School of Biotechnology and Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
J Sci Food Agric. 2013 Sep;93(12):3016-23. doi: 10.1002/jsfa.6134. Epub 2013 Apr 19.
Xylanases have attracted much attention owing to their potential applications. The applicability of xylanases, however, was bottlenecked by their low stabilities at high temperature or extreme pH. The purpose of this work was to enhance the thermostability of a mesophilic xylanase by N-terminal replacement.
The thermostability of AoXyn11, a mesophilic family 11 xylanase from Aspergillus oryzae, was enhanced by replacing its N-terminal segment with the corresponding one of EvXyn11(TS) , a hyperthermotolerant family 11 xylanase. A hybrid xylanase with high thermostability, NhXyn11⁵⁷, was predicted by molecular dynamics (MD) simulation. An NhXyn11⁵⁷-encoding gene, Nhxyn11⁵⁷, was then constructed as designed theoretically, and overexpressed in Pichia pastoris. The temperature optimum of recombinant NhXyn11⁵⁷ (re-NhXyn11⁵⁷) was 75 °C, much higher than that of re-AoXyn11. Both xylanases were thermostable at 65 and 40 °C, respectively. Additionally, the pH optimum and stability of re-NhXyn11⁵⁷ were 5.5 and at a range of 4.0-8.5. Its activity was not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²⁺ and Ag⁺.
This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn11⁵⁷ a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes.
木聚糖酶由于其潜在的应用而受到广泛关注。然而，木聚糖酶的适用性受到其在高温或极端 pH 值下低稳定性的限制。本工作旨在通过 N 端替换来提高中温嗜热木聚糖酶的热稳定性。
通过用嗜热菌 Aspergillus oryzae 来源的耐热型 11 家族木聚糖酶 EvXyn11(TS)的 N 端取代中温嗜热木聚糖酶 AoXyn11 的 N 端，提高了其热稳定性。通过分子动力学（MD）模拟预测了具有高耐热性的杂合木聚糖酶 NhXyn11⁵⁷。然后，根据理论设计构建了一个编码 NhXyn11⁵⁷的基因 Nhxyn11⁵⁷，并在毕赤酵母中过表达。重组 NhXyn11⁵⁷（re-NhXyn11⁵⁷）的最适温度为 75°C，远高于 re-AoXyn11。两种木聚糖酶在 65 和 40°C 下均具有热稳定性。此外，re-NhXyn11⁵⁷的最适 pH 值和稳定性分别为 5.5 和 4.0-8.5。其活性不受测试的金属离子和 EDTA 的显著影响，但强烈抑制 Mn²⁺和 Ag⁺。
本工作明显提高了中温嗜热木聚糖酶的热稳定性，使 re-NhXyn11⁵⁷成为工业过程的有前途的候选者。它还为提高其他中温酶的热稳定性提供了一种有效的技术策略。
