Semi-rational engineering of leucine dehydrogenase for enhanced L-tert-leucine production.

Abstract

Leucine dehydrogenase (LeuDH) is a promising enzyme for the industrial production of L-tert-leucine (L-Tle), but its catalytic activity toward trimethylpyruvate (TMP) requires enhancement. In this study, we employed a semi-rational design approach involving homology modeling of LeuDH from Exiguobacterium sibiricum (EsiLeuDH) and molecular docking with TMP to predict potential mutation sites. These sites were tested using an alanine scanning strategy to assess their impact on enzymatic activity, followed by site-saturation mutagenesis and iterative saturation mutagenesis. The resulting mutant, EsiLeuDH-M3, exhibited a remarkable 306 % increase in specific enzymatic activity (104.69 U·mg^-1), compared to the wild-type EsiLeuDH (WT). Molecular docking indicated that EsiLeuDH-M3 had an increased number of hydrogen bonds, improved stability, and an enlarged substrate-binding pocket. Moreover, molecular dynamics simulations suggested that EsiLeuDH-M3 possessed a more stable conformation but a more flexible pocket, allowing TMP to access the catalytic center more easily. Experiments examining the effects of different substrate concentrations on TMP bioconversion catalyzed by WT and EsiLeuDH-M3 indicated that EsiLeuDH-M3 tolerated higher TMP concentrations than the WT enzyme. Finally, L-Tle was produced using EsiLeuDH-M3 coupled with an NADH regeneration system, achieving a high conversion rate (91 %) of TMP at a substrate concentration of 0.7 M, which is expected to reduce production costs in the industrial application of L-Tle.