Indian Journal of Biochemistry & Biophysics Vol. 43, December 2006, pp. 391-394

Purification of L-asparaginase from a bacteria Erwinia carotovora and effect of a

dihydropyrimidine derivative on some of its kinetic parameters

V P Kamble1, R Srinivasa Rao1, Prita S Borkar1, C N Khobragade1* and B S Dawane2

1School of Life Sciences, Biotechnology Research Laboratory, Swami Ramanand Teerth Marathwada University,

Nanded (MS) 431 606 2Department of Chemistry, Yeshwant Mahavidyalaya,

Nanded (MS) 431 606 Received 19 August 2006; revised 17 November 2006

L-Asparaginase shows antileukemic activity and is generally administered in the body in combination with other anticancer drugs like pyrimidine derivatives. In the present study, L-asparaginase was purified from a bacteria Erwinia carotovora and the effect of a dihydropyrimidine derivative (1-amino-6-methyl-4-phenyl-2-thioxo, 1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester) was studied on the kinetic parameters Km and Vmax of the enzyme using L-asparagine as substrate. The enzyme had optimum activity at pH 8.6 and temperature 35C, both in the absence and presence of pyrimidine derivative and substrate saturation concentration at 6 mg/ml. For the enzymatic reaction in the absence and presence (1 to 3 mg/ml) of dihydropyrimidine derivative, Km values were 7.14, 5.26, 4.0, and 5.22 M, and Vmax values were 0.05, 0.035, 0.027 and 0.021 mg/ml/min, respectively. The kinetic values suggested that activity of enzyme was enhanced in the presence of dihydropyrimidine derivative.

Keywords: Erwinia carotovora, L-Asparaginase activity, Dihydropyrimidine derivative

L-Asparaginase (E.C. 3.5.1.1) a pyrimidine derivative has long been considered as an important compound in the management of childhood acute lymphoblastic leukemia. Its antileukemic effect is related to the blast cells incapability to synthesize asparagine from aspartic acid. Lymphatic tumor cells require large amounts of asparagine for rapid malignant growth. Therefore, the antileukemic treatment with L-asparaginase aims at depleting the asparagine level in the blood, in order to exhaust its supply1,2. In general, the leukemic treatment involves combination

chemotherapy with a number of different anticancer drugs3-5. L-Asparaginase obtained from Escherichia coli and another bacteria Erwinia carotovora possess the anti-lymphoma properties6-9 and the characteristics of E. coli L-asparaginase have been reported earlier10,11. Pyrimidines, which comprise an integral part of nucleic acids, also show anti-viral, anti-bacterial and anti-cancer activities, depending upon geometry and type of substituents attached to the pyrimidine ring12. Conjugation of L-asparaginase with anticancer drugs like dihydropyrimidine derivatives affects its affinity to its substrate L-asparagine13. However, the characteristics and effect of dihydropyrimidine derivatives on the L-asparaginase activity from E. carotovora have not been studied in detail. In the present study, attempt has been made to purify L-asparaginase from E. carotovora and the effect of a dihydropyrimidine derivative (1-amino-6-methyL-4-phenyL-2-thioxo, 1,2,3,4-tetrahydropyrimi-dine-5-carboxylic acid methyl ester) has been studied on the enzyme activity by determining the kinetic parameters Km and Vmax at variable pH and temperatures.

Materials and Methods All the chemicals were purchased from S. D. Fine-Chem. Ltd., Mumbai, India and were used without further purification unless stated otherwise.

Bacterial isolates, extraction and purification of L-asparaginase Erwinia carotovora (MTCC, 1428) was procured from the Institute of Microbial Technology, Chandigarh, India was used as a source of L-asparaginase. The bacterium was cultivated in the nutrient broth (HiMedia Mv 088) containing beef extract (3.0 g), NaCl (5.0 g) and peptone (5.0 g) in 1000 ml distilled water. The medium was sterilized at 15 Lbs for 15 min at 121C using autoclave. A slant culture of bacteria (loop full) was transferred in a 100 ml sterile cultivation medium in aseptic condition. The medium was incubated at 25C, continuously for 7 days. The pH of the culture media was measured every day and if varied, corrected at pH 6.9 with dropwise addition of alkali (0.1 N NaOH). After optimum growth of bacterial culture, nutrient broth was filtered through filter paper

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*Corresponding author Email: cnkhobragade@rediffmail.com Tel: 91(2462) 229242 Fax: 91(2462) 229245, 229325

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(Whatman No.1) and filtrate was subjected to centrifugation in cooling centrifuge at 15,000 rpm for 15 min, so as to remove the bacterial cell mass completely. Thereafter, the clear supernatant was collected and treated with ammonium sulphate of 50, 60, 80, and 100% final saturation (w/v). The chilled precipitates from different saturations of ammonium sulphate were collected by cold centrifugation at 20,000 rpm for 15 min and were allowed to settle completely by placing the medium in a refrigerator for overnight. The chilled contents were recentrifuged at 20,000 rpm for 15 min at 4C and the enzyme was pooled out completely in the form of pellets14. The pellets were dissolved in 25 ml of Tris-HCl buffer (0.1 M, pH 8.6) and dialyzed against two changes of Tris-HCl buffer (0.01 M, pH 8.6) for 48 h. The dialyzed enzyme (desalted) was collected and subjected to anion-exchange chromatography.

DEAE-cellulose anion-exchange chromatography A chromatography column (2 40 cm) was packed with DEAE-cellulose and equilibrated with 0.1 M Tris-HCl buffer containing 2 mM MgCl2 (pH 8.6). The desalted enzyme obtained from dialysis (25 ml) was loaded on to the top of the column and eluted with 1.0 L of linear gradient Tris-HCl buffer composed of NaCl (0-1 M) at room temperature. The flow rate of the column was adjusted to 1 ml/min. The fractions were collected and monitored constantly to determine the protein content in the eluant (280 nm UV/visible Schimadzu). The 15 ml fractions were pooled and assayed for the protein and enzyme activity.

Protein determination The eluted fractions from DEAE-cellulose anion-exchange column (scanned at 280 nm) were used for quantitative protein determination using bovine serum albumin as a standard15 and protein content was found to be 0.1 mg/ml. The purity was checked by applying 10% SDS-PAGE16 and molecular mass was found to be 33.5 kD.

Synthesis of dihydropyrimidine derivative The dihydropyrimidine derivative 1-amino-6-methyL- 4-phenyL-2-thioxo-1, 2, 3, 4-tetrahydropyrimi-dine-5-carboxylic acid methyl ester was synthesized essentially according to the methods described previously17,18. Briefly, a mixture of benzaldehyde derivative (0.01 mole, 1.06 g) with Z as substituents, 2-Cl, 4-Cl, acetoacetic ester (0.01 mole, 1.30 g) and thiosemicarbazide (0.01 mole, 1.06 g) in absolute

ethyl alcohol (15 ml) and catalytic amount of piperidine was refluxed for 4 h. The reaction mixture was cooled and poured over ice. The solid, thus separated was filtered under reduced pressure, dried and recrystallized from methanol to give above product (m p = 218C, yield = 85%, mol. formula = C14H16N3O2S). The product was characterized as 1-amino-6-methyL-4-phenyL-2-thioxo, 1, 2, 3, 4-tetra-hydropyrimidine-5-carboxylic acid methyl ester (Scheme 1), on the basis of spectral characteristics.

Spectral characterization

1H NMR (300 MHz, CDCl l3 + DMSO-d6): 4.8 (s, 2H, NH2), 9.6 (s, 1H, NH), 8.2-7.5 (m, 5H, Ar-H), 5.4(s, 1H, 4-CH), 4.1 (q, 2H, -CH2CH3), 2.3 (t, 3H, -OCH2CH3), 1.2 (s, 3H, CH3); IR (KBr): cm-1 3360 (NH str), 3070 (aromatic C-H str.), 1680 (C=O, str. of ester), 1120 (C = S str.), 1485 (C=C str.), 1180 (C N vib.) 1080 (C O str.)

Enzyme activity assay L-Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The test organism was grown in nutrient broth containing L-asparagine and pH indicator phenol hypochlorite. During incubation at 25C, the released ammonia reacts with phenol hypochlorite to form blue color, which indicates the production of L-asparaginase19. One unit of L-asparaginase was defined as the amount of enzyme that liberated 1 mole of ammonia per min under assay conditions20. L-Asparaginase activity was determined in Tris-HCl buffer (pH range 8.0-9.2) and at temperatures 5-55C. A solution of L-asparagine (6 mg/ml), a

CHO

Z

+ CH3 C

O

CH2 C

O

OC2H5 + H2N NH C

S

NH2

PiperidineC2H5OH4 h

N

N H

S

NH2

H3C

CO

C2H5O

Z

Scheme 1

NOTES

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substrate for L-asparaginase was prepared in double-distilled water. A reaction cocktail was prepared by taking 1.0 ml each of the enzyme, asparagine, chromogen solution (0.25% phenol red), Tris-HCl buffer and the volume was adjusted to 5 ml using distilled water. The reaction mixture was incubated at 35C for 15 min and thereafter, 1.0 ml of 0.66 N H2SO4 was added. The level of released ammonia was determined from the ammonium sulphate standard curve by recording the absorbance at 560 nm against the respective blank.

Determination of kinetic parameters Km and Vmax The Vmax and Km values were determined in the presence and absence of dihydropyrimidine derivative by increasing the asparagine volume (1.0 to 10.0 ml). In a reaction mixture, the volume of purified enzyme, chromogen, H2SO4, distilled water and dihydropyri-midine derivative (1 mg/ml) was fixed at 1.0 ml and Tris-HCl buffer was added to make up a final volume of 5.0 ml. The Vmax and Km values were determined in absence and presence of the dihydropyrimidine derivative (1-3 mg/ml) using a Lineweaver-Burk double-reciprocal plot21.

Results and Discussion L-Asparaginase, an important therapeutic enzyme, has been isolated from a number of sources. However, only E. coli and E. carotovora asparaginase has shown anti-tumour activity22,23. In the present study, the enzyme was purified from the E. carotovora using

dialysis and anion-exchange chromatography. The enzyme activity was determined by using L-asparagine as a substrate and found to be 0.31 mg/ml/min (Table 1). The effect of the pH and temperature on the enzyme activity was studied both in the absence and presence of dihydropyrimidine derivative. The activities of the enzyme were determined in a pH range 8.0 to 9.2 at interval of 0.2 and between 5C to 55C at interval of 10C. The optimum pH of the L-asparaginase was found to be 8.6 (Table 2). The enzyme was active between 5C and 55C and the optimum temperature was 35C (Table 3). Results revealed that in the absence and presence of (1-3 mg/ml) dihydropyrimidine derivative in the reaction medium, the values of Km were 7.14, 5.26, 4.0 and 3.22 M respectively, whereas the Vmax values were calculated as 0.05, 0.035, 0.027 and 0.021 mg/ml/min (Fig. 1). The decrease in Km values with increase in concentration of dihydropyrimidine derivative indicated that L-asparaginase had more affinity towards the substrate (L-asparagine) in the presence of dihydropyrimidine derivative. The decrease in Vmax values observed after the addition of dihydropyrimidine derivative indicated the change in microenvironment of the enzyme. The decrease in Km values may be attributed to the fact that dihydropyrimidine derivative might be binding possibly to the one of the monomer of L-asparaginase, a K+-dependant oligomeric enzyme24, thereby affecting its conformation favourably to

Table 2Effect of pH on L-asparaginase activity in absence and presence of dihydropyrimidine derivative

pH Enzyme activity (U/ml)

Specific activity (mg/ml)

8.0 0.33 3.3 8.2 0.46 4.6 8.4 0.66 6.6 8.6 0.80 8.0 8.8 0.60 6.0 9.0 0.46 4.6 9.2 0.40 4.0

Table 1Activity and purity of L-asparaginase from Erwinia carotovora obtained at different steps

Steps Volume Activity Total activity Protein Specific activity Purification (ml) (U/ml) (U) (%) (mg/ml) (U/mg) (fold)

Broth 100 0.06 6.0 100 1.72 0.03 --- Ammonium sulphate 30 0.17 5.1 85 0.84 0.20 6 Dialysis 25 0.20 5.0 83 0.77 0.25 8 DEAE 15 0.31 4.6 76 0.10 3.10 88

Table 3Effect of temperature on L-asparaginase activity in absence and presence of dihydropyrimidine derivative

Temp (C)

Enzyme activity (U/ml)

Specific activity (mg/ml)

05 0.40 4.0 15 0.46 4.6 25 0.53 5.3 35 0.86 8.6 45 0.50 5.0 55 0.40 4.0

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enhance its activity25,26. The stereochemistry between the aryl group and dihydropyrimidine ring was found to be one of the factors having a pronounced effect on hydrolytic activity. In addition, in an enzyme-dihydropyrimidine derivative bound conformation, the substituted aryl ring (Z = 2-Cl, 4-Cl) might be positioned axially or perpendicular to the dihydropyrimidine ring with a 4-aryl substituted Z, thereby adopting a synperiplanar (relative to C4-H) orientation27,28.

Acknowledgement The authors are thankful to Head, School of Life Sciences, SRTM University, Nanded (MS) for providing laboratory facilities during the experimental work and Dr V T Kamble, School of Chemical Sciences, SRTM University for useful discussion.

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Fig. 1Effect of dihydropyrimidine derivative on kinetic parameters Km and Vmax