Enhancing the Thermostability of Phytase to Boiling Point by Evolution-Guided Design

The good thermostability of enzymes is an important basis for their wide application in industry. In this study, the phytase APPA from Yersinia intermedia was designed by evolution-guided design. Through the collection of homologous sequences in the NCBI database, we obtained a sequence set composed of 5,569 sequences, counted the number and locations of motif N-X-T/S, and selected the sites with high frequency in evolution as candidate sites for experiments. Based on the principle that N-glycosylation modification sites are located on the protein surface, 13 mutants were designed to optimize the number and location of N-glycosylation sites. Through experimental verification, 7 single mutants with improved thermostability were obtained. The best mutant, M14, with equal catalytic efficiency as the wild-type was obtained through combined mutation. The half-life (t1/2) value of mutant M14 was improved from 3.32 min at 65°C to 25 min of at 100°C, allowing it to withstand boiling water treatment, retaining approximately 75% initial activity after a 10-min incubation at 100°C. Differential scanning calorimetry analysis revealed that while the mutants' thermodynamic stability was nearly unchanged, their kinetic stability was greatly improved, and the combined mutant exhibited strong refolding ability. The results of a in vitro digestibility test indicated that the application effect of mutant M14 was about 4.5 times that of wild-type APPA, laying a foundation for its industrial application. IMPORTANCE Due to the harsh reaction conditions of industrial production, the relative instability of enzymes limits their application in industrial production, such as for food, pharmaceuticals, and feed. For example, the pelleting process of feed includes a brief high temperature (80 to 85°C), which requires the enzyme to have excellent thermostability. Therefore, a simple and effective method to improve the thermostability of enzymes has important practical value. In this study, we make full use of the existing homologous sequences (5,569) in the database to statistically analyze the existence frequency of N-X-T/S motifs in this large sequence space to design the phytase APPA with improved thermostability and a high hit rate (~50%). We obtained the best combination mutant, M14, that can tolerate boiling water treatment and greatly improved its kinetic stability without damaging its specific activity. Simultaneously, we proved that its performance improvement is due to its enhanced refolding ability, which comes from N-glycan modification rather than amino acid replacement. Our results provide a feasible and effective method for the modification of enzyme thermostability.