title: Fluorescent assay for directed evolution of perhydrolases.
abstract: Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the µM range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4'-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5-9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased k(cat) for perhydrolytic activity compared with wild type.
authors.affiliation: RWTH Aachen University, Worringerweg, Aachen, Germany.
authors.forename: Dragana
authors.initials: D
authors.lastname: Despotovic
authors.forename: Ljubica
authors.initials: L
authors.lastname: Vojcic
authors.forename: Radivoje
authors.initials: R
authors.lastname: Prodanovic
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Maurer
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D001965:Bromides / Q000737:chemistry; D019020:Directed Molecular Evolution / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D008958:Models, Molecular; D016296:Mutagenesis; D010463:Peracetic Acid / Q000032:analysis / Q000378:metabolism; D010545:Peroxides / Q000032:analysis / Q000378:metabolism; D017670:Sodium Compounds / Q000737:chemistry; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism*; D014468:Umbelliferones / Q000378:metabolism
chemical_list: D001965:Bromides; D005456:Fluorescent Dyes; D010545:Peroxides; D017670:Sodium Compounds; D014468:Umbelliferones; D013381:Subtilisins; D010463:Peracetic Acid; C027938:sodium bromide