Abstract: The rational design of Aspergillus niger β-mannanase was carried out in order to obtain mutants with improved thermal stability and catalytic activity. The FireProt server was used to analyze the β-mannanase and four single-point mutants were selected. The mutant genes were obtained by site-directed mutagenesis, and the mutant enzymes were expressed and purified in vitro. The enzymatic properties of the mutants were determined using locust bean gum as the substrate, and the mutant E325M was selected on the basis of its improved thermal stability and catalytic activity of A. niger β-mannanase. Compared with the wild type (WT), the catalytic activity of E325M was increased by 33.8%. After treatment at 60 ℃ for 1 h, the residual activity of E325M was increased by 40.2%. The half-life of E325M at 60 ℃ was 72.2 min, which was 1.22 times longer than that of WT. The surface electrostatic charge analysis showed that the optimization of the surface charge of mutant E325M was beneficial to improve the thermal stability of the enzyme. Molecular docking analysis showed that the enhanced hydrogen bonding between the enzyme and the substrate may be responsible for the simultaneous improvement of the thermal stability and catalytic activity of E325M. Molecular dynamics simulation showed that the increased thermal stability and catalytic activity of mutant E325M could be attributed to the increased rigidity of some α-helix regions and the increased flexibility of the important catalytic residues D152 and E206. These findings provide a useful strategy for improving the thermal stability and activity of β-mannanase from A. niger, and promote understanding of the relationship between β-mannanase structure and function, as well as the industrial application of the enzyme.

Key words: locust bean gum; β-mannanase; thermal stability; catalytic activity; rational design