Improving the Thermostability of Lipase ATL by Engineering Disulfide Bonds in Flexible Regions

Lipases are versatile biocatalysts for the synthesis of functional lipids, but their industrial application is frequently constrained by limited thermostability. In this study, disulfide bond engineering targeting flexible regions identified by molecular dynamics (MD) simulations was employed to enhance the thermostability of lipase ATL from Aspergillus tamarii. The combined mutant Mut2D harboring the disulfide bonds A46C-N68C and L263C-G271C exhibited a Tm increase of 5.83 °C and a 23.9-fold improvement in half-life at 45 °C compared to the wild-type ATL. The catalytic efficiency (kcat/Km) of Mut2D for pNP-butyrate, pNP-caprate and pNP-stearate increased by 34.7%, 27.0% and 39.0%, respectively, compared to the wild-type. Comparative MD simulations showed that the introduction of the two disulfide bonds increased the overall conformational rigidity of the enzyme, which likely accounts for the increased thermostability. In diacylglycerol (DAG) synthesis, Mut2D achieved a 4.8% higher DAG content with a faster reaction rate compared to the wild-type at their respective optimum temperatures. This study paves the way for the industrial application of ATL and provides valuable insights for enhancing the thermostability of other lipases.