Ghorban_2026_Enzyme.Microb.Technol_199_110884

Thermostability is a critical limitation for industrial lipases. Rational design using stabilizing structural motifs like AXXXA offers a targeted engineering approach. This study aimed to enhance both thermostability and catalytic efficiency of Bacillus thermocatenulatus lipase (BTL2) through computational design and experimental validation. Fifty potential AXXXA-forming mutations were screened in silico using deltadeltaG calculations to predict thermostabilizing effects. The deltadeltaG criterion identifies mutations that enhance folding stability; among these, the top candidate (N60A) was selected because its location at a helix-helix interface adjacent to the active site also suggested potential catalytic benefits through active site rigidification. The N60A mutation was experimentally characterized for both stability and activity. Kinetic parameters (Km, kcat), melting temperature (Tm), optimal temperature, pH profile, and stability under industrial stressors (organic solvents, detergents) were systematically compared between wild-type and mutant enzymes. The N60A mutation was predicted to eliminate an unfavorable polar interaction, forming a stabilizing AXXXA motif. Experimentally, N60A showed a 10 degreesC higher optimal temperature (65 degreesC vs 55 degreesC) and a 4.3 degreesC increase in Tm. Crucially, it exhibited significantly improved kinetics: for tricaprylin (C8), kcat increased 1.8-fold (380 vs 210 s(-)) and catalytic efficiency (kcat/Km) doubled (905 vs 438 mM(-)s(-)). The mutant also demonstrated superior stability at 65 degreesC (55.5% vs 22% residual activity after 60 min) and enhanced tolerance to organic solvents and detergents. The N60A mutation successfully enhanced BTL2's thermostability and catalytic efficiency simultaneously, validating the AXXXA motif as a potent engineering tool. The engineered lipase presents a superior biocatalyst for high-temperature industrial applications.