title: Label-Free Surface Enhanced Raman Scattering Approach for High-Throughput Screening of Biocatalysts.
abstract: Biocatalyst discovery and directed evolution are central to many pharmaceutical research programs, yet the lack of robust high-throughput screening methods for large libraries of enzyme variants generated (typically 10(6)-10(8)) has hampered progress and slowed enzyme optimization. We have developed a label-free generally applicable approach based on Raman spectroscopy which results in significant reductions in acquisition times (>30-fold). Surface enhanced Raman scattering (SERS) is employed to monitor the enzyme-catalyzed conversion by xanthine oxidase of hypoxanthine to xanthine to uric acid. This approach measures the substrates and products directly and does not require chromogenic substrates or lengthy chromatography, was successfully benchmarked against HPLC, and shows high levels of accuracy and reproducibility. Furthermore, we demonstrate that this SERS approach has utility in monitoring enzyme inhibition illustrating additional medical significance to this high-throughput screening method.
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: Chloe
authors.initials: C
authors.lastname: Westley
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Chemistry, University of Liverpool , Liverpool L69 7ZD, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Carnell
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Turner
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: Royston
authors.initials: R
authors.lastname: Goodacre
mesh_terms: D055162:Biocatalysis; D057166:High-Throughput Screening Assays; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D013059:Spectrum Analysis, Raman; D013499:Surface Properties; D014527:Uric Acid / Q000737:chemistry / Q000378:metabolism*; D019820:Xanthine / Q000737:chemistry / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism*
chemical_list: D019820:Xanthine; D014527:Uric Acid; D019271:Hypoxanthine; D014969:Xanthine Oxidase