Rational Design of Disulfide Bonds Increases Thermostability of a β-1,3-1,4-Glucanase from Paenibacillus

Abstract β-1,3 − 1,4-gluconases can specifically hydrolyze the adjacent β-1,4 glycoside bond of β-1,3 in β-glucan, which is widely used in food, brewing and feed industries. Its sources include bacteria, fungi, and plant endosperm cell walls, most β-1,3 − 1,4-glucanases lose their activity when the temperature exceeds 65 ℃. In this study, we selected and modified the β-1,3 − 1,4-glucanase (PlicA) gene from Paenibacillus and expressed it in Escherichia coli BL21 (DE3). Adding disulfide bonds by rational design increased the optimal temperature of the enzyme from 55 ℃ to 80 ℃, and temperature stability was also improved. The optimum pH of the modified β-1,3 − 1,4-gluconanase (Eccsl69) was 9.0–10.0. The enzyme activity in 16.9 U/mL of Eccsl69 was measured at 540 nm with 0.8% gluan as the substrate, and a nickel column purified specific enzyme activity of 320 U/mg was determined. The Km and Vmax values of Eccsl69 using barley β-glucan as substrate were 1.5 mg/ml and 8.3 mol/min·mg. The structure of the β-1,3 − 1,4-glucanase Eccsl69 tended to be stable after molecular dynamics simulation for approximately 20 ns. The enzyme was successfully applied in the pulping and papermaking field for the first time, and the pulp freeness was adjusted from 55.0 °SR to 47 °SR, which enhanced water filtration. This study provides a successful strategy for improving the heat resistance of Eccsl69, which is promising for its application in pulping and paper making industries.