title: Molecularly "wired" cholesterol oxidase for biosensing.
abstract: The influence of several factors on the activity of cholesterol oxidase (ChOx) transiently exposed to a room temperature ionic liquid (RTIL) was studied. Presence of flavin adenine dinucleotide (FAD, prosthetic group of ChOx) during exposure to RTIL makes the procedure enzyme-friendly, while the use of RTIL (green reagent) makes it environmentally-friendly. Following exposure to RTIL and its subsequent removal, FAD becomes part of the molecular structure of the refolded protein (a molecular "wire"). This makes the procedure used here a molecular one. The factors studied were: FAD presence in RTIL during modification, water presence during exposure to RTIL, and ratio FAD:RTIL during "wiring". Performance parameters monitored were: enzyme activity before and after "wiring" (expressed as (dA/dt)/mg enzyme, and measured spectrophotometrically), peak current in an amperometric biosensor for cholesterol detection, and linearity of the biosensor response depending on cholesterol concentration. After RTIL removal, the modified enzyme (ME) retained a high percentage of the added FAD, which supplemented that of the native enzyme (functioning as a "wire" and enhancing electron transfer kinetics), and a fraction of the initial activity. Used in an amperometric biosensor, ME showed catalytic activity, linear behavior as a function of cholesterol concentration, and stability.
authors.affiliation: Fairleigh Dickinson University, Metropolitan Campus, 1000 River Rd., Teaneck, NJ, 07666, USA, mleonida@fdu.edu.
authors.forename: Mihaela D
authors.initials: MD
authors.lastname: Leonida
authors.forename: Benedict
authors.initials: B
authors.lastname: Aurian-Blajeni
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D001951:Brevibacterium / Q000201:enzymology*; D002384:Catalysis; D002784:Cholesterol / Q000032:analysis*; D002789:Cholesterol Oxidase / Q000737:chemistry*; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D005182:Flavin-Adenine Dinucleotide; D002784:Cholesterol; D002789:Cholesterol Oxidase