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Biotechnology and Bioprocess Engineering 2007, 12: 131-135

Immobilization of Watermelon (Citrullus vulgaris) Urease in Agarose Gel for Urea Estimation
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Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005, India

Abstract Urease from dehusked seeds of watermelon was immobilized in 1.5% agarose gel with 53.9% entrapment. There was nego ligible leaching (< 10% at 4 C) and the same gel membrane could repeatedly be used for seven days. The immobilization o exhibited no apparent change in the optimum pH but there was a significant decrease in the optimum temperature (50 C as o compared to 65 C for soluble urease). The immobilized urease revealed an apparent Km of 9.3 0.3 mM; 1.2 times lower than the soluble enzyme (11.4 0.2 mM). Unlike soluble enzyme which was inhibited at 200 mM urea, the immobilized urease was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2 M urea. The time-dependent o thermal inactivation kinetics at 48 and 52 C was found to be biphasic, in which half of the initial activity was destroyed more rapidly than the remaining half. These gel membranes were also used for estimating the urea content of the blood samples from the University hospital. The results obtained matched well with those obtained by the usual method employed in the clinical pathology laboratory. The significance of these observations is discussed. KSBB = = = = Keywords: urease, watermelon, Citrullus vulgaris, immobilization, agarose, urease immobilization

INTRODUCTION
The metabolic function of kidney is reflected in the concentration of organic compounds, such as urea, in blood or urine. Therefore, estimation of urea is frequently performed in the medical field. Analysis of urea is also important in the agricultural field, since it has become the worlds leading nitrogen fertilizer, and has been recognized as a pollutant in agricultural wastewaters [1]. Immobilized urease (urea amidohydrolase EC 3.5.1.5) has analytical and biomedical applications. Hence it has been immobilized on variety of matrices, like calcium alginate [2], gelatin [3], carboxymethyl cellulose [4], DEAE-cellulose paper strips [5], nylon tubes [6] etc. However, most of the studies pertaining to immobilization have been aimed at to develop analytical tool viz., either a biosensor or an enzyme electrode. Urease from dehusked seeds of watermelon (Citrullus vulgaris) has been purified to apparent homogeneity and partially characterized [7]. We have also presented evidence
*Corresponding author Tel: +91-0542-2307323 Fax: +91-542-2368174 e-mail: oprakash01@yahoo.co.in

for the presence of thiol groups on the active site of the enzyme. Its inactivation kinetics with thiol specific reagents showed a biphasic kinetics in that half of the initial activity was destroyed more rapidly than the remaining half [8]. This unique phenomenon of molecular asymmetry or more specifically half-site reactivity in watermelon urease, reported for the first time, was further substantiated by its thermal inactivation studies [9]. Earlier studies on jack bean urease have established that the thiols are inhibitors; -mercaptoethanol (ME) being a competitive inhibitor in the hydrolysis of urea and dithiothreitol (DTT) being a poor inhibitor [10]. But we have recently demonstrated, for the first time, that not only ME but also DTT and L-cysteine at 30oC in 50 mM Tris-acetate buffer (pH 8.5) are excellent activator of the watermelon urease and the order of effectiveness as activator was ME > DTT > L-cysteine [11]. These differences in the behaviour of watermelon urease from that of the enzymes from other sources, stimulated us to immobilize watermelon urease in agarose (a polysaccharide consisting of 1,3-linked D-galactopyranose and 1,4-linked 3,6-anhydro-aL-galactopyranose) and study its kinetics in detail and compare it with that of the soluble enzyme. To the best of our knowledge, this is the first report of urease immobilization in agarose gel wherein we demonstrate no significant change in the kinetic behaviour of urease upon immobilization.

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MATERIALS AND METHODS

Chemicals

Immobilized Urease Assay

Urease was isolated and purified from dehusked seeds of watermelon procured from the local market. Tris was obtained from Boehringer Mannheim Gmbh, Germany. Bovine serum albumin and agarose were obtained from Sigma Chemical Co., USA. Sephadex G-200 was from Pharmacia Fine Chemicals, Uppsala, Sweden. Urea (enzyme grade), Nesslers and Folin-Ciocalteau reagents were from Qualigens Fine Chemicals, Mumbai, India. All other reagents were analytical grade chemicals either from BDH or E. Merck, India.
Enzyme

For assay of immobilized enzyme, 2~3 pieces of gel were incubated at 30oC for 10 min in standard assay medium comprising of 50 mM Tris-acetate buffer (pH 8.5) containing 250 mM urea. Following incubation, an aliquot of 1.0 mL was withdrawn from the reaction mixture and assayed as described. The gel pieces were recovered from the reaction mixture, washed thoroughly with the buffer and stored at 4oC. The percentage of immobilization is defined as the (total activity in immobilized gel/total activity of the soluble enzyme loaded) 100.
Steady-state Kinetics

Urease was isolated and purified in 25 mM Tris-acetate buffer (pH 7.5) from dehusked seeds of watermelon to electrophoretic homogeneity as described earlier [7,11]. The enzyme preparation (sp. activity 3,000 550 U/mg protein), showing a single enzyme and protein band on native 7.5% PAGE (at pH 8.3), was employed for the study.
Protein Estimation

Protein was estimated by the method of Lowry et al. [12] with Folin-Ciocalteau reagent calibrated with crystalline bovine serum albumin.
Soluble Urease Assay

The optimum pH for the soluble and the immobilized urease was determined by varying the pH between 6.0 to 9.5 of 50 mM Tris-acetate buffer. For each pH value tested, fresh gel membranes were employed. The activity was determined for each buffer by the method described for enzyme assay above. Optimal temperature was studied by varying the temperature of the standard assay medium containing immobilized and/or soluble enzyme at the increasing temperature range from 0~100 ( 0.5)oC. Km and Vmax were determined by Lineweaver-Burk plot by varying the substrate concentration from 2 to 125 mM. Effect of high urea concentration on the activity was studies by varying the substrate concentration from 10 mM to 2 M.
Thermal Inactivation

Activity was assayed in 50 mM Tris-acetate buffer (pH 8.5). An aliquot (0.8 mL) of buffer and 1.0 mL of 250 mM urea in the same buffer were brought to 30oC. The reaction was started by adding 0.2 mL of suitably diluted enzyme. After 10 min, 1.0 mL of 10% trichloroacetic acid was added to stop the reaction. The total reaction mixture was transferred to a measuring flask (50 mL) and the volume was made to 50 mL with distilled water after adding 1.0 mL of Nesslers reagent as described earlier [11]. The amount of ammonia liberated was measured at 405 nm in a Spectronic 21 UVD spectrophotometer. One enzyme unit was defined as the amount of enzyme required to liberate one mole of ammonia in one minute under the test conditions defined above (30oC, 50 mM Trisacetate buffer, pH 8.5, 250 mM urea).
Urease Immobilization

Around 20~25 pieces of immobilized gel were incubated in assay buffer (50 mM Tris-acetate buffer, pH 8.5) at the desired temperatures (48 and 52oC). At specified time intervals, a single gel piece was withdrawn, immediately cooled and transferred to the assay solution (2.0 mL; containing 1.0 mL of 50 mM Tris-acetate buffer pH 8.5 and 1.0 mL of 250 mM urea). Residual activity was determined by the usual assay method described above.
Estimation of Serum Urea

A 1.5% solution of agarose was prepared in 25 mM Trisacetate buffer (pH 7.5) by warming for 10 min. Suitably diluted enzyme solution (2 mg protein/mL) was mixed in agarose solution after cooling it to room temperature and immediately casted on preassembled gel plates. After solidification at room temperature, the gel was cut into pieces (8 8 mm) and stored in 25 mM Tris-acetate buffer (pH 7.5) at 4oC.

Various blood samples (obtained from the University hospital) were tested for urea concentration using agaroseimmobilized urease. The gel pieces (one or two) were preincubated in 1.8 mL of assay buffer (50 mM Tris-acetate, pH 8.5) at 30oC. The reaction was started by the addition of 0.2 mL of serum. After 10 min of incubation, 1.0 mL of the reaction mixture was withdrawn and assayed for the amount of ammonia liberated from urea hydrolysis. The results reported are mean of 5~8 replicate experiments carried with fresh batch of purified enzyme.

RESULTS AND DISCUSSION

Urease Immobilization

The conditions for optimal immobilization of watermelon

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Fig. 1. Effect of pH on soluble watermelon and agaroseimmobilized urease gel membranes.

Fig. 2. Effect of temperature on soluble watermelon urease and agarose-immobilized urease gel membranes.

urease in agarose were explored. In order to select the suitable concentration of agarose, its concentration was varied from 1~2.5% (w/v). With 1.0% of agarose, there was only 26% immobilization. The enzyme activity was also lost after a single assay, probably due to the larger pore size of the membrane, resulting in leaching of the enzyme during assay. Moreover, the membrane was very fragile and susceptible to damage during handling. With 1.25% of agarose, 39% immobilization was achieved but the membrane was not stable enough to withstand the assay conditions. Further increase to 1.5%, resulted in stable membrane and exhibited 53.9% immobilization with uniform distribution of enzyme per piece. Agarose solution in the concentration range of 1.75 to 2.5%, solidified as soon it was allowed to cool even before addition of the enzyme. Therefore, 1.5% (w/v) agarose solution was employed for urease immobilization studies. Each piece (8 8 mm) contained 3.5~4.0 g of protein and the specific activity of the immobilized urease was 2,500~2,820 U/mg of protein. There was no leaching of the enzyme (< 10% in 25 mM Tris-acetate buffer, pH 7.5) over a period of 10 days at 4oC. The same gel membrane when used for seven days (with an interval of 24 h between each use), still retained 30~45% enzymatic activity.
Steady-state Kinetics

Effect of varying pH of the assay buffer (50 mM Trisacetate) is shown in Fig. 1. The pH optimum of the soluble and agarose-entrapped urease was 8.0. There was no apparent change in the pH value upon immobilization. However, a shift of 1.0 unit towards an acidic pH value was reported in calcium alginate immobilized watermelon urease [2]; jack bean urease immobilized on porous glass strips [13] and molecular sieve 4A [14]. In addition, pigeonpea urease immobilized on chitosan beads [15] and jack bean urease immobilized on a fixed-bed reactor [16] showed a shift towards the basic side. While a sensible modification of the activity with pH variation without any change in the pH optimum

has also been reported in case of urease immobilized on carboxymethyl-cellulose [4]. Effect of temperature on the activity of soluble and agarose immobilized urease was studied in the temperature range from 0 to 100 ( 0.5)oC. Results (Fig. 2) revealed an optimum temperature of 65oC for soluble urease and 50oC for agarose immobilized urease. There was a significant decrease in the optimum temperature upon immobilization. Interestingly, immobilized enzyme revealed activity at 10oC whereas soluble enzyme did not show measurable activity below 0oC. Moreover, there was an elevation in the rate of urea hydrolysis upon immobilization in the temperature range of 50~60oC as compared to the soluble enzyme. It may be mentioned here that under normal conditions of activity in living cells, almost all enzymes are bound to other macromolecules and are rarely found in the free state. Alteration in the optimum temperature from 47oC for soluble to 65oC for gelatin immobilized pigeonpea urease [3] and 67oC for DEAE-cellulose paper trip immobilized urease [5] has recently been reported. Chitosan-immobilized pigeonpea urease showed maximum activity at 77oC compared with free enzyme at 47oC [15]. Thus, a decrease in the optimum temperature upon immobilization in agarose gel makes the preparation suitable to be used in near physiological range of temperature. Urease immobilized in agarose gel revealed an apparent Km value of 9.3 0.3 mM, which is 1.2 times lower than that of the soluble urease (11.4 0.2 mM), and Vmax was 6,900 mol/min/mg, slightly higher than that of the soluble urease (6,440 mol/min/mg; results not shown). A slightly lower apparent Km value was also found for jack bean urease immobilized in polyacrylamide gel [17] and on carboxymethylcellulose [4] than the corresponding soluble enzyme. On the other hand, watermelon urease immobilized in alginate beads [2], pigeonpea urease immobilized on chitosan beads [15], gelatin beads [3], and DEAE-cellulose paper strips [5] have revealed an increase in apparent Km values. In addition, urease immobilized in hydrocarbon-based liquid surfactant

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Fig. 3. Activity of soluble and agarose-immobilized watermelon urease in presence of varying concentrations of substrate.

Fig. 4. Time-dependent thermal inactivation of soluble and agarose-immobilized watermelon urease at 48 and 52oC.

membrane exhibited a Km approximately 50 times greater than the soluble enzyme [18]. Ureases of jack bean [19], Cajanus indicus [20], and watermelon [8] have been reported to be inhibited by high concentration of urea. It was of interest to explore whether immobilization had any bearing on inhibition reported in urease activity at high substrate concentration. This aspect was studied by assaying activity in the urea concentration range of 10~2,000 mM. The results (Fig. 3) showed that the soluble enzyme was strongly inhibited by increasing urea concentration to 200 mM and above. While, upon immobilization the inhibition occurred at a much higher concentration i.e., 600 mM and above and the extent of inhibition was much less pronounced. Thus, at 2 M urea, the enzyme still retained about 47% of the activity. In addition, the immobilized urease-catalysed ureolysis occurred at a much faster rate at all the concentrations of urea than the soluble enzyme. High concentration effect may result from an inhibition of the reaction by ammonium ions or from the urea itself. It has been proposed that at very high concentrations, urea may occupy sites on urease surface that are normally occupied by water molecules (water sites), thus preventing access of water molecules for ureolysis, leading to an inhibition of the activity [19].
Time-dependent Thermal Inactivation

Table 1. Rate constants for the biphasic inactivation of agaroseimmobilized watermelon urease
Temperature o ( C) 48 52 Rate constant (min ) Fast phase (kfast) 0.345 0.231 Slow phase (kslow) 0.0128 0.0111
1

At = Afast exp(kfastt) + Aslow exp(kslowt)

(1)

where At is the residual activity at time t, Afast and Aslow, kfast and kslow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. In the data of Fig. 4, Afast Aslow 50% of the initial activity. The values of kfast and kslow obtained at 48 and 52oC have been shown in Table 1. Similar biphasic thermal inactivation kinetics has also been reported for soluble watermelon [9] and pigeonpea ureases [21]. Soluble urease shows molecular asymmetry with respect to thermal inactivation, i.e. half of the sites behave in a different manner than the other half (half-site reactivity) [8,9]. With the observed biphasic thermal inactivation of agarose-immobilized urease reported above, suggest that the original characteristics of the enzyme are still maintained even after entrapment.
Estimation of Serum Urea

The time-dependent thermal inactivation kinetics of the agarose-immobilized urease at 48 and 52oC was found to be biphasic (Fig. 4). The slow phase showed an exponential decay (first-order kinetics) and accounted for half of the initial activity. In order to obtain information about the initial fast phase, the slow phase was extrapolated to zero time in the semi-log plot, and the difference between the experimentally observed activity and the extrapolated slow phase was replotted. The fast phase also showed first-order kinetics and accounted for half of the initial activity. Thus, the inactivation of urease can be expressed by a biphasic rate equation containing two first-order terms:

The agarose-immobilized urease gel membranes were subsequently used to assay the blood urea of some patients from the University hospital (normal healthy persons and patients suffering from glomerulonephritis and hypertension). A calibration curve with urea concentration ranging from 5 to 400 mg/100 mL was prepared (results not shown). The values for the urea concentration in the clinical blood samples determined with these gel membranes compared favorably with those obtained by the usual chemical method used in the clinical pathology laboratory of the University

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Table 2. Estimation of blood urea of some clinical samples with agarose-immobilized urease compared with urea estimated by chemical method
Sample No. 1 2 3 4 5 6 7 8 9 10 Urea content (mg/100 mL) Immobilized enzyme Chemical method 18.9 23 80.1 79 88.5 86 104.7 109 171.0 172 177.4 183 183.6 180 190.2 194 202.8 206 230.9 234

5.

6. 7.

8. 9.

hospital (Table 2). 10.

CONCLUSION
Urease employed for the present study was from a rather non-expensive and readily available source. Upon immobilization, the urease retained most of its native characteristics, like no change in pH optima, workability at near physiological temperature, lowered Km value hence more affinity for the substrate, and ease of immobilization in agarose described in present study makes it a suitable product for future applications in diagnostics.
Acknowledgement We thank the clinical Pathology Laboratory, Institute of Medical Sciences, Banaras Hindu University, for providing us blood samples and their urea analysis results. The work was supported by a grant-in-aid No. 38(0999)/00/EMR-II from Council of Scientific and Industrial Research, New Delhi, India.

11. 12. 13. 14. 15.

Received August 12, 2006; accepted January 15, 2007

16.

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