A novel thermoacidophilic and thermostable endo-beta-1,4-glucanase from Phialophora sp G5: its thermostability influenced by a distinct beta-sheet and the carbohydrate-binding module

文献类型： 外文期刊

第一作者： Zhao, Junqi

作者： Zhao, Junqi;Shi, Pengjun;Huang, Huoqing;Li, Zhongyuan;Yuan, Tiezheng;Yang, Peilong;Luo, Huiying;Bai, Yingguo;Yao, Bin

作者机构：

关键词： Carbohydrate-binding module (CBM);Endo-beta-1,4-glucanase;Phialophora sp G5;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： 2012 年 95 卷 4 期

页码：

收录情况： SCI

摘要： An endo-beta-1,4-glucanase gene, egG5, was cloned from the fungus Phialophora sp. G5. The 1,290-bp open reading frame encodes a bimodular cellulase composed of an N-terminal family 1 carbohydrate-binding module (CBM) and a C-terminal family 5 glycoside hydrolase catalytic module. Recombinant EgG5 produced in Pichia pastoris exhibited maximal activity at pH 4.0-5.0 and 70 A degrees C, retained 40% of the maximal activity at pH 2.0, and was stable at pH 2.0-10.0. When compared with its closest homolog in Trichoderma sp. C-4 (70.6% identity), EgG5 had better thermostability (51.6% activity at 65 A degrees C for 12 h vs 10% activity at 60 A degrees C for 20 min). Sequence-structure analysis indicated that the distinct beta-sheet in EgG5 in place of a linking loop in Trichoderma sp. C-4 endoglucanase might be the reason. To verify its function, two mutants, EgG5-Mut (disrupting the beta-sheet with four amino acid substitutions) and EgG5-CBM (removing the CBM), were constructed, expressed in P. pastoris, and characterized. Both mutants had similar pH optima (pH 4.0) and temperature optima (70 A degrees C) but varied in pH stabilities (pH 2.0-10.0 and pH 2.0-7.0, respectively) and thermostabilities. The thermostability of EgG5-Mut (13.4% activity vs 52.5% of EgG5 at 65 A degrees C for 12 h) confirmed the effect of beta-sheet on enzyme thermostability. EgG5-CBM was more thermostable (94.9% activity at 65 A degrees C for 12 h and 15.5% activity at 80 A degrees C for 30 min) and had higher specific activity (711.6 vs 60.3 U mg(-1) of EgG5). This study presents an excellent endoglucanase with potential use in the bioconversion of lignocellulosic materials and provides good ideas for the improvement of enzyme thermostability.

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