An eco-friendly process for low-degree polymerization xylo-oligosaccharides production from corncob via enzyme adsorption-hydrolysis integrated approach by reconstructing fusion protein

Xylo-oligosaccharides (XOS) have gained significant attention for their extensive health-boosting characteristics. However, the tedious procedures involved in lignocellulose pretreatment and XOS purification, coupled with the low efficiency of enzymatic hydrolysis, constrain the broader commercialization of XOS. In this study, the effects of fusion order and linker composition on enzyme activity and thermostability of fusion proteins consisting of a GH11 xylanase mutant (XYNm) and an expansin (EXLX) were investigated. The results indicated that the reconstructed protein XYNm-R2-EXLX exhibited 2.6-fold activity higher than EXLX-R2-XYNm. Furthermore, the denaturation thermodynamics evaluation demonstrated a significant enhancement in the thermostability of XYNm-R2-EXLX. Additionally, the reconstructed fusion protein showed a notable affinity adsorption efficiency on corncob, reaching a remarkable 95.5%. Subsequently, the fusion enzyme-corncob complex underwent direct hydrolysis, and a yield of 58.7 g XOS/kg corncob was obtained by employing RSM optimization techniques. Finally, a membrane-assisted refining process was implemented, resulting in the recovery of 68.2% (w/w) of the initial XOS (X₂-X₄) with a purity of 96.2%. Here, a chemical free and eco-friendly bioprocess for production of low-degree polymerization XOS was developed, eliminating the need for xylan separation from corncob and simplifying the process for product purification. These findings exhibited the great potential in XOS (X₂-X₄) production.