A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases.
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.
Wong
Tuck Seng
TS
BASF Aktiengesellschaft, Fine Chemicals and Biocatalysis Research, GVF/E - A030, D-67056 Ludwigshafen, Germany.
Schwaneberg
Ulrich
U
Stürmer
Rainer
R
Hauer
Bernhard
B
Breuer
Michael
M
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
D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase; D006867:Hydrolases; D008049:Lipase