Significantly improving the thermostability of a hyperthermophilic GH10 family xylanase XynAF1 by semi-rational design

文献类型： 外文期刊

第一作者： Li, Guangqi

作者： Li, Guangqi;Liu, Yunpeng;Zhang, Ruifu;Li, Guangqi;Liu, Dongyang;Miao, Youzhi;Zhang, Ruifu;Zhou, Xuan;Li, Zhihong;Wan, Qun;Wan, Qun

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关键词： GH10 family xylanase; Enzyme catalysis; X-ray crystallography; Saturation mutagenesis; Thermostability

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

ISSN： 0175-7598

年卷期： 2021 年 105 卷 11 期

页码：

收录情况： SCI

摘要： Xylanases have a broad range of applications in industrial biotechnologies, which require the enzymes to resist the high-temperature environments. The majority of xylanases have maximum activity at moderate temperatures, which limited their potential applications in industries. In this study, a thermophilic GH10 family xylanase XynAF1 from the high-temperature composting strain Aspergillus fumigatus Z5 was characterized and engineered to further improve its thermostability. XynAF1 has the optimal reaction temperature of 90 degrees C. The crystal structure of XynAF1 was obtained by X-ray diffraction after heterologous expression, purification, and crystallization. The high-resolution X-ray crystallographic structure of the protein-product complex was obtained by soaking the apo-state crystal with xylotetraose. Structure analysis indicated that XynAF1 has a rigid skeleton, which helps to maintain the hyperthermophilic characteristic. The homologous structure analysis and the catalytic center mutant construction of XynAF1 indicated the conserved catalytic center contributed to the high optimum catalytic temperature. The amino acids in the surface of xylanase XynAF1 which might influence the enzyme thermostability were identified by the structure analysis. Combining the rational design with the saturation mutation at the high B-value regions, the integrative mutant XynAF1-AC with a 6-fold increase of thermostability was finally obtained. This study efficiently improved the thermostability of a GH10 family xylanase by semi-rational design, which provided a new biocatalyst for high-temperature biotechnological applications.

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