Enhancement of the catalytic activity of thermostable Endo-1,4-(3-glucanase B (TnCelB) from Thermotoga neapolitana by error-prone PCR

文献类型： 外文期刊

第一作者： Yuan, Hang

作者： Yuan, Hang;Ajeje, Samaila Boyi;Wen, Yunzhe;Hu, Yun;Wu, Qun;Sun, Fubao;Chio, Chonlong;Qin, Wensheng;Dou, Shaohua;Zhang, Ezhen

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关键词： Endo-1,4-beta-glucanase; Error-prone PCR; Structural model; Enzymatic hydrolysis; Cellulosic-substrate; Cellulosic-substrate

期刊名称：INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES （ 影响因子：8.5； 五年影响因子：8.7 ）

ISSN： 0141-8130

年卷期： 2025 年 308 卷

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收录情况： SCI

摘要： Endo-1,4-beta-glucanase plays a crucial role in converting cellulose from lignocellulosic biomass into fermentable sugars for biofuel production. However, its commercial utility is hindered by poor catalytic performance under extreme conditions. This study enhanced the catalytic activity of the endo-1,4-beta-glucanase (TnCelB) from Thermotoga neapolitana through error-prone PCR directed evolution. After screening >4000 colonies, three mutants with enhanced activity were obtained. Mutants TnCelB(Y88F), TnCelB(A233T), and TnCelB(W219R) displayed 52.14 U/mg, 44.90 U/mg, and 34.70 U/mg of specific activity on CMC, respectively, which is 1.9, 1.7, and 1.3 times higher than that of the wild-type (26.74 U/mg), correspondingly. Likewise, their enzyme activity on barley beta-D-glucan increased by 3.5, 2.2, and 1.8 times, respectively. Notably, TnCelB(Y88F) maintained over 90 % activity after 60 mins at high temperatures (80-100 degrees C), indicating an exceptional thermostability. Protein docking revealed that TnCelB(Y88F) had higher binding affinity, aligned with kinetic studies. TnCelB was capble of released more non-oxidized sugars from the hydrolysis of regenerated amorphous cellulose (RAC) by synergy with auxiliary action family 10 (AA10), which is potential in development of efficient lignocellulosic saccharification. This study can provide useful insights for the future engineering of other endoglucanases in the glycoside hydrolases family 12.

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