Enhancing the Catalytic Efficiency of D‐lactonohydrolase through the Synergy of Tunnel Engineering, Evolutionary Analysis, and Force‐Field Calculations

Computational design advances enzyme evolution and their use in biocatalysis in a faster and more efficient manner. In this study, a synergistic approach integrating tunnel engineering, evolutionary analysis, and force-field calculations has been employed to enhance the catalytic activity of D-lactonohydrolase (D-Lac), which is a pivotal enzyme involved in the resolution of racemic pantolactone during the production of vitamin B5. The best mutant, N96S/A271E/F274Y/F308G (M3), was obtained and its catalytic efficiency (k