Structure-guided engineering of Geobacillus kaustophilus L-asparaginase with improved functional and stability...

Acrylamide formation during high-temperature food processing poses significant food safety concerns. L-asparaginase (L-ASNase) is widely used as a processing aid to hydrolyze L-asparagine prior to thermal treatment, thereby limiting acrylamide generation. In this study, we applied a structure-guided protein engineering strategy to a thermophilic type II L-asparaginase from Geobacillus kaustophilus (GkASNase) to enhance catalytic efficiency and operational stability. Binding-pocket energy screening prioritized five single-residue variants (M59N, M59I, L61H, K164R, and D220E), which were subsequently evaluated by molecular dynamics simulations and experimental validation. All variants retained thermophilic characteristics, with an optimum pH of 8.5 and a temperature optimum of 55 °C. Specific activity increased from ∼2250 U/mg for the wild-type enzyme to 3950 and 3810 U/mg for M59N and D220E, respectively. The half-life improved from 3.56 h (wild type) to 40.86 h for D220E and 39.12 h for L61H. Catalytic efficiency increased 1.72-fold and 1.57-fold for D220E and M59N, respectively. In a starch-based model system, L61H and D220E achieved 93.5% and 94.1% acrylamide mitigation, compared with 66.7% for the wild-type enzyme after 1 h at 55 °C. Molecular dynamics analyses provided structural insight into mutation-dependent pocket stabilization, altered local flexibility, and their association with enhanced functional performance. These findings demonstrate that rational pocket-proximal substitutions effectively tune the stability-activity balance of thermophilic L-ASNase and provide a framework for engineering processing-robust biocatalysts for food safety applications.