Insights into the Catalytic Mechanism of a Novel XynA and Structure-Based Engineering for Improving Bifunctional Activities.
Insights into the Catalytic Mechanism of a Novel XynA and Structure-Based Engineering for Improving Bifunctional Activities.
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深入了解新型 XynA 的催化机制和基于结构的工程以改善双功能活动。
DOI：
10.1021/acs.biochem.1c00134
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发表时间：
2021-06
期刊：
影响因子：
2.9
通讯作者：
Wang Caiyan
中科院分区：
文献类型：
--
作者：
Xie Wei;Yu Qi;Zhang Ruiqing;Liu Yun;Cao Ruoting;Wang Sidi;Zhan Ruoting;Liu Zhongqiu;Wang Kui;Wang Caiyan
木聚糖和纤维素是许多类型的木质纤维素的两个主要成分。具有木聚糖酶/纤维素酶活性的双功能酶在生物燃料生产中引起了极大的关注。此前，芽孢杆菌的一种耐热的GH10家族酶(XynA)。KW1对木聚糖和纤维素均有降解作用。为了在结构的基础上提高XynA的双功能活性，我们首先确定了XynA的晶体结构。通过分子对接和活性分析，我们发现Gln250和His252对于双功能是必不可少的，因为它们可以与两个保守的催化残基Glu182和Glu280相互作用，同时使底物靠近活性口袋。然后，我们采用了基于结构的工程策略来提高木聚糖酶/纤维素酶的活性。虽然经过多次筛选没有获得双功能活性增强的突变体，但我们在XynA的N端36个氨基酸截断中找到了答案。XynA_ΔN36对山毛榉木聚糖、小麦阿拉伯木聚糖、滤纸和大麦β-葡聚糖的活性分别提高了0.47、0.53、2.46和1.04倍。此外，在应用时，这种截断在处理后的玉米秸秆和甘蔗渣的降解中释放出比野生型更多的还原糖。这些结果揭示了GH10家族双功能内切木聚糖酶/纤维素酶的详细分子机制。这些催化性能的基础和筛选出的XynA_ΔN36为XynA在工业上的进一步应用提供了线索。
Xylan and cellulose are the two major constituents of numerous types of lignocellulose. The bifunctional enzyme that exhibits xylanase/cellulase activity has attracted a great deal of attention in biofuel production. Previously, a thermostable GH10 family enzyme (XynA) from Bacillus sp. KW1 was found to degrade both xylan and cellulose. To improve bifunctional activity on the basis of structure, we first determined the crystal structure of XynA at 2.3 Å. Via molecular docking and activity assays, we revealed that Gln250 and His252 were indispensable to bifunctionality, because they could interact with two conserved catalytic residues, Glu182 and Glu280, while bringing the substrate close to the activity pocket. Then we used a structure-based engineering strategy to improve xylanase/cellulase activity. Although no mutants with increased bifunctional activity were obtained after much screening, we found the answer in the N-terminal 36-amino acid truncation of XynA. The activities of XynA_ΔN36 toward beechwood xylan, wheat arabinoxylan, filter paper, and barley β-glucan were significantly increased by 0.47-, 0.53-, 2.46-, and 1.04-fold, respectively. Furthermore, upon application, this truncation released more reducing sugars than the wild type in the degradation of pretreated corn stover and sugar cane bagasse. These results showed the detailed molecular mechanism of the GH10 family bifunctional endoxylanase/cellulase. The basis of these catalytic performances and the screened XynA_ΔN36 provide clues for the further use of XynA in industrial applications.