Enzyme and Microbial Technology 35 (2004) 182–189

Vitreoscillahemoglobin expressingEnterobacter aerogenesandPseudomonas aeruginosarespond differently to carbon cataboliteand oxygen repression for production ofl-asparaginase, an enzyme

used in cancer therapy

Hikmet Geckil∗, Salih Gencer, Mirac UckunDepartment of Biology, Inonu University, Malatya 44069, Turkey

Received 16 January 2004; accepted 8 April 2004

Abstract

The production of antileukemic enzymel-asparaginase in two distinctly related bacteria,Enterobacter aerogenes, Pseudomonas aerug-inosa, and in their recombinants expressing theVitresocilla hemoglobin (VHb) has been studied. Both bacteria showed a substantiallydifferent degree of carbon catabolite repression of the enzyme production.E. aerogenesgrown under catabolite repression had more than20-fold lowerl-asparaginase activity than the controls. This figure was only 1.6-fold forP. aeruginosa. In the medium with restrictednutrient content, however, the inhibitory effect of glucose on the enzyme production was less pronounced. The presence of VHb, an efficientoxygen uptake system, had also different effects in both bacteria. Under conditions of no catabolite repression, this protein caused about7-fold lowerl-asparaginase activity inE. aerogenes, but similar or even slightly stimulatory effect inP. aeruginosa. The use of a relativelypoor carbon source, mannitol, caused a lowerl-asparaginase level and no glucose type catabolite repression.© 2004 Elsevier Inc. All rights reserved.

Keywords: Vitreoscillahemoglobin; Bacterial hemoglobin;l-asparaginase; Chemotherapeutic enzymes; Catabolite repression; Oxygen-regulated genes

1. Introduction

Microbial enzymes have been used in various industriesfor many centuries. Advances in the field of molecularbiology of microorganisms have opened up new hori-zons for new enzymes with novel applications. Bacteriall-asparaginases (l-asparagine amidohydrolase EC 3.5.1.1)are among such products.l-asparaginase (also known com-mercially as Oncaspar, Colaspase, Crasnitin, Kidrolase, Er-winase and Elspar) is an enzyme of high therapeutic valuedue its use in certain kinds of cancer therapies, mainly inacute lymphoblastic leukemia[1]. Many gram-negative bac-teria contain twol-asparaginases, a high-affinity periplas-mic enzyme and a low-affinity cytoplasmic enzyme. InEscherichia coliand many other bacteria, synthesis of thecytoplasmic asparaginase I is constitutive, while expressionof the periplasmic asparaginase II is activated during anaer-obiosis. It has been suggested that the latter one probably

∗ Corresponding author. Tel.:+90 422 341 0010x3749;fax: +90 422 341 0037.

E-mail address:hgeckil@inonu.edu.tr (H. Geckil).

have a special function in anaerobic fumarate respiration byproviding aspartate, which is then converted to fumarate.Furthermore, only the type II enzyme has substantial antitu-mor activity [2]. The antileukemic effect of asparaginase ispostulated to result from the rapid and complete depletionof the circulating pool ofl-asparagine, as most cancer cellsare dependent on an exogenous source of this amino acidfor survival. Normal cells, however, are able to synthesizel-asparagine and thus are less affected by its rapid depletionproduced by treatment with this enzyme. The asparaginedeficiency rapidly impairs the protein synthesis and leads toa delayed inhibition in DNA and RNA synthesis and henceto an impairment of cellular function, resulting in cell death[3,4].

Studies regarding the molecular structure[5–8], catal-ysis [9–11], clinical aspects[1,3,4], genetic determinantsinvolved in regulation[12–14] and stabilization to enhancebiological half-life [15–18] of l-asparaginase have beenextensive, while the nutritional and environmental require-ments ofl-asparaginase biosynthesis have not been studiedin detail and only in a limited number of microorganisms.The production ofl-asparaginases has been studied in

0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.enzmictec.2004.04.005

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Serratia marcescens[19,20], Erwinia carotovora [21], E.coli [22,23], Enterobacter aerogenes[24], Pseudomonasaeruginosa [25], and Bacillus subtilis [2] with variouscarbon and nitrogen sources under both aerobic and fer-mentative conditions. The synthesis ofl-asparaginase bygram-negative bacteria is stringently regulated by envi-ronmental and nutritional factors such as carbon, nitrogensources and oxygen. The results sometime were contra-dictory in terms of the effect of carbon source (mainlyglucose)[19,23] and oxygen[24,26] on the production ofthis enzyme. Glucose, a generally preferred carbon sourcefor production of l-asparaginase, was reported to exertefficient carbon catabolite repression on the expression ofthis enzyme inE. aerogenes[24], while in a closely re-lated bacterium,Klebsiella aerogenesit did not inhibitedl-asparaginase production[27]. Regarding the oxygen ef-fect, the amount ofl-asparaginase inE. coli was markedlyincreased upon a shift from aerobic to anaerobic growth,while in E. aerogenesthe presence of dissolved oxygen hasa significant effect in increasingl-asparaginase synthesis[24].

The hemoglobin (VHb) of the bacteriumVitreoscillais the first discovered and probably best characterized ofthe microbial hemoglobins[28]. Its primary function, forwhich experimental support but not proof exists, is mostlikely to bind oxygen at low extracellular concentrationsand deliver it to the terminal respiratory oxidase, thusenhancing respiration under these conditions[29–31]. Fur-thermore, the expression of VHb gene (vgb) is regulatedby oxygen in both the native host,Vitreoscilla, and inE. coli, and is maximally induced under microaerophilicconditions [32–35]. In previous studies, we have shownthat, bacteria engineered with thevgb gene had 2.0- to10-fold higher oxygen uptake rates than thevgb− coun-terparts[30,36]. This study was carried our to determinewhether the presence of VHb in two distinctly relatedGram-negative bacteria,E. aerogenesand P. aeruginosa,could regulate the production ofl-asparaginase, an en-zyme expressed by an oxygen-regulated gene[24]. It isknown that, cultivation conditions (both chemical andphysical) strongly influence the cellular composition andmetabolic performance of microbial cells. Its productionphase (maximum in stationary cells) and location (mainlyin the periplasmic region) makesl-asparaginase produc-tion to be highly affected by the rate of oxygen tension.Limiting oxygen concentration would simultaneously al-leviate the problematic issue of adequate oxygen transferduring scale-up ofl-asparaginase production at high cellconcentrations. Furthermore, to determine the effect ofnutritional factors onl-asparaginase production, bacteriawere grown in rich and semi-synthetic media supplementedwith glucose or mannitol as rich and poor carbon sources,respectively. Thus, the aim of this study was to determinethe nutritional requirements and effect of oxygen concen-tration on the production of L-asaparaginase inP. aerug-inosa, E. aerogenesand in their recombinants utilizing

a highly efficient oxygen uptake system, theVitreoscillahemoglobin.

2. Materials and methods

2.1. Chemicals

l-asparagine, trichloroacetic acid (TCA), Nessler reagentchemicals (HgI2, KI and sodium hydroxide) were purchasedfrom Sigma Chemicals Co. All other chemicals used wereof analytical grade.

2.2. Bacterial strains, growth media andculture conditions

The bacteria used in this study wereE. aerogenes(NRRLB-427) andP. aeruginosa(NRRL B-771), both obtainedfrom the USDA culture collection in Peoria, IL. Thevgb−and vgb+ recombinants ofE. aerogeneswere designatedas “Ea[pUC8]” and “Ea[pUC8:15]”, respectively[34]. Thetransposon-mediatedvgb transferred recombinant strain ofP. aeruginosa, named PaJC was described previously[37].Both P. aeruginosaand PaJC were provided by Dr. Ben-jamin C. Stark (Illinois Institute of Technology, Chicago(USA)). Cells were maintained on LB agar plates at 4◦Cwith transfers at monthly intervals. The growth media usedfor l-asparaginase production were Luria-Bertani (LB)broth [38], and semi-synthetic (MMY) medium[24] bothat pH 7.0. LB contained (L−1) 10 g peptone, 5 g yeast ex-tract and 10 g NaCl. MMY was (L−1) 0.5 g MgSO4·7H2O,0.01 g FeSO4·7H2O, 0.5 g KCl, 1 g K2HPO4, 0.5 g yeastextract and 1.5 gl-asparagine as the nitrogen source. Whereindicated, media were also supplemented with various con-centrations of glucose or mannitol representing rich or poorcarbon sources for growth, respectively. Culture mediumand stock solution of carbohydrates were autoclaved sep-arately at 120◦C for 25 min and appropriate carbohydrateconcentration was made in final volume of cultures. A1/100 inoculum of overnight cultures grown in a specificmedium was made in 20 ml of the same medium in 125 mlErlenmeyer flasks and inocula in flasks were grown for 24 hat 37◦C in a 200-rpm water-bath.

2.3. Membrane permeabilization with potassiumphosphate-hexane phase system forl-asparaginaserelease

Cells cultivated forl-asparaginase production were har-vested by centrifugation (10,000 rpm for 5 min) at roomtemperature, washed once with 0.05 M potassium phosphate(KPi) buffer (pH 8.6), and resuspended toA600 = 5.0 in thesame buffer containing hexane at 1%. The selected hexaneconcentration was based on our preliminary experiments[39]. The suspensions were incubated at room temperaturefor 1 h, briefly vortexing every 10 min. Tube caps were

184 H. Geckil et al. / Enzyme and Microbial Technology 35 (2004) 182–189

left open for 5 min in order to evaporate volatile upperphase prior to analysis ofl-asparaginase activity in thesesuspensions.

2.4. l-asparaginase assay

The enzyme activity was measured by the method of Wris-ton [40], utilizing the Nessler reaction. This method utilizesthe determination of ammonia liberated froml-asparaginein the enzyme reaction by the Nessler reaction. Reactionwas started by adding 0.1 ml permeabilized cell suspensioninto the pre-warmed 0.9 ml 0.01 Ml-asparagine prepared in0.05 M Tris–HCl buffer, pH 8.6 and incubated for 30 min at37◦C. The reaction was stopped by the addition of 0.1 ml1.5 M TCA. The reaction mixture was centrifuged at roomtemperature (10,000 rpm for 5 min) to remove the precipi-tate and the ammonia released in the supernatant was de-termined colorimetrically (A480) by adding 0.25 ml Nesslerreagent into tubes containing 0.5 ml supernatant and 1.75 mlH2O. The content in the tubes was vortexed and incubated atroom temperature for 10 min, and theA480 values were readagainst the blanks that received TCA before the addition ofcell suspension. Onel-asparaginase unit (U) is defined asthe amount of enzyme that liberates 1�mole of ammoniaper min at 37◦C. Specific activity is expressed as units permilligram of protein released. The ammonia concentrationproduced in the reaction was determined based on a stan-dard curve obtained with ammonium sulfate as the standard.The limit of detection of ammonia by this method was about10�M. Total protein in permeabilized cell suspensions weredetermined colorimetrically[41], using bovine serum albu-min as the standard.

3. Results and discussion

The production ofl-asparaginase, an antileukemic en-zyme, in two distinctly related bacteria (E. aerogenesandP. aeruginosa) and in theirVitreoscilla hemoglobin (VHb)expressing recombinants grown under different culture con-ditions was investigated. Bacteria were grown in rich orsemi-synthetic media with different carbon sources to de-termine both the extent of catabolite repression reported forthis enzyme and the effect of an efficient oxygen uptakesystem, the VHb, onl-asparaginase production.

As it is stringently regulated by substrate induction aswell as by carbon catabolite repression and produced un-der oxygen-limited conditions (i.e. in stationary phase cells)[24], l-asparaginase production was studied in cells har-vested following 24 h cultivation. Bacteria were grown indifferent media with or without carbohydrate supplement.Both E. aerogenesandP. aeruginosashowed distinct pro-files in terms ofl-asparaginase levels. The expression ofVHb in these two bacteria had also different affects on en-zyme levels.

3.1. Production ofl-asparaginase by E. aerogenes, P.aeruginosa and their vgb+ recombinants grown in a richmedium in the presence and absence of glucose

The production ofl-asparaginase by bacteria grown inLB or LB with glucose is summarized inFig. 1. In mediumwith no glucose supplement, the level ofl-asparaginasein wild-type E. aerogeneswas 2.8-fold higher than thatin wild-type P. aeruginosa. The expression of VHb inboth bacteria, however, caused an opposite result; thevgb-bearingE. aerogenes(Ea[pUC8:15]) had 2.7-fold lowerl-asparaginase level thanvgb-bearingP. aeruginosastrain(PaJC). This might be due to different response of these two

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Fig. 1. Effect of glucose onl-asparaginase activity inE. aerogenes(Ea),P. aeruginosa(Pa) and theirvgb+ gene bearing recombinants () grownin a nutrient rich medium. Cells were grown in LB (a), LB with 0.1% (b)and LB with 1% (c) glucose. Ea[pUC8:15] and PaJC are thevgb bearingstrains ofE. aerogenesand P. aeruginosa, respectively, while the strainEa[pUC8] bears the parental plasmid with novgb insert. Each data pointis the average of three independent experiments with error bars showingSTDEVs (σn−1).

H. Geckil et al. / Enzyme and Microbial Technology 35 (2004) 182–189 185

bacteria to the presence of VHb.l-asparaginase produc-tion in Ea[pUC8:15] was substantially (7.1-fold) reducedcompared to the wild-type and the strain bearing the sameplasmid but with novgb insert (Ea[pUC8]), both of whichshowed similar level of enzyme activity. InP. aeruginosa,however, VHb did not show such effect, as strain PaJC hadsimilar l-asparaginase level to its parental strain. Thus, thepresence VHb inE. aerogenesand P. aeruginosamightdifferently regulate the production ofl-asparaginase, caus-ing a substantial inhibitory effect in the former and no (oreven a slightly stimulatory) effect in latter bacterium. Whencompared with their parental strains, much higher oxygenuptake rate of Ea[pUC8:15][30,36,42]might contribute tothis difference, asl-asparaginase is an enzyme repressedunder high oxygen[24,43]. Furthermore, both VHb[32,35]andl-asparaginase[44] are predominantly synthesized dur-ing anaerobiosis due to induction by the anaerobic Fnr (fu-marate nitrate reduction) type global regulatory factors andthe cAMP receptor protein (CRP), which might differentlyoperate inE. aerogenesandP. aeruginosa. Fnr in E. aero-genesand its homologous Anr inP. aeruginosaare activeunder oxygen-limiting conditions. It has been reported thatFnr and Anr, however, possess different binding specificitiesfor their respective promoters[45], a result that is in linewith findings here for substantially differentl-asparaginaselevels ofE. aerogenesandP. aeruginosaand with our pre-vious results for different expression levels of VHb in thesetwo bacteria[30,35,36,42]. Furthermore, different degreeof glucose repression of VHb expression in either bacteriagrown under aerobic or microaerobic conditions[30,35]might contribute to difference in theirl-asparaginase levels.

Considered as a general catabolic repressor forl-asparaginase[24,46], glucose was studied for its effect onproduction of this enzyme (Fig. 1). E. aerogenesand itsrecombinants grown in LB medium with 0.1% glucoseshowed a similar pattern of enzyme activity to that of cellsgrown in LB with no glucose. At this concentration, glu-cose showed even a slightly stimulatory effect which wasmore pronounced forP. aeruginosaand itsvgbrecombinantstrain PaJC; both strains had about 30% higher enzymeactivity than their counterparts grown in LB medium. Sim-ilar to enzyme levels in LB medium, the VHb expressingstrain Ea[pUC8:15] had more than 5.0-fold lower enzymelevel than its parental strainE. aerogenesand Ea[pUC8]when grown in 0.1% glucose conaining LB medium. At 1%glucose all three strains ofE. aerogenes, however, showedsimilar substantially reducedl-asparaginase levels, espe-cially for E. aerogenesand Ea[pUC8]. When grown in thismedium,E. aerogenesand Ea[pUC8] showed about 20-folddecrease in enzyme activity compared to the same strainsgrown in LB only or LB with 0.1% glucose. For VHb ex-pressing strain Ea[pUC8:15] this difference was 3.8-fold.Thus, the expression of VHb also causes a substantial re-duction inl-asparaginase production byE. aerogenesundernon-catabolite repression conditions. In contrast, the VHbexpressingP. aeruginosa(PaJC) had similar (or slightly

higher) l-asparaginase activity compared to its parentalstrain and the cataboilte repression ofl-asparaginase pro-duction was less pronounced.l-asaparaginase activity inP.aeruginosaand PaJC grown in LB with 1% glucose was,respectively, about 1.6-fold and 1.4-fold lower than that insame cells grown in LB with 0.1% glucose. This differencewas even smaller when compared withP. aeruginosaandPaJC grown in LB with no glucose supplement. In similarstudies, glucose has proved to be a poor carbon source forthe production ofl-asparaginase byE. aerogenes[24], S.marscences[20] and E. coli [23]. Contrary observationshave also been made on the enhancement ofl-asparaginasesynthesis by glucose inS. marcescens[19].

3.2. Effect of glucose onl-asparaginase production incultures grown in a nutrient restricted medium

Given that LB itself, without glucose supplement, is arich source of carbon and nitrogen both of which are knownfor their regulatory effect onl-asparaginase production, theinhibitory effect of glucose onl-asparaginase productionwas also determined in a semi-synthetic (MMY) medium(Fig. 2). The existing reports are contradictory regardingthe effect of glucose and different nitrogen sources on

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Fig. 2. Effect of glucose onl-asparaginase activity inE. aerogenes(Ea),P. aeruginosa(Pa) and theirvgb+ gene bearing recombinants () grownin a nutrient restricted medium. Cells were grown in the semi-syntheticMMY medium with 0.1% (a) and 1% (b) glucose. Ea[pUC8:15] and PaJCare thevgbbearing strains ofE. aerogenesandP. aeruginosa, respectively,while the strain Ea[pUC8] bears the parental plasmid with novgb insert.Error bars represent STDEVs (σn−1) for n = 3.

186 H. Geckil et al. / Enzyme and Microbial Technology 35 (2004) 182–189

l-asparaginase production in different and sometimes in thesame bacterium[12,14,19,23,24,26,47]. In general, the bac-teria in MMY medium showed lower enzyme activity thantheir counterparts in LB medium.E. aerogenesand itsvgb−strain (Ea[pUC8]) grown in this medium with 0.1% glucosehad more than 5.0-fold lowerl-asparaginase activity thanin LB medium. Thevgb+ strain (Ea[pUC8:15]), however,showed similar levels of enzyme activity both in LB and inMMY media with the same concentration of glucose. With1% glucose however,E. aerogenesand Ea[pUC8] had about3.0-fold higher enzyme activity in MMY medium than in LBmedium. Again, there was no difference inl-asparaginaselevel of Ea[pUC8:15] grown in LB or MMY with 1%glucose. Thus, when its inhibitory effect on the level ofl-asparaginase inE. aerogenesand Ea[pUC8] grown inLB or MMY is compared, glucose in the former mediumcaused a more extensive repression of this enzyme thanin latter medium; about 20-fold versus 1.4-fold. Contrary,the VHb expressing strain (Ea[pUC8:15]) grown in LB orMMY medium in the presence of 1% glucose had about3.0-fold lowerl-asparaginase activity than in the presenceof 0.1% glucose. The enzyme level of Ea[pUC8:15] grownin MMY medium with 0.1% glucose was similar to theother two strains, a result opposite to glucose effect on thecells grown in LB.

The effect of glucose onl-asparaginase activity inP.aerugionosaand its recombinant PaJC grown in MMY wasopposite to that inE. aerogenesand its recombinants. Glu-cose at 1% concentration had a slightly stimulatory effect onl-asparaginase production in the formers, while it caused asubstantial reduction in the latter ones. However, the stim-ulatory effect of glucose was absent in LB medium, inwhich glucose at 1% concentration caused a substantialreduction inl-asparaginase activity fromP. aerugionosaand PaJC. Thus,E. aerogenesand its recombinants responddifferently from P. aeruginosaand PaJC to glucose me-diated catabolite repression ofl-asparaginase productionand the inhibition extent is also depended on medium typeused. A much higher inhibitory effect of glucose in LBmedium than in MMY medium shows that, glucose in arich (LB) or relatively nutrient restricted medium (MMY)might be used for different purposes; degraded for the en-ergetic purposes in the former and converted more effi-ciently as energy and carbon source for the cell material inthe latter. Our results confirm that depending on the typeof bacteria used and in part on medium composition thepresence of VHb can significantly alter carbon metabolism[48] and the carbon catabolite repression ofl-asparaginaseproduction.

3.3. l-asparaginase production in cultures grown with apoor carbon source

l-asparaginase production was also studied in the pres-ence of mannitol (Figs. 3 and 4), a poor carbon and energysource. For both bacteria, mannitol was a poorer catabolite

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Fig. 3. Effect of a poor carbon source, the mannitol, onl-asparaginaseactivity in E. aerogenes(Ea), P. aeruginosa(Pa) and theirvgb+ genebearing recombinants () grown in a nutrient rich medium. Cells weregrown in LB medium supplemented with 0.1% (a) and 1% (b) mannitol.Ea[pUC8:15] and PaJC are thevgb bearing strains ofE. aerogenesandP. aeruginosa, respectively, while the strain Ea[pUC8] bears the parentalplasmid with novgb insert. Error bars represent STDEVs (σn−1) forn = 3.

repressor forl-asparaginase production than glucose. Aswith glucose, the enzyme levels ofE. aerogenesand P.aeruginosawere differently affected with this carbohydrateand compared to glucose, this carbohydrate was also provedto be a poor substrate for the production of the enzyme whenused as the sole source of carbon (Fig. 4). Although at dif-ferent extent, both bacteria grown in LB with 0.1% manni-tol had significantly (4–5-fold) lowerl-asparaginase levelsthan bacteria grown in the same medium with 0.1% glucose.In vgbstrains of both bacteria, however, this difference wasmuch less significant. At 1% concentrations, however, glu-cose and mannitol had opposite results on enzyme produc-tion; E. aerogenes, Ea[pUC8] and Ea[pUC8:15] grown inLB + mannitol had 3.4, 3.6 and 1.8-fold, respectively, higherenzyme levels than the cells grown in LB+ glucose. In con-trast, the enzyme level inP. aeruginosagrown in LB + 1%mannitol was 3.3-fold lower than in LB+ 1% glucose.The PaJC strain, however, had similar enzyme levels in LBmedium with mannitol or glucose. The catabolite repressionof l-asparaginase production by glucose was not observedin bacteria grown with mannitol as they showed similar en-zyme levels in the presence of 1 and 0.1% mannitol. Withthe exception of wild-typeP. aeruginosawhich showedslightly lower enzyme activity in mannitol supplemented

H. Geckil et al. / Enzyme and Microbial Technology 35 (2004) 182–189 187

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Fig. 4. Effect of mannitol onl-asparaginase activity inE. aerogenes(Ea),P. aeruginosa(Pa) and theirvgb+ gene bearing recombinants () grownin a nutrient restricted medium. Cells were grown in MMY mediumwith 0.1% (a) and 1% (b) mannitol. Ea[pUC8:15] and PaJC are thevgbbearing strains ofE. aerogenesandP. aeruginosa, respectively, while thestrain Ea[pUC8] bears the parental plasmid with novgb insert. Error barsrepresent STDEVs (σn−1) for n = 3.

LB medium, in general the level ofl-asparaginase inbacteria grown in mannitol-supplemented MMY mediumwas significantly lower than in mannitol-supplemented LBmedium.E. aerogenesand its two recombinants grown inMMY medium with mannitol (either at 0.1 or 1%) hadmore than 6.0-fold lowerl-asparaginase activity than thecorresponding cells grown in LB with mannitol. The VHb

Table 1pH-values in cultures ofE. aerogenes, P. aeruginosaand recombinant strains grown in various media for 24 h

Madia E. aerogenes Ea[pUC8] Ea[pUC8:15] P. aeruginosa PaJC

LB 8.76 8.75 8.75 8.57 8.59LB + 0.1% glucose 8.68 8.62 8.65 8.60 8.64LB + 1% glucose 4.35 4.39 4.37 8.14 8.10

MMY 8.29 8.28 8.15 8.81 8.77MMY + 0.1% glucose 8.40 8.49 8.26 8.50 8.56MMY + 1% glucose 5.33 5.36 5.38 7.80 7.80

LB + 0.1% mannitol 8.65 8.60 8.58 8.29 8.42LB + 1% mannitol 4.49 4.54 4.57 7.66 7.63

MMY + 0.1% mannitol 8.42 8.46 8.25 8.47 8.50MMY + 1% mannitol 4.49 4.48 4.27 7.44 7.15

Each value is the average of the three independent experiments. For clarity STDEVs (σn−1) are not given but they are mostly less than 1% of averagepH value.

expressingP. aeruginosa(PaJC) had more than 2.0-foldlower enzyme activity. The presence of VHb, however, hada differential effect in these two bacteria grown in mannitolmedium. Contrary to almost 2.0-fold lowerl-asparaginaseactivity of the VHb expressing Ea[pUC8:15] strain thanof the wild-typeE. aerogenesand the strain carrying thecontrol plasmid (Ea[pUC8]), the VHb expressing PaJC hadmore than 3.2- and 3.4-fold higher enzyme activity thanits parental strainP. aeruginosawhen grown in LB with0.1 or 1% mannitol, respectively. To date there is no reportabout the effect of mannitol on the production or cataboliterepression ofl-asparaginase.

3.4. Effect of culture pH on enzyme production

The inhibition ofl-asparaginase in 1% glucose or man-nitol medium, however, might not be solely due to catabolicrepression type inhibition, but the acid production. The pro-duction of acids was measured as the change in culture pHfrom the initial pH of 7.0 for all media used as summarizedin Table 1. Although similar in medium pH when they weregrown in LB and LB with 0.1% glucose,E. aerogenesandP. aeruginosashowed distinct pH values when they weregrown in the presence of 1% glucose. The average mediumpH in LB or LB with 0.1% glucose was 8.70± 0.06 forE.aerogenesand its recombinats and 8.60± 0.03 forP. aerug-inosa and its PaJC strain, while these values in LB with1% glucose were 4.37± 0.02 for the former and 8.12±0.03 for the latter. This explains the extensive reduction ofl-asparaginase level inE. aerogenescompared toP. aerug-inosa when they were grown in the presence of 1% glu-cose: 20-fold versus about 1.5-fold. Supplementation of LBmedium with 1% mannitol caused a similar pH drop to thatof glucose. The average medium pH for all threeE. aero-genesstrains grown in LB with 0.1% mannitol was 8.61±0.04, while in LB with 1% mannitol this value was 4.53± 0.04. Both strains ofP. aeruginosahad pH values 8.36± 0.09 and 7.65± 0.02, when grown in LB with 0.1 and1% mannitol, respectively. The pH values in semi-synthetic

188 H. Geckil et al. / Enzyme and Microbial Technology 35 (2004) 182–189

MMY medium supplemented with 1% glucose or manni-tol showed similar drop to glucose suplemented medium.These results are in accordance with our previous results[43] and studies of others[24,47] showing that the optimalenzyme activity was detected at neutral and alkaline pH val-ues 6.5–9.5 and the production ofl-asparaginase is downregulated as the pH of the medium decreases and almost noactivity is detected at acidic pH values<6.0. Thus, it hasbeen suggested that besides acting as a repressor when usedas a carbon source, glucose may also reduce the enzymeactivity through lowering the medium pH.

In conclusion, this study reports the findings that carboncatabolite and oxygen repression ofl-asparaginase produc-tion is distinctly regulated in two related Gram-negativebacteria and also demonstrates the novel characteristics ofVHb for the regulation of activities of genes with oxy-gen responsive nature. Although being expressed underoxygen-limited conditions,l-asparaginase is not producedin cultures under complete anoxia. Each bacterium, studiedup to date, required a critical level of oxygen for produc-tion of this enzyme. Here we showed that the presence ofa highly efficient oxygen uptake system, VHb, affects dif-ferently the production ofl-asparaginase in two distinctlyrelated bacteria,E. aerogenes(an anaerobic fermentativebacterium) andP. aeruginosa(a strict aerobe). Previousstudies showed that the oxygen-uptake rates ofE. aero-genesand P. aeruginosaexpressing VHb were quite dis-tinct [30,36,35,42]and bacterial cells (E. coli) were undera more oxidized state in the presence of VHb, as forma-tion of by-products decreases[48]. Furthermore, here weshowed that bothE. aerogenesand P. aeruginosaresponddifferently to carbon catabolite repression, which is alsoindirectly regulated by the availability of oxygen. Differen-tial regulation ofl-asparaginase expression inE. aerogenesand P. aeruginosaunder catabolite repression might be aresult of different inductive control and regulation of carbonflux as they use alternative glycolytic pathways for glucosemetabolism. The former bacterium converts glucose mostlythrough the Embden-Meyerhof-Parnas (glycolysis) pathwayto several acids (formate, acetate and lactate) and fermen-tative metabolites (such as acetoin and butanediol), whilein the latter, the Entner-Doudoroff pathway dominates andglucose is mostly dissimilated to gluconate type neutralproducts[49]. This explains why, the medium pH becomesmore acidic inE. aerogenescultures compared toP. aerug-inosa when they were grown in carbohydrate containingmedia. In this respect, it is suggested that biochemical char-acteristics of different asparaginase-producing microorgan-isms are very variable so that the data obtained in studiesfor one microorganism may be inapplicable for another andthat even in one species regulation ofl-asparaginase syn-thesis by different mechanisms is possible. Furthermore, thesubstantial difference in enzyme activity from both bacteriaunder conditions studied here might not be a sole functionof different level of expression, but the difference in kineticconstants of the enzyme. To determine both the expression

level and the reaction rate constants of the enzyme fromthese sources, the enzyme has to be purified which is ournext goal.

Acknowledgments

This study was partially supported by a grant (TBAG2267(102T197)) to H.Geckil from The Scientific and TechnicalResearch Council of Turkey (TUBITAK). Authors are grate-ful to Dr. Benjamin C. Stark (at Illinois Institute of Technol-ogy, Chicago) for providing bacterial strainsP. aeruginosaand PaJC.

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