African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12271

Full Length Research Paper

Optimization, purification and characterization of recombinant L-asparaginase II in Escherichia coli

Trang Thi Hien Nguyen
  • Trang Thi Hien Nguyen
  • Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr., Caugiay, 10000 Hanoi, Vietnam.
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Cuong Tien Nguyen
  • Cuong Tien Nguyen
  • Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr., Caugiay, 10000 Hanoi, Vietnam.
  • Google Scholar
Thanh Sy Le Nguyen
  • Thanh Sy Le Nguyen
  • Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr., Caugiay, 10000 Hanoi, Vietnam.
  • Google Scholar
Tuyen Thi Do
  • Tuyen Thi Do
  • Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr., Caugiay, 10000 Hanoi, Vietnam.
  • Google Scholar


  •  Received: 21 April 2016
  •  Accepted: 19 July 2016
  •  Published: 03 August 2016

 ABSTRACT

We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for L-asparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant L-asparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 312.8 U/mg. Kinetic parameters, Km, Vmax, Kcat and Kcat/Km of purified enzyme were found to be 0.5 mM, 500 U/mg, 14.9  103 s-1, and 29.9  103 mM-1s-1, respectively. Temperature and pH optimum were observed at 45ºC and pH 7.5, respectively. The enzyme exhibited about 20 and 60% retention of activity following 100 min incubation at 55 or 40°C, respectively. The activity of enzyme was inhibited by EDTA, Hg2+, Cu2+, Ni2+, and enhanced by Mg2+. Detergents (Tween 20, Tween 80, Triton X-100, and Triton X-114) decreased enzyme activity. DTT and DMSO at appropriate concentrations enhanced enzyme activity. In vitro anti-cancer activity was performed using different tumor cell lines. Concentration of recombinant L-asparaginase at 50 µg/ml inhibited 45.32, 48.22, 53.68, 51.22% with HL-60, P388, P3X63Ag8, SP2/0-Ag14 cell lines. Recombinant L-asparaginase was expressed successfully in Escherichia coli with high expression level, had a high specific activity and antiproliferative effect on several tumor cell lines.

 

Key words: Characterization, Erwinia chrysanthemi, L-asparaginase, purification, tumor cell line.


 INTRODUCTION

Acute lymphocytic leukemia (ALL) is a type of blood cancer that results when abnormal white blood cells (leukemia cells) grow quickly and crowd out the bone marrow preventing the normal red blood cells, white blood cells, and platelets that body needs. ALL incidences occur most frequently in people under the age of 15 or over 45. L-asparaginases are a cornerstone of treatment protocols for ALL (Silverman et al., 2001; Pieters and Carroll, 2008). L-asparaginase is also used in treatment of acute myeloid leukemia (AML) (Emadi et al., 2016; Tagde et al., 2016b). Beside, L-asparaginase induced significant growth inhibition and apoptosis in K562 and KU812 cells so it might be a promising new therapeutic strategy for chronic myeloid leukemia (CML) (Song et al., 2015). Normal cells can synthesize L- asparagine by asparagine synthetase. In contrast, tumor cells slowly synthesize L-asparagine and are dependent on an exogenous supply. So L-asparaginase destroys extracellular source of L-asparagine, inhibits protein synthesis in lymphoblasts inducing their apoptosis (Duval et al., 2002). L-asparaginase (EC 3.5.1.1, L-asparagine amidohydrolase) catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonium. The enzyme is considered to play a significant role in L-asparagine metabolism in normal cells. There are two types of L-asparaginases: L-asparaginase I, was used to reduce the level of acrylamide in food industry (Friedman, 2003; Yano et al., 2008). Whereas, L-asparaginase II was used to treat leukemia. There are many sources of L-asparaginase such as bacteria, fungi, yeast, actinomycetes, algae and plants (Verma et al., 2007). To date, L-asparaginase gene had been cloned from variety of host such as Escherichia coli (Wang et al., 2001), Erwinia chrysanthemi (Kotzia and Labrou, 2007), Erw. carotovora (Pourhossein and Korbekandi, 2014), Yesinia pseudotuberculosis (Sidoruk et al., 2011), Thermococcus kodakarensis (Hong et al., 2014), Saccharomyces cerevisiae (Ferrara et al., 2010), and expressed in different expression systems including E. coli (Kotzia and Labrou, 2007; Magdy and Mohammed, 2008), Bacillus subtilis (Jia et al., 2013), and Pichia pastoris (Ferrara et al., 2006). At present, clinically useful L-asparaginases are obtained from either E. coli or Erw. chrysanthemi. In 2002, Duval compared E. coli-asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies, the study showed that E. coli–asparaginase can be recommended for first-line therapy reserving Erwinia-asparaginase for high sensitive E. coli-asparaginase patients (Duval et al., 2002). Here, we examined optimization of rASPG expression, along with the purification and characterization of the recombinant L-asparaginase from Erw. chrysanthemi in E. coli. We used pET21a+ vector with highly-inducible T7 promoter and induced by isopropyl-β-D-thiogalactopyranoside (IPTG) to express L-asparaginase. The results suggest that rASPG was purified with high activity and had high potential for antiproliferative application.


 MATERIALS AND METHODS

Plasmid, bacterial strains and cell lines

The L-asparaginase gene based on L-asparaginase sequence from GenBank accession number X12746 was optimized codon for expression in E. coli, produced and inserted into vector pUC57 (pUaspg) by GenScript (USA). The DNA fragment (981 bp) encoding the mature L-asparaginase (without the signal peptide of 21 N-terminal amino acids) from pUaspg was inserted into vector pET21a(+) resulting in plasmid pEaspg to express in E. coli.

E. coli BL21(DE3) cells (F ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) (Fermentas). Luria-Bertani medium (LB) containing 1% (w/v) bacto tryptone; 0.5% (w/v) yeast extract; 1% (w/v) NaCl; pH 7-7.5 was used for cultivation of E. coli. The LB agar plates contained additionally 2% (w/v) agar and100 mg ampicillin/ml. Four tumor cell lines: human promyolecytic leukemia HL-60, mouse lymphocytic leukemia P388, mouse myeloma P3X63Ag8 and Sp2/0-Ag14 and a normal cell line mouse mus musculus NIH/3T3 were obtained from the Bioassay group (Institute of Biotechnology, Vietnam).

 

Chemicals

L-asparagine, Nessler reagent and RPMI-1640 media were from Sigma (Louis, USA). IPTG, trichloroacetic acid, bactotryptone and yeast extract were from Bio Basic Inc (New York, USA). DEAE- sepharose and Sephacryl S200 were supplied by Pharmacia Co. (GE Healthcare. SDS was supplied Sigma Aldrich Co. (St, Louis, USA). Tween 20 and Tween 80 were from Bio Basis Inc. (New York, NY, USA), and Triton X-100, Triton X-114 and EDTA by Merck (Darmstadt, Germany). All chemicals were used in the experiments in their purified forms.

 

Plasmid construction

The L-asparaginase gene based on L-asparaginase sequence (1044 bp) from GenBank accession number X12746 was optimized codon for expression in E. coli, synthesized and inserted into vector pUC57 (pUaspg) by GenScript. The DNA fragment (981 bps) encoding the mature L-asparaginase (without the signal peptide of 21 N-terminal amino acids) from pUaspg was amplified using pUaspg as template and two oligonucleotides, 21 ASP -F (5’- GCC ATA TGG ATA AAC TGC CGA -3’) and LASP-his R (5’- AAG CTC GAG TCA GTA GGT ATG GAA G -3’) were designed as primers for introduction of the underlined NdeI and XhoI restriction sites, respectively. The PCR mixture contained 2.5 ml 10 ´ PCR buffer; 2 ml of 2 mM dNTP; 2.5 ml of 25 mM MgCl2; 1 ml plasmid pUaspg (50-100 ng); 0.25 ml 5 unit Taq polymerase and 1 ml each primer (10 pmol), supplemented with 14.75 ml distillated water to fulfill 25 ml. The thermocycler conditions were as follows: 95°C/4¢; 30 cycles of (95°C/45², 55°C/45², 72°C/45²); 72°C/10¢. The polymerase chain reaction (PCR) products amplified from the pUaspg with both primer 21 ASP-F and L-ASP-his R were digested with NdeI and XhoI and purified using Gel Extraction Kit (Qiagen) in accordance with the manufacturer’s instructions. It was followed by ligation of the NdeI-XhoI digested aspg products with pET21(a+) linearized by the same enzymes, resulting in pEaspg under the control of the T7-promoter induced by IPTG (isopropyl-β-D-thiogalactopyranoside) and possessing the ampicillin marker. The L-asparaginase encoded by the plasmid pEaspg contains the mature L-asparaginase without the 6 ´ histidine-tag and no leader sequence. The pEaspg plasmid was transformed in E. coli DH5a and BL21 (DE3) cells by heat shock method as described previously (Quyen et al., 2007).

 

Soluble rASPG expression

The transformant E. coli BL21/pEaspg was cultivated in 5 ml of LB medium with 100 μg/ml ampicillin at 37°C with agitation at 220 rpm overnight. This culture was used to inoculate 250 ml of the same media, and grown to an optical density at 600 nm (OD600 nm) 0.4 - 0.6 at 37°C with shaking at 220 rpm. IPTG was then added to 1 mM final concentration, the culture was continuously incubated at 28°C with agitation of 220 rpm for 6 h of induction. Cells were harvested by centrifugation at 8000 rpm and 4ºC for 5 min.

 

Enzyme assay

Activity analysis of L-asparaginase II was performed according to Chung’s report (Chung et al. 2010) comprising the following steps: The 100 µl samples were mixed with 900 µl 0.01 M L-asparagine in 50 mM Tris buffer, pH=8.6, and 1 mL of assay mixture were incubated for 10 min at 37°C for enzymatic reaction. The reaction was interrupted with 100 µL of 1.5 M trichloroacetic acid and the samples were centrifuged before the addition of 100 µL Nessler’s reagent to measure the released ammonia after L-asparagine hydrolysis. All the measurements were done spectrophotometrically at 480 nm. The enzyme activity of recombinant protein was determined using an ammonium sulphate calibration curve. One unit of enzyme activity was defined as the amount of enzyme required to release 1 µM of ammonia per minute.

 

Effect of IPTG concentration

IPTG control T7 lac promoter so that IPTG concentration may be affected expression level of recombinant protein. To assess the effects of IPTG concentration on the enzyme specific activity, eight flasks 100 ml contain 25 ml per flask of the recombinant clone culture was grown to OD600nm of about 0.4 - 0.8 (for approximately 4 h), and induced by adding IPTG in final concentrations of 0; 0.2; 0.4; 0.6; 0.8; 1; 1.2; 1.4 mM, respectively. After 6 h of induction, bacterial cells were harvested and analyzed for the enzyme specific activity. The best IPTG concentration was selected and applied for the next stage.

 

Effect of amp concentration

Six flasks 100 ml were contained 25 mL LB broth were prepared. Amp with final concentrations of 25; 50; 100; 150; 200 and 250 µg/ ml were added to each flask, respectively. Half ml of the overnight culture was inoculated into each of flasks. The culture was cultivated at 37°C with agitation at 220 rpm until an OD 600 nm of 0.4 - 0.8 was reached (for approximately 3 h) then IPTG was added. After 6 h of induction, bacterial cells were harvested to analyze for the enzyme specific activity. The best Amp concentration before induction was selected and applied for the next stage.

 

Effect of inoculum size

To evaluate the effect of inoculum size on the enzyme expression, the overnight culture were inoculated, inoculum of different sizes 0.5%; 1; 2, and 5% (v/v) into four flasks 100 ml which contained 25 ml LB, the recombinant clone culture was grown at 37°C in LB medium for 3 h, and then induced by adding IPTG. After 6 h of induction, enzyme specific activity was evaluated. Inoculum size with higher protein production was determined.

 

rASPG purification

The rASPG was expressed in E. coli BL21(DE3). To purify rASPG, 0.7 g cells from a 100 ml culture in LB medium were harvested by centrifugation at 8000 rpm and 4°C for 5 min, and resuspended in 8 ml of 50 mM Tris HCl buffer pH 8.6, sonicated and centrifuged at 12000 rpm and 4°C for 15 min.

 

Gel filtration

The supernatant cell free extract containing the crude L-asparaginase was loaded into Sephacryl S-200 column (2.6 ´ 6 cm) equilibrated with 50 mM potasium phosphate (pH 8) and eluted with the same buffer at the flow rate of 0.5 ml per minute. Fractions showing L-asparaginase activity were pooled and concentrated with bench top protein concentrator at 4°C. The homogeneity of the protein was checked by SDS -PAGE.

 

DEAE chromatography

The concentrated enzyme solution was added on the top of Diethylaminoethyl Sepharose ion exchange column (DEAE - Sepharose) (2.6 ´ 6 cm) equilibrated with 50 mM Tris HCL (pH 8.6). The column was washed with 2 volumes of starting buffer and the protein was eluted with linear gradient of NaCl (0 - 1 M) prepared in 50 mM Tris HCL (pH 8.6) at the rate of 30 ml per hour. The eluate was collected with 1.5 ml per fractions. The fractions showing L-asparaginase activity were stored at 4°C.

The molecular mass of the rASPG was determined by 12.5% SDS polyacrylamide gel electrophoresis with Biometra equipment (Laemmli, 1970). Proteins were visualized by staining with 0.1% (w/v) Coomassie Brilliant Blue R-250. Protein concentrations were estimated by the method of Bradford with the bovine serum albumin as standard (Bradford, 1976).

 

Kinetic parameters determination

Aliquots of 100 µl of reconstituted enzyme were prepared and added with different concentrations of L-asparagine ranging from 1 mM to 4.5 mM prepared in 50 mM Tris HCl. The apparent kinetic parameters (Km, Vmax, Kcat and Kcat/Km) of enzyme for L-asparagine were determined by Lineweaver-Burk plots method.

 

Temperature and pH optimum

The pH and temperature optimum of rASPG were determined by measuring the activity as described above using 100 mM potassium acetate buffer (pH 4-6), potassium phosphate buffer (pH 6.5-8), and Tris HCl buffer (pH 8-10) at 37°C for 30 min, and in the temperature range of 20 - 65°C at pH 8.6 for 30 min.

 

Temperature and pH stability

For the determination of temperature and pH stability, the purified enzyme (0.7 µg for each reaction) was incubated at 40 and 50°C, and pH 6; 7; 8 the activities were measured at various time intervals of 20; 40; 60; 80 and 100 min. Percentage of residual activities was calculated based on the untreated control activity, which is taken as 100%.

 

Effect of metal ions and EDTA, detergents, DTT, DMSO

The purified enzyme (0.7 µg protein for each reaction) was preincubated in presence of 10 mM of various metal ions (Ca2+, Cu2+, Fe3+, Fe2+, Mg2+, Ni2+, Zn2+, K+, Hg+, Pb2+), ethylenediamine tetraacetic acid (EDTA), in presence of 1-5% (w/v) of various detergents (Tween 20, Tween 80, Triton X-100, and Triton X-114), in presence of 0.1-5 mM dithiothreitol (DTT) and in presence of 0.1-2% (w/v) dimethyl sulfoxide (DMSO) at 37°C for 1 h. The residual activity was then determined at pH 8.6 and 37°C.

 

Cell culture and proliferation assay

Cells were routinely cultured in RPMI 1640 media. It were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 50 units/ml penicillin, 50 µg/ml streptomycin. Cells were cultivated in a humid atmosphere (5% CO2, 37°C) (Takahashi et al., 2015; Hasegawa et al., 2016; Rajabi et al., 2016; Tagde et al., 2016a). Cells were seeded in 96-well plates at 1 × 104 cells per well. rASPG was added at concentrations of 0.4; 2; 10 and 50 µg/ml. After 72 h of continuous enzyme exposure, 10 µl of MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was added in each well. The plates were incubated for 1 - 4 h at 37°C and the formazan product was measured at 490 nm. The experiments were performed in triplicate in three independent sets. Cell survival was calculated by subtracting the background absorbance of media alone and then dividing the absorbance of experimental wells by the absorbance of the control (untreated) wells (Neisius and Moll, 1989; Sonawane et al., 2014; Tagde et al., 2014).


 RESULTS AND DISCUSSION

Optimization of rASPG expression

In previous studies, we expressed a plasmid pET22b(+) containing the full-length aspg gene in E. coli BL21(DE3) but no expression was detected by SDS-PAGE analysis, and recombinant plasmid pEaspg containing aspg gene and expression vector pET21a(+) was previously constructed without signal peptide with his tag, pEaspg was transformed in E. coli BL21(DE3) and expressed at 28°C, recombinant protein was higher levels expressed but had low activity (data not show). In this study, we constructed plasmid without the 6 ´ histidine-tag and no leader sequence. The DNA fragment encoding the mature L-asparaginase with stop codon, truncated 21 N-terminal amino acids was inserted into pET21(a+) vector resulting in the recombinant plasmid pEaspg. The transformant E. coli BL21/pEaspg was grown in LB medium for the rASPG production. After IPTG induction, the cells were collected used for enzyme activity assay. The E. coli BL21/pEaspg transformant was showed high production of L-asparaginase (data not show). To increase the specific as well as volumetric yield of recombinant L-asparaginase, a variety of independent cultivation parameters such as inducer concentration, ampicillin concentration and inoculum size were optimized.

 

Effect of IPTG concentration

IPTG concentration did not affect enzyme activity. Although, IPTG concentration increase from 0.2 to 1.4 mM but there are significant changes observed in enzyme activity (Figure 1). The maximum enzyme activity was at 0.8 mM IPTG (100%), but no significant decrease at 0.2 mM IPTG (95%). Thus, 0.2 mM IPTG was selected for the next stages. The similar results were reported in the study of Sidoruk et al. (2011) and Bahreini et al. (2014). In 2011, Vidya demonstrated that enzyme activity was decrease with the increase of IPTG from 10 µM to 50; 100 and 400 µM (Vidya et al., 2011).

 

 

 

Effect of Amp concentration

Amp was supplemented in culture medium to prevent the overgrowth of plasmid-free cells. Amp also affects the number of plasmid per cell. Chong reported that increase in the concentration of Amp in cultures causing the increase of the plasmid copy number in cells (Chong et al., 2003). Bahreini assumed that the increase in plasmid copy number corresponding to the rise of protein expression, but Bahreini’s research has demonstrated that the higher levels of Amp had no effect on the L-asparaginase activity (Bahreini et al., 2014). We have found a similar result. It seems that the increase of Amp level higher than 25 µg/ml slightly decrease specific enzyme activity (Figure 2).

 

 

 

Effect of inoculum size

After 4 h culture, four flasks with different inoculum size 0.5; 1; 2 and 5% (v/v) and the value of OD600 nm reached 0.5; 0.6; 0.7; 0.8, respectively. Our study showed that the inoculum size of 0.5% were found to be the most suitable condition for maximum enzyme activity, the increase in inoculum size is the reason for the decrease in enzyme activity (Figure 3). In general, the increase in cell density of bacterial expression enhances recombinant protein production. Khushoo et al. (2004) reported that induction IPTG during late log phase (OD600 nm= 4.5) resulted in maximum secretion of the recombinant asparaginase and specific activity (Khushoo et al., 2004). Later, Kenari et al. (2011) optimized inoculum size of 10% (Kenari et al., 2011). Bahreini et al. (2014) reported that in L-asparagi nase production level with the maximum production at the highest cell density of OD600 nm= 10 (Bahreini et al., 2014). A simple explanation of these findings that inoculum sizes can be attributed to decrease in the concentration of the medium components, such as O2 level, pH, and nutrients.

 

 

Purification of rASPG

The expression level of rASPG in optimized conditional expression was 78% of the total cellular protein by densitometry scanning, resulting in 10% increase in the production compared to the original condition. The expression level of rASPG in E. coli system was reported to be approximate 50% in JM105, TG1, DH5α, AS1.357 and 75% in JM109 (Wang et al., 2001). The rASPG was purified from the cell lysis of E. coli BL21(DE3) by filter chromatography Sephacryl S-200 and DEAE Sepharose showed only one protein band about 37 kDa on SDS-PAGE (Figure 4, lane 7-9). The specific activity of recombinant L-asparaginase after two step purification obtained by 312.8 U/mg with a yield of 17.8% and purification factor of 7.8 (Table 1). The specific activity was very different: The activity of purified recombinant L-asparaginase II from E. coli K-12 express in E. coli BLR(DE3) was 190 U/mg (Khushoo et al., 2004), recombinant L-asparaginase II from Erw. chrysanthemi 3937 express in E. coli BL21(DE3) pLysS was 118.7 U/mg (Kotzia and Labrou, 2007), L-asparaginase II from B. subtilis express in E. coli JM109 (DE3) was 45.5 U/mg (Onishi et al., 2011) L-asparaginase from Rhizomucor miehei express in E. coli was 1,985 U/mg (Huang et al., 2014) and activity of purified L-asparaginase from B. licheniformis was 697.09 U/mg (Mahajan et al., 2014).

 

 

 

 

 

Characteristic of rASPG

Temperature and pH optimum

The recombinant L-asparaginase from Erw. chrysanthemi had optimum temperature of 45°C (Figure 5A) and optimum pH of 7.5 in 100 mM Tris-HCl buffer (Figure 5B). It was similar to recombinant L-asparaginase II from the B. subtilis B11−06 in B. subtilis168 which had an optimum temperature of 45ºC and pH 7.5 (Jia et al., 2013). It was different from that of E. coli MTCC739 in E. coli BL21(DE3), which were 37°C and pH 6 (Vidya et al., 2011). The optimal temperature and pH of rASPG from a thermotolerant strain E. coli KH027 in E. coli DH5α was 43°C and pH 6 (Muharram et al., 2014). It was 37°C and pH 7.5 for rASPG from E. coli W3110 in E. coli BL21 (DE3) (Magdy and Mohammed, 2008) and that of rASP G from Withania somnifera in E. coli BL21(DE3) was 37°C and pH 8 (Oza et al., 2011). The optimum temperature and pH of the wild L-asparaginase from Cladosporium sp. were 30°C and 6.3, respectively (Kumar and Manonmani, 2013). Consequently, the optimal temperature and pH of the recombinant enzyme is not same with different bacterial sources and different expression host.

 

 

 

pH and thermo stability

The thermal stability of the purified enzyme at 40 and 55°C was studied to find out the extent of temperature resistance of the enzyme. Around 60 and 30% of the initial activity was retained by the purified enzyme after 100 min of incubation at 40 and 55°C, respectively (Figure 6A). The earlier reports on the thermostability of different L-asparaginase preparations indicate that the native enzymes were unstable at high temperatures. Wild L-asparaginase from Erw. chrysanthemi, which expressed in E. coli BL21(DE3) retains ~40% of its initial activity after 7.5 min of incubation at 50°C and on mutagenesis, around 20% increase in activity retention was achieved (Kotzia and Labrou, immobilized enzyme after similar treatment was 66.8% (Magdy and Mohammed, 2008). rASPG showed pH stability at a pH range 6-8. The residual rASPG activity was above 80% in comparison to the original activity after 100 min of treatment (Figure 6B).

 

 

 

Effect of metal ions and EDTA, detergents, DTT, DMSO

The rASPG activity was inhibited by EDTA (Table 2). This results is in agreement with results reported for L- asparaginase from Actinomycetes (Basha et al., 2009) and L-asparaginase from Thermococcus kodakarensis KOD1 in E. coli (Hong et al. 2014). But several researches reported that EDTA enhanced the enzyme activity (Raha et al., 1990; Warangkar and Khobragade, 2010).

 

 

Metal ions showed that rASPG inhibitory activity of 92-72% in order of Na+ > K+, Fe3+ > Ca2+ > Ni+ >Al3+ >Cu2+ >Hg2+ while Mn2+ enhancer activity of the enzyme by 123%. And Pb2+, Mg2+, Ba2+ showed a slightly enhance effect on rASPG activity, the residual activity was accounted for 108% -109% of the original activity and Zn2+ was not effect enzyme activity (Tab.2). The same results was also found in the report that the activity of native L-asparaginase from Cylindrocarpon obtusisporum MB-10 was inhibited by Zn2+, Fe2+, Cu2+, Hg2+ and Ni2+ (Raha et al. 1990) and the activity of rASPG from Thermococcus kodakarensis KOD1 was inhibited by Ca2+, Co2+, Cu2+, Ni2+, enhanced by Mg2+ (Hong et al., 2014). Warangkar and Khobragade, (2010) reported that L-asparaginase form Erw. carotovora was loss of activity with Hg2+, Ni2+, Cd2+, Cu2+, Fe2+ , and Zn2+, too, but Na+ and K+ acting somewhat as an enhancer and Mg2+ inhibited enzyme activity. All detergents (Tween 20, Tween 80, Triton X-100, and Triton X-114) showed an inhibitory effect on rASPG activity. The higher concentration, the detergents are more inhibited enzyme activity (Table 3).

 

 

Interestingly, the addition of DTT at low concentration of 0.1 - 0.5 mM was not significantly effected enzyme activity, but at higher concentration of 1 - 5 mM enhanced the enzyme activity by 46% and 19%, respectively (Table 4). L – asparaginase of activity from Erw. carotovora was aslo reported to enhance in presence of thiol protecting reagents like DTT and it has been explained by asparaginase possesses the thiol group binding domain with high affinity towards free-SH group containing effectors (Warangkar and Khobragade, 2010). The addition of lower concentration DMSO (0.1 and 0.5%), enzyme activity was not effect, but the rASPG activity remained much higher 152% by the addition of DMSO at higher concentration (1 mM). When concentrations of DMSO was higher (1.5; 2%), the level of increased enzyme activity were decreased (Table 5).

 

 

 

Kinetic parameters-

The Km, Vmax, Kcat and Kcat/Km obtained for rASPG Erw. chrysanthemi expressed in E. coli with L-asparagine substrate were 0.5 mM, 500 U/mg, 14.9 ´ 103 s-1, 29.9 ´ 103 mM-1s-1, respectively. The Km, Vmax, Kcat and Kcat/Km value of the recombinant L - asparaginase from Thermococcus kodakarensis KOD1 expressed in E. coli BLR(DE3) were found to be 2.6 mM, 1121 U/mg, 694 s-1, and 266.9 mM-1s-1, respectively (Hong et al. 2014), of recombinant L-asparaginase from B. subtilis in E. coli were 2.06 mM, 45.5 U/mg, 98.6 s-1, and 48 mM-1s-1 respectively (Onishi et al. 2011). The Km, Kcat and Kcat/Km value of rASPG from Erw. chrysanthemi 3937 in E. coli BL21(DE3) pLysS were 0.058 mM, 23.8 ´ 103 s-1, and 411.8 ´ 103 mM-1s-1 respectively (Kotzia and Labrou, 2007). It was found that rASPG had low Km value, high Vmax, Kcat and Kcat/Km value. So rASPG is highly specific for the substrate L-asparagine.

 

Anti-cancer activity of the rASPG

Abakumova et al. (2013) provided convincing evidence that L-asparaginase induced apoptosis as its principal process of causing death of leukemic and solid tumor cells (Abakumova et al., 2013). In our study, four tumor cell lines with increasing amounts of rASPG resulted in a significant increase in the number of dead cells (Table 6). At dose 0.4 µg/ml, rASPG did not significantly affect the growth of cells but at dose 50 µg/ml, rASPG inhibited HL-60 (45.32%), P388 (48.22%), P3X63Ag8 (53.68%), and SP2/0-Ag14 (51.22%). Normal cells were not significantly affected by rASPG at dose 50 µg/ml rASPG inhibited 18.02 % for NIH/3T3.

 

 

Muharran et al. (2014) indicated that recombinant L-asparaginase in E. coli caused a reduction of 50% in cell viability of RS4 at a dose of 100 µg/ml after 96 h of incubation, and a reduction of 50% in cell viability of HL-60 at a dose of 200 µg/ml after 72 h of incubation (Muharram et al. 2014). L- asparaginase from Streptomyces acrimycini NGP (130 U/mg) inhibits the gastric stomach cancer cells with an IC50 of 49.11 µg/ml (Selvam and Vishnupriya, 2013). L-Asparaginase from bean (Vicia faba) and white kidney bean (Phaseoulus vulgaris) seeds (2.75 and 1.47 U/mg) had low IC50 values 217.71 μg/ml and 187.86 μg/ml with Hep-G2 cells (Sanaa et al., 2012). IC50 value of L-asparaginase from Aspergillus flavus on MCF-7 cells was 120.875 µg/ml (Rani et al., 2011)


 CONCLUSION

In conclusion, L-asparaginase was expressed in E. coli BL21 (DE3) at high level expression. After purification, a single band indicative of purified protein was recorded. The purified enzyme was 7.8 folds with a final specific activity of 312.8 IU/mg protein and about 17.8% yield recovery. Temperature and pH optimum for rASPG were 45°C and 7.5, respectively. The activity of enzyme was enhanced by Mn2+, Pb2+, Mg2+, Ba2+ and inhibited by EDTA, Na+, K+, Fe3+, Ca2+, Ni+, Al3+, Cu2+, Hg2+ and detergents (Tween 20, Tween 80, Triton X-100, and Triton X-114). Also, DTT at a concentration of 1 mM and DMSO at a concentration of 1% enhanced the enzyme’s activity. Recombinant enzyme had high anti-cancer activity. The number of apoptotic cells significantly increased after rASPG treatment experimental wells by the absorbance of the control (untreated) wells.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.


 ACKNOWLEDGEMENTS

This study was supported by Vietnam Academy of Science and Technology (project VAST02.03/13-14: “Study on the production of recombinant L-asparaginase to inhibit cancer cell lines and treatment of acute lymphoblastic leukemia”. The authors are thankful to Prof. Quyen Dinh Thi for guidance. We also extend our thanks to Dr. Do Thi Thao (Bioassay group, Institute of Biotechnology) for tumor cell lines.



 REFERENCES

Abakumova O, Podobed OV, Karalkin PA, Kondakova LI, Sokolov NN (2013). Antitumor activity of L-asparaginase from Erwinia carotovora from against different leukemic and solid tumours cell lines. Biomed Khim. 59:498-513.
Crossref

 

Bahreini E, Aghaiypour K, Abbasalipourkabir R, Goodarzi MT, Saidijam M, Safavieh SS (2014). An optimized protocol for over-production of recombinant protein expression in Escherichia coli. Prep. Biochem. Biotechnol. 44(5):510-528.
Crossref

 
 

Basha NS, Rekha RR, Komala M, Ruby S (2009). Production of extracellular anti-leukaemic enzyme L-asparaginase from marine Actinomycetes by solid state and submerged fermentation: Purification and characterisation. Trop. J. Pharm. Res. 8:353-360.
Crossref

 
 

Bradford MM (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Crossref

 
 

Chong M, Leung R, Wong C, Yuen AJ (2003). The effects of ampicillin versus tetracycline on the plasmid copy numbers of pBR322. J. Exp. Microbiol. Immunol. 3:87-95.

 
 

Chung R, Der C, Kwan J, Lincez P (2010). Assessment of periplasmic enzyme isolation methods: isolating L-asparaginase from E. coli using microwave irradiation and potassium phosphate-hexane permeabilization methods. J. Exp. Microbiol. Immunol. 14:1-6.

 
 

Duval M, Suciu S, Ferster A, Rialland X, Nelken B, Lutz P, Benoit Y, Robert A, Manel AM, Vilmer E, Otten J (2002). Comparison of Escherichia coli-asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment of Cancer-Children's Leukemia Group phase 3 trial. Blood 99(8):2734-2739.
Crossref

 
 

Emadi A, Bade NA, Stevenson B, Singh Z (2016). Minimally-Myelosuppressive Asparaginase-Containing Induction Regimen for Treatment of a Jehovah's Witness with mutant IDH1/NPM1/NRAS Acute Myeloid Leukemia. Pharmaceuticals 9(1):12.
Crossref

 
 

Ferrara MA, Severino NM, Mansure JJ, Martins AS, Oliveira EM, Siani AC, Pereira N, Torres FA, Bon EP (2006). Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene. Enzyme Microb. Technol. 39:1457-1463.
Crossref

 
 

Ferrara MA, Severino NMB, Valente RH, Perales J, Bon EPS (2010) High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiae ASP3 gene. Enzyme Microbial. Technol. 47:71-76.
Crossref

 
 

Friedman M (2003) Chemistry, biochemistry, and safety of acrylamide. A review. J. Agric. Food Chem. 51:4504-4526.
Crossref

 
 

Hasegawa M, Takahashi H, Rajabi H, Alam M, Suzuki Y, Yin L, Tagde A, Maeda T, Hiraki M, Sukhatme VP, Kufe D (2016). Functional interactions of the cystine/glutamate antiporter, CD44v and MUC1-C oncoprotein in triple-negative breast cancer cells. Oncotarget 7(11):11756-11769.

 
 

Hong SJ, Lee YH, Khan AR, Ullah I, Lee C, Park CK, Shin JH (2014). Cloning, expression, and characterization of thermophilic L-asparaginase from Thermococcus kodakarensis KOD1. J. Basic Microbiol. 54(6):500-508.
Crossref

 
 

Huang L, Liu Y, Sun Y, Yan Q, Jiang Z (2014). Biochemical characterization of a novel L-Asparaginase with low glutaminase activity from Rhizomucor miehei and its application in food safety and leukemia treatment. Appl. Environ. Microbiol. 80:1561-1569.
Crossref

 
 

Jia M, Xu M, He B, Rao Z (2013). Cloning, expression, and characterization of L-asparaginase from a newly isolated Bacillus subtilis B11-06. J. Agric. Food Chem. 61:9428-9434.
Crossref

 
 

Kenari DSL, Alemzadeh I, Maghsodi V (2011). Production of l-asparaginase from Escherichia coli ATCC 11303: Optimization by response surface methodology. Chem. Eng. Res. Des. 8:315-321.
Crossref

 
 

Khushoo A, Pal Y, Singh BN, Mukherjee KJ (2004). Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. Protein Expr. Purif. 38:29-36.
Crossref

 
 

Kotzia GA, Labrou NE (2007). L-Asparaginase from Erwinia chrysanthemi 3937: cloning, expression and characterization. J. Biotechnol. 127:657-669.
Crossref

 
 

Kotzia GA, Labrou NE (2009). Engineering thermal stability of L-asparaginase by in vitro directed evolution. FEBS J. 276:1750-1761.
Crossref

 
 

Laemmli UK (1970). Clevage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Crossref

 
 

Magdy MY, Mohammed AA-O (2008). Cloning, purification, characterization and immobilization of L-asparaginase II from E. coli W3110. J. Biochem. Mol. Biol. Biophys. 3:337-350.

 
 

Mahajan RV, Kumar V, Rajendran V, Saran S, Ghosh PC, Saxena RK (2014). Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties. PLoS One 9:e99037.
Crossref

 
 

Mohan Kumar NS, Manonmani HK (2013) Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp. World J. Microbiol. Biotechnol. 29:577-587.
Crossref

 
 

Muharram MM, Abulhamd AT, Mounir MS-B (2014) Recombinant expression, purification of L-asparaginase II from thermotolerant E. coli strain and evalution of its antiproliferative activity. Afr. J. Microbiol. Res. 8:1610-1619.
Crossref

 
 

Neisius D, Moll V (1989). Renal ultrasonography in the management of calculus disease. Urol. Clin. N. Am. 16:829-840.

 
 

Onishi Y, Yano S, Thongsanit J, Takagi K, Yoshimune K, Wakayama M (2011). Expression in Escherichia coli of a gene encoding type II L-asparaginase from Bacillus subtilis, and characterization of its unique properties. Ann. Microbiol. 61:517-524.
Crossref

 
 

Oza VP, Parmar PP, Patel DH, Subramanian RB (2011). Cloning, expression and characterization of l-asparaginase from Withania somnifera L. for large scale production. 3 Biotech 1(1):21-26.

 
 

Pieters R, Carroll WL (2008). Biology and treatment of acute lymphoblastic leukemia. Pediatr. Clin. N. Am. 55:1-20.
Crossref

 
 

Pourhossein M, Korbekandi H (2014). Cloning, expression, purification and characterisation of Erwinia carotovora L-asparaginase in Escherichia coli. Adv. Biomed. Res. 3:82.
Crossref

 
 

Quyen DT, Dao TT, Thanh Nguyen SL (2007). A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization. Protein Expr. Purif. 51:133-140.
Crossref

 
 

Raha SK, Roy SK, Dey SK, Chakrabarty SL (1990). Purification and properties of an L-asparaginase from Cylindrocarpon obtusisporum MB-10. Biochem. Int. 21:987-1000.

 
 

Rajabi H, Tagde A, Alam M, Bouillez A, Pitroda S, Suzuki Y, Kufe D (2016). DNA methylation by DNMT1 and DNMT3b methyltransferases is driven by the MUC1-C oncoprotein in human carcinoma cells. Oncogene
Crossref

 
 

Rani AS, Sundaram L, Vasantha BP (2011). In vitro antioxidant and anticancer activity of L-asparaginase from Aspergillus flavus (KUFS20). Asian J. Pharm. Clin. Res. 4:174-177.

 
 

Sanaa TE-S, Amal AF, Amira MG-E (2012). Immobilization, properties and anti-tumor activity of L-asparaginase of Vicia faba and Phaseoulus vulgaris seeds. Aust. J. Basic Appl. Sci. 6(3):785-794.

 
 

Selvam K, Vishnupriya B (2013). Partial purification and cytotoxic activity of L-asparaginase from Streptomyces acrimycini NGP. Int. J. Res. Pharm. Biomed. Sci. 4:859-869.

 
 

Sidoruk KV, Pokrovsky VS, Borisova AA, Omeljanuk NM, Aleksandrova SS, Pokrovskaya MV, Gladilina JA, Bogush VG, Sokolov NN (2011). Creation of a producent, optimization of expression, and purification of recombinant Yersinia pseudotuberculosis L-asparaginase. Bull. Exp. Biol. Med. 152(2):219-223.
Crossref

 
 

Silverman LB, Gelber RD, Dalton VK, Asselin BL, Barr RD, Clavell LA, Hurwitz CA, Moghrabi A, Samson Y, Schorin MA, Arkin S, Declerck L, Cohen HJ, Sallan SE (2001). Improved outcome for children with acute lymphoblastic leukemia: results of Dana-Farber Consortium Protocol 91-01. Blood 97(5):1211-1218
Crossref

 
 

Sonawane P, Cho HE, Tagde A, Verlekar D, Yu AL, Reynolds CP, Kang MH (2014). Metabolic characteristics of 13-cis-retinoic acid (isotretinoin) and anti-tumour activity of the 13-cis-retinoic acid metabolite 4-oxo-13-cis-retinoic acid in neuroblastoma. Br. J. Pharmacol. 171:5330-5344.
Crossref

 
 

Song P, Ye L, Fan J, Li Y, Zeng X, Wang Z, Wang S, Zhang G, Yang P, Cao Z, Ju D (2015). Asparaginase induces apoptosis and cytoprotective autophagy in chronic myeloid leukemia cells. Oncotarget 6(6):3861-3873.
Crossref69

 
 

Tagde A, Rajabi H, Bouillez A, Alam M, Gali R, Bailey S, Tai YT, Hideshima T, Anderson K, Avigan D, Kufe D (2016a). MUC1-C drives MYC in multiple myeloma. Blood 127:2587-2597.
Crossref

 
 

Tagde A, Rajabi H, Stroopinsky D, Gali R, Alam M, Bouillez A, Kharbanda S, Stone R, Avigan D, Kufe D (2016b). MUC1-C induces DNA methyltransferase 1 and represses tumor suppressor genes in acute myeloid leukemia. Oncotarget.
Crossref

 
 

Tagde A, Singh H, Kang MH, Reynolds CP (2014). The glutathione synthesis inhibitor buthionine sulfoximine synergistically enhanced melphalan activity against preclinical models of multiple myeloma. Blood Cancer J. 4:e229.
Crossref

 
 

Takahashi H, Jin C, Rajabi H, Pitroda S, Alam M, Ahmad R, Raina D, Hasegawa M, Suzuki Y, Tagde A, Bronson R T, Weichselbaum R, Kufe D (2015). MUC1-C activates the TAK1 inflammatory pathway in colon cancer. Oncogene 34:5187-5197.
Crossref

 
 

Verma N, Kumar K, Kaur G, Anand S (2007). L-asparaginase: a promising chemotherapeutic agent. Crit. Rev. Biotechnol. 27:45-62.
Crossref

 
 

Vidya J, Vasudevan UM, Soccol CR, Pandey A (2011). Cloning, functional expression and characterization of L -asparaginase II from E. coli MTCC 739. Food Technol. Biotechnol. 49:286-290.

 
 

Wang Y, Qian S, Meng G, Zhang S (2001). Cloning and expression of L-asparaginase gene in Escherichia coli. Appl. Biochem. Biotechnol. 95:93-101.
Crossref

 
 

Warangkar SC, Khobragade CN (2010). Purification, characterization, and effect of thiol compounds on activity of the Erwinia carotovora L-Asparaginase. Enzyme Res. Volume 2010 (2010), Article ID 165878, 10 pages.

 
 

Yano S, Minato R, Thongsanit J, Tachiki T, Wakayama M (2008). Overexpression of type I L-asparaginase of Bacillus subtilis in Escherichia coli, rapid purification and characterisation of recombinant type I L-asparaginase. Ann. Microbiol. 58:711-716.
Crossref

 

 




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