Accelerating Enzyme Evolution with Computational Design: A Case Study on D-lactonohydrolase

Computational design can significantly speed up enzyme evolution for biocatalysis. This study used D-lactonohydrolase as a case study to demonstrate the reliability of AlphaFold2 for enzyme evolution. A novel smart combination strategy, CSFp, was implemented to enhance enzyme activity. The resulting mutant, N96S/A271E/F274Y/F308G, showed a remarkable 56-fold increase in activity with 99% enantioselectivity towards DL-pantolactone. This approach holds promise for improving enzyme activity for proteins with unknown structures or obscure catalytic mechanisms, enabling wider applications in protein engineering.