2026/01/01 by Bernard D. G. Eenink, Josephin M. Holstein, Magdalena Heberlein +7 · 1 voice
Chemistry · Engineering · Medicine · #Alkaline Phosphatase Research Studies #Chemical Reaction Mechanisms #Innovative Microfluidic and Catalytic Techniques Innovation
paper · pdf · doi:10.1039/d5an00865d
openalex publication_date 2026/01/01 · openalex created_date 2026/02/07 · openalex updated_date 2026/07/22
enzymes with desired and effective functions. Most proteins organize in families, sets of divergent sequences which share a common ancestor and have a similar structural fold. Here, we study aryl sulfatases, a subfamily of the large and evolutionary old alkaline phosphatase superfamily. We demonstrate how ultrahigh-throughput droplet microfluidics can be used for studying aryl sulfatases and their computationally reconstructed putative common ancestors. We compare the evolvability and robustness of three ancestors and three extant aryl sulfatases which all exhibit catalytic promiscuity towards a range of substrate classes. Using varying mutations rates, eleven libraries were constructed and expressed in single-cell microdroplets. In general, higher mutation rates resulted in wider distribution of active variants but fewer improved variants overall. However, the impact of mutation rate differed between enzymes, with some benefiting from higher and others from lower mutation rate, underscoring the need to test diverse mutagenesis regimes.