Critical Salt Bridges Govern the High Thermostability of a meso-2,3-butanediol Dehydrogenase with Resistance Up to 100 °C.

Chiral α-hydroxy ketones and vicinal diols are valuable intermediates in pharmaceuticals and fine chemicals. 2,3-Butanediol dehydrogenase (2,3-BDH) is important for their enantioselective synthesis, but its low thermostability limits industrial use. Here, a thermostable meso-BDH from Lactococcus lactis (LlBDH) was identified using Klebsiella pneumoniae BDH (KpBDH) as the query, retaining 30.5% activity after 10 min at 100 °C. Molecular dynamics (MD) simulations revealed three unique salt bridges (K20-E45, K28-E49, R123-E170) that enhance LlBDH thermostability, and disrupting them reduced stability. Strengthening the K28-E49 interaction by incorporating p-isothiocyanate phenylalanine (pNCSF) further improved the LlBDH stability. Introducing the three salt bridges into KpBDH generated a stabilized quadruple mutant (L20K/Q48E/A28K/R52E), which exhibited a 2.0 °C increase in the melting temperature, a 4.3-fold longer half-life at 40 °C, and 1.7-fold higher product formation at 50 °C. MD simulations confirmed that the engineered salt bridges rigidified local regions. These results provide a rational strategy for designing robust biocatalysts.