Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical industries. To achieve practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion-based dual fluorescence assay (SDFA) for high-throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescence protein (MsfGFP), which could result in secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure activity of the ADH based on a red fluorescence signal, whose increase is coupled to formation of NADH (a redox co-factor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197-fold higher kcat/km value toward (S)-2-octanol compared to its wild-type.
