PURIFICATION AND IMMOBILIZATION OF EXTRACELLULAR XYLANASE FROM Thermomyces lanuginosus ON MAGNETIC CHITOSAN NANOPARTICLES FOR PREBIOTIC XYLOOLIGOSACCHARIDE PRODUCTION

Xylanases are key biocatalysts for the degradation of hemicellulosic polysaccharides, yielding xylooligosaccharides (XOS) with recognized prebiotic properties. This study aimed to purify and immobilize extracellular xylanase from the filamentous fungus Thermomyces lanuginosus and to assess its application in producing XOS, which can stimulate probiotic growth. The microorganism was cultivated in Czapek medium supplemented with corn straw (1.5%, w/v), at 150 rpm and 45 °C for 4 days, and the crude extract exhibited a specific activity of 19.2 U mg^-1. Purification by ion-exchange chromatography using DEAE-Sephadex and DEAE-Sepharose columns increased the specific activity to 73.2 U mg^-1, resulting in a 3.81-fold purification and a 19.6% yield. Purified xylanase was immobilized onto magnetic chitosan nanoparticles using 10% glutaraldehyde as a crosslinker. Optimal conditions were achieved in sodium phosphate buffer (10 mM, pH 7.0), resulting in an immobilization yield of 88.4%, efficiency of 69.1%, and recovered activity of 61.1%. These parameters highlight the effectiveness of the immobilization strategy, which provides enhanced enzyme stability and reusability for industrial applications. The prebiotic potential of the XOS produced was evaluated using Lactobacillus curvatus as a model probiotic, previously cultivated in minimal MRS medium. Growth curves showed that cultures supplemented with XOS achieved a moderate but sustained increase in biomass (OD₆₀₀ ≈ 0.25–0.30) throughout 50 h of incubation. These results indicate that XOS promotes stable and prolonged growth of Lactobacillus, supporting its functionality as a prebiotic substrate. In conclusion, the purification and immobilization of T. lanuginosus xylanase on magnetic chitosan nanoparticles represent an efficient approach for producing prebiotic XOS. This study highlights the potential of immobilized enzymes for the sustainable production of functional oligosaccharides, applicable to the food and health industries.