Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp Strain F32 in Thermostability and Catalytic Efficiency
Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp Strain F32 in Thermostability and Catalytic Efficiency
复制标题
Caldicellulosiruptor sp 的糖苷水解酶 10 (GH10) 和 GH11 木聚糖酶的碳水化合物结合模块的不同作用。
DOI：
10.1128/aem.03677-14
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发表时间：
2015-03-01
影响因子：
4.4
通讯作者：
Li, Fu-Li
中科院分区：
文献类型：
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作者：
Meng, Dong-Dong;Ying, Yu;Li, Fu-Li
木聚糖酶对于木质纤维素生物质的解构是至关重要的，并且通常含有接近可降解聚合物的非催化碳水化合物结合模块（CBM）。了解多模块酶如何组装可以通过旨在适应各种环境条件而有益于蛋白质工程。已从热纤维素酶属菌株F32中鉴定出两种多模块木聚糖酶XynA和XynB，它们分别属于糖苷水解酶家族11（GH 11）和GH 10。在这项研究中，木聚糖酶及其截短突变体在大肠杆菌中过表达，纯化，并进行了表征。与XynA相比，缺乏CBM的GH 11 XynATM 1在比活性（215.8 U nmol（-1）对94.7 U nmol（-1））和热稳定性（在75 ℃下半衰期为48 h对5.5 h）方面表现出相当大的改善。然而，GH 10 XynB表现出更高的酶活性和热稳定性比其截短的突变体没有CBM。N-末端氨基酸的定点突变导致突变体XynATM 1-M，在75 ℃下48 h具有50%的残余活性改善，揭示了无序区域对蛋白质热稳定性产生负面影响。两种木聚糖酶及其截短突变体的热稳定性与其熔融温度（Tm）一致，这是通过使用差示扫描量热法测定。通过同源性建模和交联分析，我们证明，XynB，对热灭活的阻力一般是通过提高域的性质和域间的相互作用，而XynA，没有观察到域间的相互作用增强。优化的分子内相互作用可以加速热稳定性，这为微生物提供了强大的进化策略来组装适应各种生态条件的催化剂。
Xylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol(-1) versus 94.7 U nmol(-1)) and thermal stability (half-life of 48 h versus 5.5 h at 75 degrees C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75 degrees C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (T-m), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions.