title: Real-Time Monitoring of Enzyme-Catalysed Reactions using Deep UV Resonance Raman Spectroscopy.
abstract: For enzyme-catalysed biotransformations, continuous in situ detection methods minimise the need for sample manipulation, ultimately leading to more accurate real-time kinetic determinations of substrate(s) and product(s). We have established for the first time an on-line, real-time quantitative approach to monitor simultaneously multiple biotransformations based on UV resonance Raman (UVRR) spectroscopy. To exemplify the generality and versatility of this approach, multiple substrates and enzyme systems were used involving nitrile hydratase (NHase) and xanthine oxidase (XO), both of which are of industrial and biological significance, and incorporate multistep enzymatic conversions. Multivariate data analysis of the UVRR spectra, involving multivariate curve resolution-alternating least squares (MCR-ALS), was employed to effect absolute quantification of substrate(s) and product(s); repeated benchmarking of UVRR combined with MCR-ALS by HPLC confirmed excellent reproducibility.
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess street, Manchester, M1 7DN, UK.
authors.forename: Chloe
authors.initials: C
authors.lastname: Westley
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess street, Manchester, M1 7DN, UK.
authors.forename: Heidi
authors.initials: H
authors.lastname: Fisk
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess street, Manchester, M1 7DN, UK.
authors.forename: Yun
authors.identifier: http://orcid.org/0000-0003-3228-5111
authors.initials: Y
authors.lastname: Xu
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess street, Manchester, M1 7DN, UK.
authors.forename: Katherine A
authors.identifier: http://orcid.org/0000-0002-7028-047X
authors.initials: KA
authors.lastname: Hollywood
authors.affiliation: Department of Chemistry, University of Liverpool, Liverpool, L69 7ZD, UK.
authors.forename: Andrew J
authors.identifier: http://orcid.org/0000-0002-6353-403X
authors.initials: AJ
authors.lastname: Carnell
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess street, Manchester, M1 7DN, UK.
authors.forename: Jason
authors.identifier: http://orcid.org/0000-0001-8951-4873
authors.initials: J
authors.lastname: Micklefield
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess street, Manchester, M1 7DN, UK.
authors.forename: Nicholas J
authors.identifier: http://orcid.org/0000-0002-8708-0781
authors.initials: NJ
authors.lastname: Turner
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess street, Manchester, M1 7DN, UK.
authors.forename: Royston
authors.identifier: http://orcid.org/0000-0003-2230-645X
authors.initials: R
authors.lastname: Goodacre
mesh_terms: D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D006836:Hydro-Lyases / Q000378:metabolism*; D016018:Least-Squares Analysis; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity; D014969:Xanthine Oxidase / Q000378:metabolism*
chemical_list: D014969:Xanthine Oxidase; D006836:Hydro-Lyases; C048831:nitrile hydratase
keywords: UV resonance Raman (UVRR) spectroscopy; biotransformations; on-line monitoring; quantification; reaction monitoring