Development of a Continuous Bioconversion System Using aThermophilic Whole-Cell Biocatalyst

Pham Huynh Ninh,a Kohsuke Honda,a,b Yukako Yokohigashi,a Kenji Okano,a Takeshi Omasa,a,c Hisao Ohtakea

Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japana; PRESTO, Japan Science and Technology Agency (JST), Saitama, Japanb;Department of Life System, Institute of Technology and Science, The University of Tokushima, Tokushima, Japanc

The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation ofindigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalystscomparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakageof thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeatedand continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus(TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used asa whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealedthat the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membranepermeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying theglutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzy-matic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.

Thermophilic enzymes are promising tools for biotransforma-tion owing to their high operational stability, cosolvent com-

patibility, and low risk of contamination (1–3). The direct use ofrecombinant mesophiles (e.g., Escherichia coli) having heterolo-gous thermophilic enzymes at high temperatures results in thedenaturation of indigenous enzymes and the elimination of un-desired side reactions; therefore, highly selective whole-cell cata-lysts comparable to purified enzymes can be readily prepared.Honda et al. have demonstrated that the rational combination ofthese thermophilic whole-cell catalysts enables the constructionof in vitro artificial biosynthetic pathways for the production ofvalue-added chemicals (4). Recently, Ye et al. have successfullyconstructed a chimeric Embden-Meyerhof pathway with the bal-anced consumption and regeneration of ATP and ADP, using ninerecombinant E. coli strains, each of which overproduces a thermo-philic glycolytic enzyme (5).

The membrane structure of E. coli cells is partially or entirelydisrupted at high temperatures, and thus thermophilic enzymes,which are produced as soluble proteins, leak out of the cells (6–9).Although the heat-induced leakage of thermophilic enzymes re-sults in better accessibility between the enzymes and substrates, itlimits the applicability of thermophilic whole-cell catalysts tocontinuous and repeated-batch reaction systems. This limitationprevents us from exploiting the most advantageous feature ofthermophilic biocatalysts, namely, their excellent stability. Toovercome this limitation, one potential strategy is the integrationof thermophilic enzymes to the membrane structure of cells. Inour previous work, we found that the heat-induced leakage of athermophilic glycerol kinase from recombinant E. coli cells couldbe prevented by fusing the enzyme to an E. coli membrane-intrin-sic protein, YedZ (8). However, the specific enzyme activity of therecombinant E. coli having the YedZ-fused enzyme decreased to6% of that of the recombinant with the nonfusion enzyme. A tightintegration of the glycerol kinase to the E. coli membrane structuremight have prohibited the conformational change of the enzyme,resulting in a decreased specific activity. Thus, the screening for a

suitable membrane-anchoring protein would be essential to mit-igate the loss of the specific activity.

An alternative approach to preventing the heat-induced leak-age is the use of protein cross-linking reagents for the consolida-tion of the cell membrane as well as for the linkage of enzymes tothe membrane structure. In this approach, unlike in the integra-tion via membrane-anchoring proteins, cross-linkage level can bereadily controlled by changing the conditions for the cross-linkingreaction, and thus the best compromise between the prevention ofthe heat-induced leakage and the maintenance of the specific en-zyme activity can be achieved. Glutaraldehyde (GA) and relateddialdehydes are some of the most effective protein cross-linkingreagents and have been widely used for biocatalyst immobilization(10–13). GA is mainly used to immobilize enzymes to carrierssuch as activated charcoal, anion-exchanging resin, and glassbeads. Generally, for the cross-linkage of enzymes to these carri-ers, the enzyme has to be isolated from cells, purified to a certainlevel, attached to carriers in a suitable way, and then cross-linkedwith GA.

In this study, E. coli cells having a thermophilic fumarase weretreated with GA. GA-treated cells were heated at 70°C to inactivatethe intrinsic enzymes, and then directly used for the conversion offumarate to malate. Through this simple procedure, many stepsrequired in conventional procedures for the preparation of im-mobilized enzymes, such as protein extraction, enzyme purifica-tion, and the preparation of immobilizing carriers, could be en-tirely skipped, and a highly stable and selective immobilized

Received 5 December 2012 Accepted 9 January 2013

Published ahead of print 18 January 2013

Address correspondence to Kohsuke Honda, honda@bio.eng.osaka-u.ac.jp.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03752-12

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enzyme, of which heat-killed E. coli cells served as carriers, couldbe prepared.

MATERIALS AND METHODSBacterial strain and culture conditions. The expression vector for theThermus thermophilus fumarase (TtFTA) was obtained from the RIKENThermus thermophilus HB8 expression plasmid library (14) and desig-nated pET-TtFTA. The expression vector for the malic enzyme of Ther-mococcus kodakarensis KOD1 (TkME) was constructed as described else-where (15). E. coli Rosetta 2 (DE3) pLysS (Novagen, Madison, WI) wasused as the host cell for gene expression. Recombinant E. coli was culturedin a 500-ml Erlenmeyer flask containing 200 ml of Luria-Bertani brothsupplemented with 100 mg/liter ampicillin and 34 mg/liter chloramphen-icol. Cells were cultivated at 37°C with orbital shaking at 180 rpm. Isopro-pyl-�-D-1-thiogalactopyranoside (IPTG) was added to the culture at afinal concentration of 0.4 mM in the late-log phase. After a 3-h induction,the cells were harvested by centrifugation and washed once with 0.1 Msodium phosphate buffer (pH 7.0).

Glutaraldehyde treatment. Two hundred milligrams of wet cells wassuspended in 1 ml of 0.1 M sodium phosphate buffer (pH 7.0). GA solu-tion (25% in water; Nacalai Tesque, Kyoto, Japan) was added to the cellsuspension to give final concentrations of 0.03% to 0.15% (vol/vol). Themixture was gently stirred at 4°C for 1 h. The treated cells were harvestedby centrifugation at 8,000 � g for 10 min and then washed once with thesame buffer.

Enzyme assay. TtFTA activity was assessed at 70°C by coupling withthe Thermococcus malic enzyme, which catalyzes a NADP�-dependentdecarboxylation of malate to pyruvate. E. coli cells having either TtFTA orTkME were suspended in 0.1 M sodium phosphate buffer (pH 7.0) anddisrupted using an ultrasonicator (UD-201; Kubota, Osaka, Japan) at 40W for 3 min. The resulting lysate was heated at 70°C for 20 min. After theremoval of cell debris and denatured proteins by centrifugation, the re-sulting supernatant was used as the enzyme solution. Enzyme activity wasmeasured spectrophotometrically by monitoring the increase in the ab-sorbance of NADPH at 340 nm. A molar absorption coefficient for NA-DPH of 6.22 � 103 M�1 cm�1 was used. One unit of enzyme was definedas the amount of enzyme that catalyzes the production of 1 �mol ofNADPH per 1 min. The standard reaction mixture contained 1.25 mMsodium fumarate, 50 mM MnCl2, 0.1 mM NADP�, 0.1 U TkME, and 0.1M phosphate buffer (pH 8.0), in a total volume of 1 ml. The reaction wasstarted by adding an appropriate amount of TtFTA solution.

For the determination of the reaction rate catalyzed by whole cells, freeor GA-treated E. coli cells having TtFTA were suspended in 0.1 M phos-phate buffer at a concentration of 8 mg (wet weight)/ml. Cells were used ascatalysts after the heat treatment at 70°C for 20 min. The reaction wasinitiated by adding 2.5 ml of the heat-treated cell suspension into a screw-cap cylindrical vessel (inner diameter, 35 mm) containing 2.5 ml of 0.4 Msodium fumarate in the same buffer, which was preheated at 70°C. Thereaction was performed at 70°C with stirring. Aliquots (0.5 ml) of themixture were withdrawn after the reaction for 5, 10, 30, and 60 min,acidified with HCl to stop the reaction, and then subjected to high-per-formance liquid chromatography (HPLC).

Heat-induced leakage of TtFTA. The free and GA-treated E. coli cellshaving TtFTA were suspended in 0.1 M phosphate buffer (pH 7.0) at 200mg (wet weight)/ml and incubated at 70°C. After the incubation for 5, 10,30, and 60 min, the cells and their debris were removed by centrifugationat 15,000 � g for 10 min at 4°C. The supernatants were resubjected to heattreatment at 70°C for 20 min. Denatured proteins were removed by cen-trifugation, and the TtFTA activity in the supernatant was determined.Leakage level was expressed as the percentage of TtFTA activity in the cellextract, prepared by subjecting the same concentration (200 mg/ml) ofthe free-cell suspension to ultrasonication, followed by heat treatment at70°C for 20 min.

Electron microscopic analysis. Scanning electron microscopy (SEM)(S-5200; Hitachi, Tokyo, Japan) was used to take images of the free and

GA-treated E. coli cells before and after the heat treatment at 70°C for 20min. The suspensions of the free and GA-treated cells were centrifuged at5,000 � g for 10 min. The pellets were fixed with 2.5% (wt/wt) GA– 0.2 Mphosphate buffer for 1 h, and then stained with 5% (wt/wt) osmic acid inthe same buffer for 30 min. The samples were dehydrated in a gradedethanol series, lyophilized for 2 h, and then coated with osmium (HPC-1Shollow cathode plasma chemical vapor deposition, vacuum device; Mito,Japan).

For transmission electron microscopy (TEM), the dehydrated sampleswere mixed with an epoxy resin solution and then polymerized at 45°C for5 days. The resin-embedded samples were cut with a glass knife on aReichert-Nissei ultramicrotome, mounted on carbon-coated copper grids(Nisshin EM, Tokyo, Japan), and poststained with uranyl acetate and leadcitrate. TEM was carried out with a JEOL JEM 1200 EX (80-kV) electronmicroscope.

Effect of heat treatment on the membrane permeability of GA-treated E. coli cells. Fluorescein-labeled dextrans with average molecularmasses of 3 kDa (Dex3) and 40 kDa (Dex40) (Invitrogen, Carlsbad, CA)were used to evaluate the membrane permeability of the GA-treated cells.The GA-treated cells were suspended in 5 ml of 0.1 M sodium phosphatebuffer (pH 7.0) at 200 mg (wet weight)/ml and then heated at 70°C for 20min. The heated-cell suspension was centrifuged at 8,000 � g for 10 min,and the cell pellet was washed once with the same buffer. The cells werethen resuspended in 5 ml of the buffer containing either Dex3 or Dex40(0.05 �M each) and incubated in the dark at room temperature for 30 minwith gentle shaking. After incubation, the cells were pelleted by centrifu-gation at 8,000 � g for 10 min and then resuspended in 5 ml of the freshbuffer without dextrans. The resuspended solution was shaken with avortex mixer for 1 min and then centrifuged at 8,000 � g for 10 min. Thefluorescence intensity of the supernatant was measured at excitation andemission wavelengths of 494 and 521 nm, respectively. A control experi-ment was carried out using the GA-treated cells without the heat treat-ment.

Reusability of GA-treated cells. The reaction mixture containing 4mg/ml wet cells having TtFTA and 0.2 M sodium fumarate– 0.1 M sodiumphosphate buffer (pH 7.0) was placed in a screw-cap cylindrical vessel(inner diameter, 27 mm) and incubated at 70°C with stirring. After beingallowed to react for 30 min, the cells were removed by centrifugation at8,000 � g for 10 min. The supernatant was acidified with HCl, recentri-fuged to remove denatured proteins, and then subjected to HPLC to de-termine the concentrations of malate and fumarate. The reaction wasrepeatedly performed in the same manner to assess the reusability of thefree and GA-treated E. coli cells. After each reaction cycle, the cells wereharvested by centrifugation at 8,000 � g for 10 min, washed with thebuffer, and then resuspended in a fresh reaction mixture.

HPLC was performed using a Shimadzu Prominence LC-20 system(Kyoto, Japan) equipped with a Cosmosil 5C18-AR-II packed column(Nacalai Tesque) (4.6 by 250 mm) at 50°C. The UV detector was set at 210nm, and a 0.1% (vol/vol) phosphoric acid solution at a flow rate of 1ml/min was used as the mobile phase.

Malate production in a continuous reactor using GA-treated cells.The reaction mixture (10 ml) comprising 4 mg (wet weight)/ml of theGA-treated cells having TtFTA, 0.2 M sodium fumarate, and 0.1 M so-dium phosphate buffer (pH 7.0) was put in a stirred ultrafiltration cell(model 8010; Millipore, Bedford, MA). A polyvinylidene fluoride (PVDF)membrane filter (Toray, Kamakura, Japan) (diameter, 25 mm) (16) wasset at the bottom of the reactor to separate cells from the solution. Thereaction was performed at 70°C with stirring by a magnetic stirrer. Afterthe reaction was performed for 40 min to achieve a steady state, a freshsubstrate solution (0.2 M sodium fumarate in the same buffer) was addedand the product solution was removed using peristaltic pumps (model3385; Fisher Scientific, Fair Lawn, NJ). To determine product yield, ali-quots (0.1 ml) of the eluent were taken at 40-min intervals and subjectedto HPLC. The feeding and removal rates of the solution were maintained

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at 0.25 ml/min. Residence time and conversion rate were calculated asfollows:

Residence time � (min)

� � F ⁄ V

Conversion rate � (mmol/liter/min)

�Sin � S� � � � �

where Sin � substrate concentration in the feeding solution, S � substrateconcentration in the reactor, F � flow rate, and V � total volume ofreaction mixture.

RESULTSEffects of GA treatment on heat-induced leakage of TtFTA. Fig. 1shows the time course of the heat-induced leakage of TtFTA fromthe recombinant E. coli cells. When the free cells were incubated at70°C, more than 80% of the activity was released from the cells by5 min. This observation was in good agreement with previousreports on the thermolysis of E. coli cells (7–9). Owing to thermol-ysis, the boundary surface between the supernatant and the pel-leted free cells was unclear after the centrifugation at 15,000 � gfor 10 min. However, the heated GA-treated cells could be clearlyseparated by centrifugation. The level of TtFTA leakage from theGA-treated cells was considerably lower than that from the freecells and obviously decreased with the increasing GA concentra-tion used for the cross-linkage. The leakage from the cells pre-treated with 0.11% (vol/vol) or higher GA concentrations waskept under a detectable level after the incubation at 70°C for atleast 60 min. However, it was also possible that the decrease in theapparent level of enzyme leakage arose from the inhibitory effectof GA on the enzyme as well as the lowered substrate permeabilitydue to the consolidation of the membrane structure caused by thecross-linkage. We then assessed the performance of the free andGA-treated cells for catalyzing fumarate hydration. As predicted,the catalytic performance of GA-treated cells decreased in propor-tion to the GA concentration used for their preparation (Fig. 2).The apparent initial reaction rate was estimated on the basis of theproduct concentration after the reaction for 5 min. The reactionrates obtained using the cells pretreated with 0.03, 0.07, 0.11, and0.15% GA were 94, 80, 77, and 64% of that obtained using the freecells, respectively. However, no significant difference was ob-served in the product concentration after the reaction for 30 min.On the basis of these observations, we employed a GA concentra-tion of 0.11% for the cross-linkage as the best compromise be-

tween the prevention of heat-induced leakage and the mainte-nance of enzyme activity.

The conversion of fumarate to malate is a reversible reactionwith the equilibrium at approximately 80 mol% (13, 17) under thestandard state (25°C). In this study, the conversion yield using theseries of E. coli cells having TtFTA reached about 70 mol% after 30min and then remained constant, regardless of the difference intheir specific activities. This indicates that the equilibrium of thereaction under our experimental conditions was achieved at aconversion yield of approximately 70 mol%.

Electron microscopic analysis. The free and GA-treated cellsbefore heat treatment showed the appearance of normal healthycells in both SEM and TEM (Fig. 3A, C, E, and G). In contrast,after a 20-min incubation at 70°C, the SEM image of the free cellsdisplayed cellular structure disintegration (Fig. 3B). Blebs withvarious sizes were formed on the cell surface. The TEM of theheat-treated free cells showed the surface subsidence of the cells,probably resulting from membrane disruption followed by theleakage of intracellular components, suggesting that the heat-in-duced leakage of the enzyme was attributable to the collapse of themembrane structure of the cells (Fig. 3F).

In contrast, the overall structure of the GA-treated cells stillremained in good condition after the incubation at 70°C for 20min. Both TEM and SEM showed the appearance of a cell surfacecomparable to the surface of healthy cells, except for the forma-tion of some small blebs on the membrane (Fig. 3H). This obser-vation supported the notion that the prevention of the heat-in-duced leakage of TtFTA from E. coli cells was, at least in part,attributable to the consolidation of the membrane structure by theGA treatment.

Membrane permeability of GA-treated cells. Dextrans, poly-mers of glucose, are hydrophilic polysaccharides characterized bya high molecular mass and good water solubility. Dextrans withmolecular masses higher than 2 kDa generally cannot permeateinto intact cells (18). In this study, fluorescein-labeled dextranswith different average molecular masses were used to assess themembrane permeability of the GA-treated cells. The cells werefirst suspended in a fluorescence-labeled dextran solution and pel-leted by centrifugation, and then the pellet was resuspended in adextran-free buffer. The fluorescence intensity of the supernatantof the resulting cell suspension correlates with the amount of dex-tran involved in the cell pellet; therefore, a higher intensity can beobtained when dextran can penetrate into the cell. The slight flu-

FIG 1 Heat-induced leakage of TtFTA from free cells (filled diamonds) andcells pretreated with 0.03% (circles), 0.07% (open diamonds), 0.11% (trian-gles), and 0.15% (squares) GA. The leakage level was expressed as the percent-age of the TtFTA activity in the cell extract prepared by ultrasonication. Thedata are shown as averages standard deviations (n � 3).

FIG 2 Time course of malate production catalyzed by free cells (filled dia-monds) and cells pretreated with 0.03% (circles), 0.07% (open diamonds),0.11% (triangles), and 0.15% (squares) GA. The data are shown as averages standard deviations (n � 3).

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orescence observed in the control experiments with the non-heat-treated cells was likely attributable to the dextran remaining in theintercellular space of the cell pellets (Fig. 4, gray bars). The fluo-rescence intensity of the supernatant of the heat-treated cell sus-pension was almost double that of the non-heat-treated cell sus-pension when Dex3 was used as an indicator of membranepermeability. On the other hand, when Dex40 was used, no sig-nificant difference was observed between the fluorescence inten-sities of the heat-treated and non-heat-treated cell suspensions.Taken together with the results of electron microscopic observa-tion, these results indicated that the membrane structure of theGA-treated cells was partly disrupted by the heat treatment, whilemaintaining the overall cell structure; therefore, only relativelysmall molecules (up to at least 3 kDa) could permeate into and outof the cells.

Reusability of GA-treated cells in repeated batch reactions.To evaluate the reusability of the GA-treated cells having TtFTA,batch reactions were repeatedly performed. As a control experi-ment, the reactions were also carried out using the free cells. Al-

though a slight decrease was observed in the product concentra-tion, probably due to the thermal inactivation of TtFTA, theproduct yields of the reactions with the GA-treated cells weremaintained above 60 mol% when the reactions were repeated 6times (Fig. 5). In contrast, the product yields with the free cellslinearly decreased with repeated reactions. These results clearlydemonstrated the superiority of the GA-treated cells in the re-peated reactions.

Continuous bioconversion using GA-treated cells as whole-cell catalysts. For a further demonstration of the applicability ofthe GA-treated cells to long-term bioconversion, the cells wereused as a catalyst in a continuous reactor. The feeding rate of thesubstrate solution (0.25 ml/min) was experimentally decided toachieve a conversion yield of 60 mol%, and residence time (�) wascalculated to be 40 min. When the free cells were used as a catalyst,the filterability of the separation membrane was significantly im-paired, and product recovery at a constant rate could not beachieved (data not shown). This was likely due to the formation ofa cake layer on the separation membrane by debris of heat-dam-

FIG 3 SEM (A to D) and TEM (E to H) images of free and GA-treated E. coli cells before and after heat treatment at 70°C for 20 min. The open circle shows smallblebs on the cell surface.

FIG 4 Membrane permeabilities of GA-treated cells with (white bars) andwithout (gray bars) heat treatment. The dextran concentration was deter-mined using a calibration curve obtained by measuring the fluorescence in-tensities of the serial dilutions of the standards. The data are shown as aver-ages standard deviations (n � 3).

FIG 5 Reusabilities of free (diamonds) and GA-treated (triangles) cells. Eachreaction was performed at 70°C for 30 min. The data are shown as averages standard deviations (n � 3).

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aged cells. On the other hand, when the GA-treated cells wereused, a continuous product recovery could be maintained for atleast 640 min. The production yield decreased slightly in the first80 min but then remained stable until the end of the operation. Asa result, an overall product yield of 60 mol% could be achievedduring the operation for 640 min. Production rate was calculatedto be 3.0 mmol/liter/min, and 19.2 mmol (2.57 g) of malate couldbe produced from 32 mmol (3.71 g) of fumarate.

DISCUSSION

In this study, we demonstrated that the heat-induced leakage of athermophilic enzyme from recombinant E. coli cells could be pre-vented by the pretreatment of the cells with GA. Through theoptimization of the GA concentration, a thermotolerant whole-cell catalyst with an acceptable catalytic ability could be preparedand successfully used for a continuous bioconversion of fumarateto malate. This approach is much simpler and more universal forthe preparation of stable and selective immobilized enzymes thanconventional procedures, which typically involve many steps,such as protein extraction, enzyme purification, and the prepara-tion of immobilization carriers. The GA-treated cells exhibitedsufficient catalytic stability and reusability in both repeated-batchand continuous bioconversions at 70°C. They could catalyze fu-marate hydration in the continuous bioreactor over 10 h with aconstant product yield of 60 mol% or higher. This productivitywas considerably better than that in a previous report on the con-tinuous conversion of fumarate using permeabilized Saccharomy-ces cells, in which the malate yield was about 50% and the enzymelost 30 to 50% of its activity after 10 h (19).

The electron microscopic observation of the GA-treated cellsrevealed that heat treatment caused no apparent change in theoverall structure of the cells except for the formation of some smallblebs on their surface. GA is a five-carbon dialdehyde that canreact with several functional groups of proteins, such as amine,thiol, phenol, and imidazole, and form covalent bonds with them(20–23). The GA-mediated stabilization of the membrane struc-ture implied that GA might cross-link between protein moleculesin the outer membrane and those in the peptidoglycan layer of theE. coli membrane to make it more rigid. In fact, the cells changedto pink-red after the GA treatment; this phenomenon is typicallyobserved as a result of the interaction between membrane proteinsand GA (24, 25).

The natural barrier function of the cellular membrane oftenhampers sufficient whole-cell bioconversion since it limits the ac-cessibility of substrates to intracellular enzymes (26). Although nosignificant change was observed in the overall structure of cells,the membrane permeability test with different sizes of fluores-cently labeled dextrans revealed that relatively small molecules(up to at least 3 kDa) could penetrate the membrane barrier of theGA-treated cells heated at 70°C. The formation of small blebsobserved in the TEM analysis of the heated GA-treated cells waslikely responsible for the increased membrane permeability. Mostlipids and lipopolysaccharides comprising the cell membrane can-not be fully cross-linked to each other by GA (21). Thus, theirorientation might be disrupted at high temperatures. Conse-quently, small blebs were formed on the membrane of the GA-treated cells, and the rupture of the blebs resulted in the formationof membrane pores, through which molecules having low andmedium molecular masses can diffuse. The fact that the dextranwith an average molecular mass of 40 kDa hardly penetrated the

membrane was in good agreement with the observation that theleakage of TtFTA (approximately 51 kDa) could be prohibited byGA treatment.

The observation that the GA-mediated consolidation of themembrane structure was involved in the prevention of the heat-induced leakage of the enzyme raised the issue of whether theintracellular enzyme can freely diffuse in the cytosol of the GA-treated cells. To address this question, we attempted to extract thesoluble form of TtFTA from the GA-treated cells by disrupting thecells by lysozyme digestion followed by ultrasonication. TheTtFTA activity recovered in the resulting lysate was approximately17% of that detected in the cell extract of the free cells, indicatingthat intracellular TtFTA was, at least in part, present in solubleform and freely diffused in the cytosolic fraction.

ACKNOWLEDGMENTS

This work was in part supported by the Japan Science and TechnologyAgency (JST), PRESTO program.

We thank Y. Muranaka (Research Center for Ultra-High VoltageElectron Microscopy, Osaka University) for his technical assistance inthe electron microscopic analysis. We also thank Takashi Mimitsuka(Toray Industries, Inc.) for kindly donating the cell-separation mem-brane filter.

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