPS buffer, pH 6.75, 37°C, HRP and ABTS (Triton X-100 and taurocholic acid)
Streptomyces sp. SA-COO (P12676)
pUC19, E. coli BL21(DE3)plysS, 2YT, 28°C
Butyl-Sepharose column chromatography, DEAE-cellulose column chromatography
Formation rate of H2O2 was monitored in a coupled assay with HRP and ABTS, 37°C (Triton X-100, BSA)
The recombinant CgChoA was active between pH 4–8 with optimal activity in the neutral range similarly to other cholesterol oxidases (Table 1), e.g. at pH 6.75 using 0.011 M MOPS buffer for the coupled HRP assay. At higher concentrations of MOPS, the activity declined steadily at any of the 6 pH values measured. MOPS buffer with a pH lower than 6.75 has not been tested as it buffers only between 6.5 and 8. A temperature optimum between 32°C and 40°C was found, which is in the range of the cholesterol oxidase from Corynebacterium cholesterolicum, but lower than that of Streptomyces violascens or Brevibacterium sp. enzymes, which showed optimum activity at around 50°C . The activity data obtained when the substrate was dissolved in the presence of Triton X-100 and/or water only could not be fitted to the Michaelis-Menten equation, which is only applicable for enzymatic reactions in homogeneous solutions and therefore cannot be directly adapted to the heterogeneous reaction conditions that were applied here. The presence of the anionic surfactant taurocholate proved to affect the measured activity and an apparent Km of 0.5 mM was obtained for CgChoA and the substrate cholesterol. We cannot exclude for taurocholate an effect not only regarding an improved substrate solubilisation, and thus enhanced accessibility to the enzyme, but also an effect on the enzyme itself. In summary, the anionic surfactant taurocholate is sufficient as additive for monitoring the enzyme activity of CgChoA with regard to the natural substrate cholesterol, while the presence of the non-ionic additive Triton X-100 did not affect the general kinetic behaviour. These data may be of special interest for developing biosensors for samples with at low cholesterol content as dilution in the presence of taurocholate might provide a linear correlation between the substrate concentration and the signal measured.
The cholesterol oxidase CgChoA from C. gleum was successfully expressed in E. coli JM109 co-transformed with pCgChoA and pRARE2. The CgChoA carrying an N-terminal His-tag was purified and subjected to a pH and temperature screen. The highest specific activity was determined to be 15.5 U/mg. Michaelis-Menten type kinetics could only be observed in the presence of taurocholate as single surfactant within the enzymatic assay. The CgChoA cholesterol oxidation product was identified as cholest-4-en-3-one by direct and rapid detection via HPLC-MS. The rapid and robust HPLC-MS assay developed in this study enables a more detailed study of CgChoA and other cholesterol oxidases. The described enzyme complements the set of available cholesterol oxidases for diverse applications such as bionsensing and synthesis of intermediates for drug synthesis. As successful biotransformation employing C. gleum as host organism has already been demonstrated , the future engineering of CgChoA for a broader substrate specificity might enable the application of this enzyme for the conversion of other steroid compounds.
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).
2,2'-Azino-bis 3-ethylbenzothiazoline-6-sulphonic acid
Atmospheric pressure chemical ionization
Cholesterol oxidase from Chryseobacterium gleum
We thank Thomas Ramsauer, Flavia Zuber and Nadja Schulthess for technical assistance. This project was evaluated by the Swiss National Foundation and funded by Nano-Tera.ch with Swiss Confederation financing.
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