Significantly improving the thermostability of a hyperthermophilic GH10 family xylanase XynAF1 by semi-rational design
Significantly improving the thermostability of a hyperthermophilic GH10 family xylanase XynAF1 by semi-rational design
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通过半理性设计显着提高超嗜热 GH10 家族木聚糖酶 XynAF1 的热稳定性
DOI：
10.1007/s00253-021-11340-9
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发表时间：
2021-05-20
影响因子：
5
通讯作者：
Zhang, Ruifu
中科院分区：
文献类型：
--
作者：
Li, Guangqi;Zhou, Xuan;Zhang, Ruifu
木聚糖酶在工业生物技术中有着广泛的应用，这就要求酶能抵抗高温环境。大多数木聚糖酶在中温条件下具有最大酶活，这限制了其在工业上的应用。本研究对高温堆肥菌株Aspergillus fumigatusZ 5的嗜热GH 10家族木聚糖酶XynAF 1进行了表征和工程改造，以进一步提高其热稳定性。XynAF 1的最佳反应温度为90 °C。在异源表达、纯化和结晶后，通过X射线衍射获得XynAF 1的晶体结构。通过用木四糖浸泡脱辅基态晶体获得蛋白质产物复合物的高分辨率X射线晶体学结构。结构分析表明XynAF 1具有刚性骨架，这有助于保持超嗜热特性。同源结构分析和催化中心突变体的构建表明，XynAF 1的催化中心保守，导致其最适催化温度较高。通过结构分析确定了木聚糖酶XynAF 1表面可能影响酶热稳定性的氨基酸。将合理设计与高B值区域的饱和突变相结合，最终获得了热稳定性提高6倍的整合突变体XynAF 1-AC。本研究通过半理性设计有效提高了GH 10家族木聚糖酶的热稳定性，为高温生物技术应用提供了一种新型的生物催化剂。关键点·获得了GH 10家族超嗜热木聚糖酶XynAF 1的晶体结构。·阐明对GH 10家族木聚糖酶嗜热机制的理解。·构建了热稳定性提高6倍的重组木聚糖酶。
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 strainAspergillus fumigatusZ5 was characterized and engineered to further improve its thermostability. XynAF1 has the optimal reaction temperature of 90 °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.Key points• Obtained the crystal structure of GH10 family hyperthermophilic xylanase XynAF1.• Shed light on the understanding of the GH10 family xylanase thermophilic mechanism.• Constructed a 6-fold increased thermostability recombinant xylanase.