0). 0.1 ml of the appropriate dilution was plated, in triplicate, on Luria agar and incubated overnight at 28°C. The number of viable bacteria was recorded at different intervals and CFU/ml was calculated. The log10CFU/ml was plotted against incubation time (in h). For preparing lysate, cells grown in 50 ml LB learn more medium were harvested by centrifugation, washed twice and resuspended in 2.5 ml of 20 mM sodium phosphate buffer (pH 7.0). Cells were LY3023414 price disrupted by sonication with three cycles (2
s “”pulse on”" and 2 s “”pulse off”" for 2 min) at 25% intensity with Vibra-Cell (Sonics). The cell lysate was centrifuged at 18,000 × g for 30 min at 4°C to obtain cell-free extract. The supernatant was transferred to pre-chilled microcentrifuge tubes and used immediately for determination of urease activity. Protein concentration was estimated by Bradford [31] method using bovine serum albumin (Sigma) as standard. Urease assay Urease activity in the cell extract was assayed by measuring release of ammonia from urea in the phenol-hypochlorite assay [32]. Briefly, extract containing 2 μg of protein was added to 100 mM citrate buffer (pH 5.5) containing 50 mM urea in 200 μl of final volume. The mixture was incubated at 37°C for 15 min. A similar volume of the extract boiled for 10 min
served as negative control. The reaction was terminated by the addition of 1.5 ml of solution containing 1% phenol and 0.005% sodium nitroprusside; this was followed by the addition of 1.5 ml solution containing 0.5% (w/v) NaOH and 0.044% (v/v) NaClO, Gemcitabine purchase and the contents were mixed well. Following incubation at 37°C for 30 min, the absorbance was measured at
625 nm using a spectrophotometer (UV-1700 Pharmaspec; Shimadzu Scientific Instruments Inc., Columbia, Md.). Assays were carried out in triplicate and the amount of the ammonia released per minute was determined. The quantity of ammonia (in nmol) released was calculated from the calibration curve obtained from appropriate dilutions of freshly prepared NH4Cl solution, which was determined to be linear between 20-500 nmol. Data are presented as Methisazone specific activity of urease, defined as μmol of NH3/min/mg of protein. Stated values are the mean ± standard deviation of triplicate determinations. Biochemical characterization The optimum pH for urease was determined by measuring activity at pH 1.5 to 7.5. The assays were carried out in 20 mM sodium phosphate (for pH 1.5, 2.5, 5.5, 6.0, 6.5, 7.0 and 7.5) and 100 mM citrate (for pH 3.0, 3.5, 4.0 and 5.5) buffers. The optimum temperature for urease was determined by incubating the extract containing enzyme with substrate at different temperatures (18-75°C) in the phenol-hypochlorite assay described above. The kinetic data (Km and Vmax) of urease were calculated from Lineweaver-Burk plot of the initial rate of hydrolysis of urea in citrate buffer (100 mM, pH 5.5).