Withania somnifera L. (Ashwagandha): A novel source of L

asparaginase

Vishal P.Oza, Shraddha D. Trivedi, Pritesh P. Parmar and

R.B.Subramanian*

B.R.D.School of Biosciences, Sardar Patel University, V.V.Nagar 388 120 (Gujarat)

India

*Corresponding author:

B.R.D.School of Biosciences, Sardar Patel University, V.V.Nagar 388 120 (Gujarat)

India. Tel.: +91-2692-234402, Fax: +91-2692-236475. Email:subramanianrb@gmail.com

Abstract

Different parts of plant species belonging to Solanaceae and Fabaceae families were

screened for L -asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for

L-asparaginase enzyme, Withania somnifera was identified as potential source of the

enzyme on the basis of high specific activity of the enzyme. The enzyme was purified

and characterized from Withania somnifera, a popular medicinal plant in South East Asia

and Southern Europe. Purification was carried out by a combination of protein

precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified

enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa as estimated by SDS-

PAGE and Size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an

optimum temperature of 37°C. The Km value for the enzyme is 6.1 x 10-2 mM. It’s the

first report for L-asparaginase from Withania somnifera a traditionally used Indian

medicinal plant.

Keywords: L-asparaginase, Purification, Solanaceae, Fabaceae, Withania somnifera.

Introduction

L-asparaginases (E.C. 3.5.1.1) are enzymes that catalyze the hydrolysis of L-

asparagine to L-aspartate and ammonia. Using amino acid sequences and biochemical

properties as criteria, enzymes with asparaginase activity can be divided into several

families (Borek 2001). The two largest and best-characterized families include bacterial

and plant-type asparaginases. The bacterial-type enzymes have been studied for over 40

years (Campbell et al.1967), mostly because they are important agents in the therapy of

certain types of lymphoblastic leukemias (Gallagher et al. 1989). Their homologues are

found in some mammals and in fungi (Bonthron and Jaskolski 1997). The bacterial-type

enzymes frequently exhibit other activities as well, and this family may be significantly

larger than the collection of sequences deposited as asparaginases. In particular, enzymes

such as glutamin-(asparagin)-ases (EC 3.5.1.38) (Ortlund et al. 2000), lysophospholipases

(EC 3.1.1.5) (Sugimoto et al. 1998) and the α-subunit of Glu-tRNA amidotransferase (EC

6.3.5.-) (Tumbula et al. 2000), can also be considered part of the bacterial asparaginase

family. It has been shown on the basis of kinetic and structural studies that two conserved

amino acid motifs are responsible for the activity of the above mentioned proteins.

(Bonthron and Jaskolski 1997; Kozak and Jaskolski 2000; Lubkowski et al. 2003; Palm et

al. 1996).

L-asparaginase has been isolated from a number of sources. These include

Proteus vulgaris (Lee and Yang 1973) Erwinia carotovora (Cammack et al. 1972),

Acinatobacter (Jones at al. 1973), Serratia marcescens (Whelan and Wriston 1974),

Mycobacterium bovis (Soru et al. 1972), Streptomyces griseus (DeJong 1972),

Achromobacteraceae (Roberts et al. 1972), guinea pig liver (Mathews and Brown 1974)

and Pisum sativum (Siechiechowicz and Ireland 1989). Though L-asparaginase has been

reported in many higher plants, little work has been carried out on the purification and

characterization of L-asparaginase from higher plants. Presence of an amidase in barley

roots capable of hydrolyzing L-asparagine had been reported earlier, the distribution of

L-asparaginase in Lupinus luteus and Dolichos lab lab seedlings have also been reported

(Lough et al. 1992b).

The plant-type enzymes have been studied less thoroughly. In plants, L-

asparagine is the major nitrogen storage and transport compound, and it may also

accumulate under stress conditions (Sieciechowicz et al. 1988). Asparaginases liberate

from asparagine the ammonia that is necessary for protein synthesis. There are two

groups of such proteins, called potassium-dependent and potassium-independent

asparaginases. Both enzymes have significant levels of sequence similarity. The plant

asparaginase amino acid sequences did not have any significant homology with microbial

asparaginase but was 23% identical and 66% similar to a human glycosylasparaginase

(Lough et al. 1992b).

The L-asparaginases of Erwinia and E. coli have been reported for many years as

effective drugs in the treatment of acute lymphoblastic leukemia. Their main side effects

are anaphylaxis, pancreatitis, diabetes, leucopoenia, neurological seizures and

coagulation abnormalities. Hence an attempt has been made to find out novel sources of

this enzyme from plants. In this paper, we report the identification of three new potential

sources of L-asparaginase with a high specific activity and purification and

characterization of L-asparaginase from Withania somnifera.

Results

Potential Source of Protein

In Solanaceae and Fabaceae families, 34 plant species were screened for L-

asparaginase enzyme activity and total protein content. The quantity of L-asparaginase

enzyme and total protein in various plant parts is presented in Table 1. Results showed

that Capsicum annum L., Datura innoxia L., Lycopersicum lycopersicum L., Pisum

sativum, Withania somnifera and Vigna unguicalata L. Sub: Unguiculata contained

appreciable amounts of the enzyme, where as Solanum melongena, Tamarindus indica,

Arachis hypogaea L, Glycine wightii, Saraca asoca, Delonix regia and Cassia fistula

contained trace amount. In Nicotiana tabacum, Crotalaria juncea, and Trigonella

foenum-graecum enzyme could not be detected. Specific activity of L-asparaginase

enzyme was determined (Table 2). Since L-Asparaginase has been already reported from

Capsicum annum L. and Pisum sativum L. (Siechiechowicz and Ireland 1989, Mozeena

and Sivaramakrisnan 1980), Capsicum annum L. was used for comparison of specific

activity. Among all the plant species screened, Withania somnifera showed the highest

specific activity for L-asparaginase enzyme.

Purification of L-asparaginase

L -asparaginase was purified from Withania somnifera, using a combination of

salt precipitation and gel filtration chromatography. The recovery of enzyme is shown in

table 3. Sodium borate buffer was used as the extraction buffer as the enzyme was most

stable at pH 8.6 during purification. A single band was obtained after salt precipitation

and gel filtration chromatography. The specific activity of the enzyme increased with

every step of purification with a minimum loss in quantity, giving a final recovery of

66% (Table 3). After every purification procedure, the peak fractions with the enzyme

activity were analyzed using SDS – PAGE.

The enzyme precipitated out between 40% - 60% of ammonium sulfate saturation.

Removal of salt from the enzyme by Sephadex G – 25 filtration was found to be most

suitable as after that step enzyme loss was negligible. Peak fractions of Sephadex G – 25

chromatography were pooled together and loaded onto equilibrated Sephadex G – 75

columns for purification and in the eluant two peaks for protein and enzyme activity were

obtained. Enzyme activity was detected only in trace amount in the peak I fractions. Peak

II fractions showed a lower quantity of protein but higher enzyme activity (Fig 1).

Molecular mass determination of L-asparaginase from Withania somnifera

The molecular mass of L-asparaginase from Withania somnifera is 72 ± 0.5 kDa

as determined by molecular size chromatography on Sephacryl S-400 HR and

polyacrylamide gel electrophoresis, respectively. The molecular mass of the subunit is 36

± 0.5 kDa as observed from SDS-PAGE separation. This is consistent with a

homodimeric enzyme (Fig 2a & 2b).

Optimum pH and Temperature

The pH effect on the activity L-asparaginase enzyme was studied at 37°C. The

results are presented in Fig. 3a. The pH for maximum activity was determined as 8.5. The

change in pH affects the ionization of essential active site amino acid residues, which are

involved in substrate binding and catalysis i.e. breakdown of substrate into product. L-

asparaginase was assayed at different temperatures (04°C -70°C) at pH 8.5. Fig. 3b

shows the effect of temperature on the catalytic activity of L-asparaginase. In the bell

shaped curve, the maximum activity of the enzyme was at 37°C.

Kinetic Parameters

Kinetic Parameters were determined by using different concentrations of L-

asparagine as substrate. A Km value of 6.1 x 10-2 mM was obtained by means of the

double-reciprocal Lineweaver-Burk plots (Fig. 3c). Different kinetic parameters like Km,

kcat, kcat/Km and Vmax of the enzyme from Withania somnifera were also analyzed.

The values are as follows: kcat: 178.57sec-1, kcat/Km: 2927.37 sec-1/mM, Vmax: 714.28

µmoles/min/mg. These values clearly show the efficiency of the enzyme L-asparaginase

from Withania somnifera.

Discussion

Withania somnifera popularly known as ‘Ashwagandha’ is widely used in

Ayurveda and other Indian systems of medicine as a rejuvenating tonic. However, this is

the first report on the presence of L-Asparaginase in this medicinally important plant

species. Shah and Qadri (1990) reported the anticancerous properties of the root extract

of this plant. It is assumed that the observed anticancerous property of the drug is due to

the presence of the L-asparaginase.

It will be worthwhile to subject the crude extract of the plant for anticarcinogenic

assay to verify and confirm the fact. The specific activity of L-asparaginase purified from

Withania somnifera was found to be 1892 IU/mg. The fold purification and enzyme

recovery revealed that the two step of purification method is very efficient, since the

specific activity increased and a good amount of activity was maintained. These efficient

results of purification may be due to the low critical temperature and suitable pH for

enzyme during the purification processes.

The molecular weight of Withania somnifera L-asparaginase is approximately

half to that of prokaryotic asparaginase (Lea et al. 1984). However the molecular weight

of other higher plant asparaginase was similar to that of Withania somnifera L. The

properties of L-asparaginase from Withania somnifera showed a number of similarities

and some sharp differences when compared with the L-asparaginases from other sources.

The molecular weight of the enzyme from Withania somnifera was found to be 72 ± 0.5

kDa; which sharply differs from the enzymes of guinea pig serum and E. coli, which have

a molecular weight of around 130 kDa.

The enzyme showed some degree of thermal stability and showed activity over a

wide range of pH values. The optimum pH of 8.5 for Withania somnifera enzyme

resembled that of E. coli  which also had pH optima around 8.5.; however Withania

somnifera enzyme differed from the guinea pig serum enzyme which showed a wide

range of optimal pH from 7.5–8.5. The optimum temperature of 37° C of the enzyme

from Withania somnifera is similar to all reported bacterial sources already used in the

treatment of leukaemia. The Km value for the Withania somnifera asparaginase enzyme

is 6.1 x 10-2 mM. This is considerably similar to the enzyme of E.coli and slightly lower

than that of Erwinia caratovora. The L-Asparaginase enzyme from Withania somnifera

has striking similarities to the bacterial enzymes in pH, temperature and Km, indicating

its potential as a therapeutic protein. Further work on sequence characterization and

protein structure determination is in progress.

Materials and Methods

Plant material

Different plant parts like fruit, root and leaf from locally available plants of

Solanaceae and Fabaceae family were collected from in and around the Botanical garden

of Department of Biosciences and Anand agriculture university, Anand (India).

Extraction of Protein

All plant parts were washed thoroughly with tap water followed by sterile distilled

water to remove surface dust and other extraneous material. The whole parts were

homogenized in liquid nitrogen using a homogenizer. Extraction was carried out from

10gm of frozen plant parts with two volumes of 0.01 M Sodium borate buffer pH 8.6.

The homogenate was centrifuged at 8000g for 10 min and supernatant was collected. This

was designated as crude extract. The pellet was resuspended in one volume of 0.01 M

Sodium borate buffer, pH 8.6, centrifuged at 8000g for 10 min and supernatant was

collected and mixed with first extract. All the steps were carried out at 4ºC.

Determination of enzyme activity and Protein Concentration

The assay system consisted of 0.5 ml of enzyme extract, 0.5 ml of 4.0 mM L-

asparagine as substrate and 1.0 ml of 0.01 M Sodium borate buffer pH 8.6. After 15

minutes of incubation at 37ºC, 1.0 ml of 15% (w/v) trichloroacetic acid was added to stop

the reaction. The mixture was centrifuged and the supernatant was collected. For enzyme

assay, ammonia released by the catalysis of L-asparagine was estimated by Nessler’s

method. To 1.0 ml of supernatant in a test tube, 8.0 ml distilled water and 1.0 ml of

Nessler’s reagent was added. The reaction was allowed to proceed for 15 min at 37ºC and

the intensity of colour was measured at 450 nm using a spectrophotometer (Unicam

Alpha). The absorption was then compared to a standard curve of ammonium sulfate.

One international unit (IU) of L-asparaginase is that amount of enzyme which liberates

1µmole of ammonia in 1 min at 37ºC. The total protein was determined by Lowry’s

method (Lowry et al. 1951), using bovine serum albumin as standard.

Purification of the enzyme

The crude protein fraction was precipitated out using different levels of

ammonium sulfate saturation from 0% to 80%. The pellet were collected after each

saturation interval by centrifugation at 12000 g for 20 min at 4º C, and dissolved in 0.01

M Sodium borate buffer pH 8.6, and checked for enzyme activity and protein

concentration. From these, dissolved pellet was loaded on to Sephadex G-25 (1.5 x 10

cm) using 0.01 M Sodium borate buffer, pH 8.6 as eluant. Further purification was done

by gel filtration using Sephadex G-75 (1.5 x 15 cm). Peak fractions of G-25 gel filtration

were collected and reloaded onto a Sephadex G-75 equilibrated with 0.01 M Sodium

borate buffer, pH 8.6. The column was washed with the same buffer. All the above

purification procedure was monitored by UV spectrophotometry, enzyme activity assay

and SDS-PAGE. The eluants containing L-asparaginase activity were used for

characterization.

Electrophoresis

The subunit molecular mass of the L-asparaginase from Withaina somnifera L.

was estimated by SDS-PAGE in a discontinuous buffer system (Laemmli 1970). The

mass of the native protein was determined by native gel electrophoresis on 10%

acrylamide gels in Tris-glycine, (pH 8.5) in a GeneI Mini- V 8 x 10 vertical gel

electrophoresis system (Banglore GeneI India.). Gels were stained with Coomassie Blue

or Silver Nitrate (Blum et al. 1987). Urease monomer (91 kDa), bovine serum albumin

(67 kDa) chicken egg albumin (43 kDa), and carbonic anhydrase (30 kDa) were used as

protein standards in native gels, while phosphorylase b (94 kDa), bovine serum albumin

(67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and carbonic anhydrase (13.7

kDa) were used as standards for SDS-PAGE. In addition, the molar size of native L-

asparaginase was estimated by chromatography on Sephacryl S-400 HR (1.6 x 25.5 cm).

The column was equilibrated with 0.01 M Sodium borate buffer, pH 8.6. The proteins

were eluted with the equilibration buffer at a flow rate of 20 ml/h. The column was

calibrated with urease pyruvate kinase (237 kDa), aldolase (158 kDa) and bovine serum

albumin (67 kDa) as protein standards.

Kinetic Measurements

To determine the optimum pH, the enzyme was assayed in the following buffers,

all at 0.05 M final concentration at 37ºC with different buffers: 0.01 M sodium acetate

(pH 3.5 – 5.5), 0.01 M Na2HPO4 / NaH2PO4 (pH 6.0 – 7.0) and 0.01 M Sodium borate

buffer (pH 7.5 – 9.5). The purified enzyme was also incubating 1 hrs at different

temperatures from 4º – 70ºC in 0.01 M Sodium borate buffer pH 8.6. The determination

of apparent Km was performed according to Lineweaver-Burk’s method (Lineweaver

1934); using different L-asparagine concentration (1.0 to 5.0mM). Determination kinetic

parameters like Km, kcat, kcat/Km and Vmax.

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Table 1. Screening for L-asparaginase in various plants of Solanaceae and Fabaceae.

Nos. Plant Species Plant

Parts

Activity IU/ml

1. Capsicum annum L. F 2475.00

2. Pisum Sativum L. F 1354.00

3. Vigna unguicalata L. Sub:

Unguiculata

L 1254.16

4. Withinia somnifera L. F 1154.16

5. Datura innoxia L. F 1137.50

6. Tamarindus indica L. L 1020.83

7. Lvcopersicum lycopersicum L. L 1004.83

8. Delonix regia L 992.16

9. Samanea saman L 841.66

10. Saraca asoca L 840..33

11. Caesalpinia pulcherrima L. L 833.00

12. Indigofera cardifolia L 829.16

13. Cassia fistula L. L 816.66

14. Phaseolus valgaris L. L 750.00

15. Cyamopsis tetragnoloba L. L 745.66

16. Cicer arietinum L. L 641.66

17. Cestrum diurum L. L 641.00

18. Prosopis Cineraria L. L 600.00

19. Cestrum nocturnum L. L 550.00

20. Vigna unguicalata L. Sub: cylindrica F 537.50

21 Cajanus cajan L. L 529.16

22. Butea monospermal L. L 470.83

23. Mimosa pudica L. L 454.16

24. Lablab perpureus L. F 429.16

25. Arachis hypogaea L F 389.00

26. Physalis minima L. L 375.00

27. Trigonella foenum-graecum L. L 362.50

28. Glycine wightii L 333.33

29. Crotalaria juncea L. L 312.50

30. Acasia sinuata L. L 308.33

31. Nicotiana tabacum L. L 233.33

32. Solanum tuberosum L. L 191.66

33. Acacia nilotica L 189.16

34. Solanum melongena L. L 145.83

Where L: Leaf, F: Fruit

Table 2. Potential sources of L-asparaginase from Solanaceae and Fabaceae.

Family Plant Species Activity

IU/ml

Protein

mg/ml

Specific activity

IU/mg

Percentage

Specific activity

Capsicum annum. 2499 27.38 91.25 100.00

Pisum sativum 1354 30.16 44.89 49.20

Withania somnifera 1196 10.23 116.80 128.01

Lvcopersicum lycopersicum 1302 26.66 48.83 53.52

Solanaceae

Datura innoxia 1134 24.85 45.62 50.00

Vigna 1254 16.07 78.02 85.51 Fabaceae

Tamarindus indica L. 1020.83 22.82 44.68 48.97

Table 3. Purification of L-asparaginase from Withinia somnifera L.

Sample Volume

ml

Protein

mg

Activity

IUa

Specific

Activity

IU/mg

Fold

purification

Recovery

%

Crude 10.0 102.4 11960.00 117.25 - 100

40-60%

(NH4)2SO4

precipitation

05.0 51.4 9850.12 191.63 1.64 82

Gel filtration

(G -75)

8.0 4.2 7950.00 1892.85 16.28 66

aIU, One international unit (IU) of L-asparaginase is that amount of enzyme which

liberates 1µmole of ammonia in 1 min at 37ºC

Figure 2. Sephadex G-75 chromatography of L-Asparaginase showing (□) protein profile

and (0) Enzyme activity profile.

Sephadex G-75

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25Fraction

O.D

.

Figure 3. Kinetic analysis of L-asparaginase

(A) Effect of pH on activity of L-asparaginase from purified enzyme of Withania

somnifera L.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 2 4 6 8 10 12 14pH

O.D

@45

0nm

(B) Effect of temperature on activity of L-asparaginase from purified enzyme of Withania

somnifera L.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 10 20 30 40 50 60 70 80

Temperature (C)

O.D

.@45

0nm

(C) Lineweaver –Burk Plots for the determination of Km and Vmax for L-asparaginase

from Withania somnifera L.

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

-0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

1/[S] (1/mM)

1/Vm

ax

Captions of Figure:

Figure 2a: 10% SDS-Poly Acrylamide gel electrophoresis: (A) Medium range molecular

marker. (B) Purified protein after final step of purification.

Figure 2b: 10% Native Poly Acrylamide gel electrophoresis: (A) Medium range of

molecular marker (B) Purified protein after final step of purification.