TY - JOUR ID - 247331 TI - Highly Active F260R/T312R Double Mutant Phytase from Yersinia Intermedia for the Efficient Hydrolysis of Phytic Acid JO - Biomacromolecular Journal JA - BMMJ LA - en SN - 7280-2423 AU - Ghorbani, Mansoureh AU - Hemmati, Roohullah AU - Saffar, Behnaz AU - Mortezavi, Mojtaba AD - Department of Biology, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran AD - Department of Biology, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran. Biotechnology Research Institute, Shahrekord University, Shahrekord, Iran AD - Department of Genetics, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran AD - Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran Y1 - 2020 PY - 2020 VL - 6 IS - 2 SP - 147 EP - 154 KW - Phytic acid KW - Phytase KW - Targeted mutagenesis KW - Quickchange KW - Kinetics KW - Catalytic efficiency DO - N2 - 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. UR - https://www.bmmj.org/article_247331.html L1 - https://www.bmmj.org/article_247331_2950cd06232eeefe6ee9d8cda60f8169.pdf ER -