Urease activity was determined by using the method of Askin and Kizikaya with few modifications. Different enzyme concentrations including 1, 2, 4, 6, 8, and 10 IU were added into a specific amount of citrate buffer to each tube to make the volume 1 mL. Then 150 µL toluene was to each tube and incubated the sample for 15 min at 37 ℃. Then 1mL urea and 2 mL citrate buffer were poured into each test tube and again incubated the samples for 3 h at 37 ℃. After the incubation time, the samples were filtered using Whatman No. 1 filter paper and took 1 mL filtrate from each tube and transfer to other tubes and added 100 µL H2SO4 to each tube and placed on ice for 10 min. After 10 min, the samples were removed from the ice, and 4 mL sodium phenolate and 3 mL sodium hypochlorite solutions were added to each tube and incubated for 15 min at 37 ℃. Absorbance was measured at 580 nm using a UV-Vis spectrophotometer (V-730). The urease hydrolysis activity was calculated from the standard curve of N corresponding to the difference in optical density between the sample and the reagent blank .