Objective To improve the activity of D-lyxose isomerase under acidic conditions.Methods Solvent-accessible surface bioinformatics analysis was performed on surface residues of D-lyxose isomerase derived from Caldanaerobius polysaccharolyticus, and six mutation sites, i.e., K2D, K2E, K8D, K8E, K18D, and K18E, were designed. Mutants were constructed and subjected to recombinant inducible heterologous expression in Escherichia coli. The recombinant mutant enzymes were isolated and purified using a nickel affinity chromatography column. In vitro comparative experiments were conducted to evaluate the catalytic conversion of D-fructose to D-mannose by the recombinant mutant enzymes at acidic pH 5.5 and near-neutral pH 6.5.Results The recombinant mutant enzymes K8D and K8E showed significantly improved catalytic conversion rates of D-fructose, with the conversion rate reaching 140% under optimal conditions (pH 6.5). At pH 5.5, the conversion rate was 1.26 times higher than that of the wild-type enzyme.Conclusion Molecular modification successfully enhanced the catalytic activity of D-lyxose isomerase mutants under acidic conditions.