College of Life Sciences, Capital Normal University, Beijing, 100048, China.
College of Life Sciences, Capital Normal University, Beijing, 100048, China.
Microbiol Res. 2021 Dec;253:126886. doi: 10.1016/j.micres.2021.126886. Epub 2021 Oct 4.
Xylan is one of the major polymeric hemicellulosic constituents of lignocellulosic biomass, and its effective utilization by microorganisms is crucial for the economical production of biofuels. In this study, Paenibacillus physcomitrellae XB exhibited different xylan degradation ability on different substrates of corncob xylan (CCX), oat spelt xylan (OSX), wheat flour arabinoxylan (AX) and beech wood xylan (BWX). The RT-QPCR result showed that two genes (Pph_0602 and Pph_2344) belonging to the glycoside hydrolase family 43 were up-regulated more than 5-fold on CCX and xylose. Substrate-specific assays with purified proteins Ppxyl43A (Pph_0602) and Ppxyl43B (Pph_2344) revealed that both exhibited β-xylosidase activity toward the chromogenic substrate p-nitrophenyl-β-D-xylopyranoside, and α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside, indicating their bifunctionality. By testing their degradation characteristics on different natural substrates, it was found that both Ppxyl43A and Ppxyl43B showed similar degradation ability on CCX and OSX. Both enzymes could hydrolyze xylohexaose and xylobiose completely to xylose, but could not hydrolyze BWX and AX, suggesting they mainly hydrolyze xylo-oligosaccharides by β-xylosidase activity. Further analysis showed that both of them displayed very high pH stability and thermostability on the β-xylosidase activity, but Ppxy143B exhibited wider pH and temperature ranges, higher pH and temperature stability, was less influenced by metal ions, and had a slower start-up response than Ppxyl43A. Given their predicted structure, it is likely that the enzymatic differences between Ppxyl43A and Ppxyl43B might be related to the extra C-terminus domain (GH43_C2) in Ppxyl43B, which could enhance the enzymatic stability while restricting the substrates' or metal ions' access to the active sites of Ppxyl43B. In conclusion, both Ppxyl43A and Ppxyl43B were β-xylosidase/α-L-arabinofuranosidase bifunctional enzymes and might be useful in xylan biomass conversion, especially in the hydrolysis of xylo-oligosaccharides into xylose.
木聚糖是木质纤维素生物质中主要的聚合半纤维素成分之一，微生物对其的有效利用对于生物燃料的经济生产至关重要。在这项研究中，巨大芽孢杆菌 XB 对不同的底物（玉米芯木聚糖（CCX）、燕麦黑麦木聚糖（OSX）、小麦面粉阿拉伯木聚糖（AX）和山毛榉木聚糖（BWX））表现出不同的木聚糖降解能力。RT-QPCR 结果表明，属于糖苷水解酶家族 43 的两个基因（Pph_0602 和 Pph_2344）在 CCX 和木糖上的表达上调了 5 倍以上。用纯化蛋白 Ppxyl43A（Pph_0602）和 Ppxyl43B（Pph_2344）进行的底物特异性测定表明，这两种蛋白都对显色底物对硝基苯-β-D-木吡喃糖苷具有β-木糖苷酶活性，对 p-硝基苯-α-L-阿拉伯呋喃糖苷具有α-L-阿拉伯呋喃糖苷酶活性，表明它们具有双功能。通过测试它们在不同天然底物上的降解特性，发现 Ppxyl43A 和 Ppxyl43B 对 CCX 和 OSX 具有相似的降解能力。两种酶都能完全水解木六糖和木二糖生成木糖，但不能水解 BWX 和 AX，表明它们主要通过β-木糖苷酶活性水解木寡糖。进一步分析表明，两种酶在β-木糖苷酶活性方面均具有很高的 pH 稳定性和热稳定性，但 Ppxy143B 具有更宽的 pH 和温度范围、更高的 pH 和温度稳定性，受金属离子的影响较小，且启动反应比 Ppxyl43A 慢。鉴于它们的预测结构，Ppxyl43A 和 Ppxyl43B 之间的酶差异可能与 Ppxyl43B 中额外的 C 末端结构域（GH43_C2）有关，该结构域可以增强酶的稳定性，同时限制底物或金属离子进入 Ppxyl43B 的活性位点。综上所述，Ppxyl43A 和 Ppxyl43B 都是β-木糖苷酶/α-L-阿拉伯呋喃糖苷双功能酶，可能在木聚糖生物质转化中有用，特别是在将木寡糖水解成木糖方面。
