Rational design of ancestral L-asparaginase via dynamics-guided engineering for improvement of thermostability and acrylamide mitigation efficiency.

Abstract

L-asparaginase (ASNase) is used both for treating acute lymphoblastic leukemia and as a processing aid to hydrolyze L-asparagine prior to heating, thereby suppressing acrylamide formation in foods and certain traditional Chinese medicinal materials. Industrial pretreatment, however, typically involves high temperature, alkaline conditions, and complex ionic environments, where many enzyme preparations rapidly lose activity, and gains in thermostability often come at the expense of catalytic efficiency. Here, we developed Evolutionary Dynamic Guided Engineering (EDGE), an evolution-and-dynamics-informed strategy that integrates phylogenetic reconstruction with residue-level conformational flexibility profiling to define a target rigidity-flexibility architecture and to select an ancestral starting scaffold. Using EDGE, we performed ancestral sequence reconstruction along the hyperthermophilic branch and identified an inferred ancestral enzyme, designated Anc-ASNase, as the baseline scaffold for engineering. Furthermore, we implemented a two-stage computational pipeline screening, selecting ten single-point candidates for experimental validation. The triple mutant (S282P/A117P/D250M) exhibited a specific activity of 4892.71 U/mg (3.29-fold higher than Anc-ASNase) and a prolonged half-life of 141.6 min at 85 °C. Molecular dynamics simulations indicated that these mutations enhance hydrophobic packing and stabilize interaction networks, suppressing nonproductive fluctuations while retaining functional conformations required for catalysis. Under harsh processing conditions (100 °C, 30 min), the engineered enzyme reduced acrylamide by 70.2% in French fries and by 57.0% in herbal material samples, and it remained tolerant under alkaline conditions and in the presence of several metal ions. Together, these findings support dynamics-guided engineering as a practical route to develop hyperthermophilic ASNase for high-temperature processing and acrylamide mitigation.