Cooperative mutations reshape lid dynamics in thermophilic lipase LipT6 enhancing stability and biodiesel production.

Abstract

Enzymatic biodiesel production offers a sustainable alternative to chemical transesterification, but industrial application of lipases is limited by acidic pH and high alcohol concentrations. Here, we combined previously identified stabilizing mutations in the thermophilic lipase LipT6 from Geobacillus stearothermophilus T6 to generate the S5 variant (H86Y/E251C/A269T/G332C/R374W) and investigated the molecular basis of its improved performance. S5 retained 94% of its activity after 1h at pH 4.8, compared with 33% for wild-type LipT6, and increased conversion of waste chicken oil to fatty acid methyl esters from 30.4% to 71.3%. The variant also showed increased thermal stability under neutral, acidic, and ethanol-rich conditions. Its determined 2.4Å crystal structure closely matched the wild-type closed-lid conformation, with a Cα RMSD of 0.38Å, indicating that enhanced function did not result from major structural rearrangements. Molecular dynamics simulations revealed conformation-dependent effects: wild-type and S5 behaved similarly in the closed state, whereas S5 exhibited greater lid flexibility and broader conformational sampling in the open state. Back-to-wild-type reversions in silico produced distinct shifts in lid sampling, supporting a cooperative contribution of the engineered mutations. Structural analysis further suggested that H86Y alters the environment of the Zn²⁺-binding region and adjacent lid hinge, while core mutations provide complementary stabilization. These findings demonstrate that cooperative engineering of core stability and lid dynamics can improve lipase performance under biodiesel-relevant conditions.

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