Pisum Sativum L. seeds were obtained from faculty of agriculture, Kafr Elshaikh University, Kafrelshaikh city, Egypt. The seeds were soaked in distilled water for 6 hours, germinated in the dark at 22°C for 2, 4, 6, 8, 10, 12, 14 and 16 days. The germinated seeds were stored separately in deep freezer (−20°C) for further experimental purposes. Dextran polymer particles (Sephadex G-200), bovine serum albumin (BSA), standard proteins, and DEAE-cellulose were purchased from Sigma Chemicals Ltd., USA. All other chemicals used for this research were of analytical grade. All absorbance measurements were performed using Lambda 35 PerkinElmer UV/V is spectrometer.
Urease extraction and purification
Unless mentioned otherwise, all of the following procedures were done at 4°C. Ten grams of germinated seeds of pisum sativum were pasted in a mortar and pestle and then suspended in 40 mL of 20% chilled acetone (−20°C). Occasional stirring for 3 h was required. Double layer cheese cloth was used for filtrating of the suspension. After 15 minutes of centrifuging of the filtrate, the supernatant was isolated and used as “crude extract”.
The urease assay was performed as described by Sharma et al. . Enzyme extract (0.25 μL) was added to 10 mL of urea solution (0.4 g urea in 25 ml of phosphate buffer). One millilitre of the previous solution was added to each test tube containing 5 mL of Nessler’s reagent, and incubated at 40°C for 5 min. They were followed by the addition of 1.0 M HCl to terminate the reaction after specific time. Absorbance measurements were taken for the resulting solutions (at 405 nm). The estimation of urease was carried out using the standard curve of ammonium sulphate. One unit of urease activity is defined as “the amount of enzyme required to liberate 1.0 μM of NH3 from urea per minute at pH 7.5 and temperature 40°C” .
Proteins were determined according to Lowry et al.
 using BSA as standard material. Different concentrations of BSA were prepared ranging from (0 to 25) μg/mL. The linear calibration curve was used to determine the concentration of protein in the assay and estimated for the original sample.
The enzyme was purified to homogeneity by the following successive steps which carried out at 4°C:
The “crude extract” was adjusted to 50% saturation by addition of acetone (chilled to −20°C) under constant and gentle stirring. The resulting precipitate was centrifuged, collected, dissolved in minimum volume of pre-cold 50 mM phosphate buffer (pH = 7.4), and finally dialyzed against the same buffer for 24 h. The resulting solution was then centrifuged for 10 min and the clear supernatant was designated as “crude enzyme solution”.
The “crude enzyme solution” was dialyzed against 50 mM phosphate buffer, pH 7.4. It was then loaded on pre-equilibrated DEAE-cellulose column (15 × 3.0 cm) (with 50 mM phosphate buffer, pH 7.8). The bound proteins were eluted with a linear gradient of NaCl (100 – 500 mM), prepared with phosphate buffer, pH 7.8, at a flow rate of 0.5 mL min−1. After collecting the active fractions (the fractions that shows urease activity), the proteins binding to the column were eluted using gradient of (0–0.5 M) KCl and (20 mM) phosphate buffer, pH 7.5. The absorbance of these fractions was measured at wavelength of 280 nm. The active fractions were combined and the volume was measured for the determination of the urease activity and protein contents in the assay.
Gel filtration chromatography
The enzyme obtained from the ion exchange step was concentrated with acetone and loaded on the Sephacryl S-200 column (1.5 × 65) at a flow rate 30 mL/h using 50 mM phosphate buffer, pH = 7.8. Five millilitre eleunts were collected. The enzymatically active fractions were pooled and dialyzed against 50 mM phosphate buffer, pH 7.4 for 24 h. The absorbance measurements at 280 nm were used to determine the protein concentration and urease activity.
The isolated enzyme activity was characterized and studied as a function of pH, temperature, storage period, enzyme concentration, and substrate concentration using the following procedures:
Determination of molecular weight of urease
Two millilitres of blue dextran-2000 solution (6 mg into 3 mL of PEM buffer pH 7.5, 0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4, at pH = 6.6; where PIPES is piperazine-N, N′-bis(2-ethanesulfonic acid, and EGTA is ethylene glycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) were passed through Sephacryl S-200 column. 20 mM of PEM buffer pH 7.5 was added. Fractions of 5 ml were eluted and the absorbance at 600 nm for each fraction was measured. The column void volume (V
) was determined by estimating the total volume of the fractions characterized with the starting point movement of the dextran to climax of absorbance of the blue dextran. Same procedure was done for the standard proteins; BSA, aldolase, catalase, ferrtin, and thyroglobulin. The eluted fractions, which give a maximum absorbance at 280 nm, were determined, and the eluted volume (V
) was calculated for each standard protein. The linear calibration curve of against logarithm value of molecular weight of standard protein was plotted. The curve was used for determining the molecular weight of native urease.
Effect of pH on the activity of pisum sativum urease
The pH profile for the purified urease was estimated using urea as a substrate. The pH range used was from 3 to 10 using 50 mM phosphate buffer.
Effect of storage at −4°C on enzyme activity
To determine the effect of storage of the enzyme on the urease activity, the enzyme was stored at different time internals of 0–60 days. The enzyme activity was measured after each separate time period.
Effect of different concentration of enzymes
The optimum enzyme concentration was determined by varying the amount of the pure enzyme.
Effect of temperature
The optimum temperature for urease activity was determined over the temperatures from 10 to 40°C using the standard conditions of the assay.
The relationship between the rate of an enzymatic reaction and activation energy is given by the empirical formula of the Arrhenius equation:
are the enzyme activities at the temperatures T
is the energy of activation (kJ mol−1) which can be determined from the slope of the Arrhenius plot of ln(V) against .
The activation enthalpy (ΔH) can be calculated by eqn. 2.
Finally the entropy (ΔS) was calculated by eqn 3 (Eyring-Polanyi), which correlates ∆H, E
, and Arrhenius equation (eqn. 1);
where T, K
, h, and R are absolute temperature, Boltzmann constant, Planck constant and gas constant respectively.
Effect of different concentration of substrates
The effect of urea concentration on the activity of enzyme was examined. Urea solution of different concentration was taken in different test tubes and the enzyme activity was measured. K
for urease were calculated using Lineweaver-Burk double reciprocal plot .