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Production and characterization of asparaginase from Bacillus subtilis strain BNT isolated from natto.

Introduction

Asparaginase is the enzyme that hydrolyzes asparagine into L-aspartic acid and ammonia. Asparagine involves in cancer development in 2 ways; i.e. being an essential nutrient for growth of cancer cell [1] and being a precursor for the formation of acrylamide, a potential carcinogen in high-carbohydrate-content food [2,3]. Therefore, asparaginase has been used as chemotherapeutic agents against cancer in order to deaminate asparagines, leading to starvation in cancer cells [4]. It is also used in treatment of food raw materials in order to break down asparagines, limiting the available precursor for acrylamide formation [5,6].

Many microbial asparaginase has been isolated including those from bacteria [1], yeasts and some from fungi [7,8,9]. However, new sources of aspearaginase are still required in order to search for the one with the best property. In this study, Bacillus subtilis strain BNT, isolated from Japanese fermented soybean--natto, was found to produce asparaginase. The enzyme was purified and characterized. Moreover, a low cost medium for the enzyme production has been formulated. BNT asparaginase could be an alternative source of the enzyme for the mentioned applications.

Materials and Methods

Cultivation

A stock culture of Bacillus subtilis BNT (BNT) was grown in nutrient agar (NA; Oxoid, Co.) at 37[degrees]C for 24 hours and stored at 4[degrees]C until used. The inoculum for fermentation was prepared by cultivating the bacteria in 20 ml of nutrient broth (NB; Oxoid, Co.). The low cost medium was developed based on the modified PSB [10] which was composed of 2.0% (w/v) glucose, 0.2% (w/v) Ca[Cl.sub.2], 0.05% (w/v) KCl, 0.05% (w/v) MgS[O.sub.4] x 7[H.sub.2]O, 0.5% (w/v) N[H.sub.4]N[O.sub.3], 0.001% (w/v) FeS[O.sub.4] x 7[H.sub.2]O and 0.001% (w/v) MnS[O.sub.4] x [H.sub.2]O in distilled water. Cultivation was carried out at 37[degrees]C for 24 hours, otherwise specified. The bacterial growth was determined by measuring the turbidity at OD600 using spectrophotometer (Hitachi Co., USA).

Crude enzyme extraction

The 24-hour broth culture was centrifuged at 2000 rpm for 15 min, then, the supernatant was collected as the crude enzyme extract.

Asparaginase assay

A quantitative assay for NH3 released by asparaginase was done following Dharmsthiti and Luechai [8]. Briefly, the reaction mixture contained 0.2 ml of 0.05 M Tris-HCl buffer, pH8.6; 1.7 ml of 0.01 M L-asparagine prepared in 0.05 M Tris-HCl buffer, pH 8.6 and 0.1 ml of properly diluted enzyme. The reaction was carried out at 37[degrees]C for 30 min. The reaction was stopped by adding 0.1 ml of 1.5 M trichloroacetic acid. The mixture was centrifuged to obtain clear solution. Then, 0.5 ml of the solution was transferred to another tube containing 6.5 ml of reagent grade water and added with 1.0 ml of Nessler's reagent. The mixture was left at room temperature for 10 min. The color developed was spectrophotometrically measured at 450 nm. The activity was calculated based on standard curve prepared by mixing 1 [micro]mole ammonia [dissolved 1.179 gm [(N[H.sub.4]).sub.2]S[O.sub.4] in reagent grade water to a final volume of 100 ml, then, diluted 1.4 ml of this solution to 100 ml of water to give 1 [micro]mole N[H.sub.3]] at different volume ranging from 0 to 1.0 ml with water to a final volume of 7.5 ml. This was then added with 1.0 ml of Nessler's reagent. One unit of asparaginase activity was defined as the amount of enzyme that released 1 [micro]mole N[H.sub.3] per min.

Purification of asparaginase

The purification was carried out at 4[degrees]C followed the method of Dharmsthiti and Luechai [8]. Concentration of crude enzyme was carried out by ammonium sulfate precipitation to 80% saturation. The protein precipitate was resuspended in 0.01 M phosphate buffer, pH 8.5, and dialyzed against the same buffer. The concentrated crude enzyme was then subjected to a CM-Sephadex C50 ion-exchange chromatography that was pre-equilibrated with 0.01 M phosphate buffer, pH 8.5. The bound enzyme was eluted with the NaCl gradient, 0 to 0.5 M, in the same buffer. The active fractions were pooled and concentrated using ammonium sulfate precipitation and dialysis. This was then applied to a column of Sephadex G-100 gel filtration that was pre-equilibrated with 0.01 M phosphate buffer, pH 8.5. The protein elution was done with the same buffer. The flow rate for both CM-Sephadex C50 and Sephadex G-100 columns was 1 ml/min. The active fractions from the Sephadex G-100 column were pooled and concentrated using ultrafiration through a Vivaspin 20 membrane, 3000 MWCO PES (Generon Ltd, UK).

Polyacrylamide gel electrophoresis

Electrophoresis PAGE under non-denaturing conditions and SDS-PAGE were carried out as described by Laemmli [11]. For non-denaturing PAGE, a 6% separating gel and a 3% stacking gel were used. For SDS-PAGE, a 7.5% separating gel and a 3% stacking gel were used, each containing 5 M urea and 0.5% (v/v) Triton X-100. All gels were run at 20 mA for 120 min. The gels were stained with Coomassie blue R-250. Molecular mass markers used in this study were purchased from MBI fermentas Co., UK.

Determination of asparaginase activity on glutamine

This was carried out following the method for asparaginase assay except changing the substrate of the reaction from asparagines to glutamine.

Analytical method

The protein concentration in each fraction from column chromatography was determined by measuring the absorbance at 280 nm (Hitachi U-1900, Japan). For the pooled fractions obtained from each purification step, the protein concentration was determined using Lowry method [12]. Bovine serum albumin at 30 to 150 [micro]g was used as a standard.

Results and Discussion

Preparation of Crude enzyme extract from BNT

Asparaginase production from BNT was carried out in four of 500-ml flasks containing 250 ml of nutrient broth incubated on a rotatory shaker (200 rpm) at 37[degrees]C for 24 hours. Then, the culture broth was centrifuged and the cell pellet was discarded. The culture supernatant was assayed for the asparaginase activity which resulted as 0.4 U/ml. After concentration, the activity was increased to 2.5 U/ml and the protein concentration was 16.5 mg/ml.

Purification of BNT asparaginase

BNT asparaginase was purified using CM-Sephadex C50 ion-exchange chromatography, which was eluted with a gradient of 0-0.5 M NaCl, and then through a Sephadex G-100 gel filtration. The purification profile is shown in Table 1. The specific activity decreased after passing through the first column and increased again after the second column. This could be due to the present of an unknown substance that could inhibit the enzyme activity and, yet, the effect could have been masked by some other antagonistic substances. Then, after the first column of CM-Sephadex C50, such an "antagonistic substance" was diluted out, revealing the enzyme inhibition effect. After elution through the second column, the unknown inhibitor substance, itself, could have been separated out leading to an increase in the specific activity.

The molecular weight of BNT asparaginase was determined by SDS-PAGE as 45.6 kDa (Figure 1). This asparaginase was considered very small when compared to those of other microbes; e.g. 112 and 160 kDa of those from bacteria, Streptomyces albidoflavus [13] and [14], respectively; 480 kDa of that from a yeast, Candida utilis [7]; and approximately 91 kDA of that from a fungus, Apergillus niger (8). Nonetheless, BNT asparaginase could be a multiple-subunit-enzyme consisting of two or more identical subunits of the 45.6 kDa protein, similar to that reported for asparaginase from a strain of E.coli [15].

[FIGURE 1 OMITTED]

Effect of pH and temperature on the activity and stability of purified BNT asparaginase

The effect of pH and temperature on purified BNT asparaginase was determined. The optimum pH for the enzyme activity was at pH8.0 under the condition of 0.1 M tris-HCl buffer (Figure 2a). The enzyme was inactivated at pH2.0 to 4.0 and at pH10.0 to 11.0 and. In fact, the enzyme showed activity only in the range of pH7.0 to pH9.0.

In the case of optimum temperature, the activity of BNT asparaginase was in the range of higher than 95% of the maximum level when the reaction was carried out in the range of 37[degrees]C to 60[degrees]C (Fig. 2b). At 30[degrees]C, the enzyme activity was at 50% of the full capacity while at 70[degrees]C and 80[degrees]C, it functioned at around 67% and 45%, respectively.

[FIGURE 2 OMITTED]

The effect on the stability was expressed as the remaining activity after 2-hr incubation of the enzyme at various conditions (Table 2). The remaining activity was calculated as percentage based on the original activity of 5.7 U/ml. The effect of pH was determined only at the range of pH5.0 to pH9.0 where the enzyme was still function. During the 2-hour storage, the enzyme aliquots adjusted to different pHs were incubated at 37[degrees]C. The activity of the purified BNT asparaginase was retained at higher than 70% of the original level when stored at pH6.0 to pH7.0 and at 60% when did at pH5.0 and pH9.0.

For the effect of temperature on the stability of BNT asparaginase under a condition of 0.05M Tris-HCl buffer, pH8.6, the remaining activities of the enzyme after 2-hr storage at various temperatures revealed that the purified enzyme was most stable at 40[degrees]C to 60[degrees]C which the activity was retained at [greater than or equal to] 0% of the original. The ctivity remained 78% and 67% when stored at 70[degrees]C and 80[degrees]C, respectively.

Effect of NaCl on the stability of BNT asparaginase

Since one application of asparaginase was for reducing the asparagine content in raw materials for food manufacture, thus, the effects of NaCl or table salt was observed. The enzyme was stored in the presence of NaCl at 0.5% and 5% concentration. Samples were taken at various time intervals and assayed for the asparaginase activity. It was found that the activity of BNT asparaginase was retained at around 90% to 100% of the original activity throughout the period of 2-hour storage.

Determination of the activity against glutamine substrate of BNT asparaginase

The glutaminase activity of BNT asparaginase was determined as the two activities were commonly found on the same protein [16,17]. It was found that the enzyme functioned at only 0.5 U/ml against L-glutamine, while it did at 5.7 U/ml against L-asparagines. This clearly indicated that BNT asparaginase was highly specific to L-asparagines substrate. Thus, BNT asparaginase should to be suitable for the use as a potent antileukemic agent, since it has been suggested that glutaminase caused depletion of circulating glutamine leading to the development of some side-effects including immunosuppression in the asparaginase-treated mice [18].

Production of BNT asparaginase in a low-cost medium and its general characteristics

This was carried out based on the minimal medium, PSB, containing 2% glucose as the carbon source. The medium was tried supplemented with yeast extract at 1% concentrations, which led to approximately 25% increase in asparaginase activity (Table 3). Replacing glucose with molasses helped increasing the enzyme production by 20% and 400% when molasses was added at 1% and 2%, respectively. Concomitantly, soybean meal was found to be able to replace yeast extract. The optimum concentration of soybean meal to be used was at 1%. BNT asparaginase produced from this newly developed medium was at 1.1 U/ml.

The crude enzyme obtained was tested for the properties in activity and stability during incubation for the enzyme reaction as well as storage at different pHs and temperatures. It was shown that all the mentioned properties were indifferent from those of the purified form (Figure 3a, b). This indicated that the newly developed medium could be effectively used for production of BNT asparaginase.

[FIGURE 3 OMITTED]

Acknowledgement

This research was supported by the Mahidol University Research Grant, Mahidol University, Thailand.

References

[1] Sanches, M., Krauchenco, S., and Polikapov, I., 2007, "Structure, substrate complexation and reaction mechanism of bacterial asparaginases," Curr. Chem. Biol., 1, pp. 75-86.

[2] O'Brien, J., Nursten, H.E., Crabbe, M.J.C., and Ames, J.M., 1998, The Maillard Reaction in Foods and Medicine, Royal Society of Chemistry, Cambridge, UK.

[3] Gokmen, V., and Palazoglu, T.K., 2008, "Acrylamide formation in foods during thermal processing with a focus on frying," Food Bioproc. Technol., 1, pp. 35-42.

[4] Kent, U.M., 1999, "Purification of antibodies using ammonium sulfate fractionation or gel filtration," Meth. Mol. Biol., 115, pp. 11-18.

[5] Tareke, E., Rydberg, P., Karlsson, S., Eriksson, M., and Tornqvist, M., 2002, "Analysis of acrylamide, a carcinogen form in heated stuffs," J. Agric. Food Chem., 50, pp. 4998-5006.

[6] Biedermann, M., Biedermann-Brem, S., Noti, A., and Grob, K., 2002, "Methods for determining the potential of acrylamide formation and its elimination in raw materials for food preparation, such as potatoes," Mitt. Geb. Lebensm. Unters. Hyg., 93, pp. 653-667.

[7] Sakamoto, T., Araki, C., Beppu, T., and Arima, K., 1977, "Extracelluar asparaginase from Candida utilis, its properties as glycoprotein and antitumor activities," Agric. Biol. Chem., 41, pp. 1365-1371.

[8] Dharmsthiti, S.C., and Luechai, S., 2010, "Purification and characterization of asparaginase from solid state culture of Aspergillus niger AK10," Int. J. Biotechnol. Bi[degrees]Chem., 6, pp. 1083-1092.

[9] de Moura Sarquis, M.I., Oliveira, E.M.M., Santos, A.S., and da Costa, G.L., 2004, "Production of L-asparaginase by filamentous fungi," Mem. Inst. Oswaldo Cruz, 99, pp. 489-492.

[10] Chancharoensin, S., Luechai, S., and Banditwattanawong, C., 2010, "Media Development for Biomass Production of Antifungal Strains of Pseudomonas aeruginosa," Int. J. Appl. Agric. Res., 5, pp. 669-677.

[11] Laemmli, U.K., 1970, "Cleavage of structure of proteins during assembly of the head of bacteriophage-T4," Nature, 227, pp. 680-685.

[12] Lowry, O.H., Rosbrough, N. J., Farr, A.L., and Randall, R.J., 1951, "Protein measurement with the Folin phenol reagent," J. Biol. Chem., 193, pp. 265-275.

[13] Narayana, K.J.P.; Kumar, K.G.; and Vijayalakshmi, M., 2007, L-asparaginase production by Streptomyces albidoflavus," Ind. J. Microbiol., 48, pp. 331-336.

[14] El-Bessoumy, A.A., Sarhan, M., and Mansour, J., 2004, "Production, isolation and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation," Biocem. Mol. Biol., 37, pp. 387-393.

[15] Maita, T., Morokuma, K., and Matsuda, G., 1974, "Amino acid sequence of L-asparaginase from Escherichia coli," J. Bi[degrees]Chem., 76, pp. 1351-1354.

[16] Roberts, J., 1976, "Purification and properties of a highly potent antitumor glutaminase-asparaginase from Pseudomonas 7A," J. Biol. Chem., 251, pp. 2119-2123.

[17] Davidson, L., Brear, D.R., Wingard, P., Hawkins, J. and Kitto, G.B., 1977, "Purification and properties of an L-glutaminase-L-asparaginase from Pseudomonas acidovorans," J. Bacteriol., 129, pp. 1379-1386.

[18] Distasio, J.A., Salazar, A.M., Nadji, M. and Durden, D.L., 1982, "Glutaminase-free asparaginase from vibrio succinogenes: An antilymphoma enzyme lacking hepatotoxicity," Int. J. Cancer., 30, pp. 343-347.

Saovanee Chancharoensin * (1), Sudaporn Luechai (1) and Duangnetre Israngul (2)

(1) Science Division, Mahidol University International College, Mahidol University, Puthamonthon 4 Rd, Nakornpathom 731 70, Thailand.

(2) Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand.

* Corresponding Author E-mail: icsaovanee@mahidol.ac.th; saovanee03@yahoo.com
Table 1: Purification profile of asparaginase from B.subtilis BNT.

Step Total Total Specific
 activity protein activity
 (U) (mg) (U/mg)

Crude concentrated 420.0 277.0 1.5
 enzyme
CM-Sephadex (C50) 238.0 177.0 1.3
Sephadex G100 330.6 87.0 3.8

Step Purification Yield
 (fold) (%)

Crude concentrated 0.0 100
 enzyme
CM-Sephadex (C50) 0.7 57
Sephadex G100 2.5 79

Table 2: The remaining activity of asparaginase from B.subtilis BNT
after 2 hour storage at different pH and temperatures.

BNT Remaining activity (%) after 2-hour storage under
asparaginase different conditions

 37[degrees]C pH8.6
 pH5 pH6 pH7 pH8 pH9 40[degrees]C

Purified enzyme 60 80 98 60 45 80
Crude enzyme * 48 78 98 60 37 83

BNT
asparaginase

 pH8.6
 50[degrees]C 60[degrees]C 70[degrees]C

Purified enzyme 90 99 78
Crude enzyme * 90 100 79

BNT
asparaginase

 80[degrees]C

Purified enzyme 67
Crude enzyme * 60

* Supernatant from the culture grown on low-cost medium at
37[degrees]C for 24 hours.

Table 3: Growth and asparaginase production from BNT cultivated in
various media.

Medium Carbon source Supplements Growth Asparaginase
 ([OD.sub. activity
 600]) (U/ml)

Nutrient both none * (1) none 3.6 0.4

PSB 2% glucose none 0.3 0.3
 2% glucose 1% yeast 2.4 0.5
 extract
 1% molasses 1% yeast 2.0 0.6
 extract
 2% molasses 1% yeast 2.0 0.7
 extract
 3% molasses 1% yeast 1.9 0.6
 extract
 2% glucose 1% soybean 1.9 0.8
 meal
 2% glucose 2% soybean 2.1 0.8
 meal
 2% glucose 3% soybean 1.8 0.5
 meal
 2% molasses 1% soybean 2.1 1.1
 meal
* (1) none: No extra addition
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Author:Chancharoensin, Saovanee; Luechai, Sudaporn; Israngul, Duangnetre
Publication:International Journal of Biotechnology & Biochemistry
Date:Sep 1, 2011
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