Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.

宏基因组学（Metagenomics）已被广泛用于挖掘新型酶类与代谢通路，实现木质纤维素生物质向燃料及化学品的转化。在此研究背景下，本研究报道了从甘蔗土壤宏基因组中鉴定得到的一种新型GH10家族内切木聚糖酶（SCXyl）的分离、重组表达、生化与结构表征。重组表达的SCXyl对山毛榉木聚糖具有高活性，最适酶活条件为pH 6.0与45℃。该酶的晶体结构解析分辨率达2.75埃（Å），呈现经典的(β/α)8桶状折叠，具有保守的活性位点口袋，且Trp281-Arg291环存在固有柔性，可随底物结合呈现不同构象状态。降解产物的毛细管电泳分析显示，该酶具备降解木三糖等小分子低聚木糖的特殊能力，这与+1亚位点的疏水相互作用以及远离水解位点的亚位点低结合能相一致。木聚糖聚合物与磷酸预处理甘蔗渣（PASB）的主要反应产物为低聚木糖；延长孵育时间后，还可生成木二糖与木糖。此外，在添加商用纤维素酶复合制剂前，先用SCXyl对PASB进行预处理，可显著提升糖化效率。上述特性表明，该酶在食品与饲料工业的多项生物技术工艺，以及生物质预处理用于原料生产与乙醇制备中均具有良好的应用前景。