title: Novel technique for high throughput measurement of active monooxygenase concentration.
abstract: Absorbance measurements via transmitting light spectroscopy in microtiter plates are established for high throughput screening of biological systems. These measurements allow for the determination of important process parameters within a short time. However, absorbance determination via transmitted light measurements is not always feasible. As for carbon monoxide difference absorbance spectroscopy, used for concentration measurements of active P450 monooxygenases (P450s), security standards, and consistent gassing have to be addressed. In this study, a non-invasive online measuring principle for absorbance via scattered light is proposed. Based on optical fiber measurements, a decrease in scattered light signals at 450 nm wavelength of reflecting polymer particles is observed, and P450 concentrations are calculated. In this way, high throughput determination of P450 concentrations in a secure, gas-tight environment is realized. The designed method was successfully applied to concentration measurements and carbon monoxide (CO) saturation kinetics ranging from 0.3 to 5.0 μM P450 BM3 achieving a measurement accuracy of ±0.05 μM P450. Biotechnol. Bioeng. 2017;114: 929-933. © 2016 Wiley Periodicals, Inc.
authors.affiliation: AVT.Biochemical Engineering, Bioeconomy Science Center (BioSC), RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Welters
authors.affiliation: Lehrstuhl für Biotechnologie, Bioeconomy Science Center (BioSC), RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Horn
authors.affiliation: Lehrstuhl für Biotechnologie, Bioeconomy Science Center (BioSC), RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Anna Joelle
authors.initials: AJ
authors.lastname: Ruff
authors.affiliation: Lehrstuhl für Biotechnologie, Bioeconomy Science Center (BioSC), RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
authors.affiliation: AVT.Biochemical Engineering, Bioeconomy Science Center (BioSC), RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs
mesh_terms: D002248:Carbon Monoxide / Q000032:analysis / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004867:Equipment Design; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System
keywords: P450 monooxygenase concentration; absorbance measurement; high-throughput screening; online measurement; scattered light