Molecular Modification of D-lyxose Isomerase with Weak Acid Characteristic and D-mannose Production

The aim of this paper was to modify the weak acid characteristics of D-LI (the optimum pH was 6.5) from Thermoprotei archaeon, so as to produce D-mannose at pH5.5, thereby inhibiting the original maillard reaction and reducing the separation cost. Based on the strategy of protein sequence alignment and acid-base amino acid replacement, eight single point mutations were designed, and then three better single point mutants E82K, P105K and E165K were selected for further double-point mutation. The results showed that the optimum pH of the double-point mutant E82K/P105K was shifted from 6.5 to 6.0, and the enzyme activity at pH5.5 was 3.4 times higher than that of the original enzyme. When D-fructose and D-mannose were used as substrates, the kinetic parameters K_m and k_cat/K_m of the mutant were 78.77 mmol/L and 328.12 mmol/L, 15.37 mmol/L⁻¹·min⁻¹ and 48.92 mmol/L⁻¹·min⁻¹, respectively. After reacting with 80 g/L of D-fructose for 10 h, the conversion rate of the double point mutant E82K/P105K at pH5.5 was close to that of the original enzyme at pH6.5, and the color and degree of Maillard reaction decreased about 3~4 times compared with the original enzyme, which provided a feasible enzyme preparation for industrial production of D-mannose by D-LI.