Kaciran_2026_Int.J.Biol.Macromol__154003

GDSL lipases, members of the SGNH hydrolase superfamily, exhibit broad substrate specificity and catalytic adaptability, making them valuable model enzymes for investigating structure-function relationships under high-temperature conditions. Despite these features, residue-level determinants influencing the catalytic performance of thermophilic GDSL lipases remain insufficiently characterized. In this study, we investigated the effects of rationally selected amino acid substitutions on the catalytic properties of a thermophilic GDSL lipase using an in silico-guided strategy combined with experimental characterization methods. The gene encoding the GDSL lipase GstLipC from Geobacillus stearothermophilus ATCC 7953 was cloned into the pET28a(+) vector, and four point mutations (I184H, I184V, R196L, and R196F) were introduced based on structural modeling and molecular docking analyses. Wild-type and mutant enzymes were expressed and purified for biochemical and kinetic characterization. GstLipC, GstLipC_I184V, and GstLipC_R196F exhibited maximal activity at 90 degreesC, whereas GstLipC_I184H exhibited optimal activity at 100 degreesC. GstLipC_R196L displayed comparable activities at both temperatures. All variants showed optimal activity at pH 8 and high thermal stability. In the presence of 0.1 mM Triton X-100, the Km values of the mutant enzymes were lower than that of the wild type, suggesting altered substrate interactions, with GstLipC_R196F showing the lowest Km value. Overall, this study demonstrates that residue-level engineering can modulate the catalytic properties and thermal behavior of a thermophilic GDSL lipase, providing insights into the structure-function relationships of thermostable enzymes.