Highly Active F260R/T312R Double Mutant Phytase from Yersinia Intermedia for the Efficient Hydrolysis of Phytic Acid

Phytase hydrolyzes phytic acid. Phytic acid is the main form of phosphorus storage in plant seeds in cereals, legumes, and oilseeds. In addition, phytic acid chelates essential minerals and binds to amino acids and proteins and prevents the action of digestive enzymes leads to decrease in the digestibility of proteins. Monogastric animals are unable to hydrolyze phytic acid due to the lack of phytase in their digestive system. In the current study, based on the docking results and DynaMut webserver predictions, T312R and F260R mutants, were chosen. Then, using Quick-Change PCR, the two residues in the active site of phytase from Yersinia intermedia phytase were mutated to Arg. Moreover, a double mutant F260R/T312R was generated. Afterwards, the recombinant mutant and wild-type phytases were expressed in bacterial host, purified and their activities were measured. According to the results, the activities of T312R and F260R almost remained unchanged; however, that of T312R/F260R was increased about 2.35 folds compared with the wild-type. This indicated that incorporation of two positively-charged arginine residues in the active site led to more interactions of these residues with the negatively charged-rich substrate and increased the phytase activity. In conclusion, highly active T312R/F260R double mutant can be used as feed additive in feed industries.