L-asparagine, L-glutamine, Trichloro acetic acid and Tris were purchased from Merck (Darmstadt, Germany). DEAE-Sepharose was provided by Pharmacia (Uppsala, Sweden). L-asparaginase derived from E.coli was a gift from cancer research institute, Iran. All other chemicals were from Sigma (St. Louis, MO, USA) and were of analytical grade.

Two new bacterial strains, Bacillus PG-03 and Bacillus PG-04 were previously isolated from the Persian Gulf sediments and identified. Colonies were sub-cultured on to nutrient agar slants and kept in 20% glycerol at -20 ˚C as stock culture. The primary inoculum was grown overnight in 10 ml nutrient broth medium and was inoculated in 100 ml of M9 broth, consisting of (g/L): maltose 20% as a sole carbon source; Na₂HPO₄ 6.0; KH₂PO₄ 3; NaCl 0.5; CaCl₂ 0.011; MgSO₄.7H₂O.7H₂O 0.12; pH 7.0 and incubated at 37 ˚C with shaking at 200 rpm for 24 hours. The cell mass concentration was determined by measuring the optical density of the culture at 600 nm.

To study the effect of different nitrogen sources on L-asparaginase activity, alternative nitrogen sources consisting of L-asparagine, arginine and NaNO₃+(NH₄)₂SO₄ (1% w/v) were used. pH 7 and pH 8 were tested for maximum enzyme production.

For extraction of the intracellular enzyme, the sonication method was used. The biomass was separated out by centrifugation (8000 rpm, 10 min and 4 ˚C); the pellet was resuspended in 3mL 50mM Tris buffer pH 9 containing 10% (v/v) glycerol. The cells were disrupted by sonication and then centrifuged at 13,000 rpm and at 4 ˚C for 15 min. The supernatant used as the crude extract. The crude enzyme solution was applied to DEAE-Sepharose column (10mm×100 mm) that was pre-equilibrated with a 50mM Tris-HCl buffer, pH 9.0. The protein was eluted with the NaCl gradient (0.0-0.7 M) and 50 mM Tris-HCl buffer, pH 8. The active fractions were collected, dialyzed and concentrated. This preparation was used in the subsequent steps.

SDS-PAGE was carried out using a 12% polyacrylamide gel by the method of Laemmli [22]. A high molecular weight marker (Fermentas) was used. Protein bands were detected by Coomassie Brilliant Blue R250 and destained with a solution of methanol, acetic acid and water in the ratio of 4:1:5.

L-asparaginase activity was measured by the known method of Imada et al. [23]. The samples were mixed with 40 mM L-asparagine in 50 mM Tris-HCl buffer, pH 7.2. 200 µL of assay mixture were incubated for 15 min at 37 °C for enzymatic reaction. Reaction was stopped by the addition of 250 μL of 1.5 M Trichloro acetic acid (TCA) and samples were centrifuged before the addition of Nessler’s reagent to measure the released ammonia after L-asparagine hydrolysis. Absorbance was measured at 450 nm. One unit of L-asparaginase activity was defined as the amount of enzyme that released 1 μmol of ammonia (with ammonium sulfate as the standard) per minute under the assay conditions specified. L- glutaminase activity was determined as that of L-asparaginase using the method of Mashburn and Wriston [18]. Protein concentration was also measured according to the method of Bradford using bovine serum albumin as the standard [24]. The pH profile and pH optima were determined at room temperature in various pH using 50 mM phosphate buffer. The activity of purified enzyme was determined at several temperatures (from 25 to 50 ˚C) in 20 mM Tris-HCl buffer, pH 7.2.

In order to determine kinetic constants for L-asparaginase, measurements were carried out over different substrate concentrations including 10, 20, 30, 40 and 80 mM and the final concentration of the enzyme was 0.5 mg/mL. Different blanks were used for each L-asparagine concentration. Kinetic parameters, K_m and V_max for L-asparaginase enzymes were determined from a series of initial rates. Experimental data were analyzed graphically and numerically using the Lineweaver–Burk equation.

Half-lives and T₅₀ were determined at two temperatures (37, 40˚C). For half-life measurement, 1 mg/mL enzyme solution in 20 mM Tris–HCl buffer (pH 7.2) was used. 10 μL aliquots were removed at various time intervals, chilled on ice for 60 min and diluted into 20 μL of an assay solution (L-asparagine 40 mM) for the measurement of residual activity at 37˚C for 15 min. Plots of the log of residual activity versus time were linear, indicating a first-order decay. T₅₀ is the temperature of incubation at which 50% of the initial enzyme activity is lost during 30 min incubation [25]. The ΔG* parameter of the L-asparaginase enzyme was determined as follows [26]:

ΔG * = RTln (kBT/h) - RTlnk

No defined medium has been established for the optimum production of L-asparaginase from different microbial sources. Each organism has its own special conditions for maximum enzyme production. The prerequisite for obtaining L-asparaginase from bacteria is to grow them in a medium optimized with various nitrogen sources [27, 28]. Therefore, two bacterial strains, Bacillus PG03 and Bacillus PG04 were screened for the L-asparaginase production at various nitrogen sources and pH. Three different nitrogen sources including L-asparagine, L-arginine and NaNO₃+(NH₄)₂SO₄ were separately added to the basal medium containing maltose as carbon source, pH 7 and pH 8. The results revealed that for both bacterial strains, maximum L-asparaginase production obtained in the presence of L-asparagine as the nitrogen source at pH 7. The cells were cultivated in each media for 24 h and 48 h. The enzyme production was higher after 48 h for both strains (Fig. 1). Previous studies have shown that a nitrogen source is the limiting factor and plays key role in the L-asparaginase production [29]. It seems that L-asparagine might be an inducer for L-asparaginase production and hence the production of the enzyme is more feasible at the presence of this nitrogen source compared to L-arginine and NaNO₃ + (NH₄)₂SO₄. Maximum L-asparaginase production using L-asparagine as the sole source of nitrogen has been observed in Streptomyces gulbargensis [30], Enterobacter cloacae [31] and Aeromonas sp [32]. On the contrary, Narayana et al. reported yeast extract (2%) as the best nitrogen source for L-asparaginase production by S. albidoflavus [33].

Fig. (1). Effect of various nitrogen sources and pH on production of L-asparaginases. L-asparaginase production is shown for Bacillus PG03 and Bacillus PG04 after 24 hour (Dotted columns and dashed columns) and after 48 hour (black columns and white columns) of inoculation respectively.

After bacterial cell sonication, the crude enzyme was applied to anion-exchange chromatography on DEAE-Sepharose. The fractions of the largest peak from the elution profile that had L-asparaginase activity were pooled, dialyzed and concentrated by ultrafiltration (Fig. 2A, B). SDS-PAGE analysis of the purified enzyme estimated that the apparent molecular weight of the purified enzyme was 25 kDa for Bacillus PG03 and and 30 kDa for Bacillus PG04 (Fig. 2C). Since most of the bacterial L-asparaginases exist as a tetramer of identical subunits, then the exact estimation of the molecular weight requires a native PAGE analysis.

The most important physical factors, which influence the enzymatic reaction rate, are pH and temperature of incubation with the substrate. Each enzyme has a characteristic pH and temperature optima beyond which the reduction in activity is observed. The suitable temperature and pH of the enzyme was studied using a 50 mM phosphate buffer of different pH values, ranging from 3.0 to 10. The results showed that L-asparaginase was active over broad pH ranges from 3 to 8 for Bacillus PG03 L-asparaginase and from 3 to 10 for Bacillus PG04. The enzyme activity gradually increased until pH 6 for Bacillus PG03 L-asparaginase and pH 7 for Bacillus PG04 at which the maximum activity was observed (Fig. 3A). E. coli L-asparaginase was optimally active at pH 6 (Supplementary Data Fig. ( 1A)). These results indicated that the optimum pH and temperature lie near that of the growth condition. Along with E. coli L-asparaginase, enzymes isolated from Erwinia carotouora are now being used for the treatment of acute lymphoblastic leukemia [19]. However, Kamble et al. found that the optimum pH of the enzyme from E. carotouora was 8.6 [34]. Our results revealed that the L-asparaginase produced by Bacillus PG03 and Bacillus PG04 were maximally active around pH 7 which is near to the physiologic pH. Since the physiological pH is one of the perquisites for antitumor activity [35], hence this characteristic makes the enzyme highly attractive for both the basic research studies and the industrial and pharmacological processes. Different optimum pH, from 6 to 9.5 has been reported for enzyme produced by microbial sources [11, 36]. There are very few reports in which the L-asparaginase enzymes showed optimum pH near physiologic pH range [37-39]. The majority of L-asparaginases from Erwinia sp. showed alkaline pH optima (8.0–9.0) whereas the enzyme from E. coli exhibited an acidic pH optimum of 5.0–6.0 [40, 41]. Similarly, the effect of temperature on the activity of L-asparaginase was studied. Maximum activity was obtained at 37˚C for Bacillus PG03 L-asparaginase and 40˚C for Bacillus PG04 and E. coli L-asparaginase (Fig. 3B) and Supplementary Data Fig. ( 1B)). However the enzyme from both bacterial strains was active at a wide range of temperature condition from 25˚C to 50˚C and retained 50% (Bacillus PG03) and 60% (Bacillus PG04) activity in 50˚C. This pattern was similar to the temperature pattern of L-asparaginase derived from E. coli. This property makes the enzyme more suitable for complete elimination of asparagine in patients suffering from leukemia. Mannan et al. [35] also found 37˚C to be the optimum temperature for the enzyme activity. Mesas et al. reported 40˚C as the optimum temperature of the L-asparaginase from Corynebacterium glutamicum [42]. Anyway, a literature review from Zuo et al. and from Batool et al. reported different optimum temperatures from 30˚C to 80˚C for different microbial L-asparaginases [11, 36]. The L-asparaginase enzymes were assayed for amidohydrolase activity. No activity was determined when L-Glutamine was used as substrate. This is consistent with the results of Chityala et al. and Kumar et al. who reported glutaminase free L-asparaginase enzyme [43, 44].The enzymes from Bacillus PG03 and Bacillus PG04 revealed Michaelis-Menten kinetics when L-asparagine was used as a substrate. (Table 1) and Fig. (3C) shows the K_m and V_max values calculated based on the Lineweaver-Burk analysis. Also the kinetic parameters of the E. coli L-asparaginase was studied (Supplementary Data Fig. ( 2A)) Previous studies have reported that L-asparaginases from different microorganisms had different substrate affinities and probably played different physiological roles. Higher and lower K_m values have been reported for different L-asparaginase enzymes [8, 45]. E. coli L-asparaginase showed K_m of 3.4 mM which is similar to that of Bacillus PG04 and lower than the K_m value of Bacillus PG03.

Fig. (2). Ion exchange chromatography and SDS-PAGE of L-asparaginases. The sonicated crude extract was chromatographed on DEAE-Sepharose. Total protein was monitored at 280 nm. L-asparaginase was purified from Bacillus PG03 (A) and Bacillus PG04 (B). Dashed line represents OD 280 nm, Solid line represents enzyme activity. (C) SDS-PAGE of the purified L-asparaginases. Molecular weight marker (lane 1), L-asparaginase from Bacillus PG03 (lane 2) and Bacillus PG04 (lane 3).

Fig. (3). (A) Effect of pH and (B) temperature on activity of L- asparaginase from Bacillus PG03 (closed diamonds) and Bacillus PG04 (open circles). (C) Lineweaver–Burk plots of L-asparaginase activity purified from Bacillus PG03 (closed diamonds) and Bacillus PG04 (open circles).

Fig. (4). (A) Half-life curve of L-asparaginase from Bacillus PG03 at 37˚C (closed circles) and 40˚C (closed diamond) and Bacillus PG04 at 37˚C (open circles) and 40˚C (open diamond). (B) T50 curve of the L-asparaginase from Bacillus PG03 (open circles) and Bacillus PG04 (closed diamonds).

The method that has been used to analyze the heat inactivation rate constants of L-asparaginase requires enzyme incubation at desirable temperatures and cooling in ice before measurement of the residual activity [25, 46]. After incubation at 37˚C and 40˚C, the half-lives (t_1/2) of Bacillus PG03 and Bacillus PG04 were measured (Table 1). k_i (inactivation rate constant) values were estimated from the plots of Log of residual activity versus incubation time. As is shown in Fig. (4A) inactivation process was a first order decay. L-asparaginase from both strains showed higher half-life in 37˚C compared to 40˚C. Bacillus PG03 L-asparaginase showed the highest half -life of more than 1 hour in 37˚C. This makes the enzyme potentially suitable for future therapeutic uses. Bacillus PG04 enzyme also revealed t1/2 of 34.6 min in 37˚C. In 40˚C still both enzymes were active, although the reduction in half- life of the enzymes was clearly observed compared to 37˚C. The interrelationship between conformational stability and enzyme activity suggested that in naturally occurring enzymes the highest stability could be seen at temperatures near that of growth of an organism [28, 47]. E. coli L-asparaginase showed the half-life of more than 6 hours (Supplementary Data Fig. ( 2B). Together these results showed that although the purified enzymes had appropriate stability in 37˚C and 40˚C but when compared to E. coli L-asparaginase, the half- life of the enzyme needs to be more improved by protein engineering methods and hence these enzymes could be regarded as an interesting target for further investigations. Another criterion of thermal stability is T₅₀. As is shown in Fig. (4B) and Table 1, T₅₀ value for Bacillus PG03 is slightly higher than that of Bacillus PG04. Free energy changes of inactivation (ΔG*) was evaluated for both enzymes. L-asparaginase from Bacillus PG03 displayed a higher ΔG* values of inactivation than the enzyme from Bacillus PG04, indicating that the distance between the native state and the transition state has increased in L-asparaginase from Bacillus PG03 and, consequently, a larger input energy is required for inactivation (Table 1). Results of this study suggest that L-asparaginase from Bacillus PG03 is thermally more stable than that of Bacillus PG04.