Computer-Aided Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase AlyMc

Alginate lyase degrades alginate by the β-elimination mechanism to produce unsaturated alginate oligosaccharides (UAOS), which have better bioactivities than saturated AOS. Enhancing the thermal stability of alginate lyases is crucial for their industrial applications. In this study, a feasible and efficient rational design strategy was proposed by combining the computer-aided ΔΔG value calculation with the B-factor analysis. Two thermal stability-enhanced mutants, Q246V and K249V, were obtained by site-directed mutagenesis. Particularly, the t_{1/2, 50 °C} for mutants Q246V and K249V was increased from 2.36 to 3.85 and 3.65 h, respectively. Remarkably, the specific activities of Q246V and K249V were enhanced to 2.41- and 2.96-fold that of alginate lyase AlyMc, respectively. Structural analysis and molecular dynamics simulations suggested that mutations enhanced the hydrogen bond networks and the overall rigidity of the molecular structure. Notably, mutant Q246V exhibited excellent thermal stability among the PL-7 alginate lyase family, especially considering the heightened enzymatic activity. Moreover, the rational design strategy used in this study can effectively improve the thermal stability of enzymes and has important significance in advancing applications of alginate lyase.