Thermally stable and acidic pH tolerant mutant phytases with high catalytic efficiency from Yersinia intermedia for potential application in feed industries

Heat- and pH-stable phytase efficiently hydrolyzes phytic acid. In this research, heat- and pH-stable mutant phytases, (TR)-R-83, (LR)-R-287, and (TR)-R-83/(LR)-R-287 were generated by site-directed mutagenesis from Yersinia intermedia. After the induction and expression of recombinant wild-type and mutant phytases in E. coli BL21, the enzymes were purified using nickel sepharose affinity chromatography, and characterized kinetically and thermodynamically using spectroscopy methods. The mutants showed optimum activity at pH 5.15 and 55-61 degrees C. The catalytic efficiencies of (TR)-R-83, (LR)-R-287, (TR)-R-83/(LR)-R-287, and wild-type phytases were calculated to be 2941, 29346, 4906, and 6917 mmol/L(-1)s(-1), respectively. Moreover, after the incubation of (TR)-R-83, (LR)-R-287, wild-type, and (TR)-R-83/ (LR)-R-287 phytases at 100 degrees C for 1 h, the enzymes retained 22, 5, 4, and 2% of their initial activities, respectively. In addition, (TR)-R-83, (TR)-R-83/(LR)-R-287, (LR)-R-287, and wild-type phytases retained 82, 44, 16 as well as 11% of their initial activities after 1 h at pH 5.15, respectively. Among these mutants, (TR)-R-83 mutant showed 18% increase in thermal stability, 71% increase in pH stability, and +0.103 KJ/mole increase in Delta Delta G, while the catalytic efficiency and Delta Delta G value of (LR)-R-287 mutant increased by 4 times and +0.0903 KJ/mole, respectively. Thus, the mutants have the potential to be used in feed industries to increase the bioavailability of minerals while decreasing soil and water pollution.

Automated summary

In this study, heat- and pH-stable mutant phytases were generated using site-directed mutagenesis and their characteristics were analyzed. The mutants showed optimum activity at specific pH and temperature, and exhibited good thermal and pH stability. They also demonstrated high catalytic efficiency, making them suitable for application in the feed industry to increase mineral bioavailability and reduce soil and water pollution.