Critical Salt Bridges Govern the High Thermostability of a <i>meso</i> -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 <i>meso</i>-BDH from <i>Lactococcus lactis</i> (<i>Ll</i>BDH) was identified using <i>Klebsiella pneumoniae</i> BDH (<i>Kp</i>BDH) 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 <i>Ll</i>BDH thermostability, and disrupting them reduced stability. Strengthening the K28-E49 interaction by incorporating <i>p</i>-isothiocyanate phenylalanine (pNCSF) further improved the <i>Ll</i>BDH stability. Introducing the three salt bridges into <i>Kp</i>BDH 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.