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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 (kcat/KM) was nearly 23?fold higher than that of the wild?type. The M3 whole?cell converted 20% of DL?pantolactone into D?pantoic acid (D?PA, >99% e.e.) with a conversion rate of 47% and space?time yield of 107.1 g L?1 h?1, demonstrating its great potential for industrial?scale D?pantothenic acid production. Molecular dynamics (MD) simulations revealed that the reduction in the steric hindrance within the substrate tunnel and conformational reconstruction of the distal loop resulted in a more favourable"catalytic" conformation, making it easier for the substrate and enzyme to enter their pre?reaction state. This study illustrates the potential of the distal residue on the pivotal loop at the entrance of the D?Lac substrate tunnel as a novel modification hotspot capable of reshaping energy patterns and consequently influencing the enzymatic activity.