Engineering an α-L-rhamnosidase from Aspergillus niger for efficient conversion of rutin substrate

α-L-Rhamnosidase has great potential in the food and pharmaceutical industries, but its use in industrial applications is limited by its low catalytic efficiency. In this study, an α-L-rhamnosidase (AnRha) from Aspergillus niger CCTCC M 2018240 was engineered to enhance its catalytic efficiency towards rutin. Based on molecular docking and structural analysis, alanine scanning, saturation and combinatorial mutagenesis were performed on 16 residues involved in the substrate binding pocket. Four mutants, L234A, L234C, F345S and L234A/S339V/F345S had more than 2-fold higher catalytic efficiency than wild-type (WT) enzyme, and one mutant, S339V had a markedly improved half-life at 65 °C. F345S had 2.5 times the catalytic efficiency of WT, without loss of thermostability. Molecular docking revealed that F345 is the critical substrate entrance tunnel residue in AnRha. Molecular dynamics simulation indicated that the structure of L234A is more flexible, but less stable, whereas S339V is more rigid. Crude enzyme was produced by the F345S mutant and used to hydrolyze an aqueous suspension (130 g/L) of low water-solubility rutin, with a conversion of 95.89 %, nearly 30 % higher than WT (67.18 %). F345S is by far the most effective biocatalyst reported for conversion of rutin. This study also provides useful reference data for engineering α-L-rhamnosidase.