Production of Xylooligosaccharides (XOSs) with Anti-inflammatory and Prebiotic Activities Using Recombinant Endo-1,4-β-xylanases from <i>Bacillus subtilis</i>

The enzymatic production of xylooligosaccharides (XOSs) from xylan-rich agricultural byproducts has gained attention as a sustainable and environmentally friendly approach. In this work, two forms of recombinant <i>Bacillus subtilis</i> xylanases, MBP-<i>Bs</i>XynA11 (a fusion of a maltose-binding protein with the xylanase) and <i>Bs</i>XynA11-His (the xylanase with a 10xHis-tag), were investigated for their ability to convert xylan into value-added XOS. Both enzymes exhibited similar temperature and pH optima at 60 °C and 6.5, with specific activities of 7,900 and 4,800 U/nmol, respectively. However, MBP-<i>Bs</i>XynA11 demonstrated higher yields upon recombinant expression, as well as greater specificity and thermostability compared to <i>Bs</i>XynA11-His. Optimal hydrolysis of xylan yielded 53% and 51% XOS for MBP-<i>Bs</i>XynA11 and <i>Bs</i>XynA11-His, respectively. The resulting XOS were water-soluble, with over 90% of the products consisting of xylobiose, xylotriose, and xylotetraose, while xylose accounted for less than 0.5%. Finally, XOS generated from MBP-<i>Bs</i>XynA11 demonstrated both prebiotic and anti-inflammatory activities in human monocytes.