Liang_2025_Int.J.Biol.Macromol_307_142236

Multifunctional enzymes have the potential to enhance hydrolytic efficiency and reduce enzyme cost. In this study, by optimizing linker peptide identity and the order of fusion partners, an efficient trifunctional xylanase/glucanase/feruloyl esterase, named Xyn(Glu)A/FaeB-L1, was obtained through associating the Bacillus bifunctional xylanase/glucanase Xyn(Glu)A with its cognate feruloyl esterase FaeB using a flexible linker peptide (L1). In comparison to the parental enzymes, Xyn(Glu)A/FaeB-L1 displayed similar pH optima for enzyme activity, shifting no more than a single unit. However, the optimal reaction temperature was 5-10 degreesC lower than that of the parental enzymes. Intriguingly, its catalytic efficiencies (k(cat)/K(m)) with wheat arabinoxylan and barley beta-glucan measured 1.48- and 8.90-fold that of Xyn(Glu)A, respectively. Similarly, the Xyn(Glu)A/FaeB-L1 k(cat)/K(m) for para-nitrophenyl ferulate measured 3.84-fold that of FaeB, which may be attributed to the physical association of the fusion partners. Moreover, Xyn(Glu)A/FaeB-L1 was superior to a mixture of Xyn(Glu)A + FaeB at degrading feruloylated arabinoxylan. Furthermore, Xyn(Glu)A/FaeB-L1 surpassed a commercial cellulase (Cel) in the hydrolysis of pretreated corn stover, also showing synergistic effects when combined with Cel. These results demonstrate the feasibility of constructing efficient chimeric enzymes and the potential of Xyn(Glu)A/FaeB-L1 for the conversion of agricultural by-products into fermentable sugars, making it an attractive candidate for biorefinery applications.