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We recently reported that the transketolase from Geobacillus stearothermophilus (TKgst) upon acyl transfer to nitrosoarenes generates N‐aryl hydroxamic acids (HA). The latter are metal chelating compounds that in the presence of Fe(III) ions form deep‐red complexes. Here, we applied this principle to the development of a colorimetric assay in both solid‐ and liquid‐phase formats for the high‐throughput screening of TKgst and its variants. Screening a set of positive hits from a L382X/D470X library validated the specificity and sensitivity of the assays. The solid surface assay allows a clear distinction between positive and negative colonies by the naked eye in qualitative mode, and further also to measure activity in semi‐quantitative fashion in the liquid‐phase format. The assay will be important for engineering the TKgst enzyme towards improved conversion of aromatic aldehydes by their close structural analogy to nitrosoarenes.

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