Abstract
Alginate lyase plays a critical role in the production of alginate oligosaccharides (AOS), necessitating efficient activity and a certain level of thermal stability for industrial AOS production. In this study, through sequence alignment and structural analysis, a non-catalytic amino acid residue, Ala195, located at the entrance of the catalytic pocket was identified. This residue is crucial for the catalytic efficiency and thermal stability of Alg2944, a member of the polysaccharide lyase family 18. The specific activities of A195F, A195I, and A195L were enhanced by 1.84-, 1.39-, and 1.98-fold, respectively, while the thermal stability of mutants A195L and A195I showed significant improvements at 35 °C and 40 °C compared to the wild-type (WT) enzyme. Furthermore, molecular docking, structural analysis, and molecular dynamics (MD) simulations revealed that the enhanced specific activity and thermal stability of the A195L mutant can be attributed to the creation of a hydrophobic pocket, increased rigidity in the loop region, and a notably lower binding free energy of the A195L mutant (-45.96 ± 7.69 kcal/mol) compared to the WT (-38.40 ± 4.25 kcal/mol). These findings underscore the essential role of rigidity and hydrophobicity at the entrance of the catalytic pocket in ensuring both catalytic efficiency and thermal stability of alginate lyase.
Published Version
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