Rachna Goswami; Venkata Dasu Veeranki; Krishnamoorthy Hegde

Research Article

Austin J Biotechnol Bioeng. 2015;2(1): 1034.

Enhanced Production of Novel Glutaminase Free Recombinant L-Asparaginase II of Erwinia Carotovora Subsp. Atroseptica Scri 1043 in Escherichia Coli BL21 (DE3) in a Batch and Fed-Batch Culture

Rachna Goswami^1,2, Venkata Dasu Veeranki¹* and Krishnamoorthy Hegde¹

¹Department of Biotechnology, Indian Institute of Technology Guwahati, India

²Department of Bioscience, AP IIIT Nuzvid, Rajiv Gandhi University of Knowledge Technologies (RGUKT), India

*Corresponding author: Venkata Dasu V, Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.

Received: November 06, 2014; Accepted: December 03, 2014; Published: January 29, 2015

Abstract

L-asparaginase (E.C.3.5.1.1) is used for treatment of acute lymphoblastic leukemia. It is also used as a processing aid for reducing the formation of acrylamide in starchy foods preparations. In this study, the effects of glucose, controlled pH and Dissolved Oxygen Concentration (DOC) level on cell growth and production of novel glutaminase free recombinant L-asparaginase II of Erwinia carotovora subsp. atroseptica SCRI 1043, expressed in Escherichia coli BL21 (DE3) were investigated in a batch bioreactor using Taguchi experimental design technique. At optimum circumstances of glucose (1.5 g/l), controlled pH (7.0) and DOC (40%), the maximum dry cell weight and production of recombinant L-asparaginase II was found to be 1.84 g/l and 24.57 U/ml, respectively. Fed-batch culture is used frequently to increase expression of heterologous recombinant proteins in Escherichia coli. Therefore, the production of recombinant L-asparaginase II was performed in fed batch culture. In fed-batch fermentation, 7.35 g/l of dry cell weight and 96.78 U/ml of recombinant glutaminase free L-asparaginase II were achieved, corresponding to about four fold increase in dry cell weight and production as compared with the batch culture.

Keyword: Erwinia carotovora subsp. atroseptica SCRI 1043; Recombinant L-asparaginase II; Taguchi’s method; Batch and Fed batch

Introduction

L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been widely used for the treatment of acute lymphoblastic leukemia and non-Hodgkin’s lymphoma [1]. It is also used in food industry for acrylamide free food production [2] model enzyme for the development of new drug delivery system [3] and L-asparagine biosensor for leukemia [4]. The antileukemic effect of L-asparaginase is postulated to result from the rapid and complete depletion of the circulating pool of L-asparagine, as most of the cancer cells are dependent on an exogenous source of this amino acid for survival. However, normal cells are able to synthesize L-asparagine and thus are less affected by its rapid depletion due to treatment with this enzyme. The L-asparagine deficiency rapidly impairs the protein synthesis and leads to a delayed inhibition in DNA and RNA synthesis and hence an impairment of cellular functions, resulting in cell death [5,6]. Studies on the molecular structure [4], catalysis [7], clinical aspects [5], genetic determinants involved in regulation [8] and stabilization to enhance biological half-life [9] of L-asparaginase have been reported. Several gram-negative bacteria contain two L-asparaginases, a low affinity cytoplasmic enzyme and a highaffinity periplasmic enzyme. In Escherichia coli and several other bacteria, the synthesis of cytoplasmic asparaginase I is constitutive, while expression of periplasmic asparaginase II is activated during anaerobiosis. It has been suggested that the latter one probably has a special function in anaerobic fumarate respiration by providing aspartate, which is then converted to fumarate. Further, only the type II enzyme has shown substantial antitumor activity [10]. The various side effects of this drug are mainly due to the presence of partial glutaminase activity [11]. Hence, for successful clinical studies, glutaminase free L-asparaginase is highly desirable.

The production of L-asparaginase has been studied in Serratia marcescens [12,13], Erwinia carotovora [14], E. coli [15], Enterobacter aerogenes [16], Pseudomonas aeruginosa [17], and Bacillus subtilis [10] with various carbon and nitrogen sources. The synthesis of L-asparaginase by gram negative bacteria is regulated by environmental and nutritional factors. The results are contradictory in terms of the effect of glucose [12,15] and oxygen [16] on the production of this enzyme. On the other hand, the production of L-asparaginase from wild strain is very limited. Therefore, recombinant L-asparaginases were developed to increase the expression levels [5,8,9,18]. The interest in E. carotovora L-asparaginase II arose from its significantly lower glutaminase activity as compared to that exhibited by E. coli and E. chrysanthemi enzymes [5].

The production of any metabolite varies from shake flask to bioreactor fermentation. This might be possible due to uncontrolled pH, agitation pattern and aeration in shake flask fermentation. The strategy of studying one variable at a time and keeping all others at a predetermined level is very ineffective in many cases and also a time consuming technique [19,20]. Factorial designs are not preferred to optimize variables in bioreactors, as the experiments cannot be carried out in blocks. Additionally, they need more information to design parameter levels [21]. Taguchi’s method has been used in industrial process design, mostly in developmental trials. This method is used to produce adequate process information to set up the screening and optimal conditions of parameters for particular process using a minimum number of experiments possible. The basic principle of this technique serves as screening filters, which evaluate the effects of various process variables and identify those factors which have main effects on the process using less number of experiments [22].

Escherichia coli have been used most widely as a host system for the expression of recombinant proteins, as it was characterized in terms of its physiology, molecular genetics and expression systems [23]. The High Cell Density Culture (HCDC) techniques have been developed using E. coli systems for the production of recombinant proteins with high productivities [24-26].

The selection of nutrient feeding strategy plays a vital role in high cell density cultivations as it affects the metabolic pathway fluxes, and consequently influences the the specific productivity of recombinant proteins, formation of by-products (acetic acid) and cell growth. A significant relation was observed between the post-induction glucose feeding strategy and recombinant protein production [27,28]. The feed rate of glucose would be adjusted to control the formation of acetate in fed-batch cultures. To date, there is no rule established for selecting a particular feeding strategy to achieve maximum productivity for a given recombinant protein. Prior to carbon source, the nitrogen source is also very much important for extending the cell growth and improving protein production [29,30].

In this work, we investigated the influence of initial glucose concentration, controlled pH and Dissolved Oxygen Concentration (%) (DOC) level on the production of glutaminase free recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 in E. coli BL21 (DE3) using Taguchi’s experimental technique in a batch bioreactor. Recombinant L-asparaginase II production was also performed in fed batch culture for enhanced productivity.

Materials and Methods

Chemicals

Isopropyl- ß-d-Thiogalactopyranoside (IPTG) and ampicillin were purchased from Sigma, India. Culture media and their constituents, L-asparagine, ammonium sulfate, were procured from Hi-Media, India. Nessler’s reagent was purchased from Loba Company, India. All chemicals were procured from Sigma unless otherwise stated and were of the highest quality.

Bacterial strains and plasmid

Erwinia carotovora subsp. atroseptica SCRI 1043 was kindly provided by Dr. Paul Birch, Scotland Crop Research Institute (SCRI), Scotland. Escherichia coli BL21 (DE3) and plasmid pET 22b(+) were purchased from Novagen, USA. The gene encoding L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 was amplified by PCR, and introduced between BamHI and XhoI restriction sites of pET 22 b(+) in the downstream of the T7 promoter and resultant recombinant construct was transformed in E. coli BL21 (DE3) for recombinant L-asparaginase II expression [20]. The over-expression of cloned gene was controlled by T7 polymerase responsive promoter. The positive clone was confirmed by restriction digestion analysis and colony PCR (data not shown). It was maintained in 20% sterile glycerol at -80oC.

Inoculum development

A loopful of frozen glycerol stock culture (kept at -80°C) was streaked on a LB-agar plate containing ampicillin (100 μg/ml) and incubated at 37°C for 14–16 h. A single colony was isolated and transferred in 20.0 ml of sterile LB medium containing ampicillin (100 μg/ml) in Erlenmeyer flask (100 ml) on a rotary shaker at 37°C and 200 rpm for 6–8 h and this pre-inoculum was transferred at a rate of 2.34 % (v/v) to the inoculum medium (tryptone: 14.50 g/l, yeast extract: 5.30 g/l and NaCl: 4.03 g/l).

Optimization methodology for enhanced production of recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 in batch bioreactor

In order to minimize the number of experiments and for medium components optimization, shake flask experiments were carried out by exploiting central composite design (data not shown). It is well accounted that production of any metabolite varies from shake flask to bioreactor fermentation. This might be because of uncontrolled pH, agitation pattern and aeration in shake flask fermentation. It is reported that on addition of glucose less than 0.05%, the plasmid stability and protein yields improves [20,31,32]. Therefore, Taguchi’s method was applied to know the parameters, which significantly affect the recombinant L-asparaginase II production in a batch bioreactor. Three parameters (glucose, controlled pH and DOC) in nine experiments were used to evaluate the influence of variables on recombinant L-asparaginase II production according to Taguchi’s orthogonal array. The parameters and their levels employed in Taguchi’s experimental design are mentioned in the Table 1. The glucose concentration and levels of controlled pH and DOC changed according to the experimental plan given in the Table 2. MINITAB® Release 15.1, PA, USA was used in the current study. In each experimental run, the result was recorded as the recombinant L-asparaginase II production (U/ml) and corresponding signal-tonoise (S/N) ratio was calculated by Eq. 1 with an overall objective for estimating the effects of a range of parameters on recombinant L-asparaginase II production, where a large S/N ratio is desired. $\frac{S}{N} = - 10 \log [\frac{\frac{1}{y^{2}}}{n}]$

Table 1: Parameters and their levels employed in the Taguchi’s robust design method for recombinant L-asparaginase II production for batch study in bioreactor.




  
    Parameters 
    Constituents 
    Levels 
  
  
    1 
    2 
    3 
  
  
    A
    Glucose (g/l) 
    0.5 
    1.0 
    1.5
  
  
    B
    pH 
    6.5 
    7.0 
    7.5
  
  
    C
    DOC (%)
    10
    25
    40



Table 1:  Parameters and their levels employed in the Taguchi’s robust design
method for recombinant L-asparaginase II production for batch study in
bioreactor.

Where, Y is the outcome [enzyme activity (U/ml)] and n is the number of experimental runs. Statistical analysis of the results was performed, in the form of analysis of variance (ANOVA). Validity of the model was verified by conducting the experiment at optimal level of parameters in bioreactor.

All batch fermentations were carried out in a 3.0 L bioreactor (Applikon, Holland) with 1.0 L working volume. The bioreactor was fitted with essential controllers. With 2 M NaOH and/or 2 M HCl, the pH of the medium was adjusted. According to experimental plan by varying airflow and impeller speed, the levels of pH (controlled) and The DOC was maintained. During growth phase, microorganism was grown at 37°C whereas, for production of recombinant L-asparaginase II, temperature was decreased to 30°C (previously optimized temperature for production). When cell density at Ab_600nm reached 1.50 to 1.80 (~ after 3 h), production of recombinant protein was induced with 1mM IPTG and further cultivated for 12 h at 30°C. The samples were collected at regular intervals of time and enzyme activity was measured in triplicates and averages of the results were taken as response.

Fed-batch cultivation

To enhance the cell concentration and productivity, the production of recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 in E. coli was performed in Fed-batch culture (3.0 L Bioreactor, Applikon, Holland). Initial batch culture was started with 1.0 L of medium. After the utilization of glucose, fed-batch experiments were performed by feeding the solutions containing glucose (100 g/l), yeast extract (100 g/l) and 100 mg/l ampicillin. Concentration of glucose in the medium was observed at regular interval of time and fed by intermittent pulse feeding in relative to the cell mass. Feeding of glucose with specific feed rate of 0.5 g glucose/g DCW/ h and 0.25 g glucose/g DCW/ h was performed in the growth phase and production phase, correspondingly. The residual glucose was monitored and maintained below 1.0 g/l [20,28,33-35]. During the growth phase, microorganism was grown at 37°C and in production phase, temperature was reduced to 30°C. The biomass concentration in the bioreactor was measured throughout the fermentation, by calculating the optical density (OD) as well as the dry cell weight. The DOC was restricted between 35-40% of saturation by changing agitation (350-1200 rpm) and aeration (2.0- 3.0 vvm). The pH was maintained at 7.0 by adding 2N HCl and/or 2N NaOH. The recombinant protein expression was induced by adding 1mM IPTG at 30°C, when the cell OD at Ab_600nm reached ~10.00.

Analytical methods

Assays for L-glutaminase and L-asparaginase activity: L-asparaginase activity was measured in terms of hydrolysis rate of L-asparagine by calculating the quantity of ammonia released in the reaction assay of L-asparaginase. Assay for glutaminase and L-asparaginase was carried out as described earlier [20,36]. The ammonia produced in the reaction was determined on the basis of standard curve obtained with ammonium sulfate as standard. The amount of enzyme that liberates 1 μmol of ammonia per minute at 37°C was defined as one unit of enzyme activity. Specific activity is denoted as units per milligram of protein.

Protein determination: The total protein contents of the samples were determined according to the method described by Lowry et al. [37] using bovine serum albumin (Sigma) as standard.

Estimation of DCW: The cell density was calculated by measuring the culture’s optical density at Ab_600nm with a UV-visible spectrometer. Dry cell weight was calculated by centrifuging the sample broth at 8000g for 10 min and drying the washed cells to constant weight at 80°C.

Estimation of glucose: Residual sugar in the samples was determined by the 3,5-Dinitrosalicylic Acid (DNS) method. Glucose was used as standard [38].

Measurement of plasmid stability: Plasmid stability was measured as described earlier by Goswami et al. 2014 [20].

Results and Discussion

The results of the Taguchi’s experiments are presented in the Table 2. Depending upon the combination of the levels of chemical and physical process parameters, recombinant L-asparaginase II production altered in each run significantly, signifying the strong effect of the variables and their levels on the response. Their ranking was performed based on the calculated delta S/N ratio, to evaluate the effect of these variables on recombinant L-asparaginase II production. In general, delta value for a factor, calculated by determining the difference between the maximum and minimum characteristic average S/N ratio of the factor, indicates its relative significance over others on a particular response. For a factor, higher value of delta signifies a larger significant effect than others, while S/N ratio points to effect of factors on a response. Thus delta S/N ratio have used as an important factor for ranking the variables for their effects on the response [40]. In this study, based on the delta S/N ratio obtained for each factor, all parameters were ranked accordingly which has shown that pH had the maximum effect and followed by glucose concentration and DOC (Table 3). To validate these findings on the importance of the individual parameters and their involvement on L-asparaginase production, ANOVA was employed. Table 4 presents the ANOVA of L-asparaginase II production of E. carotovora subsp. atroseptica SCRI 1043 in E. coli). In general, low P value of a term in ANOVA specifies high importance of the term. In this study, all the parameters viz., glucose concentration, pH and DOC were observed to have a significant effect on recombinant L-asparaginase II production. The effect of parameters on L-asparaginase production by ANOVA is in good agreement with those observed previously from the factors ranking based on their delta S/N ratio.

Table 2: Taguchi’s robust experimental design matrix and recombinant Lasparaginase II production of E. carotovora subsp. atroseptica SCRI 1043 in E. coli in bioreactor.




  
    Run No. 
    Glucose (g/l) 
    pH 
    DOC (%) 
    E. carotovora subsp. atroseptica SCRI 1043 
  
  
    Activity (U/ml) 
    Sp. Activity (U/mg) 
    S/N 
  
  
    1
    0.5 
    6.5 
    10 
    3.81 
    27.41
    11.61
  
  
    2
    0.5 
    7.0 
    25 
    14.98
    49.84
    23.51
  
  
    3
    0.5 
    7.5 
    40 
    12.35
    42.62
    21.83
  
  
    4
    1.0 
    6.5 
    25 
    6.02
    41.06
    15.59
  
  
    5
    1.0 
    7.0 
    40 
    16.98
    57.95
    24.59
  
  
    6
    1.0 
    7.5 
    10 
    11.12
    35.63
    20.92
  
  
    7
    1.5 
    6.5 
    40 
    13.13
    49.02
    22.36
  
  
    8
    1.5 
    7.0 
    10 
    19.24
    52.21
    25.68
  
  
    9
    1.5
    7.5
    25
    16.99
    51.25
    24.60



Table 2:  Taguchi’s robust experimental design matrix and recombinant Lasparaginase
II production of E. carotovora subsp. atroseptica SCRI 1043 in E.
coli in bioreactor.

Table 3: Values of average S/N ratio of the process variables at various levels and their ranking on the basis of delta S/N ratio.



Table 3:  Values of average S/N ratio of the process variables at various levels
and their ranking on the basis of delta S/N ratio.

Table 4: Analysis of variance for production of recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043.




  
    Source 
    DF 
    Seq SS 
    Ads SS 
    Ads MS 
    F 
    P 
  
  
    Glucose 
    2 
    63.678 
    63.678 
    31.8392 
    120.51 
    0.008 
  
  
    pH 
    2 
    135.455 
    135.455 
    67.7275 
    256.34 
    0.004 
  
  
    DOC 
    2 
    11.477 
    11.477 
    5.7387 
    21.72 
    0.044 
  
  
    Residual Error 
    2 
    0.528 
    0.528 
    0.2642 
     
     
  
  
    Total 
    8 
    211.139 
     
     
     
     
  
  
    R-Sq    = 99.7%, R-Sq(adj) = 99.0%



Table 4:  Analysis of variance for production of recombinant L-asparaginase II of
E. carotovora subsp. atroseptica SCRI 1043.

Selection of optimal levels of parameters for enhanced recombinant L-asparaginase II production

All selected parameters (glucose, pH and DOC) were tested at different levels; particular levels of the variables caused a significant increase in the mean response as compared to other levels of the variables. For e.g. level 3 of glucose and DOC and level 2 of pH were found to exhibit a noteworthy positive consequence on the response (Figure 1). Therefore, these levels of the variables were selected as optimum for the highest production of recombinant L-asparaginase II in the medium. In the optimized conditions, production of recombinant L-asparaginase II was increased. At the most favorable levels of factors, recombinant L-asparaginase II production was carried out in a batch-operated 3 L bioreactor (Applikon, Holland) with 1.0 L medium. It was observed that the production of recombinant L-asparaginase was very high (24.57 U/ ml) in the bioreactor, as compared to any of those values given in Table 2. The production of recombinant L-asparaginase was found to be 1.40 fold higher under optimal levels of parameters as compared to un-optimized conditions (17.58 U/ml. Due to higher growth of cells in bioreactor, sp. activity of enzyme was lower (61.71 U /mg) than shake flask (64.62 U/mg). Maximum production of L-asparaginase was achieved at 7 h of fermentation in a batch bioreactor and plasmid stability analysis was carried out by comparing the number of CFUs (colony forming units) on amp+ and amp- plates formed after plating the culture, withdrawn at different time-points throughout cultivation. The presence of ampicillin resistance was used as an indicator for the existence of recombinant plasmid in the cells. It was found that 74% of the culture retained the recombinant plasmid at 24 h post-induction, comparable to the shake flask cultivation.

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Citation: Goswami R, Veeranki VD and Hegde K. Enhanced Production of Novel Glutaminase Free Recombinant L-Asparaginase II of Erwinia Carotovora Subsp. Atroseptica Scri 1043 in Escherichia Coli BL21 (DE3) in a Batch and Fed-Batch Culture. Austin J Biotechnol Bioeng. 2015;2(1): 1034. ISSN: 2378-3036

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Level	Glucose	pH	DOC
1	18.99	16.53	19.41
2	20.37	24.60	21.24
3	24.22	22.45	22.93
Delta	5.23	8.07	3.52
Rank	2	1	3