Thermally stable and acidic pH tolerant mutant phytases with high catalytic efficiency from Yersinia intermedia for potential application in feed industries
2022
Abbasi Kheirabadi, Marjan | Saffar, Behnaz | Hemmati, Roohullah | Mortazavi, Mojtaba
Heat- and pH-stable phytase efficiently hydrolyzes phytic acid. In this research, heat- and pH-stable mutant phytases, T⁸³R, L²⁸⁷R, and T⁸³R/L²⁸⁷R 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 °C. The catalytic efficiencies of T⁸³R, L²⁸⁷R, T⁸³R/L²⁸⁷R, and wild-type phytases were calculated to be 2941, 29346, 4906, and 6917 mmol/L⁻¹s⁻¹, respectively. Moreover, after the incubation of T⁸³R, L²⁸⁷R, wild-type, and T⁸³R/ L²⁸⁷R phytases at 100 °C for 1 h, the enzymes retained 22, 5, 4, and 2% of their initial activities, respectively. In addition, T⁸³R, T⁸³R/L²⁸⁷R, L²⁸⁷R, 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, T⁸³R mutant showed 18% increase in thermal stability, 71% increase in pH stability, and +0.103 KJ/mole increase in ΔΔG, while the catalytic efficiency and ΔΔG value of L²⁸⁷R 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.
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