Characterization and Molecular Engineering of a Thermostable Amino Acid Dehydrogenase for Enhanced ( S )‐2‐Amino‐1‐Butanol Synthesis

( S )‐2‐Amino‐1‐butanol, a key intermediate for ethambutol hydrochloride synthesis, is currently produced via heavy metal‐catalyzed processes or enzymatic resolution with limited theoretical yields. While amine dehydrogenases (AmDHs) offer sustainable alternatives for asymmetric reductive amination, their applicability is often restricted by narrow substrate scope. Here, we report the discovery of a thermostable amino acid dehydrogenase from Thermoactinomyces daqus ( Td AADH) that exhibits optimal activity at pH 9.0 and 55 °C with exceptional thermostability ( t _1/2  = 21 days at 40 °C). Although inactive toward the non‐native substrate 1‐hydroxy‐2‐butanone, structure‐guided engineering via site‐directed and iterative saturation mutagenesis (ISM) successfully endowed the enzyme with desired functionality: the initial double mutant K93S/N288L (M1) acquired de novo amination activity, while subsequent evolution to the quintuple mutant M4 (incorporating L136T/T139D/M141R) enhanced specific activity by 2.75‐fold (5.38 U·g ⁻¹ ) without compromising enantioselectivity ( ee  > 99%). Mechanistic investigations via molecular docking suggest that these mutations plausibly reshape the substrate‐binding pocket, alleviate steric hindrance, and optimize electrostatic interactions to accommodate the hydroxyketone substrate. Preparative‐scale biotransformation (40 °C, 30 mM substrate, coupled with GDH/glucose for cofactor recycling) achieved quantitative conversion (≥99%) within 36 h. This work expands the enzymatic toolbox for thermostable AmDHs.