Pan_2025_Int.J.Biol.Macromol_309_142646

Lipases are widely used as green industrial catalysts. Lipases from thermophilic microorganisms are particularly valuable due to their expected thermostability. However, the natural catalytic abilities and tolerance to extreme conditions of most enzymes are often not directly suited to the demands of industrial applications. Enzyme thermostability is closely associated with its structure, making it a target for improving enzyme thermostability. Therefore, we obtained the crystal structure of lipase from Caldibacillus thermoamylovorans (CtLip) with a resolution of 2.2 A using X-ray diffraction and identified its optimal temperature at 50 degreesC, with a half-life (t(1/2)) of 21.59 min at 50 degreesC. Mutants B1 (R269E/G270S/V271I/V272L), A335I and the stacked mutant B1/A335I (R269E/G270S/V271I/V272L/A335I) in loop region were constructed under the guidance of molecular dynamics analysis. Optimal temperature of mutant B1/A335I increased by 5 degreesC, with a half-life 8.36 times longer than that of the wild-typed. Our findings provide strategies to improve lipase thermostability by modification of the loop region of the enzyme.