Appl Biochem Biotechnol (2014) 173:2140–2151 DOI 10.1007/s12010-014-1016-x
Effect of Vitreoscilla Hemoglobin and Culture Conditions on Production of Bacterial L-Asparaginase, an Oncolytic Enzyme Sebnem O. Erenler & Hikmet Geckil
Received: 28 February 2014 / Accepted: 16 June 2014 / Published online: 27 June 2014 # Springer Science+Business Media New York 2014
Abstract L-asparaginase is a widely used cancer chemotherapy enzyme. The source for the enzyme with this property is mainly bacterial and its synthesis is strongly regulated by oxygen. In this study, we utilized two recombinant systems: one carried the gene (vgb) for the Vitreoscilla hemoglobin (VHb), a protein of prokaryotic origin which confers a highly efficient oxygen uptake to its host and the other carried the L-asparaginase gene (ansB). The host bacteria were Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa. Of these three bacteria, all gram-negative, E. coli and its recombinant strain showed up to sevenfold higher Lasparaginase activity in lactose than in other carbon sources. Although, in this bacterium glycerol was the poorest source for L-asparaginase synthesis, it supported the highest biomass production. In glucose medium, L-asparaginase activity of E. aerogenes was about threefold higher than its vgb and ansB recombinants. ansB recombinant showed significantly higher enzyme levels than both host and vgb recombinants in glycerol and lactose media. In this bacterium, VHb/vgb clearly caused a decrease in the enzyme synthesis under all conditions. As seen for E. aerogens, glycerol was the most favorable carbon source for P. aeruginosa and its vgb strain in terms of both L-asparaginase synthesis and biomass production. The cultures grown in glycerol had more than two- and threefold biomass than in glucose and lactose, respectively, and up to elevenfold than in mannitol. Indeed, the highest biomass production for all bacteria and their recombinants was in glycerol. The VHb/vgb system is clearly advantageous for production of L-asparaginase in P. aeruginosa. The same, however, does not hold true for E. aerogenes. Keywords
L-asparaginase . Vitreoscilla hemoglobin . Chemotherapeutic enzymes . ansB . vgb
Introduction L-asparaginase (EC 3.5.1.1), commercialized under the brand name Elspar, is an enzyme of high therapeutic value given its use in certain kinds of cancer therapies, mainly in lymphoblastic
S. O. Erenler (*) Department of Biology, Inonu University, Malatya 44280, Turkey e-mail: [email protected] H. Geckil Department of Molecular Biology and Genetics, Inonu University, Malatya 44280, Turkey
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leukemia [1]. Although it has been isolated and characterized from various microorganisms, the enzyme with above potential has only been determined in a few gram-negative bacteria [2–6]. These bacteria produce two types of L-asparaginases: L-asparaginase I and L-asparaginase II. L-asparaginase I is a constitutive cytoplasmic enzyme, and its synthesis is almost unaffected by the growth conditions. L-asparaginase II, however, is an inducible periplasmic enzyme, and its synthesis is largely dependent on growth conditions [5]. However, only the periplasmic form of this enzyme (i.e., L asparaginase II) has cancer therapeutic activity given its kinetic properties for its main substrate L-asparagine [7]. The study here is concerned with the synthesis of the periplasmic form of the enzyme and unless otherwise stated, the term “L-asparaginase” is used interchangeably for this form of the enzyme (i.e., L-asparaginase II). L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia, resulting in the depletion of serum L-asparagine (Fig. 1). Plasma L-asparagine is undetectable throughout the entire period in which L-asparaginase is present. Leukemic lymphoblasts and certain other tumor cells, which lack or have very low levels of L-asparagine synthetase, do not synthesize L-asparagine de novo, and they rely on this amino acid supplied in the serum. In early studies, L-asparaginase was speculated to selectively kill leukemic cells without affecting normal cells. Because normal cells have the ability to synthesize L-asparagine de novo via induction of the enzyme L-asparagine synthetase. Thus, the anti-leukemic effect of L-asparaginase is postulated to result from the rapid and complete depletion of the circulating pool of L-asparagine. Because only tumor cells rely on an exogenous supply of this amino acid for their proliferation. Although, a large number of investigations have been performed to clarify the molecular structure, the mechanism of catalysis, clinical aspects, biosynthesis, and regulation of L-asparaginase [8] [9–12], there have been relatively few reports concerning the improvement of enzyme production. In this context, it is known that the nutritional requirements for maximal L-asparaginase synthesis vary from one microorganism to other, and rate of synthesis varies even in the same organism under different culture conditions [2, 3]. These probably occur because of the interactions of various culture factors such as pH, amount of dissolved oxygen, culture phase strongly influence the cellular composition, and metabolic performance of microbial cells. As with a number of other enzymes, L-asparaginase formation is also inhibited by the addition of certain sugars, particularly glucose. It was shown that the inhibition of L-asparaginase synthesis by glucose was due to both catabolite repression (by lowering cAMP level in the cell) [13] and acid production [14]. Thus, L-asparaginase synthesis mainly occurs under fermentative conditions, due to induction by the fumarate nitrate reduction (FNR) protein and positive regulation by the cAMP receptor protein [13]. So, L-asparaginase is produced for commercial purposes mainly by submerged or solid-state fermentation [6, 15]. The importance of oxygen supply for microbial growth and product formation by microorganisms in these types of fermentations is well known. Since microorganisms growing in submerged culture utilize dissolved oxygen
Fig. 1 Hydrolysis of L-asparagine to L-aspartic acid and ammonia
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in the fermentation medium, a critical oxygen transfer rate is essential for microbial biosynthesis of specific products. In this regard, previous studies showed that Vitreoscilla hemoglobin (VHb) expressing heterologous cells had better growth characteristics, ATP production, and oxygen uptake rates than their counterparts [16–18]. Thus, development of conditions that would support a relatively stable oxygen transfer rate and a high cell population without a substantial decrease in culture pH is important for L-asparaginase production. VHb is the first discovered and probably best characterized microbial hemoglobin [19]. Its primary function is most likely to bind oxygen at low extracellular concentrations and deliver it to the terminal respiratory oxidases, thus enhancing respiration under such conditions [17, 20, 21]. Furthermore, the expression of VHb gene (vgb) is regulated by oxygen in both native host (Vitreoscilla) and in Escherichia coli, and is maximally induced under microaerophilic conditions [22–25]. In previous studies, we have shown that, bacteria engineered with the vgb gene had 2.0- to 10-fold higher oxygen uptake rates than the vgb− counterparts [16, 17]. Given all these, this study was carried out to determine how the presence of VHb in distinctly related gram-negative bacteria (E. coli, Enterobacter aerogenes, and Pseudomonas aeruginosa) with different metabolic preferences regulates the production of L-asparaginase.
Materials and Methods Chemicals L-asparagine,
TCA, and Nessler reagent chemicals (HgI2, KI, and sodium hydroxide) were purchased from Sigma Chemicals Co. All other chemicals used were of analytical grade. Strains and Culture Conditions
The bacterial hosts used throughout this study were E. coli (NRRL JM103), E. aerogenes (NRRL B-427), and P. aeruginosa (USDA B771). The vgb− and vgb+ recombinants of E. aerogenes were designated as “Ea[pUC8]” and “Ea[pUC8:15]”, respectively. E. aerogenes and its ansB recombinant constructed in this study was designated as “Ea[pBPGA]”. E. coli and its ansB recombinant, “E. coli pAHZ12” was a gift from Prof. Socorro-Duran Vargas (Departamento de Biotecnologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico) [26]. The transposon-mediated Vitreoscilla hemoglobin gene (vgb) bearing recombinant strain of P. aeruginosa, “PaJC,” was from the Laboratory of Benjamin C. Stark [27]. The pB-PGA plasmid, a pBluescript SK(+) containing the ansB, was from the laboratory of Prof. Klaus-Heinrich Röhm (Philipps-Universitat, Institut für Physiologische Chemie, Karl-Von-Frisch-StraMe, D-35033 Marburg, Germany). The growth medium used in their work was either LB or L-asparaginase supplemented M1 medium [28]. The growth medium used for L-asparaginase production was Minimal Medium Yeast (MMY) containing (l−1) 0.5 g MgSO4⋅7H2O, 0.01 g FeSO4⋅7H2O, 0.5 g KCl, 1.0 g K2HPO4, and 0.5 g yeast extract at pH 7.0. Cells were maintained on LB agar plates at 4 °C with transfers at monthly intervals. A 1/100 inoculum of overnight cultures grown in Luria–Bertani medium (LB) was made in 20 ml MMY in 125 ml Erlenmeyer flasks. Inocula in flasks were grown for 24 h at 37 °C in a 200 rpm water bath.
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Membrane Permeabilization with Potassium Phosphate-Hexane Phase System for L-Asparaginase Release Cells cultivated for L-asparaginase (i.e., L-asparaginase II) production were harvested by centrifugation (10,000 rpm for 5 min) at room temperature, washed once with 0.05 M potassium phosphate (KPi) buffer (pH 8.6), and re-suspended to A600 =5.0 in the same buffer containing hexane at 1 %. The selected hexane concentration was based on our preliminary experiments [29]. The suspensions were incubated at room temperature for 1 h, briefly vortexing for every 10 min. Tube caps were left open for 5 min to allow the volatile upper phase to evaporate prior to analysis of L-asparaginase activity in the cell-free aqueous phase. Cloning Studies Two types ansB vectors were used in this study: pB-PGA was introduced into E. aerogenes (designated as Ea[pB-PGA]) and pAHZ12 was introduced into E. coli (designated as Ec[pAHZ12]) using CaCl2 method [30]. The presumptive transformant colonies showing a satellite formation after 18 h of incubation on antibiotic (ampicillin) plates were selected and plasmid isolation was carried out by alkali lysis method [30]. The presence of respective plasmids was confirmed through restriction mapping (i.e., minipreps). P. aeruginosa and its vgb harboring strain (PaJC) were from the laboratory of Benjamin C. Stark at Illinois Institute of Technology (Chicago, IL, USA) [27]. The capacity of the recombinants in terms of Lasparaginase activity was compared with host and vgb-carrying strains.
Results Establishing the effect of various carbon and nitrogen sources on the production of Lasparaginase requires their use as the sole nutritional agents in the growth medium. Thus, standard minimal medium (MM) was used as the base growth medium. However, since MM supports formation of a limited biomass and so too low an enzyme activity to measure, it has been enriched with 0.05 % yeast extract (MMY). Overnight MMY cultures of E. aerogenes and its vgb and ansB recombinants were inoculated (at 1/100 ratio) into new MMY containing 0.01 % of one of the selected carbon (i.e., glucose, mannitol, lactose, glycerol) or nitrogen (i.e., ammonium chloride, L-asparagine, glutamine, urea) sources. Effect of Various Carbon Sources in L-Asparaginase Synthesis The highest L-asparaginase activity was determined in host E. aerogenes grown with glucose, which was, however, the second (after mannitol) least favorable carbon source in recombinants (Fig. 2). Both recombinants showed the highest (more than twofold compared to host strain) enzyme activity with glycerol. The biomass (measured as OD600 of cultures) in glycerol medium was about twofold higher than in glucose and lactose and sevenfold higher than in mannitol. Similar experiments were run for E. coli (Fig. 3), P. aeruginosa (Fig. 4), and their recombinants [pAHZ12] and PaJC, respectively. Both E. coli and its [pAHZ12] strain showed the higher (between two- to sevenfold) Lasparaginase activity when they were cultivated in lactose-supplemented MMY. Although,
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Fig. 2 Effect of carbon sources on L-asparaginase activity in E. aerogenes (Ea) and its vgb+ (Ea[pUC:15]) and ansB (Ea[pBPGA]) gene-bearing recombinants. Cells were grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
glycerol was the poorest carbon source for E. coli and the second poorest (after mannitol) for its ansB strain ([pAHZ12]), as for E. aerogenes, this carbon source supported the highest biomass production in both strains. Causing a significantly high activity of L-asparaginase in PaJC strain, glycerol was the most favorable carbon source for P. aeruginosa, while glucose was one of the least (after mannitol) preferred carbon source. Glycerol has also supported a better cell growth as it was apparent from total biomass. The cultures grown in glycerol had more than two- and threefold biomass than in glucose and lactose, respectively, and up to elevenfold than in mannitol (data not shown). 7 Glucose
Mannitol
Lactose
Glycerol
-1
L-Asparaginase (U mg )
6 5 4 3 2 1 0 E.coli
E.coli [pAHZ12]
Fig. 3 Effect of carbon sources on L-asparaginase activity in E. coli and its ansB gene bearing recombinant (E.coli[pAHZ12]) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
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Fig. 4 Effect of carbon sources on L-asparaginase activity in P. aeruginosa (Pa) and its vgb+ gene-bearing recombinant (PaJC) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
Effect of Various Nitrogen Sources in L-Asparaginase Synthesis Four nitrogen sources (ammonium chloride, asparagine, glutamine, and urea) were used. While asparagine and glutamine are natural substrates for L-asparaginase, ammonium chloride and urea are convenient nitrogen sources used by bacteria. The ansB-bearing E. aerogens (i.e., Ea[pBPGA]) showed the highest L-asparaginase activity when it was grown with asparagine and glutamine. Both nitrogen sources caused similar and high activity in L-asparaginase profiles in this strain (Fig. 5). Ammonium chloride was the poorest source in all three strains. 4
A.Chloride
Asparagine
Glutamine
Urea
L-Asparaginase (U mg -1)
3,5 3 2,5 2 1,5 1 0,5 0 Ea
Ea[pUC8:15]
Ea[pBPGA]
Fig. 5 Effect of nitrogen sources on L-asparaginase activity in E. aerogenes (Ea) and its vgb+ and ansB genebearing recombinants (Ea[pUC:15] and Ea[pBPGA]) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn − 1)
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Asparagine and glutamine were also favorable nitrogen sources in host E. coli strain. But, its recombinant which carries ansB, had the highest enzyme activity in urea-supplemented MMY (Fig. 6). Similar to E. aerogenes and its recombinants, the least preferred nitrogen source was ammonium chloride for both strains. As determined for the ansB recombinants of E. coli and E. aerogenes, the vgb+ recombinant of P. aeruginosa (PaJC, which does not have extra ansB gene) had high L-asparaginase activity when it was cultivated in asparagine and glutamine media (Fig. 7). Similar to previous two bacteria (E. coli and E. aerogenes), P. aeruginosa and its recombinant had the lowest enzyme activity when grown with ammonium chloride.
Discussion It is well established that ansB and vgb genes are strongly regulated by oxygen [2, 3, 22, 31–33]. Oxygen regulation of these genes is under complex array of mechanisms including various transcription factors (ArcAB, FNR, and CRP). Furthermore, ansB and vgb are regulated by nutritional and environmental factors like carbon and nitrogen sources, and oxygen [34–36]. Given this regulation, three host bacteria (E. coli, E. aerogenes, and P. aeruginosa) carrying either ansB or vgb were studied for the production of L-asparaginase, a well-known oncolytic enzyme. Results showed that a carbon or nitrogen source favorable for one strain may be considerably inconvenient for the other strains of the same bacteria. Glucose was the most preferred sugar to support enzyme synthesis in E. aerogenes, while it was the second poorest source (after mannitol) for the recombinant strains (i.e., vgb and ansB recombinants) of the same bacterium. It is interesting that even lactose (a disaccharide) was a more effective sugar than glucose in these recombinants, while the host strain utilized it poorly considering L-asparaginase synthesis. It is known that transcription factors such as ArcAB, FNR, and CRP are strong effectors for expression of genes involved in aerobic and anaerobic catabolic pathways, and given the VHb’s role in oxygen uptake, these global 4
A.Chloride
Asparagine
Glutamine
Urea
-1
L-Asparaginase (U mg )
3,5 3 2,5 2 1,5 1 0,5 0 E.coli
E.coli [pAHZ12]
Fig. 6 Effect of various nitrogen sources on L-asparaginase activity in E. coli and its ansB gene-bearing recombinant (E.coli[pAHZ12]) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
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6 A.Chloride
Asparagine
Glutamine
Urea
L-Asparaginase (U mg-1)
5
4
3
2
1
0 Pa
PaJC
Fig. 7 Effect of nitrogen sources on L-asparaginase activity in P. aeruginosa (Pa) and its vgb+ gene-bearing recombinant (PaJC) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
regulators may considerably be affected in cells expressing this protein (i.e., VHb). What is also known, the plasmid presence itself puts a heavy burden on host cells both metabolically and physiologically. In this regard, the ansB strain (Ea[pBPGA]) which carried L-asparaginase gene on the plasmid pBluescript II SK + had a considerably higher L-asparaginase level (up to 2.2 times) in glycerol and lactose media compared to the wild type and vgb strains. This newly formed strain may have potential for production of L-asparaginase in future studies. Although glucose is the most commonly used carbon source in production of this enzyme, previous reports showed that it causes a high catabolite inhibition especially in some bacteria [2, 35, 37], paralleled to our findings here. The vgb+ strain (i.e., Ea[pUC8:15]] displayed a lower enzyme activity in all carbon sources. This finding is also in line with an earlier report showing the negative effect of VHb on L-asparaginase synthesis [2]. The higher oxygen by Ea[pUC8:15] may cause a lower L-asparaginase synthesis, given the repression of L-asparaginase synthesis under high oxygen conditions [3]. Both VHb and L-asparaginase are induced by global transcription factors fumarate nitrate reduction (FNR) and cAMP receptor protein (CRP) expressed under low oxygen conditions [3, 5, 38]. Therefore, higher expression of VHb in late cultures may cause repression of L-asparaginase. Compared to E. aerogenes, in E. coli, the effect of carbon compounds on L-asparaginase synthesis was considerably different. Glycerol, determined as a favorable carbon source in the former was determined as the least favorable in latter, although this carbon source supported highest biomass production in both bacteria and in their recombinants. Clearly, the correlation between biomass production and L-asparaginase synthesis was not same in these two bacteria (i.e., E. aerogenes and E. coli). Further, the only meaningful difference in L-asparaginase synthesis level between E. coli and its recombinant (carrying [pAHZ12]) was in glycerol medium, where recombinant strains showed about two times greater enzyme activity than the host strain. The level of the enzyme in other media was similar in both strains. The highest enzyme levels were with lactose and a glucose repression was the case in both strains. A previous study showed that in E. coli L-asparaginase synthesis was almost completely repressed in the medium containing 5 % glucose, and it was suggested that the most significant
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factor inhibiting L-asparaginase synthesis was the decline in intracellular cAMP level and increase in acidity [39]. The level of catabolite repression displayed by glucose can also be linked to its flux into cell. This sugar is taken into cell through phosphotransferase system (PTS), which operates with a series of carriers transferring phosphate group from phosphoenol pyruvate to membrane where it phosphorylates glucose. However, the final phosphate carrier of this system is also the activator of adenylate cyclase that converts ATP to cAMP, an ubiquitous secondary messenger. So, since adenylate cyclase is not sufficiently activated when there is an active influx of phosphorylated glucose, intracellular cAMP level decreases. This causes downregulation of cAMP-regulated promoter of ansB and thus lowered L-asparaginase synthesis. Given the active transport of lactose into cells (without involvement of PTS system), glucose liberated from the breakdown of this disaccharide did not show the same catabolite regulatory effect. Thus, lactose had not a significant repression of L-asparaginase. Since PTS is not used in P. aeruginosa, such a situation is not concerned here. P. aeruginosa and its vgb recombinant (PaJC) generally displayed a higher L-asparaginase activity in all carbon sources than the other two bacteria (E. aerogenes and E. coli) and their recombinants. This difference is particularly more pronounced in PaJC grown in glycerolsupplemented medium, where it displayed two times higher enzyme levels than its wild-type host and up to six times higher than other bacteria. Interestingly, repressive effect of VHb on Lasparaginase synthesis observed in other bacteria was not seen in PaJC. In this context, our previous study [35], showed that carbon catabolite repression in vgb recombinants of E. aerogenes and P. aeruginosa were differently regulated. Substantial differences in carbon catabolite repression and VHb effect (i.e., higher oxygen uptake) on L-asparaginase synthesis in these three bacteria can be attributed to their different metabolic characteristics. Although E. coli and E. aerogenes are facultative anaerobes, it is known that the latter makes fermentation of butandiol and acetoin which are more neutral products, while the former carries out mixed-acid fermentation. Global regulators E. aerogenes and P. aeruginosa are also activated/deactivated differently. P. aeruginosa, however, is an obligate aerobe and mainly uses Entner–Doudoroff pathway as alternative to glycolysis for catabolism of glucose to pyruvate and as alternative to pentose phosphate pathway for generation of NADPH, the anabolic coenzyme. Therefore, these three gram-negative bacteria represent a great range of metabolic activities. Results of this study showed that the difference in L-asparaginase synthesis under same conditions is not only restricted two bacteria at species level, but also strain level. There are significant differences in L-asparaginase levels among different strains of the same species (host bacteria ansB or vgb recombinants). Therefore, conditions required for high L-asparaginase synthesis of one bacterium cannot be applied in other closely related (but metabolically distinct) bacteria. Given the plasmid burden, effect of gene (i.e., vgb and ansB) dosage, these conditions may also differ among different strains of the same bacteria. Thus, once a bacterium or strain is selected for the L-asparaginase synthesis (here P. aeruginosa or its vgb strain), it should be dealt separately for environmental conditions supporting optimal enzyme production. Similar to results of the study held with carbon sources, the highest L-asparaginase levels with any nitrogen sources were also determined in P. aeruginosa. Particularly the vgb recombinant strain (PaJC) of this bacterium showed the highest enzyme level among all bacteria and strains. Glutamine was the most appropriate nitrogen source in this strain followed by asparagine. Thus, VHb had a clear positive effect for L-asparaginase production in P. aeruginosa. In E. aerogens, however, the same effect of VHb was not observed. The host and vgb recombinant showed similar L-asparaginase profiles. The ansB strain of this bacterium, however, had higher L-asparaginase levels than the host and vgb strain. This is thought to be a result of gene (i.e., ansB) dosage effect. Thus, our newly constructed ansB strain is an
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encouraging strain for L-asparaginase production. In host E. coli, the highest L-asparaginase level was provided with asparagine, while in its ansB strain (pAHZ12), the favorite nitrogen source was urea, where this strain displayed approximately 2.7 times higher enzyme activity compare with E. coli. Thus, the metabolic burden inflicted by plasmid presence may be a factor in determining which nitrogen source the bacteria will prefer. All bacteria had the lowest enzyme activity in medium-containing ammonium chloride, the only inorganic nitrogen source. Given their organic nature, other nitrogen sources (asparagine, glutamine, and urea) may be utilized not only as nitrogen sources but also as carbon sources. In this context, the lowest biomass (as OD600 of cultures) was observed in ammonium chloride-supplemented medium. Some previous studies reported that while organic nitrogen sources, such as Lasparagine, L-glutamine, L-glutamic acid, and urea, induce L-asparaginase synthesis, in media-containing ammonium compounds the level of enzyme was considerably decreased [40, 41]. How the data acquired here for shake-flask experiments can be translated into large scale industrial one (e.g., bioreactors) remain to be seen. In this context, the majority of studies have been at the shake-flask level and very few has been successfully scaled up. However, some recent studies have reported bioprocess strategies for the scale up of such systems, employing fed-batch techniques [42, 43]. Our results show that even closely related bacteria possess considerably different biochemical characteristics, which forms the basis of their difference in response to same nutritional and physical variables. Therefore, it is imperative that optimal conditions (chemical or physical) for producing a product be determined case by case. The study here showed this is true even for two strains of the same bacterium harboring different plasmids. Collectively, our results show that the most favorable carbon source for L-asparaginase synthesis in E. aerogenes and P. aeruginosa (including their recombinants) is glycerol. Glycerol-containing medium has also supported a better biomass production in these two strains (compared to other carbon sources, up to sevenfold in E. aerogenes and more than tenfold in P. aeruginosa). For E. coli and its recombinant strain, however, lactose clearly is a better source for the enzyme synthesis and biomass production. The data in this study suggest that in a scale-up study using E. aerogenes, the VHb/vgb system be used with a caution, as better oxygen uptake causes a reduction in L-asparaginase synthesis. However, for P. aeruginosa, the opposite seems to be true. The VHb/vgb system is clearly advantageous for production of L-asparaginase using P. aeruginosa.
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The Journal of Biological Chemistry, 276, 24781–24789. 22. Joshi, M., & Dikshit, K. L. (1994). Oxygen-Dependent Regulation of Vitreoscilla globin gene: evidence for positive regulation by FNR. Biochemical Biophysical Reseach Communication, 202, 535–542. 23. Dikshit, R. P., Dikshit, K. L., Liu, Y. X., & Webster, D. A. (1992). The Bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases. Archives of Biochemistry and Biophysics, 293, 241–245. 24. Dikshit, K. L., & Webster, D. A. (1988). Cloning, Characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene, 70, 377–386. 25. Erenler, S. O., Gencer, S., Geckil, H., Stark, B. C., & Webster, D. A. (2004). Cloning and expression of the Vitreoscilla hemoglobin gene in Enterobacter aerogenes: effect on cell growth and oxygen uptake. Appl Biochem Micro, 40, 241–248. 26. Ortuno-Olea, L., & Duran-Vargas, S. (2000). The L-asparagine operon of Rhizobium etli contains a gene encoding an atypical asparaginase. Fems Microbiology Letters, 189, 177–182. 27. Chung, J. W., Webster, D. A., Pagilla, K. R., & Stark, B. C. (2001). Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability. Journal of Industrial Microbiology & Biotechnology, 27, 27–33. 28. Huser, A., Kloppner, U., & Rohm, K. H. (1999). Cloning, sequence analysis, and expression of ansB from Pseudomonas fluorescens, encoding periplasmic glutaminase/asparaginase. Fems Microbiology Letters, 178, 327–335. 29. Geckil, H., Ates, B., Gencer, S., Uckun, M., & Yilmaz, I. (2005). Membrane permeabilization of gramnegative bacteria with a potassium phosphate/hexane aqueous phase system for the release of L-asparaginase: an enzyme used in cancer therapy. Process Biochemistry, 40, 573–579. 30. Sambrook, J., & Russell, D. W. (2001). 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31. Narta, U., Roy, S., Kanwar, S. S., & Azmi, W. (2011). Improved production of L-asparaginase by Bacillus brevis cultivated in the presence of oxygen-vectors. Bioresource Technology, 102, 2083–2085. 32. Agarwal, A., Kumar, S., & Veeranki, V. D. (2011). Effect of chemical and physical parameters on the production of L-asparaginase from a newly isolated Serratia marcescens SK-07. Letters in Applied Microbiology, 52, 307–313. 33. Boerman, S., Webster, D. A., & Roush, A. H. (1981). Control of heme content in Vitreoscilla by oxygen. Federation Proceedings, 40, 1864–1864. 34. Khleifat, K. M. (2006). Correlation between bacterial hemoglobin and carbon sources: their effect on copper uptake by transformed E. coli strain alpha DH5. Current Microbiology, 52, 64–68. 35. Geckil, H., Gencer, S., & Uckun, M. (2004). Vitreoscilla hemoglobin expressing Enterobacter aerogenes and Pseudomonas aeruginosa respond differently to carbon catabolite and oxygen repression for production of Lasparaginase, an enzyme used in cancer therapy. Enzyme and Microbial Technology, 35, 182–189. 36. Hymavathi, M., Sathish, T., Brahmaiah, P., & Prakasham, R. S. (2010). Impact of carbon and nitrogen sources on L-asparaginase production by isolated Bacillus circulans (MTCC 8574): application of saturated Plackett-Burman Design. Chemical and Biochemical Engineering Quarterly, 24, 473–480. 37. Wei, D. Z., & Liu, H. (1998). Promotion of L-asparaginase production by using n-dodecane. Biotechnology Techniques, 12, 129–131. 38. Wriston, J. C., Jr., & Yellin, T. O. (1973). L-asparajinase: a review. Advances in Enzymology and Related Areas of Molecular Biology, 39, 185–248. 39. Willis, R. C., & Woolpolk, C. A. (1974). Asparagine utilization in Escherichia coli. Journal Bacteriology, 118, 231–41. 40. Golden, K. J., & Bernlohr, R. W. (1985). Nitrogen catabolite repression of the L-asparaginase of Bacillus licheniformis. Journal of Bacteriology, 164, 938–940. 41. Treshalina, H. M., Lukasheva, E. V., Sedakova, L. A., Firsova, G. A., Guerassimova, G. K., Gogichaeva, N. V., & Berezov, T. T. (2000). Anticancer enzyme L-lysine α-oxidase. Applied Biochemistry Biotechnology, 88, 267–273. 42. Roth, G., Nunes, J. E. S., Rosado, L. A., Bizarro, C. V., Volpato, G., Nunes, C. P., Renard, G., Basso, L. A., Santos, D. S., & Chies, J. M. (2013). Recombinant Erwinia carotovora L-asparaginase II production in Escherichia coli fed-batch cultures. Brazilian Journal of Chemical Engineering, 30, 245–256. 43. Uppuluri, K. B., & Reddy, D. S. R. (2009). Optimization of L-asparaginase production by Isolated Aspergillus niger using sesame cake in a column bioreactor. Journal Pure Applied Microbiology, 3, 83–90.
Effect of Vitreoscilla Hemoglobin and Culture Conditions on Production of Bacterial L-Asparaginase, an Oncolytic Enzyme Sebnem O. Erenler & Hikmet Geckil
Received: 28 February 2014 / Accepted: 16 June 2014 / Published online: 27 June 2014 # Springer Science+Business Media New York 2014
Abstract L-asparaginase is a widely used cancer chemotherapy enzyme. The source for the enzyme with this property is mainly bacterial and its synthesis is strongly regulated by oxygen. In this study, we utilized two recombinant systems: one carried the gene (vgb) for the Vitreoscilla hemoglobin (VHb), a protein of prokaryotic origin which confers a highly efficient oxygen uptake to its host and the other carried the L-asparaginase gene (ansB). The host bacteria were Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa. Of these three bacteria, all gram-negative, E. coli and its recombinant strain showed up to sevenfold higher Lasparaginase activity in lactose than in other carbon sources. Although, in this bacterium glycerol was the poorest source for L-asparaginase synthesis, it supported the highest biomass production. In glucose medium, L-asparaginase activity of E. aerogenes was about threefold higher than its vgb and ansB recombinants. ansB recombinant showed significantly higher enzyme levels than both host and vgb recombinants in glycerol and lactose media. In this bacterium, VHb/vgb clearly caused a decrease in the enzyme synthesis under all conditions. As seen for E. aerogens, glycerol was the most favorable carbon source for P. aeruginosa and its vgb strain in terms of both L-asparaginase synthesis and biomass production. The cultures grown in glycerol had more than two- and threefold biomass than in glucose and lactose, respectively, and up to elevenfold than in mannitol. Indeed, the highest biomass production for all bacteria and their recombinants was in glycerol. The VHb/vgb system is clearly advantageous for production of L-asparaginase in P. aeruginosa. The same, however, does not hold true for E. aerogenes. Keywords
L-asparaginase . Vitreoscilla hemoglobin . Chemotherapeutic enzymes . ansB . vgb
Introduction L-asparaginase (EC 3.5.1.1), commercialized under the brand name Elspar, is an enzyme of high therapeutic value given its use in certain kinds of cancer therapies, mainly in lymphoblastic
S. O. Erenler (*) Department of Biology, Inonu University, Malatya 44280, Turkey e-mail: [email protected] H. Geckil Department of Molecular Biology and Genetics, Inonu University, Malatya 44280, Turkey
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leukemia [1]. Although it has been isolated and characterized from various microorganisms, the enzyme with above potential has only been determined in a few gram-negative bacteria [2–6]. These bacteria produce two types of L-asparaginases: L-asparaginase I and L-asparaginase II. L-asparaginase I is a constitutive cytoplasmic enzyme, and its synthesis is almost unaffected by the growth conditions. L-asparaginase II, however, is an inducible periplasmic enzyme, and its synthesis is largely dependent on growth conditions [5]. However, only the periplasmic form of this enzyme (i.e., L asparaginase II) has cancer therapeutic activity given its kinetic properties for its main substrate L-asparagine [7]. The study here is concerned with the synthesis of the periplasmic form of the enzyme and unless otherwise stated, the term “L-asparaginase” is used interchangeably for this form of the enzyme (i.e., L-asparaginase II). L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia, resulting in the depletion of serum L-asparagine (Fig. 1). Plasma L-asparagine is undetectable throughout the entire period in which L-asparaginase is present. Leukemic lymphoblasts and certain other tumor cells, which lack or have very low levels of L-asparagine synthetase, do not synthesize L-asparagine de novo, and they rely on this amino acid supplied in the serum. In early studies, L-asparaginase was speculated to selectively kill leukemic cells without affecting normal cells. Because normal cells have the ability to synthesize L-asparagine de novo via induction of the enzyme L-asparagine synthetase. Thus, the anti-leukemic effect of L-asparaginase is postulated to result from the rapid and complete depletion of the circulating pool of L-asparagine. Because only tumor cells rely on an exogenous supply of this amino acid for their proliferation. Although, a large number of investigations have been performed to clarify the molecular structure, the mechanism of catalysis, clinical aspects, biosynthesis, and regulation of L-asparaginase [8] [9–12], there have been relatively few reports concerning the improvement of enzyme production. In this context, it is known that the nutritional requirements for maximal L-asparaginase synthesis vary from one microorganism to other, and rate of synthesis varies even in the same organism under different culture conditions [2, 3]. These probably occur because of the interactions of various culture factors such as pH, amount of dissolved oxygen, culture phase strongly influence the cellular composition, and metabolic performance of microbial cells. As with a number of other enzymes, L-asparaginase formation is also inhibited by the addition of certain sugars, particularly glucose. It was shown that the inhibition of L-asparaginase synthesis by glucose was due to both catabolite repression (by lowering cAMP level in the cell) [13] and acid production [14]. Thus, L-asparaginase synthesis mainly occurs under fermentative conditions, due to induction by the fumarate nitrate reduction (FNR) protein and positive regulation by the cAMP receptor protein [13]. So, L-asparaginase is produced for commercial purposes mainly by submerged or solid-state fermentation [6, 15]. The importance of oxygen supply for microbial growth and product formation by microorganisms in these types of fermentations is well known. Since microorganisms growing in submerged culture utilize dissolved oxygen
Fig. 1 Hydrolysis of L-asparagine to L-aspartic acid and ammonia
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in the fermentation medium, a critical oxygen transfer rate is essential for microbial biosynthesis of specific products. In this regard, previous studies showed that Vitreoscilla hemoglobin (VHb) expressing heterologous cells had better growth characteristics, ATP production, and oxygen uptake rates than their counterparts [16–18]. Thus, development of conditions that would support a relatively stable oxygen transfer rate and a high cell population without a substantial decrease in culture pH is important for L-asparaginase production. VHb is the first discovered and probably best characterized microbial hemoglobin [19]. Its primary function is most likely to bind oxygen at low extracellular concentrations and deliver it to the terminal respiratory oxidases, thus enhancing respiration under such conditions [17, 20, 21]. Furthermore, the expression of VHb gene (vgb) is regulated by oxygen in both native host (Vitreoscilla) and in Escherichia coli, and is maximally induced under microaerophilic conditions [22–25]. In previous studies, we have shown that, bacteria engineered with the vgb gene had 2.0- to 10-fold higher oxygen uptake rates than the vgb− counterparts [16, 17]. Given all these, this study was carried out to determine how the presence of VHb in distinctly related gram-negative bacteria (E. coli, Enterobacter aerogenes, and Pseudomonas aeruginosa) with different metabolic preferences regulates the production of L-asparaginase.
Materials and Methods Chemicals L-asparagine,
TCA, and Nessler reagent chemicals (HgI2, KI, and sodium hydroxide) were purchased from Sigma Chemicals Co. All other chemicals used were of analytical grade. Strains and Culture Conditions
The bacterial hosts used throughout this study were E. coli (NRRL JM103), E. aerogenes (NRRL B-427), and P. aeruginosa (USDA B771). The vgb− and vgb+ recombinants of E. aerogenes were designated as “Ea[pUC8]” and “Ea[pUC8:15]”, respectively. E. aerogenes and its ansB recombinant constructed in this study was designated as “Ea[pBPGA]”. E. coli and its ansB recombinant, “E. coli pAHZ12” was a gift from Prof. Socorro-Duran Vargas (Departamento de Biotecnologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico) [26]. The transposon-mediated Vitreoscilla hemoglobin gene (vgb) bearing recombinant strain of P. aeruginosa, “PaJC,” was from the Laboratory of Benjamin C. Stark [27]. The pB-PGA plasmid, a pBluescript SK(+) containing the ansB, was from the laboratory of Prof. Klaus-Heinrich Röhm (Philipps-Universitat, Institut für Physiologische Chemie, Karl-Von-Frisch-StraMe, D-35033 Marburg, Germany). The growth medium used in their work was either LB or L-asparaginase supplemented M1 medium [28]. The growth medium used for L-asparaginase production was Minimal Medium Yeast (MMY) containing (l−1) 0.5 g MgSO4⋅7H2O, 0.01 g FeSO4⋅7H2O, 0.5 g KCl, 1.0 g K2HPO4, and 0.5 g yeast extract at pH 7.0. Cells were maintained on LB agar plates at 4 °C with transfers at monthly intervals. A 1/100 inoculum of overnight cultures grown in Luria–Bertani medium (LB) was made in 20 ml MMY in 125 ml Erlenmeyer flasks. Inocula in flasks were grown for 24 h at 37 °C in a 200 rpm water bath.
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Membrane Permeabilization with Potassium Phosphate-Hexane Phase System for L-Asparaginase Release Cells cultivated for L-asparaginase (i.e., L-asparaginase II) production were harvested by centrifugation (10,000 rpm for 5 min) at room temperature, washed once with 0.05 M potassium phosphate (KPi) buffer (pH 8.6), and re-suspended to A600 =5.0 in the same buffer containing hexane at 1 %. The selected hexane concentration was based on our preliminary experiments [29]. The suspensions were incubated at room temperature for 1 h, briefly vortexing for every 10 min. Tube caps were left open for 5 min to allow the volatile upper phase to evaporate prior to analysis of L-asparaginase activity in the cell-free aqueous phase. Cloning Studies Two types ansB vectors were used in this study: pB-PGA was introduced into E. aerogenes (designated as Ea[pB-PGA]) and pAHZ12 was introduced into E. coli (designated as Ec[pAHZ12]) using CaCl2 method [30]. The presumptive transformant colonies showing a satellite formation after 18 h of incubation on antibiotic (ampicillin) plates were selected and plasmid isolation was carried out by alkali lysis method [30]. The presence of respective plasmids was confirmed through restriction mapping (i.e., minipreps). P. aeruginosa and its vgb harboring strain (PaJC) were from the laboratory of Benjamin C. Stark at Illinois Institute of Technology (Chicago, IL, USA) [27]. The capacity of the recombinants in terms of Lasparaginase activity was compared with host and vgb-carrying strains.
Results Establishing the effect of various carbon and nitrogen sources on the production of Lasparaginase requires their use as the sole nutritional agents in the growth medium. Thus, standard minimal medium (MM) was used as the base growth medium. However, since MM supports formation of a limited biomass and so too low an enzyme activity to measure, it has been enriched with 0.05 % yeast extract (MMY). Overnight MMY cultures of E. aerogenes and its vgb and ansB recombinants were inoculated (at 1/100 ratio) into new MMY containing 0.01 % of one of the selected carbon (i.e., glucose, mannitol, lactose, glycerol) or nitrogen (i.e., ammonium chloride, L-asparagine, glutamine, urea) sources. Effect of Various Carbon Sources in L-Asparaginase Synthesis The highest L-asparaginase activity was determined in host E. aerogenes grown with glucose, which was, however, the second (after mannitol) least favorable carbon source in recombinants (Fig. 2). Both recombinants showed the highest (more than twofold compared to host strain) enzyme activity with glycerol. The biomass (measured as OD600 of cultures) in glycerol medium was about twofold higher than in glucose and lactose and sevenfold higher than in mannitol. Similar experiments were run for E. coli (Fig. 3), P. aeruginosa (Fig. 4), and their recombinants [pAHZ12] and PaJC, respectively. Both E. coli and its [pAHZ12] strain showed the higher (between two- to sevenfold) Lasparaginase activity when they were cultivated in lactose-supplemented MMY. Although,
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Fig. 2 Effect of carbon sources on L-asparaginase activity in E. aerogenes (Ea) and its vgb+ (Ea[pUC:15]) and ansB (Ea[pBPGA]) gene-bearing recombinants. Cells were grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
glycerol was the poorest carbon source for E. coli and the second poorest (after mannitol) for its ansB strain ([pAHZ12]), as for E. aerogenes, this carbon source supported the highest biomass production in both strains. Causing a significantly high activity of L-asparaginase in PaJC strain, glycerol was the most favorable carbon source for P. aeruginosa, while glucose was one of the least (after mannitol) preferred carbon source. Glycerol has also supported a better cell growth as it was apparent from total biomass. The cultures grown in glycerol had more than two- and threefold biomass than in glucose and lactose, respectively, and up to elevenfold than in mannitol (data not shown). 7 Glucose
Mannitol
Lactose
Glycerol
-1
L-Asparaginase (U mg )
6 5 4 3 2 1 0 E.coli
E.coli [pAHZ12]
Fig. 3 Effect of carbon sources on L-asparaginase activity in E. coli and its ansB gene bearing recombinant (E.coli[pAHZ12]) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
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Fig. 4 Effect of carbon sources on L-asparaginase activity in P. aeruginosa (Pa) and its vgb+ gene-bearing recombinant (PaJC) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
Effect of Various Nitrogen Sources in L-Asparaginase Synthesis Four nitrogen sources (ammonium chloride, asparagine, glutamine, and urea) were used. While asparagine and glutamine are natural substrates for L-asparaginase, ammonium chloride and urea are convenient nitrogen sources used by bacteria. The ansB-bearing E. aerogens (i.e., Ea[pBPGA]) showed the highest L-asparaginase activity when it was grown with asparagine and glutamine. Both nitrogen sources caused similar and high activity in L-asparaginase profiles in this strain (Fig. 5). Ammonium chloride was the poorest source in all three strains. 4
A.Chloride
Asparagine
Glutamine
Urea
L-Asparaginase (U mg -1)
3,5 3 2,5 2 1,5 1 0,5 0 Ea
Ea[pUC8:15]
Ea[pBPGA]
Fig. 5 Effect of nitrogen sources on L-asparaginase activity in E. aerogenes (Ea) and its vgb+ and ansB genebearing recombinants (Ea[pUC:15] and Ea[pBPGA]) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn − 1)
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Asparagine and glutamine were also favorable nitrogen sources in host E. coli strain. But, its recombinant which carries ansB, had the highest enzyme activity in urea-supplemented MMY (Fig. 6). Similar to E. aerogenes and its recombinants, the least preferred nitrogen source was ammonium chloride for both strains. As determined for the ansB recombinants of E. coli and E. aerogenes, the vgb+ recombinant of P. aeruginosa (PaJC, which does not have extra ansB gene) had high L-asparaginase activity when it was cultivated in asparagine and glutamine media (Fig. 7). Similar to previous two bacteria (E. coli and E. aerogenes), P. aeruginosa and its recombinant had the lowest enzyme activity when grown with ammonium chloride.
Discussion It is well established that ansB and vgb genes are strongly regulated by oxygen [2, 3, 22, 31–33]. Oxygen regulation of these genes is under complex array of mechanisms including various transcription factors (ArcAB, FNR, and CRP). Furthermore, ansB and vgb are regulated by nutritional and environmental factors like carbon and nitrogen sources, and oxygen [34–36]. Given this regulation, three host bacteria (E. coli, E. aerogenes, and P. aeruginosa) carrying either ansB or vgb were studied for the production of L-asparaginase, a well-known oncolytic enzyme. Results showed that a carbon or nitrogen source favorable for one strain may be considerably inconvenient for the other strains of the same bacteria. Glucose was the most preferred sugar to support enzyme synthesis in E. aerogenes, while it was the second poorest source (after mannitol) for the recombinant strains (i.e., vgb and ansB recombinants) of the same bacterium. It is interesting that even lactose (a disaccharide) was a more effective sugar than glucose in these recombinants, while the host strain utilized it poorly considering L-asparaginase synthesis. It is known that transcription factors such as ArcAB, FNR, and CRP are strong effectors for expression of genes involved in aerobic and anaerobic catabolic pathways, and given the VHb’s role in oxygen uptake, these global 4
A.Chloride
Asparagine
Glutamine
Urea
-1
L-Asparaginase (U mg )
3,5 3 2,5 2 1,5 1 0,5 0 E.coli
E.coli [pAHZ12]
Fig. 6 Effect of various nitrogen sources on L-asparaginase activity in E. coli and its ansB gene-bearing recombinant (E.coli[pAHZ12]) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
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6 A.Chloride
Asparagine
Glutamine
Urea
L-Asparaginase (U mg-1)
5
4
3
2
1
0 Pa
PaJC
Fig. 7 Effect of nitrogen sources on L-asparaginase activity in P. aeruginosa (Pa) and its vgb+ gene-bearing recombinant (PaJC) grown in MMY. Each data point is the average of three independent experiments with error bars indicating standard deviations (σn−1)
regulators may considerably be affected in cells expressing this protein (i.e., VHb). What is also known, the plasmid presence itself puts a heavy burden on host cells both metabolically and physiologically. In this regard, the ansB strain (Ea[pBPGA]) which carried L-asparaginase gene on the plasmid pBluescript II SK + had a considerably higher L-asparaginase level (up to 2.2 times) in glycerol and lactose media compared to the wild type and vgb strains. This newly formed strain may have potential for production of L-asparaginase in future studies. Although glucose is the most commonly used carbon source in production of this enzyme, previous reports showed that it causes a high catabolite inhibition especially in some bacteria [2, 35, 37], paralleled to our findings here. The vgb+ strain (i.e., Ea[pUC8:15]] displayed a lower enzyme activity in all carbon sources. This finding is also in line with an earlier report showing the negative effect of VHb on L-asparaginase synthesis [2]. The higher oxygen by Ea[pUC8:15] may cause a lower L-asparaginase synthesis, given the repression of L-asparaginase synthesis under high oxygen conditions [3]. Both VHb and L-asparaginase are induced by global transcription factors fumarate nitrate reduction (FNR) and cAMP receptor protein (CRP) expressed under low oxygen conditions [3, 5, 38]. Therefore, higher expression of VHb in late cultures may cause repression of L-asparaginase. Compared to E. aerogenes, in E. coli, the effect of carbon compounds on L-asparaginase synthesis was considerably different. Glycerol, determined as a favorable carbon source in the former was determined as the least favorable in latter, although this carbon source supported highest biomass production in both bacteria and in their recombinants. Clearly, the correlation between biomass production and L-asparaginase synthesis was not same in these two bacteria (i.e., E. aerogenes and E. coli). Further, the only meaningful difference in L-asparaginase synthesis level between E. coli and its recombinant (carrying [pAHZ12]) was in glycerol medium, where recombinant strains showed about two times greater enzyme activity than the host strain. The level of the enzyme in other media was similar in both strains. The highest enzyme levels were with lactose and a glucose repression was the case in both strains. A previous study showed that in E. coli L-asparaginase synthesis was almost completely repressed in the medium containing 5 % glucose, and it was suggested that the most significant
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factor inhibiting L-asparaginase synthesis was the decline in intracellular cAMP level and increase in acidity [39]. The level of catabolite repression displayed by glucose can also be linked to its flux into cell. This sugar is taken into cell through phosphotransferase system (PTS), which operates with a series of carriers transferring phosphate group from phosphoenol pyruvate to membrane where it phosphorylates glucose. However, the final phosphate carrier of this system is also the activator of adenylate cyclase that converts ATP to cAMP, an ubiquitous secondary messenger. So, since adenylate cyclase is not sufficiently activated when there is an active influx of phosphorylated glucose, intracellular cAMP level decreases. This causes downregulation of cAMP-regulated promoter of ansB and thus lowered L-asparaginase synthesis. Given the active transport of lactose into cells (without involvement of PTS system), glucose liberated from the breakdown of this disaccharide did not show the same catabolite regulatory effect. Thus, lactose had not a significant repression of L-asparaginase. Since PTS is not used in P. aeruginosa, such a situation is not concerned here. P. aeruginosa and its vgb recombinant (PaJC) generally displayed a higher L-asparaginase activity in all carbon sources than the other two bacteria (E. aerogenes and E. coli) and their recombinants. This difference is particularly more pronounced in PaJC grown in glycerolsupplemented medium, where it displayed two times higher enzyme levels than its wild-type host and up to six times higher than other bacteria. Interestingly, repressive effect of VHb on Lasparaginase synthesis observed in other bacteria was not seen in PaJC. In this context, our previous study [35], showed that carbon catabolite repression in vgb recombinants of E. aerogenes and P. aeruginosa were differently regulated. Substantial differences in carbon catabolite repression and VHb effect (i.e., higher oxygen uptake) on L-asparaginase synthesis in these three bacteria can be attributed to their different metabolic characteristics. Although E. coli and E. aerogenes are facultative anaerobes, it is known that the latter makes fermentation of butandiol and acetoin which are more neutral products, while the former carries out mixed-acid fermentation. Global regulators E. aerogenes and P. aeruginosa are also activated/deactivated differently. P. aeruginosa, however, is an obligate aerobe and mainly uses Entner–Doudoroff pathway as alternative to glycolysis for catabolism of glucose to pyruvate and as alternative to pentose phosphate pathway for generation of NADPH, the anabolic coenzyme. Therefore, these three gram-negative bacteria represent a great range of metabolic activities. Results of this study showed that the difference in L-asparaginase synthesis under same conditions is not only restricted two bacteria at species level, but also strain level. There are significant differences in L-asparaginase levels among different strains of the same species (host bacteria ansB or vgb recombinants). Therefore, conditions required for high L-asparaginase synthesis of one bacterium cannot be applied in other closely related (but metabolically distinct) bacteria. Given the plasmid burden, effect of gene (i.e., vgb and ansB) dosage, these conditions may also differ among different strains of the same bacteria. Thus, once a bacterium or strain is selected for the L-asparaginase synthesis (here P. aeruginosa or its vgb strain), it should be dealt separately for environmental conditions supporting optimal enzyme production. Similar to results of the study held with carbon sources, the highest L-asparaginase levels with any nitrogen sources were also determined in P. aeruginosa. Particularly the vgb recombinant strain (PaJC) of this bacterium showed the highest enzyme level among all bacteria and strains. Glutamine was the most appropriate nitrogen source in this strain followed by asparagine. Thus, VHb had a clear positive effect for L-asparaginase production in P. aeruginosa. In E. aerogens, however, the same effect of VHb was not observed. The host and vgb recombinant showed similar L-asparaginase profiles. The ansB strain of this bacterium, however, had higher L-asparaginase levels than the host and vgb strain. This is thought to be a result of gene (i.e., ansB) dosage effect. Thus, our newly constructed ansB strain is an
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encouraging strain for L-asparaginase production. In host E. coli, the highest L-asparaginase level was provided with asparagine, while in its ansB strain (pAHZ12), the favorite nitrogen source was urea, where this strain displayed approximately 2.7 times higher enzyme activity compare with E. coli. Thus, the metabolic burden inflicted by plasmid presence may be a factor in determining which nitrogen source the bacteria will prefer. All bacteria had the lowest enzyme activity in medium-containing ammonium chloride, the only inorganic nitrogen source. Given their organic nature, other nitrogen sources (asparagine, glutamine, and urea) may be utilized not only as nitrogen sources but also as carbon sources. In this context, the lowest biomass (as OD600 of cultures) was observed in ammonium chloride-supplemented medium. Some previous studies reported that while organic nitrogen sources, such as Lasparagine, L-glutamine, L-glutamic acid, and urea, induce L-asparaginase synthesis, in media-containing ammonium compounds the level of enzyme was considerably decreased [40, 41]. How the data acquired here for shake-flask experiments can be translated into large scale industrial one (e.g., bioreactors) remain to be seen. In this context, the majority of studies have been at the shake-flask level and very few has been successfully scaled up. However, some recent studies have reported bioprocess strategies for the scale up of such systems, employing fed-batch techniques [42, 43]. Our results show that even closely related bacteria possess considerably different biochemical characteristics, which forms the basis of their difference in response to same nutritional and physical variables. Therefore, it is imperative that optimal conditions (chemical or physical) for producing a product be determined case by case. The study here showed this is true even for two strains of the same bacterium harboring different plasmids. Collectively, our results show that the most favorable carbon source for L-asparaginase synthesis in E. aerogenes and P. aeruginosa (including their recombinants) is glycerol. Glycerol-containing medium has also supported a better biomass production in these two strains (compared to other carbon sources, up to sevenfold in E. aerogenes and more than tenfold in P. aeruginosa). For E. coli and its recombinant strain, however, lactose clearly is a better source for the enzyme synthesis and biomass production. The data in this study suggest that in a scale-up study using E. aerogenes, the VHb/vgb system be used with a caution, as better oxygen uptake causes a reduction in L-asparaginase synthesis. However, for P. aeruginosa, the opposite seems to be true. The VHb/vgb system is clearly advantageous for production of L-asparaginase using P. aeruginosa.
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