biostudies-literatureYuan YNatural Science Foundation of Shanxi Provincethe Natural Science Foundation of Shanxi ProvinceKey technology and application of alkaline protease industrialization for detergent in 1331 Pro-vincial Engineering Research CenterKey technology and application of alkaline protease industrialization for detergent in 1331 Provincial Engineering Research Center1160https://www.ebi.ac.uk/biostudies/studies/S-EPMC11901772biostudies-literatureUnknown30(5)The alkaline protease from <i>Bacillus licheniformis</i> strain 2709 (AprE 2709) is widely used in Chinese industries but faces stability challenges under high-temperature conditions. This study employed molecular modeling and mutagenesis to identify Asn residues at positions 61, 160, and 211 as key sites affecting the stability of AprE 2709. By leveraging the additive and cooperative effects of mutations, the mutant enzyme AprE 2709 (N61G/N160G/N211G) was engineered, exhibiting enhanced thermostability and catalytic activity. The mutant demonstrated a 2.89-fold increase in half-life at 60 °C and a 1.56-fold improvement in catalytic efficiency compared to the wild-type enzyme. Structural analysis revealed that the improved thermostability was due to altered electrostatic interactions and sMolecules (Basel, Switzerland)Enhancing the Thermostability of &lt;i&gt;Bacillus licheniformis&lt;/i&gt; Alkaline Protease 2709 by Computation-Based Rational Design.PMC11901772PT20181220210302123458Zhang JLu JZhao GYuan YWang LShi YfalseEnhancing the Thermostability of &lt;i&gt;Bacillus licheniformis&lt;/i&gt; Alkaline Protease 2709 by Computation-Based Rational Design.The alkaline protease from <i>Bacillus licheniformis</i> strain 2709 (AprE 2709) is widely used in Chinese industries but faces stability challenges under high-temperature conditions. This study employed molecular modeling and mutagenesis to identify Asn residues at positions 61, 160, and 211 as key sites affecting the stability of AprE 2709. By leveraging the additive and cooperative effects of mutations, the mutant enzyme AprE 2709 (N61G/N160G/N211G) was engineered, exhibiting enhanced thermostability and catalytic activity. The mutant demonstrated a 2.89-fold increase in half-life at 60 °C and a 1.56-fold improvement in catalytic efficiency compared to the wild-type enzyme. Structural analysis revealed that the improved thermostability was due to altered electrostatic interactions and s2025-01-01T00:00:00Z2025 Mar2026-04-08T19:48:20.23Z2025-04-04T09:07:57.251ZS-EPMC119017724007638410.3390/molecules30051160