Abstract

The thermostability of lipases represents a key advantage for expanding their application potential in the food industry. In this study, a thermotolerant lipase, rTaLip, was identified from Baiju Daqu and successfully expressed in Komagataella phaffii. After determining its enzymatic properties, we developed two thermostable mutants through rational engineering (V219C/D222C/S73F/V141T and V219C/D222C/S73F/V141D). Compared to the wild-type, the mutants showed 2.2-fold and 4.1-fold longer half-lives at 65 °C, and their catalytic efficiency increased to 455% and 119%, respectively. Molecular dynamics simulations elucidated the structural basis underlying the increased thermal stability and catalytic activity. In application tests, the mutant V219C/D222C/S73F/V141T improved palm oil hydrolysis efficiency from 63.01% to 88.77%. This study demonstrates that combining the mining of thermostable lipases from Baijiu Daqu with rational design-based engineering constitutes a valuable strategy for improving the industrial applicability of lipases in harsh processing environments.