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Journal of Bioscience and BioengineeringVOL. xx No. xx, xxx–xxx, 2009

JBIOSC-00221; No. of pages: 6; 4C:

www.elsevier.com/locate/jbiosc

ARTICLE IN PRESS

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Oxidative/heat stress enhanced production of chitosanase from Streptomyces griseuscells through its interaction with liposome

Kien Xuan Ngo, Hiroshi Umakoshi, Haruyuki Ishii, Huong Thi Bui,Toshinori Shimanouchi, and Ryoichi Kuboi⁎

⁎ CorrespondE-mail add

1389-1723/$ -doi:10.1016/j.jb

Please citeits interact

O

Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka, 560-8531, Japan

Received 23 April 2009; accepted 15 June 2009

R TEDPThe effective production and secretion of chitosanase from S. griseus cells, in the presence of hydrogen peroxide, were

studied by the treatment of the cells with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes underheat condition. The variation of the conformation and activity of the chitosanase caused by the interaction of liposomes withtarget chitosanase under stress condition was systematically investigated by using circular dichroism (CD) spectra anddielectric dispersion analysis (DDA). The effect of the oxidative stress (hydrogen peroxide) on the lipid and protein peroxide ofcell membrane of S. griseus pretreated with and without liposomes under heat stress at 41 °C was further carried out. Thepossible utilization of membrane–membrane interaction between liposomes and cell membrane induced by the heattreatment was further investigated to enhance the production of chitosanase from S. griseus under oxidative stress condition.

© 2009, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Membrane stress biotechnology; Chitosanase; Liposome; Oxidative stress]

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RRECChitosanase produced by Streptomyces griseus is secreting and

non-membrane protein with 33.8 kDa in molecular weight (M.W).Chitosanase can be classified as glycosyl hydrolase that catalyzes thedegradation of chitosan. The three-dimensional structure of chitosa-nase from S. griseus species has previously been analyzed (1).Chitosanase can be classified into three groups according to theirspecificity for the hydrolytic reaction of β-glucosidic linkage in par-tially N-acetylated chitosan molecules (2, 3).

“Stress” can often reveal the potential roles of bacterial cells. Heat isknown to be sensitive and useful stimuli. A series of previous reportson heat shock protein (HSP), which is known as the molecularchaperone in bacteria, has described the important roles of HSPs forprotein refolding (4, 5) and translocation (6, 7). Oxidative stress,induced by the reactive oxygen species (ROS), has beenwidely studied.It has been reported that the hydrogen peroxide (H2O2) could playimportant roles as second messenger in the initiation and amplifica-tion of signaling at the antigen receptor (8). A several excellent reviewsabout the functions of hydrogen peroxide as secondary messengerhave already appeared (8–13). It has been also reported that theproduction rates of superoxide radical (.O2

−) and hydrogen peroxideare linearly related to the number of the active respiration chains thatreaches to the maximal values during the exponential growth andsignificantly decreased at the stationary phase (14).

Much effort to overcome the above restrictions has recently beenconducted by several researchers. A stress-mediated bioprocess asanother designed strategy enhances the productivity of the biological

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ing author. Tel./fax: +81 6 6850 6285.ress: MSB@cheng.es.osaka-u.ac.jp (R. Kuboi).

see front matter © 2009, The Society for Biotechnology, Japan. All rights resiosc.2009.06.010

this article as: Ngo, K.X., et al., Oxidative/heat stress enhanceion with liposome, J. Biosci. Bioeng. (2009), doi:10.1016/j.jbi

target (15, 16). The stress-mediated bioprocess utilizing the stressresponse function of bacterial cells is a preferable strategy to enhancethe target production. It has been reported that the heat stress couldenhance the periplasmic recovery of cytoplasmic β-galactosidasethrough the translocation of this enzyme across the inner membraneof Escherichia coli cells (17–19). In addition, under stress condition, thebiological membrane and liposome induce so many sophisticatedproperties. The addition of the liposome into the cell culture has beenreported to enhance the production and release of the chitosanasefrom S. griseus under heat stress condition at the specific temperature(20–22). On the basis of the recently reported concept of membranestress biotechnology, a conventional bioprocess is further improved.

In this study, the effective production and secretion of chitosanasefrom S. griseus cells were studied by the treatment of these cells withthe liposome prepared by 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-phatidylcholine (POPC) under the heat and/or oxidative stresses. Theinteraction of the POPC liposomes with target chitosanase and thevariation of the conformation and activity of chitosanase were sys-tematically investigated by using circular dichroism (CD) spectra anddielectric dispersion analysis (DDA). The possible utilization ofmembrane–membrane interaction between liposomes and cell mem-brane induced by the heat treatment was further characterized underoxidative stress condition to enhance the production of chitosanasefrom S. griseus.

EXPERIMENTAL

Materials The 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)was purchased from Avanti Polar Lipid (Alabaster City, AL, USA). Other lipids to preparethe cell-mimicing liposome, such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic

erved.

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FIG. 1. Production of chitosanase by S. griseus cells treated with heat stress andliposomes under the oxidative stress condition. The cultivation was carried out at 37 °Cand the heat stress at 41 °C for 20minwas applied to cell broth twice after incubation of10 and 24 h. The oxidative of 5 mM H2O2 was added to above cell culture together withand without 1 mM POPC liposomes. The cultivationwas continuous at 37 °C after abovetreatment. Symbols: “open circle”: Control, “open square”; with H2O2, “double opencircle”; with H2O2/Liposome, “closed circle”; with H2O2/Liposome under heat stress.

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acid (POPA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1-palmi-toyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), were also purchasedfrom Avanti Polar Lipid. Chitosanase from S. griseus was purchased from Sigma Aldrich.Other chemical reagents of highly analytical grade were purchased from Wako PureChemical (Osaka, Japan).

Preparation of liposomes The POPC was solubilized in chloroform. Usingvacuum rotary evaporator, a phospholipid thin layer was prepared in the round bottomflask. Further dried process was operated with nitrogen flow for at least 5 h. In order tocreate the multi lamellar vesicles, the phospholipid thin dried layer was hydrated with10 mM phosphate buffer containing 150 mM NaCl (pH 7) for overnight. The freezing–thawing cycle was repeated 5 times with hydrated POPC solution for the formation ofunilamellar vesicles. This vesicles solution was extruded through the polycarbonatemembrane with pore size of 100 or 200 nm to form unilamellar vesicles prior to use.The liposome containing lipids mimicking cell membrane (LMCM, DMPG/DOPE/POPC/POPA (56:22:20:2 in molar ratio)) was prepared by previously reported method (23).

Evaluation of secondary structure of chitosanase using CD spectra Confor-mational transition of chitosanase under oxidative stress conditions was analyzed inthe presence and absence of liposomes by using Jasco J-820 SFU spectropolarimeter.The molecular ellipticity of chitosanase as the function of the wavelength in range 250to 190 nmwas fitted based on the previous results (21) in order to get the informationof the secondary structure of chitosanase. In the experiment shown the efficiency of theheat treatment, an amount 4.3 μM chitosanases was dissolved in 10 mM phosphatebuffer containing 150 mM NaCl, pH 7.0. The effect of oxidative stress on theconformation of chitosanase with and without liposomes was also evaluated by co-incubation of 4.3 μM chitosanases with 5 mM hydrogen peroxide in the presence andabsence of 1 mM POPC liposomes. In order to minimize the noise on the CD spectra ofchitosanase, 1 mM POPC liposomes solution was well prepared to be transparent in thesame buffer and their CD spectra were measured as the blank sample. The quartz cellwith 1 mm in path length, the bandwidth of spectra at 0.1 nm and scanning rate at10 nm/min were applied to measure CD spectra of chitosanase.

Dielectric dispersion analysis An impedance analyzer (RF impedance analyzer4291B, Agilent Technology) was equipped with handmade brass electrode cell in orderto measure the dielectric spectra of liposomes in the frequency range between 1 MHzand 100 MHz. The shift of relative permittivity (or dielectric dispersion) of POPCliposomes as function of frequency may show the orientation of the molecules, such asamplitude of the dielectric dispersion (Δɛ), the lateral diffusion (fc1) and the rotation(fc2) of dipolar head groups of individual phospholipid molecules in the liposomes.Such dielectric parameters can be obtained based on the fitting analysis using Debye'sequation as shown below (24). 30 mM POPC liposome sample (125 μl) was applied tothe electrode in order to obtain capacitance and conductance values. The interactionbetween POPC liposomes and chitosanase was evaluated as a shift in the aboveparameters of the dielectric dispersion (15 mM POPC and 4.3 μM chitosanase as thefinal concentration). The samples of pure POPC and POPC/chitosanase mixture wereexposed to oxidative stress condition (5 mM hydrogen peroxide) for 20 min beforebeing applied into a brass electrode cell. The normalized fc1 and fc2 frequencies aredescribed as following formula as (fc1,p− fc1,0)/ fc1,p and (fc2,p− fc2,0)/ fc2,p; where fc1,p,fc2,p are characteristic frequencies of POPC/chitosanase mixture and fc1,0, fc2,0 arecharacteristic frequencies of pure POPC liposomes.

Debye's equations:

DeV= eV� eVw =De1

1+ f =fc1ð Þ2+

De2

1+ f =fc2ð Þ2

DeW= eW� eWw � Gdc

2pfC0=

De1 f =fc1ð Þ1+ f =fc1ð Þ2

+De2 f =fc2ð Þ1+ f =fc2ð Þ2

eV= CTC�10

eW=G

2pfC0

In these equations, the data were corrected in the form of parallel connection ofcapacitance C (F) and conductance G (S) as the function of frequencies. The dielectricconstant (ɛ′) and its loss (ɛ″) were calculated based on Eqs. (3) and (4). Similarly, thedielectric constant and loss of water (ɛ′w, ɛ″w, respectively) were calculated to evaluatethe increment of ɛ′, ɛ″ from the values of water (Δɛ′, Δɛ″) and the amplitudes ofdielectric dispersion (Δɛ1, Δɛ2) using Eqs. (1) and (2); where the f, fc1, fc2 arefrequencies (MHz); C0 is cell constant (F) and Gdc is direct current conductivity ofsample.

Analysis of lipid and protein peroxides The amount of protein and lipidperoxides was measured by ferric-xylenol orange method (25). After the mixture wasvigorously mixed for 5 min, the sample was centrifuged at 6500 ×g for 6 min.Chloroform layer containing target lipids was collected and dried by oxygen-freenitrogen and then the following reagents were added immediately; 250 μl ChCl3, 4 mMbutylated hydroxytoluen (BHT); 460 μl MeOH/4 mM BHT; 30 μl xylenol orange (XO)and 20 μl Fe2+. After incubation for 60 min in covered test tubes, the sample wasmeasured absorbance at 560 nm. A blank sample containing the extracted lipid,reduced with 1 mM triphenylphosphine was subjected to identical protocol andmeasured the absorbance at 560 nm.

The protein peroxide was assayed by G-PCA-FOX method. Briefly, 10 μl sample ofextracted protein and 500 μl 0.2 M PCA were mixed vigorously in ice, and thencentrifuged at 6500 ×g for 5 min. After 1100 μl of 6 M GuHCl solution was added topellet, the sample was vortexed to dissolve pellet and washed the solution with 1100 μl

Please cite this article as: Ngo, K.X., et al., Oxidative/heat stress enhanceits interaction with liposome, J. Biosci. Bioeng. (2009), doi:10.1016/j.jbi

ROOF

chloroform containing 4 mM BHT. Finally, the obtained sample was added to theaqueous solution in the following order; 40 μl of 0.5 M perchloric acid (PCA), 25 μl of5 mM XO, 25 μl of H2O, and 10 μl of 5 mM Fe2+. After the incubation of this sample for60 min at room temperature, the absorbance at 560 nm was measured. A blankcontaining extracted protein, reduced with 1 mM sodium dithionite, was subjected toidentical protocol and measured the absorbance at 560 nm.

Infrared spectroscopy of cell surface under various heat and pH condi-tions The spheroplast S. griseus cells were prepared by lysozyme hydrolysis (1 mg/ml as final concentration) to remove outer peptidoglycan layer of intact S. griseus cellsas our previous method (20). The concentration of these cells was adjusted at 106/ml indistilled water prior to use. The above spheroplast suspension was treated with thetemperature at 25 °C for 60 min in the presence and absence of 5 mM hydrogenperoxide before applying to FTIR to observe the surface hydration of cell membranethrough the infrared spectra of phosphate group of lipid membrane. The sample of 30 μlspheroplast S. griseus cells suspension at each condition of temperature and pH wasapplied in 50 μm thick-cell with CaF2 window. The infrared spectraweremeasuredwitha FTIR 4100 spectrometer (JASSCO, Japan) equipped with an Hg-Cd-Te detector. Theresolution was set up at 4 cm−1; the frequency range from 1700 to 1000 cm−1 wascollected for each sample. The infrared spectra of samples were subtracted to that ofwater or buffer. The accuracy of the frequency reading is better than ±0.1 cm−1.

Measurement of chitosanase activity Chitosanase activity was measured withglycol chitosan as a substrate (26). The reaction mixture containing 0.5 ml of 2 wt.%glycol chitosan in 0.1 M phosphate buffer (pH 5.6) and 0.5 ml of enzyme solution wasincubated at 37 °C for 10 min and then the enzymatic reaction was stopped by boilingfor 4 min. This solution was cooled down and added to 1 ml of acetylacetone diluted in0.5 N Na2CO3 (1:50 v/v) and 1 ml of distilled water and boiled for 20 min. An amount

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5 ml of ethanol and 1 ml of enrich reagent (p-dimethylamino-benzaldehyde, DMAB)were then added and incubated at 65 °C for 10 min. The absorbance of the assaysolution is measured at 530 nm using UV spectrophotometer (UV-1600A, Shimadzu,Japan). One unit of chitosanase is defined as the amount of enzyme that hydrolyzedglycol chitosan in order to release 1 μM glucosamine per min at given assay conditions.

RESULTS AND DISCUSSION

Enhanced production of chitosanase from S. griseus cellstreated by liposomes and specific heat under oxidative stresscondition The growth of S. griseus cells and the production ofchitosanase were investigated after the pretreatment by heat stress at41 °C in the presence of POPC liposomes under oxidative stresscondition. As shown in Fig. 1a, the growth of S. griseus cells treatedwith 5 mM hydrogen peroxide at 37 °C after incubation of 24 h wassignificantly reduced as compared with that of the control (cells wereincubated at 37 °C without any treatment). This result implies that theoxidative stress was severe to the growth of S. griseus cells. In anotherexperiment, 5 mM hydrogen peroxide was added to the suspension ofS. griseus cells pretreated with the heat stress at 41 °C in the presenceand absence of the liposomes. It has been reported that the heat stressenhanced the chitosanase release and, slightly, the cell growth (21).The result obtained above shows that the heat treatment of S. griseuscells at 41 °C after incubation of 10 and 24 h was also found to increasethe cell growth even in the presence of the oxidative stress ascompared with that of the control. The addition of the liposomefurther enhanced the growth of S. griseus cells pretreated with theheat stress at 41 °C even under oxidative stress condition. Thisobservation shows the importance of the heat stress and/or theliposome for the cell growth even under oxidative stress conditions.

Fig. 1b shows the chitosanase production by S. griseus pretreatedheat stress at 41 °C with and without the liposomes under oxidativestress condition of hydrogen peroxide. The production of chitosanasewas evaluated based on the chitosanase activity in fermented broth.The results show that the chitosanase activity obtained in the case S.griseus cells grown at 37 °C under oxidative stress without theliposome was significantly reduced, being 40% lower than that of thecontrol. Interestingly, the addition of the liposomes to the S. griseus

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FIG. 2. Frequency dependencies of dielectric parameters of POPC liposome in the presence and1 with H2O2, 3; 1 with H2O2 and chitosanase, #; 1 with chitosanase at 41 °C (data from (21

Please cite this article as: Ngo, K.X., et al., Oxidative/heat stress enhanceits interaction with liposome, J. Biosci. Bioeng. (2009), doi:10.1016/j.jbi

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cells pretreated heat stress at 41 °C increased the chitosanase pro-duction even under oxidative stress condition and its chitosanaseproductionwas 1.5 times higher than that of the control. Similar to ourprevious paper (20, 21), it was confirmed that almost all chitosanases(more than 95%) synthesized by S. griseus cell were released toaqueous medium. The above results imply that the heat stressexposurement and the liposome addition could play important rolefor the enhanced production of chitosanase from S. griseus cells evenunder oxidative stress condition.

As previously reported (20, 21), the treatment of heat stress(41 °C) or parallel treatment of heat stress (41 °C) and POPCliposomes enhanced the growth of the S. griseus cells and chitosanaseproduction to 1.4 and 2 times, respectively, higher than those of thecontrol. Although the production of chitosanase by S. griseus cellscould be carried out through the pretreatment of liposomes and heatstress at 41 °C under the “oxidative” stress condition, the reducedgrowth and lower production of chitosanase imply the severe effectsof oxidative stress. However, these results also reflect the importanceof heat stress and liposomes, which are able to protect the growthand chitosanase production of S. griseus cells under the oxidativestress condition.

Enhanced interaction between POPC liposome and chitosanaseunder oxidative stress condition by using dielectric dispersionanalysis The dielectric dispersion analysis of the POPC liposomesuspensionwas carried out in the presence and absence of chitosanaseunder oxidative conditions in the frequency range of 1 to 100 MHz todetect the possible oxidative stress-mediated interactions betweenproteins and phospholipid membrane. The variation of electrostaticinteractions at the bilayer surface may exercise an influence on thelateral area per lipid molecule and may be mediated by the rotationalisomerization of the acyl chain on the microdomain structure of themembrane (27). Dielectric dispersion analysis is a powerful techniqueto investigate the re-orientation of the molecules on the surface ofphospholipid membranes (18, 24, 27, 28). Fig. 2a shows the fc1 values,which are the characteristic frequencies of the lateral diffusion ofindividual phospholipid molecules in a liposome. These characteristicfrequencies of lateral diffusionwere varied from 1 to 10MHz. The data

absence of chitosanase under oxidative stress condition. 1; Control (POPC Liposome), 2;)).

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FIG. 3. Lipid and protein peroxides of S. griseus cell under various oxidative stresses withand without liposomes.

FIG. 4. Infrared (IR) spectra of lipid mimicking cell membrane giant vesicle (LMCM-GUV)and spheroplast Streptomyces griseus cells under oxidative stress (hydrogenperoxide)withand without POPC liposomes. IR spectra of LMCM-GUV (a); spheroplast S. griseus (b);spheroplast S. griseus/H2O2 (d); and spheroplast S. griseus/H2O2/POPC liposomes (c). Thelipidmimicking cellmembrane giant unilamellarvesicles (LMCM-GUVs)werepreparedbylipid composition as following: DMPG/DOPE/POPC/POPA (56:20:22:2 in mol%). TheLMCM-GUVs (10 mM) and S. griseus spheroplast cells (106/ml) were exposed to 5 mMH2O2 for 1 h at room temperature.

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show that the fc1 values are decreased under oxidative stressconditions as compared with that of the control. The addition ofchitosanase to liposome suspension under oxidative stress conditionsfurther decreased the fc1 values as compared with those of pureliposomes and liposomes/chitosanase mixture. These results suggestthat the lateral diffusion of lipid molecules in liposomes was reducedunder oxidative stress condition. Under oxidative stress conditions,the liposomes and chitosanase could interact with each other,resulting in the decrease of the lateral area of lipid molecule.Consequently, a decrease of lateral diffusion of lipid molecule wasobserved.

Fig. 2b shows the fc2 value of POPC liposome in the presence andabsence of chitosanase under oxidative stress conditions. The fc2 valueof the liposome was lower under oxidative stress conditions than thatof pure liposome. The addition of chitosanase to the liposomesuspension under oxidative stress conditions further decreased thefc2 values. This observation may indicate that hydrogen peroxideoxidizes the phospholipids in the liposome membrane and mediatesthe restriction of rotation of dipolar head groups of phospholipidmolecules in the membrane. It has previously been reported that theoxidation of unsaturated phospholipids in membrane bilayer caused adecrease of membrane fluidity (29). In the case of POPC/chitosanase/hydrogen peroxide, the fc2 values are significantly decreased to30 MHz and much lower than that of other conditions (both onlyliposome and liposome with hydrogen peroxide). The shift in bothfrequencies of chitosanase/liposome under oxidative stress was foundto be similar with those under heat stress condition at 41 °C (21). Thisobservation may imply that hydrogen peroxide induces the interac-tion between chitosanase and the liposome. In other words, thebinding of hydrogen peroxide to unsaturated sites of lipid acyl chainsin the liposomemay create several chemical groups such as keto (fO),hydroperoxide (–OOH), hydroxyl (–OH) (8), which could much moreeasily assist the interaction of the oxidized liposomes with chitosa-nase. It has also been reported that hydrogen peroxide residues canmimic the function of the ligand receptors of bio-molecules includingproteins and lipids (13). The creation of hydrogen bond donor andacceptor in the hydrophobic region of POPC liposome under oxidativestress conditions, such as the keto group (fO), the hydroxyl group (–OH), the hydroperoxide group (–OOH), may lead to the hydrogenbond transition between the liposome and chitosanase. It has alsobeen reported that the hydrogen bond defects, which are formed inproteins and lipid membrane, may be a signal for their interactionunder oxidative stress conditions (30). The target of this evaluation isto elucidate the diverse roles of hydrogen peroxide on the biomem-brane model (liposomes) and the responsive behavior and chitosa-nase against oxidative stress conditions that possibly reflect theresponse of bacterial cell membranes when they are exposed tooxidative stress.

Please cite this article as: Ngo, K.X., et al., Oxidative/heat stress enhanceits interaction with liposome, J. Biosci. Bioeng. (2009), doi:10.1016/j.jbi

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Interaction of liposomes with cell membranes and chitosanasecan significantly prevent the severe damage of oxidative stress oncell membrane The possible roles of POPC liposomes for theprevention of oxidative stress damage on the chitosanase and cell lipidmembrane of S. griseus were further studied. The efficiency of theliposome on the prevention of protein and lipid peroxides of cellmembrane under oxidative stress condition (hydrogen peroxide) wasinvestigated. Fig. 3 shows the formation of the lipid peroxide of cellmembranemediated by various kinds of oxidative stresses. The resultsshow that the formation of the lipid peroxide per gram of lipid of cellmembrane of S. griseus cells was 2.2 nmol/g after the treatment withhydrogen peroxide. However, the addition of the liposome signifi-cantly prevented the lipid peroxidation of cell membrane under suchoxidative stress conditions. The amount of lipid peroxide of cellmembrane in the presence of liposomes was reduced to 1.2 nmol/g.The efficiency of the liposomes on the prevention of protein peroxideof cell membrane under oxidative stress was also investigated.Although the amount of protein peroxide formation of cell membranecaused by hydrogen peroxide was 3.1 μmol/g, the addition of theliposomes significantly prevented the protein peroxides formationunder oxidative stress conditions (1.8 μmol/g). The above resultsimply that the liposomes could play very important roles to preventthe damage of oxidative stress on lipids and proteins of cellmembranes of S. griseus cells.

The variation of the characteristics of the liposome membrane wasalso analyzed by the infrared (IR) analysis. The IR spectra of lipidmimicking cell membrane (LMCM) liposomes are shown in Fig. 4a.The IR spectra of these liposomes were almost similar to those of cellmembrane of S. griseus. This observation implies the similarity of lipidcompositions between LMCM liposomes and cell lipid membranes.The spheroplast S. griseus cells pretreated with the POPC liposomeunder oxidative stress at 5 mM hydrogen peroxide after incubation of1 h showed similar IR spectra with that of spheroplast cells withoutany treatment, as shown in Fig. 4b and c. On the contrary, the IRspectra of spheroplast cells without the treatment of the POPCliposome were varied under oxidative stress condition, as shown inFig. 4d. These observations imply that the oxidative stress damaged

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the lipid cell membrane, resulting in the disappearance of IR spectrumat the frequency approximate 1231 cm−1 that corresponds to IR bandsof phosphatidylcholine head group in the aqueous region, in contrastto the LMCM liposomes (model of lipid biomembrane of S. griseuscell). As a consequence, the surface properties of spheroplast S. griseuscells might significantly change the surface properties, such as surfacenet charge and hydrophobicity (22), and surface hydration althoughthe quantity of these variations should furthermore be characterized.It is thought that the POPC liposomes interacted with cell membranesof S. griseus after heat treatment to prevent the damage of oxidativestress on the lipid cell membranes. The above results, together withthe results on the peroxide formation of lipid and protein, support thepossibility that the interaction between liposomes and cell lipidmembranes plays very important roles for the growth and chitosanaseproduction of S. griseus cells under oxidative stress conditions,although the interaction of different kinds of liposomes (positiveand/or negative liposomes) with cell membranes and how theresponses of those cells after such interaction should be dedicated.

In order to verify the efficiency of liposomes for the enhancedproduction of chitosanase even under oxidative stress condition, theconformational change of chitosanase under strong oxidative stresscondition with and without liposomes was investigated. The resultsshow that the conformation of chitosanase under strong oxidativestress at 5 mM hydrogen peroxide was completely destroyed and itcould not be detected by CD spectra as shown in Fig. 5. However, theaddition of the POPC liposome could significantly prevent the damageof the oxidative stress on conformation of chitosanase. The secondarystructure of chitosanase under oxidative stress in the presence andabsence of the liposome was also analyzed, where those ofchitosanase with and without liposomes under non-stressed condi-tions were almost same; α-helix (39%), β-sheet (6%), turn (36%) andrandom (19%). Under oxidative stress condition, the addition of theliposome was found to maintain the content of α-helix (23%) ofchitosanase and to slightly increase its β-sheet content (14%). It hasalso been shown that the oxidative stress also damages the structureof chitosanase, resulting in the increase of the β-turn and randomconformation of these enzymes even in the presence of the liposome.The efficiency of the liposome on the structure of chitosanase underthe oxidative stress conditions could be clarified into severalpossibilities (i) interaction between liposomes and chitosanase occurseffectively under the oxidative stress condition, (ii) the rearrange-ment of the hydrogen bonds between liposomes and chitosanase thatcould stabilize surface hydration of chitosanase, and (iii) the liposome

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FIG. 5. Conformational transition of chitosanase under oxidative stress condition in thepresence and absence of POPC liposomes; chitosanase (4.3 μM) was incubated with5 mM H2O2 in the presence and absence of 1 mM POPC liposomes.

Please cite this article as: Ngo, K.X., et al., Oxidative/heat stress enhanceits interaction with liposome, J. Biosci. Bioeng. (2009), doi:10.1016/j.jbi

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has scavenger-like functions under oxidative stress condition. In fact,the interaction between liposome and chitosanase could stabilize theconfiguration of chitosanase under oxidative stress condition (Fig. 5).In relation to scavenger function of POPC liposomes, it has beenreported that liposome vesicles made by phosphatidylcholine lipidscould decompose the hydrogen peroxide to water and oxygen (31).

Interactionof liposome and cellmembraneunderheat/oxidativestress for enhancedproductionandreleaseof chitosanase The roleof oxidative stress on the enhanced production and release ofchitosanase from S. griceus cells under heat/oxidative stress conditionwas finally discussed. The potential functions of S. griseus cells underoxidative stress condition could be elucidated through the interaction ofS. griseus cells and liposome membrane. It has recently been reportedthat the surface net charge of S. griseus cells is varied by the treatment ofthese cells by oxidative stress rather than the heat treatment as thecharacterization using aqueous two-phase partitioning method (22). Ithas also been reported that the interaction of the neutral POPC liposomewith cell membranes occurs more effectively at higher temperatures inspecific heat range (21, 22). The surface hydration of S. griseus cells isdependent on the heat, pH and oxidative treatments. The treatment ofheat at 41 °C and extremepH (strong acidic and alkaline) causes the lossof water on cell membrane surface. As a consequence, the surfacehydration of cell membrane is reduced under the extreme heat and pHconditions. This reduction could possibly contribute to the increasedHFS, as well as to the destabilization of hydrogen bonds of cell surfacehydration, which are mediated by the water molecules.

The cell lipid membrane is very susceptible with the oxidativestress. The oxidative stress severely damages the lipids of cellmembranes (Fig. 3). However, the membrane–membrane interactionbetween the POPC liposome and cell membrane is able to prevent thedamage of oxidative stress on the cell membranes (Fig. 1), as theresults of IR spectra of real spheroplast S. griseus cell and model of cellmembrane using LMCM-GUV (Fig. 4). It has been recently shown thatthe externally-added liposome could interact with cell membrane ofboth intact and spheroplast S. griseus cells (20). The variation ofsurface hydration of the liposome surface could be induced by heatand oxidative stresses, consequently caused by the interaction of theliposome and cell membrane. The interaction of those membranescould be controlled by considering the above physicochemicalcharacteristics of the cells under the heat/oxidative stress condition.It has been reported that the variation of the cellular membrane ormodeled biomembrane could induce the response of bacterial cells(15–22). The heat stress has also been reported to induce theadsorption, fusion and/or internalization of POPC liposomes by S.griseus cells (20).

In this paper, it was thus shown the importance of the interactionbetween liposomes and cell membrane of S. griseus under heat/oxidative stress condition for the enhanced cell growth and itsproduction of chitosanase through the prevention of lipid and proteinperoxides in cell membrane (IR spectra investigation) althoughfurther investigation is needed.

ACKNOWLEDGMENTS

The fundamental concept of this study was supported by theResearch Group of “Membrane Stress Biotechnology.” It was sup-ported in part by a Grant-in-Aid for Scientific Research (No. 17656268,19656203, 19656220, 20360350, and 21246121) from the Ministry ofEducation, Science, Sports, and Culture of Japan, a grant from the 21stCentury COE program “Creation of Integrated EcoChemistry” and theGlobal COE program “Bio-Environmental Chemistry” of the JapanSociety for the Promotion of Science (JSPS). The authors are grateful tothe Research Center for Solar Energy Chemistry of Osaka Universityand the Gas hydrate Analyzing System of Osaka University. K. X. Ngoand H. T. Bui acknowledge financial support from the Ministry of

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