Development of some properties of recombinant phosphotriesterase homology protein obtained from thermophilic geobacillus caldoxylosilyticus TK4 by mutation

In this study, supported by TUBITAK (Project number is 109T985), site specific mutations in the G. caldoxylosilyticus TK4 PHP gene cloned into pET-28a(+) vector was performed, mutant genes were expressed in E. coli BL21(DE3)pLysS and the mutant proteins were purified. Protein expression and purification were confirmed by SDS-PAGE. Whereas the enzyme did not have any activity in the presence of PTE substartes; paraoxon, parathion and malathion, the highest activities were observed with pNPA as an esterase substrate. The optimum pH were determined to be 8,0 for TK4PHP, TK4PHPM3-M4, TK4PHPM3-M4-M5 and 8,5 for TK4PHPM3. The recombinant TK4PHP and the mutant TK4PHPM3-M4, TK4PHPM3-M4-M5 enzymes exhibited optimum activity at 50 °C and TK4PHPM3's activity was optimal at 60 °C. Km and Vmax values were calculated as 1.27 mM and 7.59 U/mg protein for TK4PHP, 1.43 mM and 14.53 U/mg protein for TK4PHPM3, 0.59 mM and 3.34 U/mg protein for TK4PHPM3-M4 and 0.46 mM and 4,04 U/mg protein for TK4PHPM3-M4-M5, respectively. When the enzymes were separately incubated at 4 °C in the buffer solutions (pH ranging from 3,0- 9,0) for 7 days, the activity was mainly retained. The TK4PHPM3-M4-M5 mutant enzyme retained about 75% of its original activity after 7 days of incubation at 90 °C. It is clear from the present results that the enzymes, which are mutated from TK4PHP showing an extremely pH- and thermo- stable esterase activity, might have a very important potential for industrial applications.