Construction and characterization of bifunctional cellulases: Caldicellulosiruptor-sourced endoglucanase, CBM, and exoglucanase for efficient degradation of lignocellulose

Amorphous cellulose can be deconstructed by endoglucanase of bifunctional CelA, which created nicks in the crystalline regions for exoglucanase to engage. To improve the degradation efficiency of un-pretreated lignocellulose, endoglucanase (Endo5), exoglucanase (Exo5) and different carbohydrate-binding modules (CBMs), were fused to yield several bifunctional cellulases, containing Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5, and Endo5-CBM28-Exo5. The fusion enzymes displayed potential for a wide spectrum of substrates, and efficiently hydrolyzed filter paper, microcrystalline cellulose, steam-pretreated rice straw, rice straw, and wheat straw substrates into cellobiose and glucose. Specific activities of Endo5-2CBM-Exo5 on microcrystalline cellulose reached 0.167 U/mg, which was increased by 2.2- to 14.0-fold compared to parental CBM3b-Exo5 and Endo5CBM28, respectively. Moreover, the optimal conditions for fused enzymes were pH 4.0 to 4.5 and 60-80 degrees C, while optimal conditions for parental Endo5-CBM28 and CBM3b-Exo5 were pH 5.0, 75 degrees C and pH 4.7 to 5.5, 80 degrees C, respectively. After incubation at 85 degrees C for 2 h, these fused enzymes relative activities maintained 45-68%, while relative activity of parental Endo5-CBM28 just remained < 10%. The fused enzymes were acid-resistant stable enzymes and showed stability from pH 3.5 to 8.0. When 10 mM MnCl2 was added, the fusion enzymes activities were increased to 2.2 to 2.4 times. In conclusion, lignocellulose saccharification ability, high pH and temperature stability, make the CBMs-fused bifunctional cellulases attractive candidate for natural substrates deconstruction.