Hydrolysis of softwood by Aspergillus mannanase: role of a carbohydrate-binding module

Endo beta-1,4-mannanases (beta-mannanases, EC 3.2.1.78), belonging to CAZy GH5 and GH26 families, catalyze the hydrolysis of structurally different mannans. In this study, the mannanase encoding gene of Aspergillus aculeatus VN was expressed in Aspergillus niger D15#26 using pAN 52-4 vector, under the control of PgpdA promoter and TtrpC terminator. In order to improve the hydrolytic capacity of this GH5 on lignocellulosic substrate, the family 1 carbohydrate-binding module (CBM1) of Aspergillus niger cellobiohydrolase B was artificially fused at the C-terminal end of this enzyme with a natural linker. Both mannanase and mannanase-CBM genes were successfully expressed in A. niger D15#26, producing proteins with molecular masses of 54 and 79 kDa, respectively. The Michaelis-Menten constants, pH activity profiles and temperature optima of three enzymes (wild-type mannanase, recombinant mannanase and recombinant mannanase-CBM) were similar, but the fused mannanase-CBM enzyme was more thermostable. Cross-comparison of the three enzymes for softwood hydrolysis in association with Trichoderma reesei enzymatic cocktail showed that mannanase-CBM improved the glucose yield compared to wild-type and recombinant mannanases.