Enhanced activity and stability of thermostable endoglucanase Dictyoglomus turgidum DtCelA with multiple strategies

Thermophilic endoglucanases are of big promise in enzymatic degradation of lignocellulosic substrate, yet their limited catalytic efficiency remains a major barrier to industrial application. This study focused on the improvement of activity and stability of thermostable endoglucanase Dt CelA from Dictyoglomus turgidum with a three-stage cascade engineering strategy that integrated model-guided site-directed mutagenesis, disulfide bond introduction, and carbohydrate-binding module (CBM) fusion. Among the variants, the triple mutant S25D/T265C/A305C fused with CBM2 (S25D/T265C/A305C-A-CBM2) exhibited a 90.56 % increase in specific activity toward CMC relative to the wild type, together with a 2.97-fold longer half-life at 75 °C and a 5.3-fold longer at 80 °C. This mutant also displayed 3.0- and 5.4-fold higher hydrolytic activity against Avicel and konjac glucomannan, respectively. On natural lignocellulosic substrates including filter paper, sugarcane bagasse, and corn stover, the mutant also showed potential application. The mutated endoglucanase Dt CelA was powerful with enhanced catalytic performance and thermostability after multicascade engineering, which can provide a robust framework for the rational design of cellulases that are optimized for high-temperature biorefinery process.