Cloning, expression and biochemical characterization of the cholesterol oxidase CgChoA from Chryseobacterium gleum

Abstract

Background: 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.

Results: 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 K(m) = 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.

Conclusion: 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.

Figure 1

Reaction scheme for the oxidation of cholesterol catalyzed by CgChoA.

Figure 2

Phylogenetic tree of CgChoA and selected characterized cholesterol oxidases. The unrooted tree was based on the average distance PID and reports the percentage of identity between each protein sequence vs. the consensus sequence generated from the alignment of all the protein sequences considered. In addition to the enzyme CgChoA (subject of this study), the cholesterol oxidases considered are indicated by their PDB identifier and are the (1COY, cofactor non-covalently bound) and the (2I0K, cofactor covalently bound) flavoproteins from Brevibacterium sterolicum, the flavoprotein from Streptomyces sp. SA-COO (1B4V, cofactor non-covalently bound), and the flavoprotein from Chromobacterium sp. DS-1 (3JS8 cofactor covalently bound). The tree was produced with the software Jalview ( http://www.jalview.org).

Figure 3

UV–vis absorbance spectrum of purified CgChoA. The typical absorption spectra for FAD was obtained (peaks at 370 nm and 470 nm; dashed line: 10 fold magnification) [17]. Inset: SDS-PAGE before and after purification of CgChoA. Lane 1: cell free extract; lane 2: protein after IMAC purification; lane 3: purified sample after SEC. The band corresponding to CgChoA is indicated by the arrow.

Figure 4

Data obtained from a pH and buffer screen using partially purified cholesterol oxidase from C. gleum. (A) pH and buffer screen using partially purified cholesterol oxidase from C. gleum at 25°C. The effect of pH and buffer composition upon initial rates of cholesterol oxidation using the HRP coupled cholesterol oxidation assay with ABTS as electron donor was investigated. All data points represent mean values ± SEM from triplicate determinations. 100% corresponds to 3.8 U/L. (A) pH and buffer screen at 25°C. (B) MOPS buffer screen using partially purified cholesterol oxidase from C. gleum at 25°C The effect of pH and molarity upon initial rates of cholesterol oxidation using the HRP coupled cholesterol oxidation assay with ABTS as electron donor was investigated. All data points represent mean values ± SEM from triplicate determinations. 100% corresponds to 29.6 U/L. (C) Temperature dependency study of cholesterol oxidase activity from C. gleum in 0.11 M MOPS buffer, pH 6.75. The oxidation of cholesterol as a function of temperature is given relative to the highest activity recorded (47.0 U/L) that was taken as 100%. All data points represent mean values ± SEM from triplicate determinations.

Figure 5

Kinetic plot obtained with recombinant CgChoA from C. gleum. The specific activity (U/mg) was plotted versus the substrate concentration. An enzyme concentration of 3.57 mg/L was used and the cholesterol stock solution/dispersion was prepared and serially diluted in (●) water only, (▲) water and taurocholate, (♦) water containing 5% Triton X-100 and taurocholate, or in (■) water containing 5% (v/v) Triton X-100.

Figure 6

Chromatograms after subjecting the extracted samples to HPLC-MS measurements: cholesterol (1) and oxidation product cholest-4-en-3-one (3). Shown profiles are extracted at 200 and 250 nm from the DAD-signal for the reaction with CgChoA (A and B, respectively) and without enzyme (C and D, respectively); m/z signals are indicated.