Abstract
LeuDH catalyzes the reversible deamination of L-leucine and certain branched-chain aliphatic amino acids, resulting in the corresponding α-keto acids. This process has received considerable attention in the food and pharmaceutical industries. Although LeuDH from Planifilum fimeticola shows higher catalytic efficiency, its lack of thermostability limits its widespread use in industrial applications. This study identified a mutant, S220P/T347K, with enhanced thermostability through rational design. The experimental results show that under optimal temperature conditions of 65 °C, the half-life (t1/2) of the mutant is 2.94 times longer than that of the wild type, and it does not affect the catalytic activity. Molecular dynamics and 3D structural analysis revealed that the S220P/T347K mutant's increased thermostability mainly results from decreased flexibility, enhanced hydrophobicity, and a favorable electrostatic potential. Furthermore, the mutant's increased catalytic efficiency and specific activity may result from its tighter binding with small molecule substrates. This study is the first to use rational design to modify PfleuDH, effectively enhancing its thermostability and catalytic activity. It provides a simple and effective method for strengthening LeuDH in industrial applications.
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