Xylo-Oligosaccharide Production from Wheat Straw Xylan Catalyzed by a Thermotolerant Xylanase from Rumen Metagenome and Assessment of Their Probiotic Properties

A novel xylanase gene (<i>RuXyn854</i>) was identified from the rumen metagenome and was heterologously expressed in <i>Escherichia coli</i> to produce xylo-oligosaccharides (XOSs) as a prebiotic in this study. RuXyn854, a member of glycosyl hydrolase family 10, demonstrated peak enzymatic activity at pH 7.0 and 50 °C. RuXyn854 retains more than 50% of its activity after treatment at 100 °C for 10 min, highlighting the enzyme's excellent heat resistance. RuXyn854 showed a preferential hydrolyzation of xylan, especially rice straw xylan. RuXyn854 activity was significantly increased in the presence of 15 mM Mn²⁺, 0.25% Tween-20, and 0.25% Triton X-100 (125%, 20%, and 26%, respectively). The reaction temperature (30, 40, and 50 °C), dosage (0.20, 0.27, and 0.34 U), and time (90, 120, and 150 min) of RuXyn854 affected the XOS yield and composition, with a higher yield at 0.27 U, 50 °C, and 120-150 min. Xylobiose, xylotriose, and xylotetraose were characterized as the predominant XOS products resulting from the enzymatic hydrolysis of wheat straw xylan by RuXyn854, with xylose present at a mere 0.49% of the total yield. The prebiotic potential of XOSs was assessed through in vitro fermentation with established probiotic strains of <i>Bifidobacterium bifidum</i> and <i>Lactobacillus brevis</i>. The results showed that, regardless of incubation time, XOSs stimulated the growth and xylanolytic enzyme secretion of the two probiotics compared to the controls. These results demonstrate that the feature of RuXyn854 to withstand temperatures up to 100 °C is impressive, and its ability to hydrolyze wheat xylan into XOSs promotes the growth of probiotics.