Engineered lipase from Thermomyces dupontii with improved thermostability for preparation of long-medium-long structured lipids

Lipase from Thermomyces dupontii (TDL) shows great potential to prepare long-medium-long structured lipids (LML-SLs), but its poor thermostability represents a significant obstacle to further industrial application. Herein, a rational design based on structure and sequence analysis (including disulfide bond calculation and sequence alignment) was used to generate a more thermostable mutant. Compared with wild-type TDL, mutant M3 displayed a 9.1-fold, 7.2 °C and 10 °C increase in half-life ( t 1/2 ) at 60 °C, the half-loss temperature ( T 50 ) at 15 min and optimum temperature, respectively. Remarkably, the specific activity of mutant M3 was 49.1% higher than that of wild-type TDL. Molecular dynamics (MD) simulations showed that the structural flexibility of mutant M3 was reduced, which might contribute to the enhanced thermostability. Additionally, LML-SLs were synthesized from ethyl linoleate and tricaprylin with wild-type TDL and mutant M3. Compared with the wild-type TDL group, the LML-SL content of the mutant M3 group increased by 8.1%. This study will pave the way for the industrial application of TDL and provide valuable insights for enhancing the thermostability of other lipases. • The mutation design of TDL lipase was based on structure and sequence analysis. • Mutant M3 showed greatly increased thermostability. • Molecular dynamics simulations showed the rigidity of the mutant M3 was enhanced. • Mutant M3 showed enhanced effect in producing Long-medium-long structured lipids.