Xylo-oligosaccharide (XO) is a promising pre-biotic with applications in food, feed and healthcare products. XO can be produced by enzymatic digestion of xylan with xylanase. In this study, we aimed to improve the biochemical properties relevant to catalysis and kinetics of X11, a thermophilic glycosyl hydrolase (GH) family 11 endo-β-1,4-xylanase derived from a metagenomic library isolated from sugarcane bagasse, under high-temperature conditions preferred for XO synthesis. Removal of a carbohydrate-binding module (X11C) resulted in 6.5 fold greater catalytic efficiency. X11C was further improved by a Pro71Thr mutation in the X11P variant obtained from a random mutagenesis library, which exhibited 15.9 fold greater catalytic efficiency compared with wild-type X11 under the enzyme's optimal conditions of 80°C and pH 6.0. Homology modeling suggested that the improved performance of X11P could be attributed to formation of an extra H-bond between Thr71 and Ser75, which stabilizes the key catalytic residue Glu180 at the active pocket and β-sheet layers and agrees with the respective increase in melting temperature (T_m) where X11P >X11C >X11 as determined by differential scanning fluorimetry. The X11P variant was tested for hydrolysis of beechwood xylan, which showed X6 as the major product followed by X3 and X4 XOs. The highest yield of 5.5 g total XOs product/mg enzyme was observed for X11P, equivalent to 3.7 fold higher than that of wild-type with XO production of >800 mg/g xylan. The X11P enzyme could be developed as a thermophilic biocatalyst for XO synthesis in biorefineries.

低聚木糖（XO）是一种有前途的益生元，可用于食品，饲料和保健产品。XO可以通过用木聚糖酶酶解木聚糖而产生。在这项研究中，我们旨在改善与X11的催化和动力学有关的生化特性，X11是一种从甘蔗渣中分离出来的宏基因组库中衍生的嗜热糖基水解酶（GH）家族11内切β-1,4-木聚糖酶， XO合成首选的温度条件。除去碳水化合物结合模块（X11C），催化效率提高了6.5倍。通过从随机诱变文库获得的X11P变体中的Pro71Thr突变，进一步改善了X11C，在酶的最佳条件80°C和pH 6.0下，与野生型X11相比，其催化效率是野生型X11的15.9倍。T _m），其中通过差示扫描荧光法测定的X11P＞ X11C＞ X11。测试了X11P变体对山毛榉木聚糖的水解作用，结果表明X6是主要产物，其次是X3和X4 XO。X11P的最高XO总产物产量为5.5 g XOs / mg酶，相当于XO产量> 800 mg / g木聚糖的野生型的3.7倍。X11P酶可作为生物精炼厂XO合成的嗜热生物催化剂。