Significant Improvement of Both Catalytic Efficiency and Stability of Fructosyltransferase from Aspergillus niger by Structure-Guided Engineering of Key Residues in the Conserved Sequence of the Catalytic Domain

Fructosyltransferase is a key enzyme in fructo-oligosaccharide production, while the highly demanding conditions of industrial processes may reduce its stability and activity. This study employs sequence alignment and structural analysis to target three potential residues (Gln38, Ile39, and Cys43) around the active center of FruSG from Aspergillus niger, and mutants with greatly improved activity and stability were obtained through site-directed mutagenesis. The Km values of C43N and Q38Y were, respectively, reduced to 60.8 and 93.1% compared to those of WT. Meanwhile, the kcat of C43N was increased by 21.2-fold compared to that of WT. These imply that both the affinity and catalytic efficiency of C43N were significantly enhanced compared to WT. The Glide docking score of sucrose inside C43N was calculated to be -5.980, which was lower than that of WT (-4.887). What is more, the proposed general acid/base catalyst Glu273 with a lower pKa value of C43N calculated by PROPKA might contribute to an easier catalytic reaction compared to that of WT. The thermal stability and pH stability of the mutant C43N were significantly enhanced compared to those of WT, and more hydrogen bonds formed during molecular dynamics simulations might contribute to the improved stability of C43N.