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

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 t_1/2 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 t_1/2 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.