Endo-xylanase from Thermophilic Humicola brevis var. thermoidea Immobilized on MANAE-Agarose: A Sustainable Biocatalyst for Prebiotic Xylooligosaccharide Production from Agro-Industrial Residues

The sustainable production of xylooligosaccharides (XOS) from lignocellulosic residues represents an essential approach for adding value to agro-industrial by-products, given their well-established prebiotic, antioxidant, and immunomodulatory properties. In this study, an endo-β-1,4-xylanase (XylHb) from the thermophilic fungus Humicola brevis var. thermoidea was produced through solid-state fermentation, purified by ethanol precipitation and chromatographic steps, and subsequently immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose. Immobilization was highly efficient, with yields exceeding 95% and full activity recovery, indicating a strong interaction between the enzyme and support. The immobilized derivative (MANAE-XylHb) displayed enhanced biochemical properties compared to the free enzyme, remaining catalytically active within a broad pH range (4.5–6.5) and at temperatures up to 90 °C. In comparison, the free enzyme was inactivated entirely above 75 °C. Thermostability tests confirmed 100% activity retention after 24 h at 50 °C and significant residual activity at 70 °C. Operational stability assays further showed that MANAE-XylHb preserved approximately 95% of its initial activity after ten consecutive hydrolysis cycles, highlighting its potential for reuse in industrial processes. Hydrolysis of different xylans revealed that the enzyme preferentially cleaved less-substituted substrates, releasing mainly low-degree oligomers, such as xylobiose (X2) and xylotriose (X3), which are highly valued in the food and nutraceutical sectors for their prebiotic properties. Moreover, the robust catalytic performance of MANAE-XylHb at elevated temperatures supports its application in lignocellulosic biomass processing for biofuel production, where higher solubility and lower contamination risks are advantageous. In summary, immobilization on MANAE-agarose significantly improved the catalytic stability, operational robustness, and reusability of XylHb, enabling efficient production of XOS under industrially relevant conditions. These results position MANAE-XylHb as a versatile and sustainable biocatalyst, contributing to the development of eco-friendly biotechnological processes and reinforcing the transition toward a circular bioeconomy.