title: 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.
  
  
  CONCLUSIONS
  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.
authors.forename: Renate
authors.initials: R
authors.lastname: Reiss
authors.forename: Greta
authors.initials: G
authors.lastname: Faccio
authors.forename: Linda
authors.initials: L
authors.lastname: Thöny-Meyer
authors.affiliation: Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials, Lerchenfeldstr, 5, 9014 St, Gallen, Switzerland. michael.richter@empa.ch.
authors.forename: Michael
authors.initials: M
authors.lastname: Richter
mesh_terms: D055162:Biocatalysis; D002784:Cholesterol / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000235:genetics / Q000378:metabolism*; D045247:Chryseobacterium / Q000145:classification / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002784:Cholesterol; D002789:Cholesterol Oxidase