title: A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases.
abstract: Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.
authors.affiliation: BASF Aktiengesellschaft, Fine Chemicals and Biocatalysis Research, GVF/E - A030, D-67056 Ludwigshafen, Germany.
authors.forename: Tuck Seng
authors.initials: TS
authors.lastname: Wong
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
authors.forename: Rainer
authors.initials: R
authors.lastname: Stürmer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
authors.forename: Michael
authors.initials: M
authors.lastname: Breuer
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D019020:Directed Molecular Evolution; D006867:Hydrolases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D008049:Lipase / Q000378:metabolism; D010088:Oxidoreductases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
chemical_list: D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase; D006867:Hydrolases; D008049:Lipase