Abstract:Objective This study aims to enhance the catalytic activity, thermal stability, and pH stability of α-L-rhamnosidase (EC 3.2.1.40).Method Three combined mutants (K423R-R307Y-D508N, K423R-R307Y-N561D, and K389R-R307Y-N561D) were constructed based on improved single-point mutants.Result Compared with the wild type (WT), the combined mutants K423R-R307Y-D508N exhibited an increased half-life of 0.24 min at both 65 ℃ and 70 ℃. Additionally, its half-life improved by 145.64 min and 4.91 min at pH 7.0 and pH 8.0, respectively. However, its relative enzyme activity was only 41.01% of that of the WT. The combined mutant K423R-R307Y-N561D exhibited an increased half-life of 51.42 min and 20.65 min at pH 7.0 and pH 8.0, respectively, and its relative enzyme activity were 63.26% of that of the WT. The combined mutant K389R-R307Y-N561D achieved an relative enzyme activity 167.20% of that of the WT, with an increased half-life of 1.60 min and 1.34 min at 65 ℃ and 70 ℃, respectively. However, its stability in alkaline environments did not show significant improvement. Molecular docking, molecular dynamics simulations, and MM-PBSA analyses revealed that the increased flexibility of the catalytic domain Loop region and partial α helix structure at the bottom of K389R-R307Y-N561D enhanced substrate binding and affinity. However, the R307 mutation induced local perturbations, disrupting the hydrogen bond network and π-π hydrophobic interactions, leading to decreased structural stability.Conclusion The site compensation mechanism triggered by combined mutations is the primary reason for the difficulty in simultaneously enhancing the catalytic activity and structural stability of α-L-rhamnosidase.
