Process Biochemistry 40 (2005) 573–579

Membrane permeabilization of gram-negative bacteria with apotassium phosphate/hexane aqueous phase system for the

release ofl-asparaginase: an enzyme used in cancer therapy

Hikmet Geckila,∗, Burhan Atesb, Salih Gencera, Mirac Uckuna, Ismet Yilmazb

a Department of Biology, Inonu University, Malatya 44069, Turkeyb Department of Chemistry, Inonu University, Malatya 44069, Turkey

Received 29 September 2003; received in revised form 8 December 2003; accepted 17 January 2004

Abstract

A fast, efficient and reproducible recovery procedure for periplasmicl-asparaginase from two distinctly related gram-negative bacteria,Enterobacter aerogenesandPseudomonas aeruginosa, is presented. As the method uses inexpensive organic solvent hexane and an aqueoussalt solution, it is also highly cost-effective in comparison with the currently available techniques used for the release of this enzyme. Ashexane is a highly water immiscible organic solvent, it can be removed easily from the top of the aqueous phase by a simple evaporation.Also, various organic solvents and other membrane partitioning compounds were compared for their efficiency onl-asparaginase/proteinrelease. The degree to which the enzyme was released was different for two bacteria, suggesting that they possess different permeabilitycharacteristics. The most efficient enzyme release from both bacteria was determined to be in 50 mM potassium phosphate with 1% hexane.Enzyme recoveries up to three-fold with respect to sonication have been achieved with this system.© 2004 Elsevier Ltd. All rights reserved.

Keywords:l-Asparaginase; Periplasmic enzymes; Membrane permeabilization; Organic solvents; Hexane

1. Introduction

Bacteriall-asparaginases (l-asparagine amidohydrolaseEC 3.5.1.1) are enzymes of high therapeutic value due theiruse in certain kinds of cancer therapies, mainly in acutelymphoblastic leukemia (reviewed in[1]). These enzymes insome gram-negative bacteria accumulate mainly in periplas-mic space[2–4]. An ideal method for the release of thisenzyme would be rapid, inexpensive, gentle, and compatiblewith downstream steps of its purification. In most studiesregarding this enzyme, however, cells were broken by me-chanical methods[5–9], such as sonification, which sufferfrom several drawbacks. These methods are labor-intensive,time-consuming and require expensive apparatus, which canbe used only at laboratory scale. Furthermore, because cellsare broken completely, all intracellular materials are re-leased into the surrounding medium contributing to impurity

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

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

of the enzyme and making the separation ofl-asparaginasea highly cumbersome task from a complex mixture ofproteins, nucleic acids, and cell wall fragments. Releasednucleic acids may increase the viscosity of the solution andmay complicate subsequent processing steps and especiallychromatography, in case of laboratory experiments. Theenzymatic permeabilization[10] also suffers from similardrawbacks, such as its high cost in large-scale processes andthe necessity of removing the lytic enzyme from the product.

The cell envelope of bacteria basically consists of cell walland one or two lipid membranes. For the bacteria, two majorgroups can be discerned on the basis of their cell envelopecomposition: the gram-positive and gram-negative bacteria.Gram-positive bacteria have only one membrane, the cyto-plasmic membrane, which is surrounded by a thick, rigidcell wall. Gram-negative bacteria have, in addition to thecytoplasmic cell membrane, an outer membrane that con-sists of phospholipids and lipopolysaccharides. Between theouter membrane and the cytoplasmic membrane is a thinpeptidoglycan layer, forming the support for the cell enve-lope[11]. The periplasmic space in gram-nagative bacteria is

0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.procbio.2004.01.033

574 H. Geckil et al. / Process Biochemistry 40 (2005) 573–579

in between the plasma membrane and the outer membrane,mainly between the plasma membrane and peptidoglycanlayer. This space contains enzymes and proteins functioningin detection of the environment, acquisition of nutrients intothe cell, enzymatic hydrolysis and detoxification of harmfulcompounds[12]. In order to release the enzymes and pro-teins in the periplasmic space, the permeability barrier (outermembrane) must be weakened. Like other gram-negativebacteria, the cell envelope ofPseudomonas aeruginosaandEnterobacter aerogenesis in a double membrane structureand the antileukemicl-asparaginase has been shown to belocated mainly in the periplasmic space[6,13].

The use of organic chemicals, such as solvents (hexane,toluene, xylene, benzene, ether), non-ionic (Triton) andionic (SDS) detergents and chelating agents (e.g. ethylenediamine tetraacetic acid, EDTA) for the release of periplas-mic enzymes might be advantageous as they are cost effec-tive and cause selective permeabilization of the outer cellwall barriers allowing large-scale preparation of periplas-mic enzymes in a relatively pure form[14,15]. In thisstudy, we describe a fast, inexpensive and highly efficientmethod forl-asparaginase release from two gram-negativebacteria,E. aerogenesandP. aeruginosa, known to accumu-late antileukemicl-asparaginase in the periplasmic space.In this method we utilize a potassium phosphate/hexaneaqueous phase system for the selective release of thisenzyme.

2. Materials and methods

2.1. Chemicals

Anhydrous l-asparagine, trichloroacetic acid (TCA),Nessler reagent chemicals (HgI2, KI and sodium hy-droxide), hexane, xylene, Triton X-100, ethylene diaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS)were purchased from Sigma Chemicals Co. Toluene andbenzene were from Reidel-de Haën GmbH, diethyletherwas from Merck Chemicals. All other chemicals used wereof analytical grade.

2.2. Strains and culture conditions

The bacteria used in this study wereE. aerogenes(NRRLB-427) andP. aeruginosa(USDA B771), both obtainedfrom the USDA culture collection in Peoria, IL. Thegrowth medium used forl-asparaginase production wasLuria-Bertani (LB) containing (l−1) 10 g peptone, 5 g yeastextract and 10 g NaCl at pH 7.0[16]. Cells were main-tained on LB agar plates at 4◦C with transfers at monthlyintervals. A 1/100 inoculum of overnight cultures grownin LB was made in 20 ml LB in 125 ml Erlenmeyer flasks.Inocula in flasks were grown for 24 h at 37◦C in a 200 rpmwater-bath. The enzyme release, defined as the ratio ofunits of l-asparaginase activity to that of protein content

in milligram, was measured in cell-free supernatants ofpermeabilized and ultrasonified cells. The enzyme activityof whole-cells, a common application for measuring theenzyme activity in cell wall loosened intact cells, was notmeasured.

2.3. Permeabilization with solvents and other membranedestabilizing agents

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 inthe same buffer containing organic solvents, ionic (SDS),non-ionic (Triton X-100) detergents or a chelating agent(EDTA). The selected concentrations were based on our pre-liminary experiments. SDS, Triton X-100 and EDTA wereused at concentrations 0.01–0.001% (w/v), 1.0–0.01% (v/v)and 5 mM, respectively. All solvents were used at 1% (v/v)concentration, which was the optimal concentration of hex-ane forl-asparaginase release. The suspensions were incu-bated at room temperature for 1 h, briefly vortexing for every10 min. Tube caps were left open for 5 min in order to evap-orate volatile upper phase prior to analysis ofl-asparaginaseactivity in the cell-free aqueous phase. Cell suspensions werealso made in distilled water (dH2O) or 0.05 M KPi with nosolvents or detergents as controls. In both types of controls,cells were first suspended in KPi for 30 min then they wereharvested and resuspended either in dH2O or KPi for addi-tional 30 min. The dH2O controls were included to deter-mine whether the osmotic pressure caused by water influxhas any effect on enzyme release.

2.4. Cell disruption by ultrasonication

The ultrasonic extract preparation used for the release ofl-asparaginase was described previously[17]. Sonic cell ex-tracts were prepared from the same cells used for perme-abilization. Cells were washed once with 0.05 M Tris–HClbuffer (pH 8.6) and resuspended in the same buffer toA600 =5.0. The suspended cells were cooled in an ice bath andbroken with six 20 s ultrasonic pulses at 40 s intervals witha microtip ultrasonifier (Model 450 Sonifier, Branson Ul-trasonics Corporation, Danbury, CT, USA). The extract wascentrifuged (15,000 rpm, 10 min, 4◦C) and cell-free super-natant was subjected to enzyme assay immediately.

2.5. l-Asparaginase assay

l-Asparaginase catalyzes the hydrolysis ofl-asparagineto l-aspartic acid and ammonia. The enzyme activity wasmeasured by the method of Wriston[18], utilizing the Ness-lerization reaction. This method utilizes the determinationof ammonia liberated froml-asparagine in the enzyme reac-tion by the Nessler reaction. Reaction was started by adding0.1 ml permeabilized cell suspension or 0.1 ml supernatant

H. Geckil et al. / Process Biochemistry 40 (2005) 573–579 575

of cell suspensions from aqueous phase into the pre-warmed0.9 ml 0.01 M l-asparagine prepared in 0.05 M Tris–HClbuffer, pH 8.6 and incubated for 30 min at 37◦C. The re-action was stopped by the addition of 0.1 ml 1.5 M TCA.The reaction mixture was centrifuged at room temperature(10,000 rpm for 5 min) to remove the precipitate and the am-monia released in the supernatant was determined colorimet-rically (A480) by adding 0.25 ml Nessler reagent into tubescontaining 0.5 ml supernatant and 1.75 ml dH2O. The con-tent in the tubes was vortexed and incubated at room tem-perature for 10 min, and theA480 values were read againstthe blanks that received TCA before extract addition. Onel-asparaginase unit (U) is defined as the amount of enzymethat liberates 1�mol of ammonia per min at 37◦C. Specificactivity is expressed as units per milligram of protein re-leased. The ammonia concentration produced in the reactionwas determined on the basis of a standard curve obtainedwith ammonium sulfate as the standard. The limit of detec-tion of ammonia by this method was about 10�M. Totalprotein was determined colorimetrically[19], using bovineserum albumin as the standard.

3. Results

3.1. l-Asparaginase activity in permeabilizedcells versus sonic extracts

A comparison of KPi/hexane aqueous phase system andmechanical cell disruption onl-asparaginase release (de-termined as specific activity) fromP. aeruginosaand E.aerogenesis given in Fig. 1a. Cells were suspended in50 mM KPi/hexane or 50 mM Tris–HCl buffer solutions(both at pH 8.6) toA600 = 5.0 for permeabilization or sonicextract preparation, respectively.P. aeruginosacells sub-jected to ultrasonication for 2 min released 11.06 mg ml−1

of total proteins and 125 units ml−1 of enzyme activitycorresponding to a specific activity of 11.3 units mg−1 pro-tein. These values forE. aerogeneswere 8.15 mg ml−1, 350units ml−1 and 42.9 units mg−1. In KPi/hexane, permeabi-lized cells, however, these values were 1.67 mg ml−1, 56.8and 34.3 units mg−1 for P. aeruginosaand 2.72 mg ml−1,263 units ml−1 and 96.7 units mg−1 for E. aerogenes. Twobacteria expressedl-asparaginase at different levels andshowed different permeability characteristics under thesame conditions. The specific activity ofl-asparaginase insonic extracts ofE. aerogeneswas about four-fold higherthan that ofP. aeruginosa. A similar difference was alsodetermined in the level ofl-asparaginase in cell-free super-natants of permeabilized cell suspensions. On per cell basisthis difference was even more pronounced in the favor ofE. aerogenes; about 10-fold and 7.5-fold higher in sonicextracts and permeabilized cells, respectively than that ofP. aeruginosa. OneA600 unite value of cultures used herecorresponded to 4× 109 cells/ml for P. aeruginosaand1.5 × 109 cells/ml for E. aerogenes. Total cell protein in

(a)

0

20

40

60

80

100

120

P.aeruginosa E. aerogenes

L-as

para

gina

se (

U x

mg

-1)

x 10

00

(b)

0

2

4

6

8

10

12

14

P.aeruginosa E. aerogenes

Tot

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ell p

rote

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Fig. 1. l-Asparaginase (a) and total protein release (b) fromP. aerugi-nosaand E. aerogeneswith mechanical disruption (�) and KPi/hexaneaqueous phase system (�). Extracts were prepared by sonication, whilepermeabilization was carried out at room temeperature on cell suspen-sions made in 50 mM KPi, containing hexane to 1%. In both cases, cellswere harvested and suspended in respective buffers to equalA600 = 5.0.Each value is the average of three independent experiments with errorbars indicating STDEVs (σn−1).

permeabilized cell suspensions and sonic extracts is givenin Fig. 1b. Total protein in sonic cell extracts ofP. aerugi-nosawas 6.6-fold higher than total protein in supernatantsof permeabilized cells, while inE. aerogenesthis figurewas about three-fold. All permeabilization experimentswere performed at room temperature (25–27◦C), as it wasdetermined thatl-asparaginase from both bacteria is ahighly thermostable enzyme and also stable in KPi at roomtemperature after 24 h. The enzyme even showed a gradualincrease in activity with up to 60◦C and retained more than90% of its activity in supernatants left at room temperaturefor 24 h (data not shown).

3.2. Effect of salt concentration onl-asparaginaserelease

Effect of salt concentration in aqueous pool of KPi/1%hexane system on recovery ofl-asparaginase fromP. aerug-inosa and E. aerogenesis shown in Fig. 2. The releaseof enzyme activity was inversely proportional with bufferstrength. With increasing salt concentration the enzymeactivity recovered decreased gradually to the lowest level12.3 mU mg−1 at 0.75 M Kpi concentration forP. aerugi-nosaand to 34.2 mU mg−1 at 0.5 M KPi concentration forE. aerogenes. Furthermore, at concentrations≥0.50 M, KPi

576 H. Geckil et al. / Process Biochemistry 40 (2005) 573–579

Fig. 2. Effect of salt concentration on the recovery ofl-asparaginasefrom E. aerogenesand P. aeroginosa. Cells were permeabilized withKPi/hexane aqueous phase system in different ionic strength and 1%hexane. Each data point is the average of three independent experimentswith error bars indicating STDEVs (σn−1).

heavily interfered with absorbance reading after Nessleriza-tion reaction. The highest enzyme activity was determinedin the phase with 50 mM KPi for both bacteria; 37.1 and91.6 mU mg−1 for P. aeruginosaandE. aerogenes, respec-tively. There was almost no enzyme activity in supernatantsof KPi only cells.

3.3. Effect of hexane concentration on membranepermeabilization forl-asparaginase release

The effect of hexane at different concentrations and itscombination with an anionic detergent (SDS) on the releaseof l-asparaginase activity is given inFig. 3. In both bacte-ria higher hexane concentration caused a higher total cellprotein release and unit activity, but slightly lower specificactivity. Total cell protein was 1.53 and 2.68 mg ml−1 for P.aeruginosaandE. aerogenes, with 36.6 and 84.3 mU mg−1

l-asparaginase, respectively in permeabilized cell suspen-sions atA600 = 5.0 prepared in 50 mM Kpi, pH 8.6 with in1% hexane. In the presence of 4% hexane, these values were2.08 and 3.51 mg ml−1 for P. aeruginosaandE. aerogenes,corresponding to 32.5 and 79.3 mU mg−1 l-asparaginase,respectively. In KPi/1% hexane/0.001% SDS system the

Fig. 4. Effect of various membrane destabilizers on the release ofl-asparaginase activity fromP. aeruginosaandE. aerogeneswith Kpi with 1% solvents.Cells were grown in LB for 24 h and resuspended in 50 mM KPi toA600 = 5.0 and solvents were added to 1%.∗Triton (1%, v/v) heavily interferedwith absorbance (A480) reading after Nesslerization. Each data point is the average of three experiments with error bars indicating STDEVs (σn−1).

Fig. 3. l-Asparaginase release fromP. aeruginosaandE. aerogenesper-meabilized with various concentrations of hexane and SDS. All prepara-tions were made in 50 mM KPi buffer, pH 8.6. Each value is the aver-age of three independent experiments with error bars indicating STDEVs(σn−1).

total protein released fromP. aeruginosaandE. aerogeneswas 1.66 and 3.07 mg ml−1 with l-asparaginase activity25.5 and 53.4 mU mg−1, respectively. In the same systembut with 0.01% SDS the enzyme activity was almost in-hibited completely, although there was slightly higher totalprotein release; 1.78 and 3.26 mg ml−1 for P. aeruginosaandE. aerogenes, respectively.

3.4. Comparison of various membrane destabilizingcompounds onl-asparaginase release

The effect of aliphatic and aromatic organic solvents,non-ionic (Triton X-100) and ionic (SDS) detergents and achelating agent (EDTA) on the membrane permeabilizationfor the release ofl-asparaginase fromP. aeruginosaandE.aerogenesis given inFig. 4. Organic solvents and aqueoussolution of Triton X-100 were prepared at 1% concentra-tion of these compounds in 50 mM KPi buffer, pH 8.6. Theaqueous solutions of SDS and EDTA were also prepared inthe same buffer at concentrations indicated. Hexane was themost effective agent for the enzyme release in both bacte-ria. The specific activity of the enzyme released was 43 and92 mU mg−1 for P. aeruginosaand E. aerogenes, respec-tively. The unite activity of enzyme in toluene treated cells

H. Geckil et al. / Process Biochemistry 40 (2005) 573–579 577

Table 1Some physical properties of the solvents∗ and their effect on the release ofl-asparaginase activity fromP. aerugionosaand E. aerogenes

logP Density (g ml−1 at 25◦C) MSW (mmol l−1 at 25◦C) Molecular toxicity l-Asparaginase activity (%)

P. aerugionosa E. aerogenes

Hexane 3.5 0.659 0.14 N 226 242Toluene 2.6 0.865 5.60 Y 179 171Xylene 3.1 0.864 2.02 Y 121 108Benzene 2.0 0.879 22.9 Y 110 124Diethylether 0.9 0.715 815 Y 116 121dH2O – 1.000 – N 100 100

Asterisk (*): Solvents were used at 1% concentration in 50 mM potassium phosphate buffer, pH 8.6. Physical characteristics of solvents taken from[15,21]. The logP values are derived from the partition coefficient of the solvent between 1-octanol and water. MSW: maximum solubility in water; Y:yes; N: molecular toxicity not expected.

of both bacteria was comparatively higher than any solventtested. This solvent, however, caused more protein release(3.02 and 4.65 mg ml−1 versus 1.20 and 2.38 mg ml−1 ofhexane treated cells ofP. aeruginosaand E. aerogenes,respectively), thus reducing the specific activity of therecovered enzyme to 34 and 65 mU mg−1 for P. aerugi-nosaandE. aerogenes, respectively. Structurally similar totoluene but slightly more hydrophobic xylene was a poorerenzyme releaser. Benzene and diethyl ether showed similarl-asparaginase release in both bacteria. The recovery ofthe enzyme was the least with EDTA forE. aerogenesandwith benzene and ether forP. aeruginosa. Triton X-100reacted with Nessler reagents causing a heavy turbiditythat interfered with absorbance (A480) reading. Even 0.01%Triton X-100 heavily interfered with absorbance readings.SDS and EDTA alone also caused enzyme release, but toa much lower rate compared to hexane treated cells. Acomparison of physical properties of solvents used hereand their effect onl-asparaginase release is summarized inTable 1. Hexane was the most effective solvent on selec-tive release ofl-asparginase. In both bacteria, KPi/hexanesystem caused more than 200% increase in specific activityof the enzyme relative to controls that were first suspendedin KPi then in dH20. These controls also showed enzymeactivity and protein release. The controls, suspended in KPionly, however, did not show any activity and almost noprotein was determined in their supernatants. Toluene wasalso highly effective on release of the enzyme, but it causedsubstantially higher total cell protein release than hexane.

4. Discussion

Before discussing the results and their implications itis necessary to comment on the nature of the system usedhere, which allows simple examination and highly effi-cient release ofl-asparaginase from two distinctly relatedgram-negative bacteria, namelyP. aeruginosaandE. aero-genes. The system utilized here is based on the membranepermeabilization of cells by an aqueous salt/organic solventphase system; KPi/hexane. The aqueous phase was analyzed

for enzyme activity and total protein content after separa-tion of organic phase, which required a simple evaporationof highly water immiscible hexane. Membrane permeabi-lization and sonic extract preparations were carried out onbacterial cells grown to late stationary phase (at 24 h incu-bation), sincel-asparaginase activity in bothP. aeruginosaandE. aerogeneshas been determined as a function of cellage, with maximum activity occurring in cells harvested ator after 24 h of incubation[13,17,20]. There was no need tocarry out the permeabilization process forl-asparaginaserelease on ice or in cold as we have determined that theenzyme from these two mesophilic bacteria is stable atroom temperature and shows a gradual increase in activitywhen assayed at temperatures up to 60◦C, a result that iswell in accordance with previously published studies[3,20].The enzyme from both bacteria showed more than 50%of its activity when assayed at 80◦C compared to controlat 37◦C (100% activity). Although the enzyme specificactivity was highest at temperatures 50–60◦C we have as-sayed it at 37◦C, a common application for enzymes fromother mesophilic bacteria[3,20]. Also, high temperatureswould not be applicable for the enzyme assay in immobi-lized whole-cell systems, where intact cells are used andmembrane immobilized-enzyme systems. Furthermore, thespecific activity increase is due to better substrate acces-sibility (by increasing enzyme-substrate collision rate) athigh temperatures compared to ambient temperatures.

The membrane permeabilization system (50 mM KPi/1%hexane) used here was highly effective on specific release ofl-asaparaginase from both bacteria.l-Asparaginase activityin supernatant of permeabilized cell suspensions was sub-stantially higher than in cell-free sonic extracts for both bac-teria, while total cell protein released was considerably lowin the formers.l-Asparaginase activity in supernatants ofpermeabilized cell suspensions, however, was three-fold and2.3-fold higher than in sonic extracts forP. aeruginosaandE. aerogenes, respectively. Among solvents and other mem-brane permeabilizers (e.g., Triton X-100, EDTA and SDS)tested, the KPi/hexane system gave the highest enzyme re-covery. This might be due to its ability to permeabilize thecells better. In this regard, it has been suggested that hexane,

578 H. Geckil et al. / Process Biochemistry 40 (2005) 573–579

because of its small size, penetrates the surfactant layers eas-ily compared to larger aliphatic hydrocarbons in the sameclass, such as decane and dodecane and causes a disorgani-zation of the outer membrane sufficient enough for secretionof penicillin acylase along with other few periplasmic pro-teins out of the cell[14,15]. The salt concentration in aque-ous phase was a determinative factor for enzyme release byKPi/hexane system. As the concentration of KPi increasedthe enzyme recovery was decreased. Furthermore, at concen-trations≥0.5 M, KPi heavily interfered with the absorbance(A480) reading as it caused a heavy turbidity by reactingwith Nessler reaction reagents. In this context, Triton X-100also strongly interfered with such reading even at concentra-tion 0.01%. The supernatants of cells suspended in KPi thenin dH2O also showed some enzyme activity comparable tothat of xylene, benzene and ether. No activity, however, wasdetermined in supernatants of cells suspended in KPi only,suggesting that water influx caused some enzyme releasethrough membrane by the osmotic pressure. Since, the KPibuffer used for enzyme assay was also 50 mM, the lowestconcentration of same buffer for permeabilization was cho-sen as 50 mM; as further lowering the salt concentration orthe use of dH2O instead of KPi buffer with hexane would bedetrimental for long time stability of enzyme in the solution.Furthermore, our preliminary studies showed that, in bothbacteria the release of enzyme activity in dH2O/hexane sys-tem was less than 20% that of in KPi/hexane system (datanot shown), a result contrary to good efficiency of enzymerelease in cell suspensions made in dH2O only. A possibleexplanation is that, the cell envelope of these two bacte-ria is resistant to hexane when water is used, but with salt(KPi) some interactions in the lipopolysaccharide layer areweakened, making the solvent diffusion into the membranelayer possible and thus loosening cell envelope for enzymerelease.

Some physical characteristics of solvents and their effecton the release ofl-asparaginase activity fromP. aeruginosaandE. aerogenescells are summarized inTable 1. The hy-drophobicity characteristics of solvents are commonly in-dicated as logP, whereP is the partition coefficient of thesolvent between 1-octanol and water. In general, solventswith low logP values are regarded as poorer enzyme re-leasers than solvents with high logP values (P > 2.0)[15]. In this study, however, we found no direct relationbetween the logP values of the solvents and the extent ofenzyme activity release. Although, hexane with the highestlogP value (logP = 3.5) was the most efficient solvent forl-asparaginase release, toluene (logP = 2.6) was a betterenzyme releaser than structurally similar xylene (logP =3.1). Furthermore, although they possessed highly differentsolubility characteristics, benzene and diethyl ether showedsimilar effect on enzyme release, a result in accordance withthat of[15] showing that the more polar solvents drasticallyreduces penicillin acylase activity. Thus, high enzyme re-lease by hexane might not be only a function of its highlogP value, but also its size. Among the solvents with logP

values >2.0, hexane is the smallest molecule which makesit penetrate more efficiently into the outer cell membrane,resulting disorganization of the outer membrane sufficientenough to cause the secretion ofl-asparaginase. In a relatedstudy[14], a visual observation showed that, cells were moreuniformly dispersed in the hexane system as compared tohigher hydrocarbons and hexane was a better penicillin acy-lase releaser than other solvents tested.

In conclusion, besides being a time saving, no appara-tus requiring and highly effective method for the release ofperiplasmicl-asparaginase, this system could be utilized aspromising first step for purification of this enzyme. Due tolow affinity for water, hexane can be removed from aqueousphase by a simple evaporation causing almost no contam-ination or denaturation, which are common problems withmechanical cell disruption or other polar solvents. Further-more, contrary to mostly toxic effect of surfactants and othermembrane destabilizers on cells, hexane is non-toxic and canbe used as suitable medium for microbial processes, such asin whole-cell catalysis.

Acknowledgements

This study was partially supported by a grant (TBAG2267-(102T197)) to H. Geckil from The Scientific and TechnicalResearch Council of Turkey (TUBITAK).

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