Achieving thermostability of a phytase with resistance up to 100 ° C

文献类型： 外文期刊

第一作者： Tu, Tao

作者： Tu, Tao;Wang, Qian;Dong, Ruyue;Tian, Jian;Wang, Yaru;Luo, Huiying;Yao, Bin;Huang, Huoqing;Liu, Xiaoqing;Zhang, Wei;Penttinen, Leena;Hakulinen, Nina

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期刊名称：JOURNAL OF BIOLOGICAL CHEMISTRY （ 影响因子：3.9； 五年影响因子：4.3 ）

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年卷期： 2024 年 300 卷 12 期

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收录情况： SCI

摘要： The development of enzymes with high-temperature resistance up to 100 degrees C is of significant and practical value in advancing the sustainability of industrial production. Phytase, a crucial enzyme in feed industrial applications, encounters challenges due to its limited heat resistance. Herein, we employed rational design strategies involving the introduction of disulfide bonds, free energy calculation, and B-factor analysis based on the crystal structure of phytase APPAmut4 (1.90 & Aring;), a variant with enhanced expression levels derived from Yersinia intermedia, to improve its thermostability. Among the 144 variants experimentally verified, 29 exhibited significantly improved thermostability with higher t 1/2 values at 65 degrees C. Further combination and superposition led to APPAmut9 with an accumulation of fi ve additional pairs of disulfide bonds and six single-point mutation sites, leading to an enhancement in its thermostability with a t 1/2 value of 256.7 min at 65 degrees C, which was more than 75-fold higher than that of APPAmut4 (3.4 min). APPAmut9 exhibited a T 50 value of 96 degrees C, representing a substantial increase of 40.9 degrees C compared to APPAmut4. Notably, approximately 70% of enzyme activity remained intact after exposure to boiling water at 100 degrees C for a holding period of 5 min. Significantly, these advantageous modifications were strategically positioned away from the catalytic pocket where enzymatic reactions occur to ensure minimal compromise on catalytic efficiency between APPAmut9 (11,500 +/- 1100/mM/s) and APPAmut4 (12,300 +/- 1600/mM/s). This study demonstrates the feasibility of engineering phytases with resistance to boiling using rational design strategies.

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