Cloning, expression and biochemicalcharacterization of the cholesterol oxidaseCgChoA fromChryseobacterium gleum
Open access
Datum
2014-05-21Typ
- Journal Article
ETH Bibliographie
yes
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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 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.
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. Mehr anzeigen
Persistenter Link
https://doi.org/10.3929/ethz-b-000087160Publikationsstatus
publishedExterne Links
Zeitschrift / Serie
BMC BiotechnologyBand
Seiten / Artikelnummer
Verlag
BioMed CentralThema
Chryseobacterium gleum; Cholesterol oxidase; Recombinant expression in; Escherichia coli; Biocatalysis; TaurocholateETH Bibliographie
yes
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