Table_3_Highly efficient synergistic activity of an α-L-arabinofuranosidase for degradation of arabinoxylan in barley/wheat.docx

Here, an α-L-arabinofuranosidase (termed TtAbf62) from Thermothelomyces thermophilus is described, which efficiently removes arabinofuranosyl side chains and facilitates arabinoxylan digestion. The specific activity of TtAbf62 (179.07 U/mg) toward wheat arabinoxylan was the highest among all characterized glycoside hydrolase family 62 enzymes. TtAbf62 in combination with endoxylanase and β-xylosidase strongly promoted hydrolysis of barley and wheat. The release of reducing sugars was significantly higher for the three-enzyme combination relative to the sum of single-enzyme treatments: 85.71% for barley hydrolysis and 33.33% for wheat hydrolysis. HPLC analysis showed that TtAbf62 acted selectively on monosubstituted (C-2 or C-3) xylopyranosyl residues rather than double-substituted residues. Site-directed mutagenesis and interactional analyses of enzyme–substrate binding structures revealed the catalytic sites of TtAbf62 formed different polysaccharide-catalytic binding modes with arabinoxylo-oligosaccharides. Our findings demonstrate a “multienzyme cocktail” formed by TtAbf62 with other hydrolases strongly improves the efficiency of hemicellulose conversion and increases biomass hydrolysis through synergistic interaction.

本研究报道了一株来自热毁丝霉（Thermothelomyces thermophilus）的α-L-阿拉伯呋喃糖苷酶（命名为TtAbf62），该酶可高效切除阿拉伯呋喃糖侧链，促进阿拉伯木聚糖的降解。相较于所有已表征的糖苷水解酶家族62（glycoside hydrolase family 62, GH62）酶，TtAbf62对小麦阿拉伯木聚糖的比酶活（179.07 U/mg）为所有已报道同类酶中的最高值。将TtAbf62与内切木聚糖酶、β-木糖苷酶复配使用，可显著强化大麦与小麦生物质的水解过程。三酶复配体系的还原糖释放量显著高于单酶处理的总和：大麦水解的还原糖释放率达85.71%，小麦水解则达33.33%。高效液相色谱（High Performance Liquid Chromatography, HPLC）分析结果显示，TtAbf62可选择性作用于单取代（C-2或C-3位）吡喃木糖残基，而非双取代残基。定点突变及酶-底物结合结构相互作用分析表明，TtAbf62的催化位点可与阿拉伯木寡糖形成多种不同的多糖催化结合模式。本研究结果证实，由TtAbf62与其他水解酶构成的多酶鸡尾酒（multienzyme cocktail）体系可通过协同作用，显著提升半纤维素转化效率与生物质水解效果。