Many toxic insecticides used worldwide are organophosphates (OPs) derivatives. Phosphotriesterase (PTE) has been rewarding to protect against OP poisoning in vivo or in vitro, associated with advanced catalytic efficiency and stereoselectivity toward the hydrolysis of OPs. Phosphotriesterase homology protein (PHP) exhibits high sequence identity and similarity to PTE. In this study, site-directed mutagenesis on recombinant Geobacillus caldoxylosilyticus TK4 PHP (TK4PHP) was performed for improving the existing esterase activity, even gaining a new PTE activity. After eliminating the deficiencies in the recombinant TK4PHP gene, mutant proteins were purified and characterized biochemically. Considering all the data obtained, it was determined that the major sequence differences between PTE and TK4PHP were removed by three site-directed mutations. However the mutant TK4PHPs did not have PTE activity, it was informed that mutant esterases were more resistance to some metal ions and organic solvents and more thermal stable when it was compared with recombinant type.