Extracellular L-Asparaginase from a Protease ...

Appl Biochem Biotechnol DOI 10.1007/s12010-013-0455-0

Extracellular L-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro Yogendra Singh & Ravi Kumar Gundampati & Medicherla V. Jagannadham & S. K. Srivastava

Received: 19 June 2013 / Accepted: 20 August 2013 # Springer Science+Business Media New York 2013

Abstract An extracellular L-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg−1. The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified L-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the Km, Vmax, and kcat values of the enzyme were 0.257 mM, 1.537 U μg−1, and 993.93 s−1, respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α+β class of proteins with approximately 74 % αhelices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-KK-I-A of purified L-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the Lasparaginase has significant antineoplastic properties. Keywords L-Asparaginase . Bacillus aryabhattai ITBHU02 . Circular dichroism . Biochemical characterization . Antileukemic activity . N-terminal sequence

Y. Singh : S. K. Srivastava (*) School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi 221005, India e-mail: [email protected] S. K. Srivastava e-mail: [email protected] R. K. Gundampati : M. V. Jagannadham Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India

Appl Biochem Biotechnol

Introduction L-asparaginase (L-aspartate amidohydrolase, EC 3.5.1.1) is enzyme that catalyses free amino acid L-asparagine into L-aspartate and ammonia. For over 30 years, bacterial L-asparaginases have been a mainstay of multidrug chemotherapeutic regimens extensively used for the treatment of malignancies of lymphoid systems, acute lymphoblastic leukemia, Hodgkin's lymphoma, and melanosarcoma [1, 2]. Apart from its antileukemic activity, L-asparaginase has also been utilized successfully in food industries as an agent for producing acrylamide-free food products [3]. L-Asparaginase has been applied for constructing a diagnostic biosensor as the amount of ammonia generated by the enzyme action directly correlates to the level of L-asparagine in a patient's blood [4]. The enzyme has recently received meticulous attention of the scientists, since it has been detected in human body [5]. Unlike normal cells, the malignant cells are extensively dependent on exogenous supply of L-asparagine for survival as these are devoid of L-asparagine synthetase activity. L-Asparagine is a crucial amino acid for protein, DNA, and RNA synthesis [5] and its requirement is cell cycle specific for G1 phase at cell division [6]. Upon administration, L-asparaginase discontinues the supply of this nonessential amino acid, L-asparagine, to the tumor cells by cleaving it to L-aspartate and ammonia. Starvation for L-asparagine disturbs the metabolic status of malignant cells and finally leads them to apoptotic death [1, 7]. To date, L-asparaginase from two microbial sources, viz, Escherichia coli and Erwinia caratovora, is being used for clinical preparations. However, therapeutic output of these enzymes has been rarely reported without any incidence of toxicity [1]. To overcome the immunogenic toxicity associated with the clinical preparations of asparaginases from current microbial sources, a new serologically different enzyme, having same therapeutic effect, is required. To obtain a better and alternative source of L-asparaginase, there is an ongoing interest to screen new organisms from different biodiversities [8]. The presence of protease activity in fermented medium might cause the degradation of L-asparaginase or other proteins of interest [9, 10]. Purification of a protein of interest from protease-free source strains has an added advantage of economical downstream processing of the synthesized proteins. In this context, we have isolated an efficient L-asparaginase-producing- and protease-deficient bacterial strain identified as Bacillus aryabhattai ITBHU02 in our laboratory [11]. The present study deals with the purification, biochemical characterization, circular dichroic spectroscopy, and kinetic studies of L-asparaginase from B. aryabhattai ITBHU02. Moreover, the study was further extended to prove the antineoplastic activity of purified L-asparaginase against multiple leukemic cell lines (viz, HL60 and MOLT-4).

Materials and Methods Chemicals Diethylaminoethyl (DEAE)-Sepharose fast flow and Hi-Load Superdex 200 16/60 were purchased from GE Healthcare Biosciences, USA, while Seralose CL-6B was from Sisco Research Laboratories, Mumbai, India. Most of the substrates used in the experiments (L-asparagine, D-asparagine, DL-asparagine, D-aspartic acid, L-aspartic acid βhydroxamate, L-asparagine-t-butyl ester hydrochloride, FMOC-L-asparagine, and N-αacetyl-L-asparagine) were Sigma-Aldrich products (St. Louis, MO, USA). Protein molecular standard and biomarkers were from Genei, Bangalore. The rest all the chemicals used in the study were of analytical grade and purchased from Himedia, Merck and


Sisco Laboratories, India. All the experiments were performed independently in triplicate and their values were averaged to minimize the experimental error. Microorganism and Culture The isolation, screening, and identification of a protease-deficient bacterium B. aryabhattai ITBHU02 (accession #: JQ673559), capable of producing extracellular L-asparaginase, were performed from the soil sample contaminated with hospital waste [11]. The strain was maintained over nutrient agar slants (pH 7.0) and stored at 4 °C. Stock culture was transferred to fresh NA medium every 3–4 weeks. Inoculum Preparation and L-Asparaginase Production For inoculum preparation, the bacterial culture was grown in modified M9 medium containing the following (in gram per liter): beef extract, 2.5; glucose, 5.2; Na2HPO4·2H2O, 6.0; KH2PO4, 4.07; MgSO4·7H2O, 0.82; NaCl, 0.5; CaCl2·2H2O, 0.015; and L-asparagine, 4.91 (pH 7.5) optimized previously [11]. A loopful of cells from a slant was transferred into 100 mL of above sterile medium in 500 mL Erlenmeyer flasks and incubated at 30 °C in a rotary shaking incubator (160 rpm). A 7.5-L Bentchtop fermentor (BioFlo/Celligen 115, New Brunswick, USA) with a working volume of 3.0 L was then inoculated with inoculum (2 %v/v) from 24 to 25 h grown seed culture and allowed to grow with optimum fermentation conditions at 37 °C and stirring speed 200 rpm with constant aeration rate of 0.6 VVM [2]. After 25 h of inoculation, the cells were removed by centrifugation (10,000×g). The resulted supernatant was used as crude enzyme preparation. L-Asparaginase

Purification and Quantification

All the purification steps were performed at 4 °C unless stated otherwise. All the chromatographic runs were observed for protein at 280 nm. Ammonium Sulfate Precipitation L-Asparaginase

produced by B. aryabhattai ITBHU02 was fractionated by graded precipitation using ammonium sulfate. Initially, the crude supernatant was subjected to 30 % ammonium sulfate saturation and precipitated protein was removed by centrifugation 15,000×g for 10 min. Thereafter, the ammonium sulfate saturation was increased to 80 %, and resulting protein was collected and resuspended in minimal volume of 0.05 M Tris–HCl buffer, pH 8.6. This enzyme solution was dialyzed for 24 h against three changes of the same buffer. DEAE-Sepharose Chromatography

The dialysate from the previous step was loaded onto a DEAE-Sepharose column (2.0×10.0 cm) pre-equilibrated with 0.05 M Tris–HCl buffer, pH 8.6, and was eluted with a linear gradient of 0– 0.8 M NaCl in the same buffer. The fractions with L-asparaginase activity were pooled, desalted, and diafiltrated using an Amicon membrane concentrator having 10 kDa cutoff. Seralose CL-6B Chromatography The active fractions from DEAE-Sepharose column were further purified with a Seralose CL-6B gel (separation range# 104–106 Da; particle size# 40–190 μm) column (2.0×15.0 cm),


pre-equilibrated with 0.05 M Tris–HCl buffer, pH 8.6. The protein elution was done with the same buffer until no protein was seen in the elute. All the fractions were analyzed for the activity and enzyme concentration. The active fractions were pooled, concentrated, and dialyzed against the 0.05 M Tris–HCl buffer, pH 8.6. The dialysate was further vacuum-dried using a lyophilizer and stored at 4 °C for further studies. L-Asparaginase

Activity Assay

The enzyme of different preparation was assayed by direct nesslerization method of Kumar et al. [12] with some modifications. L-Asparaginase activity was measured in a reaction mixture containing 0.9 mL of 0.01 ML-asparagine prepared in 0.05 M Tris–HCl buffer (pH 8.6) and 0.1 mL of the enzyme at 37 °C for 30 min. Subsequently, the reaction was terminated by adding 0.1 mL of trichloroacetic acid (15 %w/v). The reaction mixture was centrifuged at 10,000×g for 5 min at 4 °C to remove any precipitate. The ammonia liberated in the supernatant was estimated spectrophotometrically at 436 nm by adding 0.1 mL Nessler's reagent into the sample containing 0.1 mL supernatant and 0.8 mL distilled water. One international unit (U) of asparaginase activity is defined as the amount of enzyme that liberates 1.0 μmol of NH3 per min at specified conditions. The specific activity of L-asparaginase was expressed in terms of units per milligram of protein. Protein Quantification and Determination of Molecular Mass Protein concentration was determined by absorbance at 280 nm as well as by Bradford's method [13] using BSA as a standard protein. The molecular weight of the purified B. aryabhattai Lasparaginase was estimated by SDS-PAGE as described by Laemmli [14] using a 12 % separating gel (pH 8.8) and a 5 % stacking gel (pH 6.8). A native PAGE was performed using 10 % resolving gel using standard protein markers (range, 29–205 kDa; PMWH, Genei). Protein bands were visualized with Coomassie Brilliant Blue R-250 staining. For molecular weight determination of the native enzyme, a gel filtration chromatography using Hi-Load Superdex-200 16/60 column was equilibrated with 0.05 M Tris–HCl buffer (pH 8.6) containing 0.05 M NaCl and 10 % glycerol. The column was calibrated with ferritin (440 kDa), aldolase (158 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa) procured from GE Healthcare. The elution volume (Ve) of each marker protein and void volume (Vo) of the column were evaluated. A plot of Ve/Vo against log of molecular weight was used to calculate the molecular weight of L-asparaginase. Determination of Isoelectric Point The isoelectric point (pI) of the purified enzyme was determined as described by El-Sayed [15]. The enzyme preparation was incubated at different pH levels (5.0–9.0) using different buffers at 4 °C. After 12 h of incubation, the enzyme was precipitated by centrifugation at 12,000×g for 10 min. The quantitative measurement of the precipitated protein was performed by using Folin's reagent. The isoelectric point was expressed as the pH at which the maximum enzyme precipitation occurred. Carbohydrate Content The structural glycosyl moieties in the enzyme molecule for glycoprotein detection were estimated by phenol sulfuric acid method [16]. The gel was also stained with Schiff's reagent, which is specific for glycoproteins [17].


Tyrosine and Tryptophan Content The tyrosine and tryptophan contents of the enzyme were determined spectrophotometrically in alkaline condition using the method as described in [18]. Briefly, the absorbance spectra of the enzyme in 0.1 M NaOH was measured between 220 and 320 nm, and the absorbance values at 280 and 294.4 nm were deduced from the spectra. The tryptophan and tyrosine contents were calculated by the following expression: w ¼ A280nm −xεy =εw −εy where w is estimated molar concentration of tryptophan in moles per liter. A280nm is absorbance at 280 nm from the protein spectra, and εw and εy are molar extinction coefficients of tryptophan and tyrosine in 0.1 M NaOH at 280 nm (εw=5225 M−1 cm−1; εy=1,576 M−1 cm−1), respectively. The total tyrosine and tryptophan content in the protein, x, was calculated using ε294.4=2,375 M−1 cm−1. The number of a particular amino acid residue per molecule of the protein was calculated from the ratio of the molar concentrations of the amino acid residues to that of the total protein. Validation of results was done by estimating the tyrosine and tryptophan contents of papain, ribonuclease, BSA, and lysozyme under similar conditions. Free and Total Sulfhydryl Content The exposed and total cysteine residues of the enzyme were estimated by Ellman's method [19], where the release of thionitrobenzoate (TNB) due to reduction of thiol with dithionitrobenzoate was determined by increase in the absorbance at 412 nm. Molar extinction coefficient of TNB anion at 412 nm is 14,150 M−1 cm−1 [16]. In order to estimate exposed sulfhydryl group, the enzyme was activated with 0.01 M β-mercaptoethanol in 0.05 M Tris–HCl buffer (pH 8.0) for 15 min and then dialyzed against 0.1 M acetic acid at 4 °C for 24 h with frequent changes. The total sulfhydryl content was quantified by reducing the enzyme in presence of 6 M GuHCl for 15 min at 37 °C and dialyzed against 0.1 M acetic acid. Validation of results was done by estimating the tyrosine and tryptophan contents of papain, ribonuclease, BSA, and lysozyme under similar conditions. Extinction Coefficient The extinction coefficient of the enzyme was estimated by spectrophotometric method [20] using the formula ε1% 280nm ¼ 10 5; 690nw þ 1; 280ny þ 120nc =M where nw, ny, and nc denotes the number of tryptophan, tyrosine, and cystine residues in the protein; M is the molecular mass of the protein; and 5,690, 1,280, and 120 are the extinction coefficients of tryptophan, tyrosine, and cystine, respectively. pH and Temperature Optima The activity of the purified B. aryabhattai L-asparaginase was measured as a function of varying pH and temperature to determine the pH and temperature optima of the enzyme. The optimum pH of the enzyme was measured by using following buffers at 0.05 M concentration: sodium acetate buffer (pH 5.0–6.0), potassium phosphate buffer


(pH 6.5–7.5), Tris–HCl buffer (pH 8.0–9.0), and glycine-NaOH buffer (pH 9.5–11.0). Blank determinations were done simultaneously at all respective pH levels without enzymes. Effect of temperature on the activity of purified enzyme was investigated by preincubating the enzyme for 15 min at desired temperature (25–70 °C) and the residual activity was measured at the same temperature. At each temperature, a control assay was carried out without adding the enzyme and used as a blank. Stability As the applicability of an enzyme is totally dependent on its stability, the effect of pH (5.0– 11.0), temperature (25–70 °C), as well as the presence of different metal ions and organic solvents such as ethanol, methanol, and acetonitrile on the activity of L-asparaginase was evaluated. The enzyme was preincubated for 24 h at specified conditions of pH, metal ions, and organic solvents, whereas for the assessment of temperature stability, the sample was preincubated for 15 min before the activity assay. The measurement of residual activity of the enzyme was performed as previously described. Effect of Various Modulators on L-Asparaginase Activity The effects of SDS, EDTA, GuHCl, β-mercaptoethanol, ascorbic acid, iodoacetamide, thiourea, and urea on L-asparaginase activity were examined after the purified enzyme had been preincubated with them at 30 °C for 30 min, individually. Residual activity was determined under optimal enzyme assay conditions. Activity assayed in the absence of any additives was taken as 100 %. Substrate Specificity The specificity of purified L-asparaginase towards different substrates was estimated. The amidohydrolytic activity of the enzyme was determined separately towards D-asparagine, DL-asparagine, L-glutamine, L-glutamic acid, D-aspartic acid, L-glutamic acid, L-aspartic acid β-hydroxamate, L-asparagine-t-butyl ester hydrochloride, FMOC-L-asparagine, and N-αacetyl-L-asparagine at a final concentration 10 mM, using L-asparagine as the standard substrate. The relative activity was expressed as the percentage ratio of the enzyme activity calculated against afore-mentioned amino acids and their analogues to enzyme activity with L-asparagine. Secondary Structure Elucidation by Far-UV Circular Dichroism The circular dichroism (CD) spectra of B. aryabhattai L-asparaginase were recorded at protein concentration 0.1 mg mL−1 in 0.05 M Tris–HCl buffer (pH 8.5) at 37 °C on a JASCO 500A spectropolarimeter, pre-calibrated with 0.1 % d-10-camphorsulfonic acid solution. The enzyme was most active and stable at pH 8.5; therefore, all the readings were collected at this pH. CD-based evaluation of molecular denaturation was performed by incubating purified enzyme with 6.0 M GuHCl dissolved in 0.05 M Tris–HCl buffer (pH 8.5) for 24 h. Secondary structures in the purified enzyme were deconvoluted from far-ultraviolet CD (far-UV CD) spectra in the wavelength range 180–260 nm by implementing K2D2 software [21]. Average three scans were recorded using a 1-mm


path-length quartz cuvette for the collected spectrum. The acquired spectra were corrected for the baseline and expressed as mean residue ellipticity [θ], using the equation ½θ ¼ θobs

MRW 10cl

where θobs, c, and l denote, respectively, the observed ellipticity in degrees, protein concentration in gram per milliliter, and the path-length of the light in centimeter. The mean weight of amino acid residue (MRW) was assumed as 110 for calculations. Kinetic Analyses For kinetic analyses, the enzyme activity was assayed by determining the rate of ammonia formation using a coupled assay with glutamate dehydrogenase, according to Balcao et al. [22]. The standard reaction mixture contained 0.05 M Tris–HCl buffer (pH 8.6), 1 mM αketoglutare, 0.2 mM NADH, 20 U glutamate dehydrogenase, and variable concentrations of L-asparagine, in a final assay volume of 1 mL. The reaction was started by adding the enzyme solution (5 μg). Finally, the concentration of liberated ammonia is estimated by determination the oxidation rate of NADH, by measuring the absorbance at 340 nm. One international unit (U) is defined as the amount of enzyme catalyzing the oxidation of 1 mmol NADH per min under the above-mentioned conditions. The kinetic parameters of purified Lasparaginase were evaluated using Eadie–Hofstee plot. Calculated regression lines from the plots were used to determine the Km and Vmax values. The value of turnover number (kcat) and specificity constant (kcat/Km) of the enzyme were calculated on the basis of one active site per subunit (Subunit Mr~38.8 kDa) and 38.8 mg of subunit protein was considered as 1 μmol of enzyme as well [7, 23]. N-terminal Amino Acid Sequencing The amino terminal sequencing of the purified enzyme was carried out at the N-terminus by automated Edman degradation method using a PROCISE amino acid sequencer (Applied Biosystems, USA) at the National Institute of Immunology, India. N-terminal sequence homology was analyzed with other known L-asparaginase sequence using the BLAST database (http://www.ncbi.nlm.nih.gov/BLAST). Further, CLUSTALW (1.82) program (http://www.ebi.ac.uk/clustalw) was used for multiple sequence alignment. Determination of Antitumor Property In Vitro The effect of variable concentrations of L-asparaginase of B. aryabhattai ITBHU02 on the survival of human leukemic cells (HL60 and MOLT-4) was evaluated at ACTREC, Mumbai. A semi-automated Sulforhodamine B (SRB) dye-based colorimetric assay was used for determination of cell survival [24]. Viable cells counting were carried out in a hemotocytometer chamber using Trypan Blue (0.4 %w/v) before each cytotoxicity experiment. The stock solution of the lyophilized L-asparaginase was prepared in 10 % (v/v) DMSO and diluted accordingly with RPMI 1640 medium to achieve a final concentration in the range of 0.1–100 μM. Aliquots of 10 μL from each drug dilutions were added to each compound well of a 96-well tissue culture plate followed by addition of 90 μL of cell suspension (cell density≈1.9×104 cells per well). Appropriate positive control (Adriamycin) and controls were also run. The plates were incubated at 37 °C for 24 h in a 5 % CO2


humidified atmosphere. The termination of the assay was achieved by adding 100 μL cold 10 % (w/v) TCA to each well and incubating the cells for 1 h at 4 °C. After three to four steps of washing with water and drying at room temperature, the fixed cells were subjected to 100 μL of 0.057 % SRB solution. After 30 min, the stained cells were rinsed thrice with 1 % (v/v) acetic acid. Cell bound SRB dye was solubilized by shaking with 10 mM Tris (pH 10.5). The absorbance was measured with a microplate reader at 540 nm with 690 nm as reference wavelength. All the experiments were repeated in triplicate.

Results and Discussion L-Asparaginase

Purification and Assessment of Physical Properties

The extracellular L-asparaginase from B. aryabhattai ITBHU02 was extracted and purified to apparent homogeneity from cell-free supernatant using following separation steps: ammonium sulfate fractionation, ion exchange, and gel filtration chromatography (Table 1). The enzyme was partially purified using ammonium sulfate fractionation at 30–80 % saturation. Subsequently, ion exchange and gel filtration chromatography on DEAE-Sepharose and Seralose CL6B columns, respectively, were implemented resulting into 68.9-fold enzyme purification and 27.9 % recovery with a specific activity of 680.47 U mg−1. The enzyme has been purified to homogeneity as judged by the presence of one single band by SDS-PAGE displaying the subunit molecular weight approximately 38.8 kDa (Fig. 1a). The native molecular weight of the purified enzyme was estimated to be 155.0±1.0 kDa by both native PAGE and gel filtration chromatography (Fig. 1b, c). These results indicated that the enzyme migrated as a tetrameric form in native PAGE and gel filtration. Several tetrameric L-asparaginases of bacterial origin with molecular mass in the range of 140–160 kDa has been reported by other researchers [23, 25, 26]. However, L-asparaginases purified from Thermus thermophilus and Cladosporium sp. have shown homohexameric [27] and heterotrimeric [28] native states, respectively. The isoelectric point for purified L-asparaginase was 7.8 based on pH–precipitation profile (Fig. 2). Most of the L-asparaginases reported with pI value ranging from 4.9 to 8.8 [23, 29, 30]. L-Asparaginase purified from B. aryabhattai was not found to be bounded with any structural glycosyl moiety. The absence of glycosyl residues from the purified enzyme indicated a lack of glycosylation for this enzyme. A comparison of physical and biochemical properties of L-asparaginases from different sources is summarized in Table 2.

Table 1 Summary of purification steps of L-asparaginase produced by B. aryabhattai ITBHU02 Purification steps

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Recovery (%)

Crude extract

3,164.9

31,269

9.88

(NH4)2SO4 precipitation

1,155.3

23,013

19.92

73.6

100

Fold purification

1 2.02

DEAE-Sepharose Fast Flow

70.96

16,040

226.02

51.3

22.9

Seralose CL-6B

12.8

8,710

680.47

27.9

68.9


Fig. 1 Assessment of homogeneity and molecular weight estimation of extracellular L-asparaginase produced by B. aryabhattai ITBHU02. a SDS-PAGE (12 % gel) showing: Marker, 1—crude extract, 2—DEAESepharose purified, and 3—Seralose CL-6B purified enzyme. b Native PAGE showing: Marker and 1— purified enzyme. c Gel filtration chromatography using Superdex-200 16/60 column

Specific Amino Acid Residues The tryptophan and tyrosine contents of the native L-asparaginase were 8 (8.11±0.021) and 44 (measured value 43.78±0.07), respectively. The total sulfhydryl content of the enzyme was found to be 8 (measured value 8.04±0.012) with no exposed cysteine residues (measured value 0.04), thus forming four disulphide bridges. The extinction coefficient of Lasparaginase was estimated 6.63 M−1 cm−1 by spectrophotometric method. Under similar experimental conditions, papain, ribonuclease, BSA, and lysozyme gave the reported values. pH and Temperature Optima The pH profile for L-asparaginase activity was evaluated from pH 5.0 to 11.0 and temperature in a range of 25–70 °C using L-asparagine as a substrate. The enzyme was shown to be active at pH range 8.0–9.0 with a maximum L-asparaginase activity at the pH 8.5 (Fig. 3a). The optimum temperature was achieved at temperature of 40 °C, on either side of which there was a decline in enzyme activity (Fig. 3b). 2.5 Precipitated protein (mg/ 2mL)

Fig. 2 Estimation of isoelectric point of L-asparaginase by measuring the precipitated protein at different pH

2.0

1.5

1.0

0.5

0.0 5.0

5.5

6.0

6.5

7.0 pH

7.5

8.0

8.5

9.0

Appl Biochem Biotechnol Table 2 Comparison of physical and biochemical properties of L-asparaginases produced by different microorganisms Source

Molecular weight (kDa)

Oligomeric state

Isoelectric point (pI)

pH optima

Temperature optima (°C)

References

Bacillus aryabhattai

155

Homotetramer

7.8

8.5

40

Current work

Pectobacterium carotovorum

144.4

Homotetramer

8.4

8.5

39.3

[23]

Bacillus circulans

140

Homotetramer

N.R.

8.6

N.R.

[25]

Thermus thermophilus

200

Homohexamer

6.0

9.2

77

[27]

Cladosporium sp.

121

Heterotrimer

N.R.

6.3

30

[28]

Pseudomonas stutzeri

34

Monomeric

6.4

9.0

37

[29]

Vibrio succinogenes

146

Tetrameric

8.74

7.3

37

[30]

Escherichia coli

150

Homotetramer

4.9

8.6

37

[37]

N.R. not reported

Fig. 3 Influence of different a pH and b temperature on activity and stability of L-asparaginase


Stability The stability of the enzyme at extreme environmental conditions is a significant factor for its substantial applications among industries. L-Asparaginase from B. aryabhattai showed an alkali-stable nature since it retained more than 80 % activity over a pH 7.5– 10. The enzyme showed 86 % activity of its original value at the pH 7.4 (pH of circulatory fluids in human body), which was a therapeutically remarkable feature of the enzyme. There is a sharp decrease in the activity on the left side of this optimal range and more than 60 % activity was observed at pH 11.0 (Fig. 3a). The L-asparaginase showed full activity up to the temperature of 40 °C followed by an abrupt decrease until it retained 11.8 % at 70 °C (Fig. 3b). The absence of protease enzyme within the strain might be a cause for better stability of this L-asparaginase in alkaline and high temperature environment. L-Asparaginases activity increased in presence of Na+ and K+ ions, while with other metal ions, viz, Ca2+, Co2+, Cu2+, Mn2+, Hg2+, Mg2+,Fe2+, Sn2+, Pb2+, and Ba2+, significant loss in activity occurred. The enzyme showed considerable stability against ethanol (86.3 %) and DMSO (87.4 %), while the stability was not significant in others organic solvents (Table 3). Organic solvents, in general, lack

Table 3 Effect of different metal ions, organic solvents, and modulators on L-asparaginase activity

N.D. not detected a

Residual activity shown in the table as mean±SD (n=3)

Addition

Concentration

Relative activity (%)a

CaCl2

10 mM

18.7±1.13

CoCl2 CuCl2

10 mM 10 mM

76.4±2.71 22.3±0.85

MnCl2

10 mM

31.5±1.29

HgCl2

10 mM

64.1±0.54

KCl

10 mM

108.4±0.35

MgCl2

10 mM

52.1±1.37

FeCl2

10 mM

72.2±1.71

SnCl2

10 mM

23.8±2.24

PbCl2 BaCl2

10 mM 10 mM

48.3±0.45 34.2±0.22

NaCl

10 mM

117.7±0.61

Ethanol

50 %

86.3±1.49

Methanol

50 %

51.1±2.31

Acetonitrile

50 %

62.2±1.03

Isopropane

50 %

54.6±1.31

DMSO

50 %

87.4±0.06

SDS EDTA

1% 10 mM

24.4±0.68 103.3±1.16

GuHCl

10 mM

N.D.

β-mercaptoethanol

2 mM

N.D.

Cysteine

2 mM

Ascorbic acid

2 mM

Iodoacetamide

1 mM

84.7±0.24

Thiourea

1 mM

N.D.

Urea

2.5 M

N.D.

100.5±1.21 N.D.


Table 4 Substrate specificity of L-asparaginase produced by B. aryabhattai ITBHU02

Substrate

Concentration

Relative activity (%)a

L-Asparagine

10 mM

100.0

D-Asparagine

10 mM

N.D.

DL-Asparagine

10 mM

2.7±0.32

L-Glutamine

10 mM

8.2±1.14

L-Glutamic

acid

10 mM

N.D.

acid L-Glutamic acid

10 mM 10 mM

N.D. N.D.

D-Aspartic

L-Aspartic

acid β-hydroxamate

L-Asparagine-t-butyl

ester

10 mM

98.2±1.31

10 mM

N.D.

10 mM 10 mM

N.D. N.D.

hydrochloride N.D. not detected a Residual activity shown in the table as mean±SD (n=3)

FMOC-L-asparagine N-α-Acetyl-L-asparagine

water's ability to engage in multiple hydrogen bonds, and because of their lower dielectric constants, they lead to offer stronger intramolecular electrostatic interaction between protein atoms. This phenomenon tends an enzyme to get more rigid structure with restricted conformational mobility [31]. GuHCl, β-mercaptoethanol, ascorbic acid, urea, and thiourea showed 100 % inhibition. Loss in activity with reducing agent (β-mercaptoethanol) indicated the presence of S-S bridges. The presence of ascorbic acid produces a highly acidic environment (pH~4.0). Cysteine and iodoacetamide did not show a significant decrease in activity due to absence of free thiol groups. Presence of the metal ion chelating agent EDTA improved the stability of the enzyme, designating asparaginase was not metaloenzyme. Results for the effect of SDS and urea on L-asparaginase from B. aryabhattai are quite comparable to that of the enzyme from Pectobacterium carotovorum [23]. Our results differ from some earlier studies in which β-mercaptoethanol [29], urea, and SDS [28] were reported for not affecting the enzyme activity.

0

Ellipticity (mdeg)x103

Fig. 4 Circular dichroism spectrum of L-asparaginase produced by B. aryabhattai ITBHU02 in native (squares) and denatured conditions (circles) at protein concentration 0.1 mg mL−1 in 0.05 M Tris–HCl (pH 8.5) and temperature 37 °C

-10

-20

-30

-40

200

210

220

230

240

Wavelength, nm

250

260


Fig. 5 Estimation of Km and Vmax of purified L-asparaginase produced by B. aryabhattai ITBHU02. a Plot between substrate concentration (0.1–5.0 mM) and catalytic velocity fitted to Michaelis–Menten equation (R2=0.963); b subsequent Eadie–Hofstee plot

Substrate Specificity Relative activities of purified L-asparaginase towards various substrates at 10 mM are summarized in Table 4. The enzyme exhibited the highest reactivity towards L-asparagine, and a high level of activity with an amino acid analogue, L-aspartic acid β-hydroxamate. The enzyme exhibited much decreased activity against L-glutamine (relative activity 100

>100

15

>100

>100

100

>100

58.6

59.2