The development of enzymes with high-temperature resistance up to 100 °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 Å), 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 t1/2 values at 65 °C. Further combination and superposition led to APPAmut9 with an accumulation of five additional pairs of disulfide bonds and six single-point mutation sites, leading to an enhancement in its thermostability with a t1/2 value of 256.7 min at 65 °C, which was more than 75-fold higher than that of APPAmut4 (3.4 min). APPAmut9 exhibited a T50 value of 96 °C, representing a substantial increase of 40.9 °C compared to APPAmut4. Notably, approximately 70% of enzyme activity remained intact after exposure to boiling water at 100 °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.

中文翻译：

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实现植酸酶的热稳定性，耐温高达 100 °C


开发耐高温高达 100 °C 的酶对于促进工业生产的可持续性具有重要的实用价值。植酸酶是饲料工业应用中的重要酶，由于其耐热性有限而面临挑战。在此，我们采用了合理的设计策略，包括引入二硫键、自由能计算和基于植酸酶 APPAmut4 （1.90 Å） 晶体结构的 B 因子分析，植酸酶 APPAmut4 （1.90 Å） 是一种源自中间耶尔森菌的表达水平增强的变体，以提高其热稳定性。在实验验证的 144 种变体中，有 29 种表现出显著改善的热稳定性，在 65 °C 时具有更高的 t1/2 值。 进一步的结合和叠加导致 APPAmut9 积累了 5 对额外的二硫键和 6 个单点突变位点，导致其热稳定性增强，在 65 °C 时 t1/2 值为 256.7 min，比 APPAmut4 （3.4 min） 高 75 倍以上。APPAmut9 的 T50 值为 96 °C，与 APPAmut4 相比大幅提高了 40.9 °C。值得注意的是，大约 70% 的酶活性在 100 °C 的沸水中保持 5 分钟后保持完整。值得注意的是，这些有利的修饰被战略性地放置在远离发生酶促反应的催化口袋的位置，以确保 APPAmut9 （11,500 ± 1100/mM/s） 和 APPAmut4 （12,300 ± 1600/mM/s） 之间的催化效率受到最小的损害。本研究证明了使用合理的设计策略设计具有抗沸腾性的植酸酶的可行性。