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Real‐Time Monitoring of Enzyme‐Catalysed Reactions using Deep UV Resonance Raman Spectroscopy
Author(s) -
Westley Chloe,
Fisk Heidi,
Xu Yun,
Hollywood Katherine A.,
Carnell Andrew J.,
Micklefield Jason,
Turner Nicholas J.,
Goodacre Royston
Publication year - 2017
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201701388
Subject(s) - substrate (aquarium) , chemistry , raman spectroscopy , resonance raman spectroscopy , spectroscopy , analytical chemistry (journal) , reproducibility , combinatorial chemistry , chromatography , optics , oceanography , physics , quantum mechanics , geology
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.