Liu Jiawen, Zhu Jingrong, Xu Qian, Shi Rui, Liu Cong, Sun Di, Liu Weijie
Jiangsu Key Laboratory of Phylogenomics & Comparative Genomics, School of Life Science, Jiangsu Normal University, No.101, Shanghai Road, Tongshan New District, Xuzhou, 221116, Jiangsu, China.
Biotechnol Biofuels Bioprod. 2023 Mar 8;16(1):40. doi: 10.1186/s13068-023-02290-7.
Glycoside hydrolase (GH) family 30 xylanases are a distinct group of xylanases, most of which have a highly specific catalytic activity for glucuronoxylan. Since GH30 xylanases do not normally carry carbohydrate-binding modules (CBMs), our knowledge of the function of their CBMs is lacking.
In this work, the CBM functions of CrXyl30 were investigated. CrXyl30 was a GH30 glucuronoxylanase containing tandem CBM13 (CrCBM13) and CBM2 (CrCBM2) at its C terminus, which was identified in a lignocellulolytic bacterial consortium previously. Both CBMs could bind insoluble and soluble xylan, with CrCBM13 having binding specificity for the xylan with L-arabinosyl substitutions, whereas CrCBM2 targeted L-arabinosyl side chains themselves. Such binding abilities of these two CBMs were completely different from other CBMs in their respective families. Phylogenetic analysis also suggested that both CrCBM13 and CrCBM2 belong to novel branches. Inspection of the simulated structure of CrCBM13 identified a pocket that just accommodates the side chain of 3(2)-alpha-L-arabinofuranosyl-xylotriose, which forms hydrogen bonds with three of the five amino acid residues involved in ligand interaction. The truncation of either CrCBM13 or CrCBM2 did not alter the substrate specificity and optimal reaction conditions of CrXyl30, whereas truncation of CrCBM2 decreased the k/K value by 83% (± 0%). Moreover, the absence of CrCBM2 and CrCBM13 resulted in a 5% (± 1%) and a 7% (± 0%) decrease, respectively, in the amount of reducing sugar released by the synergistic hydrolysis of delignified corncob whose hemicellulose is arabinoglucuronoxylan, respectively. In addition, fusion of CrCBM2 with a GH10 xylanase enhanced its catalytic activity against the branched xylan and improved the synergistic hydrolysis efficiency by more than fivefold when delignified corncob was used as substrate. Such a strong stimulation of hydrolysis resulted from the enhancement of hemicellulose hydrolysis on the one hand, and the cellulose hydrolysis is also improved according to the lignocellulose conversion rate measured by HPLC.
This study identifies the functions of two novel CBMs in CrXyl30 and shows the good potential of such CBMs specific for branched ligands in the development of efficient enzyme preparations.
糖苷水解酶（GH）家族30木聚糖酶是一类独特的木聚糖酶，其中大多数对葡糖醛酸木聚糖具有高度特异性的催化活性。由于GH30木聚糖酶通常不携带碳水化合物结合模块（CBM），我们对其CBM功能的了解尚缺。
在本研究中，对CrXyl30的CBM功能进行了研究。CrXyl30是一种GH30葡糖醛酸木聚糖酶，在其C末端含有串联的CBM13（CrCBM13）和CBM2（CrCBM2），此前在一个木质纤维素分解细菌群落中鉴定得到。这两种CBM均可结合不溶性和可溶性木聚糖，CrCBM13对带有L - 阿拉伯糖基取代的木聚糖具有结合特异性，而CrCBM2则靶向L - 阿拉伯糖基侧链本身。这两种CBM的这种结合能力与它们各自家族中的其他CBM完全不同。系统发育分析还表明，CrCBM13和CrCBM2均属于新的分支。对CrCBM13模拟结构的检查发现了一个刚好容纳3(2)-α-L-阿拉伯呋喃糖基-木三糖侧链的口袋，该侧链与参与配体相互作用的五个氨基酸残基中的三个形成氢键。CrCBM13或CrCBM2的截短均未改变CrXyl30的底物特异性和最佳反应条件，而CrCBM2的截短使k/K值降低了83%（±0%）。此外，缺失CrCBM2和CrCBM13分别导致以阿拉伯糖基葡糖醛酸木聚糖为半纤维素的脱木质素玉米芯协同水解释放的还原糖量减少5%（±1%）和7%（±0%）。此外，将CrCBM2与GH10木聚糖酶融合可增强其对分支木聚糖的催化活性，并在以脱木质素玉米芯为底物时将协同水解效率提高了五倍以上。这种对水解的强烈促进一方面源于半纤维素水解的增强，另一方面根据通过HPLC测量的木质纤维素转化率，纤维素水解也得到了改善。
本研究确定了CrXyl30中两种新型CBM的功能，并表明此类对分支配体具有特异性的CBM在开发高效酶制剂方面具有良好潜力。
