Through the search tool of BLASTP (https://blast.ncbi.nlm.nih.gov/), it is found that five sequences in PDB database have homology with phyS, and the similarity between them is not less than 95%. It is worth mentioning that among the searched homologous sequences, pdbid: 1 POO [11], pdbid: 1 QLG [11], pdbid: 3 AMR [12] and 1 CVM are all 3-phytase, so it’s reasoned that phyS is 3-phytase. Meanwhile, the crystal structures of these four homology proteins were selected as the template Formwork construction. ClustalW, a multi sequence alignment program, was used to perform multi sequence alignment for the above sequences, and the default setting of parameters were selected. Then, based on the prediction results of the secondary structure of DSSP, the results are fine tuned by comparison. The result graph of sequence alignment is completed on the online server of espript 2.2 [13]. The protein structure is displayed by the graphic software PyMOL [14].

The tertiary structure of phyS was constructed by SWISS-MODEL [15]. Using the optimization program and scoring program of modeller to optimize and sort the generated structures, and finally output the structure model in the top 5 in PDB format. In order to avoid the deviation of the results caused by the preliminary scoring, we select PROCHECK [16] and verify 3D [17] to further evaluate the quality of the generated models.

Determination of phytase activity: the method for determination of phytase activity is ferrous sulfate molybdenum blue method [18]. Take 100 μl phyS solution, incubate it in 37˚C water bathed for 5 min, add 400 μl substrate phytic acid sodium solution, and react in 37˚C water bathed for 15 min. Add 500 μl of termination solution to terminate the reaction. Add 500 μl developer, leave it at room temperature for 10 min, make it fully develop color, with the temperature of 37˚C centrifugate it at the speed of 4000 r/min for 10 min, remove some invisible precipitated impurities, and measure the OD value of the solution at 700 nm wavelength. A control experiment was set up. In the control group, 100 μl phytase solution was first added to 500 μl termination solution to inactivate the enzyme, and then the same method was used for subsequent determination.

Determination of the optimum reaction temperature: the enzyme activity of phyS was determined by enzymatic reaction of phyS protein solution in buffer system with pH of 8.0 and at different temperatures (10˚C - 65˚C).

Determination of optimum pH: under the optimum temperature, the solution of phyS was used for enzymatic reaction under the condition of pH 3.0 - 9.0 to determine the enzyme activity of phyS.

Measurement of temperature tolerance: phyS was incubated at different temperatures (20˚C - 65˚C) for different times, and then immediately cooled in ice. The enzyme activity of phyS was measured under the optimum reaction conditions

pH stability: after treating phyS in a series of different pH buffers for 30 min, the enzyme activity of phyS was determined under the optimum temperature and pH condition.

Determination of Michaelis constant and maximum reaction rate (Km value, V_max): prepare different concentrations (0.1 mmol/L - 10.0 mmol/L) of substrate solution, and determine the enzyme activity of phyS under the optimum reaction conditions. The apparent Km and V_max are calculated by the method of line weave burk double reciprocal curve [19].

The recombinant plasmid containing pet-28b-phys stored in the laboratory was sequenced, and the protein sequence was measured to have 373 residues. Color alignment was conducted on clustalw server. From the comparison results (Figure 1), we can see that the sequence identity of phyS with 1PO0, 1QLG, 3AMR and 1CVM was about 95%, and not identity region is mainly concentrated in the first 29 residues at the n-terminal. Therefore, it is effective and credible to construct the tertiary structure of phyS with the above four proteins as templates. Meanwhile, by using the online server of swiss-model, sites of L71, L80, Y83-L85, R76, V101, E187, D211, L227, I258, D288, T279, D336, H342, Q367-N369 and D379 were predicted as the interaction sites of Ca²⁺. The interactions were shown in Figure 2(b).

The tertiary structure of phyS was constructed by using the homologous modeling program SWISS-MODEL [15]. By using the optimization program and scoring program of SWISS-MODEL, the generated structure is preliminarily optimized and sorted, and finally the structure model ranking in the top 5 is output in PDB format. The structure of phyS belongs to five-blade β-propeller family, each blade was formed by four β-strands (Figure 3). In order to avoid the deviation of the results caused by the preliminary scoring, we select PROCHECK [16] and verify 3D [17] to further evaluate the quality of the generated model.

Through the analysis of Ramachandran map (Figure 4(a)) showed that 89.9% of the Ф and ψ corners of the model protein residues are in the best region, 9.1% of the residues fall into other licensing areas, 0.6% fall in the barely licensed areas, 0.4% of the residues fall in the disallowed areas. The above results show that the dihedral angle of module construction is very reasonable. At the same time, using profile_3D (Figure 4(b)), it is not difficult to find that except for V101, the scores of all other residues are greater than that of other residues, and the scores of all other residues are greater than 0, and the average 3D-1D scores of 99.7% of the residues are greater than 0.2, which indicates that the side chain environment is acceptable.