Xylanases have drawn much attention owing to possessing great potential in various industrial applications. However, the applicability of xylanases, exemplified by the production of bioethanol and xylooligosaccharides (XOSs), was bottlenecked by their low stabilities at higher temperatures. The main purpose of this work was to improve the thermostability of AuXyn11A, a mesophilic glycoside hydrolase (GH) family 11 xylanase from Aspergillus usamii E001, by N-terminus replacement.

The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeA^A126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeA^A126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (k_cat/K_m) of re-AuFaeA^A126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.

The AuMan5A, an acidophilic glycoside hydrolase (GH) family 5 β-mannanase derived from Aspergillus usamii YL-01-78, consists of an only catalytic domain (CD). To perfect enzymatic properties of the AuMan5A, a family 1 carbohydrate-binding module (CBM) of the Trichoderma reesei cellobiohydrolase I (TrCBH I), having the lowest binding free energy with cellobiose, was selected by in silico design, and fused into its C-terminus forming a fusion β-mannanase, designated as AuMan5A-CBM. Then, its encoding gene, Auman5A-cbm, was constructed as it was designed theoretically, and expressed in Pichia pastoris GS115. SDS-PAGE analysis displayed that both recombinant AuMan5A-CBM (reAuMan5A-CBM) and AuMan5A (reAuMan5A) were secreted into the cultured media with apparent molecular masses of 57.3 and 49.8 kDa, respectively. The temperature optimum of the reAuMan5A-CBM was 75°C, being 5°C higher than that of the reAuMan5A. They were stable at temperatures of 68 and 60°C, respectively. Compared with reAuMan5A, the reAuMan5A-CBM showed an obvious decrease in K_m and a slight alteration in V_max. In addition, the fusion of a CBM of the TrCBH I into the AuMan5A contributed to its cellulose-binding capacity.