Wang Kui, Cao Ruoting, Wang Meiling, Lin Qibin, Zhan Ruoting, Xu Hui, Wang Sidi
Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine, Guangzhou Higher Education Mega Center, No.232 Outer Ring West Rd., Panyu District, Guangzhou, 510006 Guangdong China.
Key Laboratory of Chinese Medicinal Resource from Lingnan, Ministry of Education, Guangzhou University of Chinese Medicine, Guangzhou Higher Education Mega Center, No.232 Outer Ring West Rd., Panyu District, Guangzhou, 510006 Guangdong China.
Biotechnol Biofuels. 2019 Mar 9;12:48. doi: 10.1186/s13068-019-1389-8. eCollection 2019.
Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals.
A novel GH10 enzyme (XynA) identified from a xylanolytic strain sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65 °C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25 °C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60 °C for 60 h, and its half-lives at 65 °C and 70 °C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, -nitrophenyl-β-d-cellobioside (NPC), and -nitrophenyl-β-d-glucopyranoside (NPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%.
XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.
纤维素和半纤维素是木质纤维素生物质中的两大主要成分。对纤维素和木聚糖具有活性的酶在木质纤维素生物质的生物转化中引起了极大关注，因为它们在提高水解性能和降低酶成本方面具有潜力。探索具有良好热稳定性以及对木聚糖和纤维素有活性的糖苷水解酶（GHs）将有利于生物燃料和生物基化学品的工业生产。
从一株木聚糖分解菌KW1中鉴定出的一种新型GH10酶（XynA）被克隆并表达。其最适pH和温度分别确定为pH 6.0和65℃。稳定性分析表明，XynA在25℃孵育24小时后在较宽的pH范围内（pH 6.0 - 11.0）稳定。此外，XynA在60℃热处理60小时后仍保留超过95%的活性，其在65℃和70℃的半衰期分别约为12小时和1.5小时。更重要的是，就底物特异性而言，XynA对木聚糖、微晶纤维素（滤纸和微晶纤维素）、羧甲基纤维素（CMC）、纤维二糖、对硝基苯基-β-D-纤维二糖苷（NPC）和对硝基苯基-β-D-吡喃葡萄糖苷（NPG）表现出水解活性。此外，在预处理玉米秸秆的水解过程中，向商业纤维素酶中添加XynA导致还原糖释放量显著增加（相对增加可能高达90%）。最后，值得一提的是，XynA与对CMC无活性的GH10木聚糖酶rXynAHJ14仅显示出88%的高氨基酸序列同一性。与其他已表征的GH10酶（包括木聚糖酶和双功能木聚糖酶/纤维素酶）的相似性不超过30%。
XynA是一种新型的具有广泛底物谱的热稳定GH10木聚糖酶。它在较宽的pH范围和高温下表现出良好的稳定性，并且对木聚糖和多种纤维素底物具有活性，这在其他GH10酶中未发现。该酶在预处理玉米秸秆的糖化方面也具有很高的能力。这些特性使XynA不仅是协助纤维素酶进行木质纤维素生物质水解的良好候选者，也是研究双功能木聚糖酶/纤维素酶结构-功能关系的良好候选者。
