The enormous screening effort is a bottleneck in the directed evolution of enzymes Computational design has the potential to accelerate the use of enzymes in biocatalysis faster and more powerfully He

The screening process in directed enzyme evolution is a significant bottleneck. However, computational design has the potential to accelerate the use of enzymes in biocatalysis in a faster and more efficient manner. As a case study, the calcium-dependent enzyme D-lactonohydrolase, which lacks a crystal structure, was utilized to demonstrate the reliability of the structure predicted by AlphaFold2 as a starting point for enzyme evolution. To enhance the catalytic efficiency of the enzyme, a novel, intelligent combination strategy was devised, known as the CSFp strategy, which integrated Steered Molecular Dynamics (SMD), Functional Library (FuncLib), Protein Strain, Unsatisfactoriness, and Frustration findER (pSUFER). The most promising mutant, N96S, exhibited a remarkable 56-fold increase in activity towards DL-pantolactone compared to the wild-type, with an enantioselectivity of 99%. This approach has the potential to rescue enzymes that lack crystal structure information and have an obscure catalytic mechanism, facilitating their application in protein engineering where improved enzyme activity is required