Structural Basis And Rational Design Of Thermostability Of Yersinia Intermedia Phytase APPAmut4

As the core enzyme in feed enzymes,phytase is of great value to improve the utilization rate of phosphorus in feed and the production performance of animals.In view of the process requirements of instantaneous high temperature in feed pelleting,thermostability has become one of the bottleneck problems restricting the industrial application of phytase.phytase APPA from Yersinia intermedia with high activity under acidic p H conditions and good resistance to pepsin and trypsin has potential application in feed industry.In the previous work,the mutant phytase APPAmut4 with significantly increased expression was obtained based on directed evolution,which greatly reduced the production cost of the enzyme.The half-life t_1/2 of APPAmut4 at 65°C is 3.4 min,and the temperature of half-inactivation T₅₀³⁰ is 55.1°C,which is far from the requirements of feed industrial application.First,the crystal structure of APPAmut4 was analyzed with a resolution of 1.90（?）.The enzyme belongs to histidine acid phosphatases（HAP）family,which exists in the form of monomer and consists of two domains:conservedα/βdomain and variableαdomain.The active site is located in the middle of the two domains,and its eight cysteines form four highly conserved disulfide bonds to maintain the structural stability.It provides a theoretical basis for the rational design of thermostability based on structure.Then,based on the structure of APPAmut4,three rational design strategies are used to study its thermostability.First,the thermostability of APPAmut4 was rationally designed by using the disulfide bond introduction.By calculating the parameters such as energy change,adverse contact,residue depth and steric hindrance after virtual mutation of all amino acids into cysteine in APPAmut4,19 mutants were designed combined with the bond length and angle of disulfide bond.The results showed that the thermostability of 7 mutants was significantly improved,the half-life t_1/2 at 65°C was increased by2.1-15.2 min,and T₅₀³⁰ was increased by 0.5-5.8°C.The mutant APPAmut5 with further improved thermostability and the equivalent catalytic efficiency was obtained by combining the above mutation sites.The T₅₀³⁰ and t_1/2 at 65°C were increased to 81.1°C and 192.5 min respectively,26.0°C and 56.6times higher than that of APPAmut4.By unfolding free energy（ΔG）analysis found that theΔG of APPAmut5 is-20.63 kcal/mol,which is significantly lower than that of APPAmut4（-5.78 kcal/mol）,which makes the protein structure more stable.Second,the calculation of unfolding free energy was used to design the thermostability of APPAmut4.Using Fold X、Deep DDG、CUPSAT and Hot Spot Wizard 3.0 to calculate and analyze the unfolding free energy of APPAmut4,105 mutants that energy decreased were designed and verified by experiments.The results showed that the thermostability of 15 mutants from 9 sites was significantly improved,the half-life t_1/2 at 65°C was increased by 0.4-5.7 min,and T₅₀³⁰ was increased by 0.5-5.4°C.The mutant APPAmut6 with further improved thermostability was obtained by combining the above mutation sites.The t_1/2 at 65°C was increased to 35.5 min,10 times higher than that of APPAmut4,which catalytic efficiency is unchanged.Through the analysis of local force,the enhancement of hydrophobic force and the increase of hydrogen bond number are the main reasons for the improvement of thermostability.Third,the thermostability of APPAmut4 was designed by coevolutionary information analysis.GREMLIN-LH model was used to extract the contact information of key residues in the evolution of APPAmut4 sequence,and Hamiltonian parameters were introduced to evaluate the thermostability.20mutants were designed and verified by experiments.The thermostability of 7 mutants were improved,the t_1/2 at 65°C was increased by 0.6-4.1 min,and T₅₀³⁰ was increased by 0.6-5.0°C.The combination mutant APPAmut7 which catalytic efficiency undamaged was obtained,the T₅₀³⁰ and t_1/2 at 65°C were increased to 71.7°C and 38.7 min respectively,16.6°C and 11.6 times higher than that of APPAmut4.Combined with the structure analysis,by optimizing the amino acid pairs in the coevolution process,the hydrophobic interaction network can be significantly enhanced,resulting in the improvement of the thermostability of phytase.Finally,the mutant APPAmut9 with significantly improved thermostability was obtained by iterative combination mutation of the above three strategies without loss of activity.The T₅₀³⁰ and t_1/2 at 65°C were increased to 96.0°C and 256.7 min respectively,40.9°C and 75.5 times higher than that of APPAmut4.APPAmut9 still has 70%residual activity after heating at 100°C for 5 min,exhibiting excellent advantages in thermostability,which laid a foundation for its application in feed industry.The results of molecular dynamics simulation showed that the RMSD of APPAmut9 is significantly lower than that of APPAmut4,indicating that the improvement of thermostability was due to the enhancement of overall structural rigidity.In addition,the mechanism of N-glycosylation site modification on the thermostability of phytase was studied.The sequence set was obtained by homologous sequence collection.The number and location of N-glycosylation site motif NXT/S were counted.13 mutants were designed and verified by experiments.The results showed that the thermostability of 7 mutants was significantly improved.Furthermore,the mutant M14 with the equal catalytic efficiency as wild-type was obtained by combined mutation,and its t_1/2 was increased to 25.0 min at 100°C.Differential scanning calorimetry analysis showed that the kinetic stability of mutant M14 was significantly improved due to its strong refolding ability.Phytase APPAmut4 from Y.intermedia has been used as the main research material in this study,and its crystal structure has been successfully analyzed to provide a structural basis for subsequent rational design.After that,many strategies such as disulfide bond introduction,energy calculation,coevolutionary analysis and N-glycosylation site modification were used to improve its thermostability,and the mutants with significantly improved thermostability were obtained,which enriched the varieties of phytase in industrial production.In addition,the molecular mechanism of mutants with improvement of thermostability was clarified,and the relationship between structure and thermostability was explored,which provided a theoretical basis for the modification of similar enzymes.