Computer-aided evolution and experimental validation of a novel L-asparaginase from Bacillus sp. SD3 toward enhanced...

L-asparaginase, a key biocatalyst for therapeutic and industrial applications, is often limited by poor thermal stability. A novel L-asparaginase gene from Bacillus sp. SD3 was identified, cloned, expressed, and characterized. Initial characterization of the native enzyme revealed two unique amino acid substitutions (W89 and A102) not typically found in other Bacillus species L-asparaginases, conferring a 2.11 kcal mol^-1 increase in thermodynamic stability compared to its closest homolog. The native enzyme exhibited mesophilic properties, with an optimal temperature of 45 °C and moderate thermal stability. To enhance thermotolerance, a computer-aided directed evolution strategy was employed. Computational predictions integrating alanine scanning, targeted mutagenesis, and 200 ns molecular dynamics simulations identified G129F and D291W as key stabilizing mutations. The G129F&D291W double mutant was constructed and characterized. This mutant demonstrated a 10 °C increase in optimal operating temperature (to 55 °C) and retained activity up to 70 °C. Thermal stability was also significantly improved, retaining 61 % activity after 180 min at 60 °C, compared to 15 % for the native enzyme. While a slight reduction in substrate affinity was observed (Km increased from 0.21 mM to 0.34 mM), the enhanced thermotolerance positions the mutant enzyme as a promising candidate for industrial applications.