This review summarizes how ultrahigh-throughput screening methods employ cells and biomimetic compartments to access the vast, unexplored diversity of biocatalysts with novel functions derived from directed evolution and metagenomics libraries.
Incorporating artificial metal‐cofactors into protein scaffolds results in a new class of catalysts, termed biohybrid catalysts or artificial metalloenzymes. Biohybrid catalysts can be modified chemically at the first coordination sphere of the metal complex, as well as at the second coordination sphere provided by the protein scaffold. Protein‐scaffold reengineering by directed evolution exploits the full power of nature's diversity, but requires validated screening and sophisticated metal cofactor conjugation to evolve biohybrid catalysts. In this Minireview, we summarize the recent efforts in this field to establish high‐throughput screening methods for biohybrid catalysts and we show how non‐chiral catalysts catalyze reactions enantioselectively by highlighting the first successes in this emerging field. Furthermore, we shed light on the potential of this field and challenges that need to be overcome to advance from biohybrid catalysts to true artificial metalloenzymes.
Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However,n ative enzymes often have lowa ctivity and resistance in OSs and at elevated temperatures.H erein, we report as mart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme,B acillus subtilits Lipase A( BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems.I terative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance towardt hree OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and halflife up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50-100 8 8C. The salt bridge redesign provides protein engineers with ap owerful and likely general approach to design OSsand/or thermal-resistant lipases and other a/b-hydrolases.
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