Production and characterization of novel glutaminase free recombinant L-asparaginase II of Erwinia carotovora subsp. atroseptica SCRI 1043 in E. coli BL21 (DE3).

Aims: To clone and express, the gene encoding L-asparaginase II (ansB2) from Erwinia carotovora subsp. atroseptica SCRI 1043 in E. coli BL21 (DE3). Further, the work is also comprised of purification and detailed biochemical characterization of L-asparaginase II. Place and Duration of Study: Biochemical Engineering laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Assam India. Experiments conducted as a part of project and a PhD thesis from December 2010 to January 2014. Methodology: The gene encoding L-asparaginase II (ansB2) from Erwinia carotovora subsp. atroseptica SCRI 1043 was cloned and expressed in E. coli BL21 (DE3). Affinity chromatography was employed to purify the enzyme to homogeneity. Detailed biochemical characterization, such as substrate specificity, operational stability in various effect or molecules, effect of pH and temperature, kinetic parameters were investigated. Results: The enzyme was found to be highly specific towards its natural substrate, L-asparagine. Original Research Article Goswami et al.; BMRJ, 6(2): 95-112, 2015; Article no.BMRJ.2015.062 96 The activity of recombinant L-asparaginase II was activated by various effect or compounds, such as mono cations, L-cystine, L-histidine, 2-mercaptoethanol and glutathione. However, considerable inhibitory effect was observed with divalent cations and iodoactamide. Kinetic parameters (Vmax, Km, kcat and Kcat/Km) of purified recombinant L-asparaginase II were found to be 0.656 mM, 312.50 IU mg, 1.38×10 s and 2.11×10 Ms, respectively. Optimum range of pH and temperature for the hydrolysis of L-asparagine by purified recombinant L-asparaginase II were found to be 6.5-9.5 and 47-52oC, respectively. Under optimal levels of medium components and physical process parameters (pH, inoculum size, agitation), the production of recombinant L-asparaginase II was increased by 1.95 fold. The purified recombinant L-asparaginase II has shown no glutaminase activity. Conclusion: The present characterization experiments of the L-asparaginase II from Erwinia carotovora subsp. atroseptica SCRI 1043 showed very high specificity for its natural substrate, Lasparagine and shown no glutaminase activity which makes it a better alternative in therapeutic applications like an anticancer drug.

The activity of recombinant L-asparaginase II was activated by various effect or compounds, such as mono cations, L-cystine, L-histidine, 2-mercaptoethanol and glutathione. However, considerable inhibitory effect was observed with divalent cations and iodoactamide. Kinetic parameters (V max , K m , k cat and K cat /K m ) of purified recombinant L-asparaginase II were found to be 0.656 mM, 312. 50

INTRODUCTION
L-asparaginase (L-asparagine amidohydrolase, E.C. 3.5.1.1) is used in chemotherapy regimens for the treatment of acute lymhoblastic leukemia and non-Hodgkin's lymphoma [1]. The enzyme has also been used to develop L-asparagine biosensor for leukemia [2] and in food industry for the production of acrylamide free food [3]. The application of L-asparaginase in anti-cancer therapy largely depends on its capability to cleave L-asparagine to ammonia and L-aspartic acid. In view of the fact that, lymphoblasts are not able to produce endogenous L-asparagine, starvation of this amino acid causes death of these cells [4,5].
Although L-asparaginase is produced by various microorganisms [2], L-asparaginase from Erwinia chrysanthemi and Escherichia coli are used for clinical purpose [6.7]. L-asparaginases from E. coli and Erwinia show different immunological specificities [8] and comparison of the two enzymes has revealed that E. coli-asparaginase can be prescribed to the patient for therapy, reserving Erwinia-asparaginase for allergic patients, because most patients allergic to the E. coli asparaginase are not immediately allergic to the Erwinia-asparaginase, which is reported to be less toxic [7,9]. However, the occurrence of Lglutaminase activity in L-asparaginase is known to be the major cause of the various side effects associated with this drug [10,11]. In addition, presence of glutaminase activity is one of the causes of toxicity [12,13]. Unfortunately, a therapeutic response by patients rarely occurs without some evidence of toxicity [1]. Erwinia asparaginase shows lower glutaminase activity [4]. The development of recombinant strain for higher production of glutaminase free Lasparaginase II plays a vital role to commercialize the product because Lasparaginases with high specificity for Lasparagine and low-to-negligible activity for Lglutamine are found to be less troublesome during the course of anti-cancer therapy [13]. Therefore, there is a need to find novel sources for higher production of L-asparaginase that is free of glutaminase. There are two types of Lasparaginases found in bacteria (type I and type II). Type II L-asparaginase has higher affinity towards L-asparagine than type I. Hence, Lasparaginase II is used as anticancer agent [14].
The increase in productivity of any metabolite would be attained through manipulation of nutritional and physical parameters. Often, conventional optimization studies involving onefactor-at-time strategy which is tiresome and has a tendency to overlook the effects of interaction between and among the factors and might guide to misinterpretation of results. In contrast, statistically designed experiments reduce the error in determining the effect of factor in an economical manner. Response surface methodology (RSM) is an efficient statistical tool for optimization of multiple variables to find out the best conditions using a minimum number of experiments. Therefore, it is superior to the traditional approach of studying one variable at a time [15]. The economic feasibility of commercialization of recombinant Lasparaginase also depends on various other parameters such as stability during isolation, easy purification using affinity tags [16], storage as well as its robustness under environmental conditions.
There are three genes' sequences available for L-asparaginase (L-asparaginase, L-asparaginase I and L-asparaginase II) of E. carotovora subsp. atroseptica SCRI 1043 in NCBI gene bank. To know whether all L-asparaginase genes encode functional L-asparaginase proteins or not, in the present study L-asparaginase genes encoding L-asparaginase (L-asparaginase, L-asparaginase I and L-asparaginase II) from E. carotovora subsp. atroseptica SCRI 1043 were cloned separately into pET22b(+) and expressed with and without C-terminal His-tag in E. coli BL21 (DE3). Since, type II L-asparaginase has higher affinity towards L-asparagine and therapeutic importance than type I therefore, L-aspargainase II was selected for further study. The production, purification and characterization of recombinant L-asparaginase II (with His-tag) was performed as L-asparaginase II is used as anticancer agent. The recombinant L-asparaginase II was purified using Ni-NTA column. The effect of pH, ionic strength of Tris-HCl buffer and NaCl, temperature, incubation time, various metals ions and inhibitors on the activity of L-asparaginase II was studied. In addition, substrate specificity of L-asparaginase with various substrate analogues has been studied and kinetic parameters were also determined [17]. The optimal levels of medium components and physical parameters (pH, inoculum size, agitation) were determined for enhanced production of recombinant Lasparaginase II (with His-tag) in E. coli.

Chemicals
All restriction enzymes, Phusion DNA polymerase, T 4 DNA ligase were purchased from New England Biolabs (NEB), USA. Isopropyl-βd-thiogalactopyranoside (IPTG), ampicillin and agarose were purchased from Sigma, Bangalore, India. L-asparagine and ammonium sulfate were purchased from Hi-Media, Mumbai, India. Nessler's reagent was purchased from Loba Company, Mumbai, India. All other chemicals were purchased from Sigma and were of analytical grade unless otherwise stated.

Cloning of L-asparaginase Genes
Genomic DNA was isolated using genomic DNA isolation kit (Sigma) from E. carotovora subsp. atroseptica SCRI 1043. The segments encoding the L-asparaginase (ans), L-asparaginase I (ansA) and L-asparaginase II (ansB2) were amplified by PCR using gene specific primers, designed according to the sequence of the asparaginase genes of E. carotovora subsp. atroseptica SCRI 1043 (NCBI accession No: BX950851). Each gene was cloned with and without stop codon for expressing the protein without and with C-terminal His-tag, respectively. Amplification of L-asparaginase was carried out using forward primer 5′ggaattcggatccaatgacgaaacccgtgattgtg-3′ (BamHI site underlined) and reverse primer 5′gaagcttctcgagacgatagatatcggcgacggg-3′ (XhoI site underlined) or with same forward and reverse primer with stop codon to express with or without C-terminal His-tag fusion. Amplification of L-asparaginase I was carried out using forward primer 5′ -ggagctcggatccaatgcaaaagaaatccat-3′ (BamHI site underlined) and reverse primer 5′ggaagcttctcgagatctttatcgctcaattc-3′ (XhoI site underlined) or with above mentioned forward and reverse primer with stop codon to express with or without C-terminal His-tag fusion. Amplification of L-asparaginase II was carried out using forward primer 5′-ggaattcggatccaatgcaactctcatttatcgcc-3′ (BamHI site underlined) and reverse primer 5′gaagcttctcgagctgctcgaaataggtacggatt-3′ (XhoI site underlined) or with same forward and reverse primer with stop codon to express with or without C-terminal His-tag fusion. The PCR reaction was carried out in a total volume of 50 µl contained 0.5 mM of each primer, 0.2 mM of each dNTP, 10 µl of 5× Phusion Taq buffer and 2 units of Phusion Taq DNA polymerase (NEB, USA). 1.0, 1.5 ng and 2.0 ng template genomic DNA was used for amplification of Lasparaginase, L-asparaginase I and Lasparaginase II, respectively. The PCR procedure comprised 30 cycles of 30 s at 96ºC, 45 s at 55ºC and 60 s at 72ºC have used for amplification of genes. A final extension time at 72ºC for 10 min was performed after the 30 cycles. The amplified DNA fragment was ligated into the T7 expression vector pET22b (+) inframe with pel B leader sequence between BamHI and XhoI restriction sites. From this ligation mixture, ~10 ng of DNA was transformed aseptically in to competent cells of E. coli (DH5α) by heat shock method [15]. Clones were analyzed by restriction digestion with BamHI and XhoI, further confirmed by sequencing and transformed into expression host E. coli BL21 (DE3). The gene cassette was expressed in frame with pelB signal sequence for periplasmic localization under IPTG inducible T7 promoter [18].

Expression of L-asparaginase Genes
The active culture was prepared by streaking frozen glycerol stock of recombinant strain (kept at -80ºC) on a LB agar plate (yeast extract 5 g l -1 , tryptone 10 g l -1 , NaCl 10 g l -1 , 2% agar, pH 7.0), supplemented with ampicillin (100 µg ml -1 ) and incubated at 37ºC for 12 h. A single isolated colony was transferred in to 20 ml of LB medium containing ampicillin (100 µg ml -1 ) and incubated on a rotary shaking incubator at 37ºC and 200 rpm for 10 h. This pre-inoculum was transferred (2% vv -1 ) in to 50 ml of LB medium supplemented with ampicillin (100 µg ml -1 ) in 250 ml conical flasks and incubated in a rotatory shaking incubator at 37°C and 200 rpm. When cell OD at 600 nm was reached to ~0.8, the expression of recombinant proteins (Lasparaginase, L-asparaginase I and Lasparaginase II) were induced by adding 1 mM IPTG, at different temperatures (25ºC, 30ºC and 37ºC). Samples were collected at different time intervals of post-induction and expression profile of recombinant protein was analysed. All the experiments were conducted in duplicates unless otherwise mentioned. The SDS-PAGE of recombinant proteins was performed in 12.5% polyacrylamide gel under reducing conditions [14]. Proteins were reduced by treatment with 10% of 2-mercaptoethanol at 95°C for 10 min and stained with Coomassie brilliant blue R-250 [19].

Assay of L-asparaginase and Lglutaminase
Some of the common assay methods used for measuring L-asparaginase activity include the Nesslerization reaction and Indooxine method [20], a coupled enzyme assay with excess glutamic-oxaloacetate transaminase and malic dehydrogenase [21], and fluorometric assay using L-aspartic acid β-(7-amido-4methlcoumarin) as a substrate [22]. However, the assay mentioned latter show some drawbacks, such as multistep operation, requirement for the highlytoxic reagent and inapplicability for real time. Therefore, in the present study, Nesslerization method was used because it is economical and faster method. For asparaginase assay, samples were centrifuged at 10,000 g for 10 min at 4±1ºC and washed twice with 0.05 M Tris-HCl buffer (pH 8.5) and ultrasonicated (VC 505 Microprocessor based cell Ultrasonic processor, Sonics & Materials Inc, CT, USA) at 20 MHz, 35% amplitude, 4 cycles (2 min per cycles with 3 s on and 1 s off). The contents were centrifuged at 20,000 g for 15 min (4±1ºC) and the supernatant was analyzed for intracellular L-asparaginase activity as described by Kumar et al. [23]. L-asparaginase catalyzes the conversion of L-asparagine to L-aspartic acid and ammonia. Ammonia would react with the Nessler's reagent to produce an orange product. The enzyme assay mixture consisted of 900 µl of L-asparagine (10 mM) in Tris HCl buffer (pH 8.5) and 100 µl of crude extract of the enzyme. The reaction mixture was incubated at 37ºC for 30 min and 100 µl of 15% trichloroacetic acid (TCA) was added to stop the reaction. The reaction mixture was centrifuged at 10,000 g for 5 min at room temperature to remove the precipitates. Ammonia released in the supernatant was determined colorimetrically by adding 100 µl Nessler reagent into sample containing 100 µl of supernatant of above reaction mixture and 800 µl distilled water. The contents in the sample were vortexed and incubated at room temperature for 10 min and OD at 425 nm was measured against the blank that received TCA before the addition of enzyme. The ammonia produced in the reaction was determined based on a standard curve obtained with ammonium sulfate as standard. Standard curve was prepared by making different concentration of (NH 4 ) 2 SO 4 solution in 50 mM Tris-HCl buffer of pH 8.5.

Calculation of L-asparaginase Activity
L-asparaginase activity in the test sample was calculated by the following equation.
L-asparaginase activity (U ml  Where, Abs 425 = test sample absorbance at 425 nm against appropriate blank. L-glutaminase activity was determined as described above for L-asparaginase activity using modified method of Mashburn and Wriston [23,24]. L-asparaginase activity (IU ml -1 ) was defined as the micromoles of ammonia released in one minute by one ml of enzyme and specific activity is expressed as units per milligram of protein.

Protein Estimation
The total protein contents of the samples were determined according to the method described by Lowry et al. [25] using bovine serum albumin (Sigma) as standard.

Purification of Recombinant Lasparaginase II of E. carotovora subsp. atroseptica SCRI 1043 from E. coli BL21 (DE3)
After 6 h of post induction, cells were separated from culture broth by centrifugation and washed twice with Tris-HCl buffer (50 mM, pH 8.5) and the pellet was suspended in 50 mM sodium phosphate buffer (pH 7.0, 10 mM imidazole, 500 mM NaCl,), and suspended cells in above buffer was ultrasonicated on ice. The contents were centrifuged at 20,000 g for 15 min (4±1°C) after ultrasonication. After separation of pellet, the supernatant was loaded onto a Ni affinity column, which was pre-equilibrated with 50 mM sodium phosphate buffer (pH 8.0, 500 mM NaCl and 10 mM imidazole). The column was washed with 10 times volume of buffer containing 20 mM imidazole after 30 min. Elution of the protein was carried out with 200 mM imidazole and dialyzed in Tris-HCl buffer (50 mM ) for 24 h. The enzyme activity and protein concentration of the purified enzyme was calculated according to the method described in the section 2.5 and 2.6, respectively [26].

Effect of pH, incubation temperature and time on activity of purified enzyme
The effect of pH on the activity of L-asparaginase II was evaluated under assay conditions at different levels of pH. For pH stability experiments, enzyme preparations were incubated at a pH range of 5.5-10.5 for 24 h at 4±1°C and residual activity was determined as mentioned in section 2.5. To evaluate the effect of temperature on the activity of L-asparaginase II, assay was carried out at a temperatures ranging from 27 to 57°C. To find out the optimum incubation time, enzyme was incubated with substrate for 15-90 minutes under the standard conditions and then enzyme activity was measured [17,27].

Effect of ionic strength on Lasparaginase activity
To determine the effect of ionic strength on the activity of L-asparaginase II, the activity of the enzyme was evaluated at different levels of ionic strength of Tris-HCl and NaCl (5 mM, 10 mM, 25 mM, 50 mM, 75 mM and 100 mM) [17,27].

Effect of various effectors on Lasparaginase activity
After 30 min exposure to the different effectors at their concentrations as shown in Table 4, the enzyme activity was evaluated. The most favorable concentration of different effectors mentioned by various researchers for characterization of L-asparaginase has been selected in this study [12,23,28]. The relative activity was denoted as the percentage ratio of the activity of the enzyme with the effectors to that of the untreated enzyme.

Substrate specificity
Activity was determined with L-asparagine, Dasparagine, DL-asparagine, L-glutamine, Laspartic acid, D-aspartic acid, DL-aspartic acid, L-aspartic acid amide, L-glutamic acid, succinamic acid, L-asparagine-t-butyl ester HCl, BOC-L-asparagine and N-a-acetyl-L-asparagine as substrate at a final concentration of 10 mM. Method used for L-asparaginase activity was used for determination of enzyme activity with other substrates. Activity of enzyme for other substrate was determined by same method. The relative activity was expressed as the percentage ratio of the enzyme activity determined against different structure analogs of L-asparagine to enzyme activity with L-asparagine [12,23].

Determination of kinetic parameters
The Michaelis constant (K m ), turnover numbers (k cat ) and maximal velocity (V max ) of the purified recombinant L-asparaginase II was evaluated using L-asparagine as substrate in the range of 0.05 to 2.5 mM. The kinetic parameters K m ,V max and k cat were calculated as reported earlier by Kumar et al. [23]. Turnover numbers were determined on the basis of one active site per 37.5 kDa subunit by SDS-PAGE.

Electrophoresis
Native PAGE of the purified L-asparaginase II was carried out using 7.5% polyacrylamide gel in glycine buffer (pH 10.0) at 5±1ºC [29]. SDS-PAGE was done by following the modified method of Laemmli (1970)with a 12.5 and 5 % acrylamide gel (pH 8.8) and stacking gel (pH 6.8) respectively, containing 0.1% SDS. The gel was stained with Coomassie brilliant blue R-250 and subunit molecular weight and intact molecular weight of L-asparaginase were identified using standard markers in the SDS-PAGE and native PAGE, correspondingly.

Experimental Design and Optimization
To find out the best medium for the maximum production of recombinant L-asparaginase II, six media [Luria Burtani (LB) medium, 2x Yeast extract and Tryptone (2x YT) medium, Terrific broth (TB) medium, Super broth (SB) medium, M9 Minimal medium, and Reseinberg medium)] were screened. Highest expression of recombinant L-asparaginase II (with His-tag) of E. carotovora subsp. atroseptica SCRI 1043 in E. coli was observed in the LB medium as compared to other media (data not shown). Therefore, LB medium was selected for further optimization study. The central composite design [30] was applied to optimize the levels and analysis of the combined effect of the medium constituents (tryptone, yeast extract and NaCl) and physical process parameters (pH, agitation, and inoculum size (%) on the production of recombinant L-asparaginase II. Each variable (medium component and physical variables) was assessed at five coded levels (−1.682, −1, 0, +1, and +1.682). The optimization of chemical and physical parameters was performed based on 2 3 full factorial central composite designs (CCD) with 6 axial points, resulting in a total of twenty experiments [31]. The full experimental plan with regard to their values in actual and coded form is provided in Tables 1 and 2, for optimization of medium components and physical parameters, respectively. All experiments were performed in duplicates and the specific activity of recombinant L-asparaginase II was considered as response. For statistical calculations, the relation between the coded values and real values were described in the following Eq. 3: Where, χ i is the independent variable coded value, X i is the real value of the independent variable, X o is the real value of the independent variable on the center point, ∆X i is the step change and the central point was set with a α of 1.682 for optimization of medium components and physical parameters. The quadratic model for predicting the optimal levels was expressed according to the Eq. 4: Where, Y is the predicted response (Sp. activity), k is the number of factor variables. X i and X j are independent variables, β 0 is the offset term, β i is the i th linear coefficient, β ii is the i th quadratic coefficient, and β ij is the ij th interaction coefficient. Statistical analysis of the data was performed by statistical software package MINITAB® Release 15.1, PA, USA. P-value less than 0.05 indicated that the model terms are significant and adequacy of the method developed was further analyzed. The significance of the model equation and model terms was estimated by F test. The quality of the polynomial model equation was expressed by determination of R 2 , adjusted R 2 and adequate precision. Analysis of variance (ANOVA) was carried out to evaluate the statistical significance of the model. By solving the regression equation, the most favorable combination of parameters was obtained.
In order to verify validity of the model, experiments were carried out at optimal levels of variables in a 250 ml Erlenmeyer flask with 50 ml of medium. All experiments were conducted in duplicates and averages of the results were taken as response. The samples were drawn at regular interval of time and specific activity was measured in duplicates.
There are several reports on positive as well as negative effect of His-tag on recombinant protein expression and activity [32][33][34]. In the present investigation, higher expression was observed in presence of His-tag for L-asparaginase I and II. L-asparaginase (ans), L-asparaginase I and Lasparaginase II with His-tag showed activity of 3.79 U mg -1 , 72.23 U mg -1 and 34.49 U mg -1 , respectively, whereas L-asparaginase (ans), Lasparaginase I and L-asparaginase II without His-tag showed the activity of 6.46 U mg -1 68.76 U mg -1 and 30.60 U mg -1 , respectively, which might be due to improved production of tagged protein [35][36]. The positive effect of the His-tag on the expression of recombinant protein might be due to the hydrophilic nature of the His-tag as generating a more hydrophilic protein, which is more compatible with the host cell [34]. Furthermore, the presence of affinity tag helps in improving the solubility and stability of the recombinant protein [32,33]. In contrast, Khushoo et al. [37] observed no effect on the expression due to C-terminal His-tag which shows that the effect of His-tag on expression varies with nature and structure of protein.
The presence of glutaminase activity would lead to the allergic reactions during therapy by Lasparaginase hence, glutaminase free Lasparaginase are preferred for therapeutic purpose [12]. The presence of L-glutaminase activity in all the recombinant proteins (Lasparaginase, L-asparaginase I and Lasparaginase II with/without His-tag) was analyzed and none of them shown L-glutaminase activity. Among all the three L-asparaginases studied, only L-asparaginase II is used as anticancer agent [14]. Therefore, all further studies were carried out with recombinant Lasparaginase II fused with C-terminal His-tag. . The recombinant L-asparaginase II was purified by 4.20 fold with 82 % of yield. SDS-PAGE and native PAGE analysis of the purified recombinant L-asparaginase II have shown single protein band of ~37.5 kDa and ~150.0 kDa, respectively (Fig. 3) which confirmed that recombinant Lasparaginase II is a tetramer.

Effect of pH, temperature and incubation time on enzyme activity
Recombinant L-asparaginase II showed a wide range of activity between pH 7.5-9.5 with optimum pH of 8.5. In acidic pH, L-aspartic acid has a greater affinity towards active site of the enzyme and becomes a competitive inhibitor [38].
In alkaline pH, the equilibrium was shifted towards the aspartate, which has lower affinity to the active site [39]. At pH above 9.5 and below 6.5, the enzyme lost 45 and 65% activity, respectively (Fig. 4A). Though maximum activity at a physiological pH is one of the prerequisites for antitumor activity of L-asparaginase, due its broad pH activity profile, ~85% of the enzyme activity was retained at pH 7.5. The enzyme displayed considerable stability at alkaline pH range (pH 7.5-9.5) with retention of ~80% of its original activity after incubation for 24 h.Most of the Lasparaginases from Erwinia species are reported to have optimal pH in the alkaline range (8.0-9.0). However, L-asparaginase from E. coli has shown optimum pH in the acidic range from 5.0-6.0 [10].
The purified enzyme showed maximum activity at 47-52ºC and pH of 8.6. The activity of enzyme was lost by 75% at 57ºC (Fig. 4B). Maladkar et al. [40] have also observed that the optimum temperature for Erwinia L-asparaginase at 50ºC. At lower temperature, the activity of the enzyme was found to be less due to slow reaction rate. Incubation time shows inverse effect on activity of enzyme. Maximum enzyme activity was observed after 30 minutes of incubation (Fig.  4C). It may be due to product inhibition. Products of enzymatic reactions are reversible inhibitors of the enzymes. A decrease in the rate of an enzyme caused by the accumulation of its own product plays an important role in the balance and most economic usage of metabolic pathways. It prevents one enzyme in a sequence of reactions from generating a new product more than the capacity of the next enzyme in that sequence, e.g., inhibition of hexokinase by accumulating glucose 6-phosphate [41]. Goswami et al. [17,27] and El-Sayed et al. [42] have also observed the decrease in Lasparaginase activity after incubating for 90 minutes.

Effect of ionic strength of buffer
As the activity of an enzyme would vary with ionic strength of buffer in the assay mixture, the assay of recombinant L-asparaginase II was performed at different ionic strengths of buffers. Ionic strength of buffer might affect activity of enzyme as inorganic ions of buffer may bind to some of the ionic side chains of a protein. Although this kind of interaction was not affecting the three dimensional configuration of the enzyme in a considerable manner, it could increase substrate binding to the active site of the enzyme [43]. Minimum and maximum L-asparagine hydrolysis was observed at concentration of 5 and 50 mM, respectively (Fig. 4D). Drop in the enzyme activity at lower and higher ionic strength of buffer was due to the inability of enzyme to form non-covalent interaction with the substrate [43]. We could not compare the effect of NaCl result as assay of L-asparaginase was performed by various researchers at a constant ionic strength of 50 mM Tris-HCl [4,23,26,38]. However, Goswami et al. [17,27] have observed similar type of effect of Tris-HCl on enzyme activity.

Effect of various effectors and substrate specificity
Activity analysis of L-asparaginase II was performed with various effector molecules as given in the Table 3 , and Zn 2+ indicated that presence sulfhydryl group(s) is essential for catalysis. The role of sulfhydryl groups in the catalytic activity of the enzyme was also evaluated by the stimulation of activity by the reducing source viz., 2-mercaptoethanol and glutathione, and inhibition by thiol group blocking agent, iodoactamide. L-cysteine and L-histidine were observed to be stimulators for Lasparaginase II activity. The L-asparaginase II has lost 38% of its activity with 2.5 M urea and 17% of activity was remained at 2.5 M sodium dodecyl sulfate (SDS) indicating the fact that Lasparaginase has the thiol group binding domain with high affinity towards free-SH group containing effectors and these effectors change the asparaginase from one conformation to other to favor catalytic activity. The L-asparaginase activation stimulated by GSH and Cys supports the hypothesis that all thiol group having compounds and amino acids may interact with the same activator site on L-asparaginase. Thiol reactivity has been mentioned with the purified Lasparaginase from P. carotovorum MTCC 1428 and E. carotovora [23,28]. The substrate specificity of the recombinant L-asparaginase II is given in the Table 3. No hydrolysis was observed when L-glutamine, D-aspartic acid, DL-aspartic acid, L-aspartic acid, and L-glutamic acid were used individually as substrates. After purification of recombinant L-asparaginase II, no glutaminase activity was observed. Therefore, it is predicted that recombinant L-asparaginase II will have no secondary glutaminase activity. The lack of glutaminase activity would minimize the risk factor for successful clinical studies [8]. This typical quality makes the recombinant L-asparaginase II highly suitable for remedial applications.

Determination of kinetic parameters
The K m , V max , turnover number (k cat ) and specificity constants (k cat /K m ) of purified recombinant L-asparaginase II were found to be 0.656 mM, 312.50 IU mg , respectively (Fig 5, Table 4). Higher K m values of 2.5 mM and 3.5 mM have been reported for L-asparaginase from C. glueamicum and cytoplasmic L-asparaginase (Type1) of E. coli, respective [38]. The K m value of L-asparaginase from PseudornonasstutzeriMB-405A was reported to be 0.145 mM [12]. The lower K m value of 0.074 mM and 0.09 mM were reported for L-asparaginase from V. succinogenes and E. carotovora, respectively [28,44].K m , V max , turnover number (k cat ) and specificity constant (k cat /K m ) of purified L-asparaginase of P. carotovorum MTCC 1428 were reported to be 0.657 mM, 4.45 Uµg , respectively [23]. The kinetic parameters determined in this study were comparable with those reported for many bacterial recombinant L-asparaginases [4,17,44,45].

Optimization of Medium Components Using Central Composite Design (CCD)
Among the media tested, the maximum production of recombinant L-asparaginase II from E. coli BL21 (DE3) was achieved in the LB medium (data not shown). Hence, experiments were performed to optimize the levels of medium components (tryptone, yeast extract and NaCl) using central composite design (CCD). The observed and predicted responses (production of recombinant L-asparaginase II) are shown in Table 1. By applying the multiple regression analysis on the experimental data, the following second order polynomial equation (3)  +0.0047 X 1 X 2 -0.0178 X 1 X 3 + 0.0237 X 2 X 3 (5) Where, X 1 is tryptone, X 2 is yeast extract and X 3 is NaCl.
The results inferred that tryptone (X 1 ) has shown maximum effect on recombinant L-asparaginase II production as it had largest coefficient (3.7113) followed by yeast extract (X 2 ) (0.7158) and NaCl (X 3 ) (0.5537). Positive coefficients of X 1 , X 2 and X 3 indicated a linear effect on increase in recombinant L-asparaginase II production. Negative coefficients were observed for quadratic terms of all the three variables. Among the interactions, X 1 X 2 and X 2 X 3 had positive coefficients, while, X 1 X 3 had negative coefficient. A positive sign indicates that a higher-level variable setting consequences in a higher response than the lower-level variable setting, while a negative sign indicates that the lowerlevel variable setting results in a higher response than the high-level variable setting. According to the ANOVA of the quadratic regression model, it was highly significant, as evident from the Fisher, F-test (The F value is the ratio of the mean square due to regression to the mean square due to error). The F values for recombinant Lasparaginase II of E. carotovora subsp. atroseptica SCRI 1043 was found to be 589.49 (the confidence interval is 0.05), indicating that the model was adequate (Table 5). For recombinant L-asparaginase II production, Pvalue for 'lack of fit' was greater than 0.05 (P= 0.739). In other words, the model was fit with the responses data collected and R 2 value was found to be 99.81%. This implies that more than 99.8% for recombinant L-asparaginase II production is attributed to the chemical parameters and 0.2% of the total variation is not explained by the model. The maximum predictable response for the production of recombinant L-asparaginase II was calculated by applying the regression analysis to the Eq. 5 using 'response optimizer' in Minitab software. The optimal levels of tryptone, yeast extract and NaCl for recombinant Lasparaginase II production were found to be 14.50 g l -1 , 5.30 g l -1 and 4.03 g l -1 , respectively.
Though, yeast extract is a complex nitrogen source, there was no remarkable difference in production of recombinant L-asparaginase due to difference in concentration of yeast extract in the medium (Table 1). There was a steep increase in the production of recombinant L-asparaginase II with an increase in tryptone concentration. It is well known that nitrogen source is mainly utilized to synthesize protein, nucleic acid and metabolites of nitrogen and supports enzyme production [46]. Relatively lower concentration of nutrient is the limited factor for cell growth and protein synthesis [47]. Since, osmoregulation is a significant biological process by which it can avert the cells from dehydration, studies on the osmotic regulation is significant for cell growth [48] and production of proteins [49].

full-factorial central composite circumscribed design matrix in uncoded units and coded values (in parenthesis) with experimental and predicted values of recombinant L-asparaginase II production for chemical parameters optimization
Run order Tryptone (X1 ) (g l -1 ) Yeast Extract (X2 ) (g l -1 ) NaCl (X3) (g l -1 ) Sp. Activity (U mg -1 ) Activity (U ml -1 ) Dry cell weight (g l -1 ) Observed Predicted      NaCl is a commonly used osmoticum. Therefore, NaCl was chosen as variable for medium optimization. The concentration of NaCl is lower in the optimized medium than un-optimized medium. Probably, higher NaCl concentration cause inhibitory effect for cell growth [48] and enzyme production [50].

full-factorial central composite circumscribed design matrix in uncoded units and coded values (in parenthesis) with experimental and predicted values of recombinant L-asparaginase II production for physical parameters optimization
To verify validity of the model, experiments were performed at optimal levels of medium components. The observed production (specific activity) of recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 was found to be 57.06 U mg -1 is in good agreement with the value predicted by the model, 54.77 U mg -1 . After optimization of medium components, 1.65 fold higher production of recombinant Lasparaginase II was achieved as compared to un-optimized medium (34.49 U mg -1 ).

Optimization of Physical Process Parameters Using CCD
In order to optimize the physical process parameters (initial pH of the medium, rpm of the shaking incubator and inoculum size) for enhanced production of recombinant Lasparaginase II, experiments were performed according to the CCD (Table 2). By applying the multiple regression analysis on the experimental data, the following second-order polynomial equation was found to explain the production of recombinant L-asparaginase II.
Y specific activity = -917.896 + 207.171 X 1 +1.915 Where X 1 is pH, X 2 is inoculum size (%) and X 3 is agitation (rpm). According to the ANOVA of the quadratic regression model, the model is highly significant, as evident from the Fisher F test. F value was determined to be 767.99, corresponding to very low probability value (P< 0.05). R 2 values for recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 was found to be 99.86%. Both, lower and higher agitation (rpm) was not found to be favorable for cell growth and enzyme production. Oxygen limitation has been found to influence the cell growth and expression of heterologous proteins in E. coli and oxygen supply is directly proportional to shaking speed [51,52]. Due to this, lower agitation was not found to be favorable. Probably, higher agitation is causing shear effect on bacterial cells therefore, 240 rpm was not found to be optimal for higher production of recombinant L-aspraginase II. The best combination of pH, agitation, inoculum size for the maximum production of recombinant Lasparaginase II was found to be 7.1, 216 rpm and 2.3%, respectively.
The model was validated by performing the experiments under the optimal levels of pH, agitation and inoculum size. The maximum production of recombinant L-asparaginase II was found to be 67.26U mg -1 (16.05 U ml -1 ) with productivity of 2675.00 U l -1 h -1 , resulting an overall 1.95 fold increase in the production as compared to the un-optimized production conditions (34.49 U mg -1 ). After optimization of medium component and process conditions, production of recombinant L-asparaginase II was 6.84 folds higher than L-asparaginase produced by Erwinia carotovora subsp. atroceptica SCRI (9.84 U mg -1 ).
These parameters are optimized at shake flask level where pH was not controlled. Under controlled pH condition, expression of recombinant L-asparaginase was increased from 16.05 U ml -1 to 24.57 U ml -1 (unpublished data).
Pectobacterium carotovorum MTCC 1428 17 and Vibrio succinogenes [53] are found to be glutaminase free but scientists have used genetic engineering [4,26,37,44,54,55] and enzyme engineering [55,56]to develop a novel Lasparaginase with reduced or negligible glutaminase activity. L-asparaginases from E. coli, E. carotovora, Erwinia crysanthemi, Helicobacter pylori and S. cerevisiae have been cloned and successfully expressed in bacterial and yeast expression systems [4,26,37,44,54,55]. According to several literature reports the specific activity of recombinant L-asparaginases (production) varied in a wide-range from 0.72 to 95.7 U mg -1 of protein in crude extract [26,37,44,54]. Derst et al. [56] has engineered the substrate specificity of E. coli asparaginase II by replacing the asparaginse at 248 position (Asp248) with some other residue and after replacement observed the reduced glutaminse activity. Offman et al.
[57] engineered Lasparaginase of E. coli for resisting proteolytic cleavage, for improving activity and for reducing glutaminase activity. L-asparaginase from Recombinant L-asparaginase II developed in present study is having high sp. activity 67.26U mg -1 after optimizing the medium and process condition and not shows any glutaminase activity. Therefore, it might be effective for treatment of leukemia patients and expected not to cause any toxic effects associated with glutaminse activity of L-asparaginase of E. coli [58]. Devi and Azmi [59] have purified glutaminase free L-asparaginase from E. carotovora MTCC 1428 and reported better performance than commercial L-asparaginase obtained from E. coli. In the present study purified recombinant L-asparaginase II reported is owing no glutaminase activity and sequence of asparaginase II gene has shown 100 % similarity with L-asparaginase II gene of Erwinia carotovora MTCC 1428. Therefore, it is expected that recombinant L-asparaginase II will have no secondary glutaminase activity and would be better choice for therapeutic purpose.

CONCLUSION
In the present study, the genes encoding Lasparaginase (L-asparaginase (ans), Lasparaginase I (ansA) and L-asparaginase II (ansB2)) of Erwinia carotovora subsp. atroseptica SCRI 1043 were cloned and expressed in E. coli BL21 (DE3). The production optimization including medium components and physical process parameters increased the recombinant L-asparaginase II yield by 1.95 fold as compared to unoptimized conditions. The biochemical characterization showed that the optimum range of pH and temperature for the hydrolysis of purified recombinant Lasparaginase II was 6.5-9.5 and 47-52ºC, respectively. The activity of recombinant Lasparaginase II was found to be activated by mono cations and inhibited by divalent cations and iodoactamide. The enzyme was found to be very specific for its natural substrate, Lasparagine and shown no glutaminase activity which makes it a better alternative in therapeutic applications like an anticancer drug.