Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as aBiocatalyst for CO2 Sequestration

Byung Hoon Jo,a Im Gyu Kim,b Jeong Hyun Seo,b Dong Gyun Kang,b Hyung Joon Chaa,b

School of Interdisciplinary Bioscience and Bioengineeringa and Department of Chemical Engineering,b Pohang University of Science and Technology, Pohang, SouthKorea

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recentlythat this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alterna-tive for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neis-seria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investi-gated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store largequantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial celldisplayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3)formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. Itwas also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate thatthe engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration.

As the worldwide population and energy demands continue togrow, there have been increasing concerns about various

problems, such as environmental pollution and the depletion offossil fuels. Along with alternative energy and fuels, significanteffort has been concentrated on reducing anthropogenic carbondioxide (CO2) from fossil fuels, one of the major greenhouse gasescausing global warming (1).

Carbon capture and sequestration (CCS) is a promising tech-nology that has the potential to greatly mitigate CO2 emissionsfrom large industrial sources (2). CCS by mineral carbonation hasthe potential to capture much more carbon than other forms ofCO2 storage, such as geological sequestration of compressed gas(3). It also has other advantages, such as a thermodynamicallyfavorable reaction, raw mineral abundance, and the high stabilityof the final carbonate minerals. The major drawback for this se-questration pathway is the very slow reaction rate of natural car-bonation; the uncatalyzed hydration of CO2 has a rate constantbelow 10�1 under ambient conditions (4). Great expense and en-ergy usage are required at the industrial point sources of CO2 tospeed up this process (5). Therefore, acceleration of the reaction inan environmentally and economically viable way is the criticalpoint for CO2 mitigation by mineral carbonation.

In biology, the processes that convert CO2 into bicarbonate (orvice versa) can occur rapidly in a regulated manner under mildconditions. Carbonic anhydrase (CA), a zinc metalloenzyme, hasa central role in these processes, catalyzing the hydration of CO2,the rate-limiting step of carbonation (6). CA is one of the fastestknown enzymes, with a kcat value of up to �106, almost 10 milliontimes faster than the noncatalyzed natural reaction. This enzymehas been found in various organisms and is ubiquitous through-out the three domains of life, participating in distinct physiologi-cal functions, such as pH modulation, carbon concentration, andbiomineralization (7).

Recently, biomimetic utilization of CA as a green route for CO2

mitigation has been proposed and attempted for postcombus-tion CO2 capture by various entities, including CO2 Solutions

(Canada) and Carbozyme (United States) (1, 7, 8). Purified CAenzymes from natural sources or from recombinant strains havebeen used in those studies (9–13). Immobilization techniques toenhance stability and reusability have also been investigated (14,15). However, sequestration processes using CA would be of pri-mary use at facilities emitting large quantities of CO2, such assteelworks and power plants, where the use of costly purified en-zymes would be unnecessary and economically unfeasible. Thus,for the economical industrial use of CA technology, it may bepossible to employ a microbial whole-cell biocatalyst system har-boring efficient CA activity to eliminate the need for cell disrup-tion and enzyme purification, each of which is a potentially high-cost process (16, 17). Also it provides advantages, such as enzymestabilization and ease of handling, including simple separation ofenzyme (18).

The concomitant decrease in pH caused by CO2 hydration candisrupt intracellular pH homeostasis and interfere with cell viabil-ity and integrity (19, 20). This can happen when CA works withinthe cytoplasmic space of a microbial cell without a continuousenergy supply to pump protons out (21). More importantly, masstransfer problems occur in whole-cell systems containing cyto-plasmically expressed enzyme because the cellular membraneworks as a diffusion barrier, which can reduce cellular catalyticactivity to a significant level (22–24). To overcome these limita-tions, strategies were devised to place the enzyme in an outer re-gion of the cell. A bacterial whole-cell CA system that exploited

Received 18 July 2013 Accepted 21 August 2013

Published ahead of print 23 August 2013

Address correspondence to Hyung Joon Cha, hjcha@postech.ac.kr.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02400-13.

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

doi:10.1128/AEM.02400-13

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cell surface display of recombinant CA was recently proposed toreduce mass transfer resistance and enhance structural stability(25). In another study, researchers built engineered yeast (Saccha-romyces cerevisiae) cells displaying recombinant CAs or mineral-ization peptides for enhanced CO2 mineralization (26). However,the resulting activities were quite low, awaiting great progress forthe industrial application of the biomimetic whole-cell CA systemto be economically competitive.

In the present work, we have developed a highly efficient whole-cell biocatalyst by localizing a CA originating from Neisseria gonor-rhoeae (ngCA) to the periplasm of the bacterium Escherichia coli.ngCA is the first prokaryotic CA known from the �-class CAs, awidely studied family of CAs (27). Its catalytic activity is known to becomparable to that of human CA II, the fastest known CA (6). Addi-tionally, as ngCA is a monomeric enzyme, it is expected to be readilytranslocated into the periplasm by the secretory machinery of E. coliwhen tagged with an appropriate signal sequence. We investigatedthe catalyst’s capabilities and characteristics, demonstrating the effi-cient sequestration of gaseous CO2 in the form of calcium carbonate(CaCO3) as a proof of concept (Fig. 1).

MATERIALS AND METHODSStrains and plasmids. E. coli TOP10 (Invitrogen) was used for DNA ma-nipulation and maintenance. E. coli BL21(DE3) (Novagen) was employedas a host strain for recombinant protein expression. The gene encodingngCA was amplified from the genomic DNA of N. gonorrhoeae by PCRusing forward primer 5=-CCATGGGACATGGCAATCACACCC-3= andreverse primer 5=-AAGCTTTTCAATAACTACACGTGCATT-3=, whichcontain the restriction sites NcoI and HindIII (underlined). The PCRproduct was ligated into the pGEM-T Easy vector (Promega), and theinsert sequence was confirmed by direct sequencing. The insert was cutout of the vector with NcoI and HindIII and was then subcloned into thevectors pET-22b(�) (Novagen) and pETat (22) digested by the samerestriction enzymes. The resulting plasmids were named pPelB-ss::NCAand pTorA-ss::NCA, respectively. The plasmid pET-NCA (11) was usedfor cytoplasmic expression of ngCA. For expression of the truncated form

of ngCA, the gene was amplified using forward primer 5=-CATATGTCAGAAGAATTCCGTTTGTGC-3= and reverse primer 5=-AAGCTTTTCAATAACTACACGTGCATT-3=, which contain the NdeI and HindIII re-striction sites (underlined), and subcloned into the pET-22b(�) plasmid.The ngCA genes in the constructed recombinant plasmids had hexahisti-dine (His6)-tagged sequences at their 3= termini.

Preparation of whole cells and purification of periplasmic ngCA.The constructed recombinant plasmids were transformed into E. coliBL21(DE3). Each recombinant strain was grown in 200 ml Luria-Bertani(LB) medium (USB Corp., United States) supplemented with 50 �g/mlampicillin in a 500-ml flask at 37°C and shaking at 220 rpm. ngCA expres-sion was induced at mid-log phase (�0.7 cell density at the optical densityat 600 nm [OD600]) by adding 1 mM (final concentration) isopropyl-�-D-thiogalactopyranoside (IPTG; Carbosynth, United Kingdom) and 0.1mM ZnSO4. Cells were incubated for 12 h and then harvested by centrif-ugation at 4°C and 4,000 � g for 10 min. The harvested cells were washedwith Tris-sulfate buffer (20 mM [pH 8.3]) and resuspended in the samebuffer at the same OD600 (�10). The different recombinant cells with thesame OD600 showed essentially identical absorption spectra at least be-tween 400 and 800 nm. The cell numbers were counted with hemocytom-eter, and the number of cells per ml · OD600 was estimated to be from 9.06� 108 to 9.31 � 108.

Periplasmic ngCA was purified by immobilized metal affinity chroma-tography (IMAC) using Ni-nitrilotriacetic acid (NTA) agarose beads(Qiagen, United States) according to the manufacturer’s instructions. Theeluted protein was dialyzed against Tris-sulfate buffer (20 mM [pH 8.3]).The protein concentration was determined using the Bradford reagent(Bio-Rad, United States) and bovine serum albumin (BSA) (Sigma-Al-drich, United States) as a standard.

Cell fractionation and Western blot analysis. Cell lysates were pre-pared by disrupting the cells using an ultrasonic dismembrator (Sonics &Materials) for 3 min at 20% amplitude (3-s pulse on and 10-s pulse off) onice. They were centrifuged at 4°C and 10,000 � g for 20 min, and theresulting supernatant was designated the soluble (S) fraction. The remain-ing pellet was designated the insoluble (IS) fraction and resuspended inthe same volume of Tris-sulfate buffer (20 mM [pH 8.3]) for the subse-quent analysis. For membrane fractionation, the soluble fractions werefurther centrifuged at 4°C and 150,000 � g for 2 h using an ultracentrifuge

FIG 1 Design concept for the engineered periplasmic whole-cell system and its biocatalytic function for CO2 sequestration. Recombinant CA transforms CO2

into HCO3� in the periplasm of E. coli. The anions are transported out of the cell through outer membrane porins or transporters and then react with Ca2� ion

to form CaCO3 precipitate. The solid black lines indicate the reaction or transfer of substrates that are experimentally confirmed or are simply deduced. Thedotted black lines represent the reactions or transfers that may depend on the extracellular pH. The symbols and lines in gray show the CO2 pathway viacytoplasmic CA that is hindered by physical (cytoplasmic membrane) or physiological (e.g., pH regulation) barriers.

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(Beckman Coulter, United States). The resultant supernatant was desig-nated the “soluble-minus-membrane (S*)” fraction. The remaining pelletwas designated the membrane (M) fraction and resuspended in the samevolume of Tris-sulfate buffer. Intact cells were partially lysed and fraction-ated into periplasm (P) and spheroplasts (Sp) by osmotic shock as previ-ously described (22). Protein expression was analyzed by Westernblotting. Proteins in the samples were separated using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted ontoa nitrocellulose membrane (Whatman, United States). Sequential treat-ments of primary and secondary antibodies were applied. Monoclonalanti-His6 antibody (ABM, Canada), polyclonal anti-�-lactamase anti-body (Millipore, United States), polyclonal anti-ATP synthase (AtpB) an-tibody (Agrisera, Sweden), and polyclonal anti-GroEL antibody (Sigma-Aldrich, United States) were used as the primary antibodies. Alkalinephosphatase-conjugated anti-mouse IgG and anti-rabbit IgG (Sigma-Al-drich, United States) were used as the cognate secondary antibodies whereappropriate. Target proteins were chromogenically detected with ni-troblue tetrazolium–5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Sigma-Aldrich, United States).

N-terminal sequencing of the recombinant ngCA. N-terminal aminoacid sequencing of the protein was performed by Edman degradation.After SDS-PAGE of the purified ngCA, the gel was blotted onto a polyvi-nylidene difluoride (PVDF) membrane (Millipore, United States), andthe membrane was stained with Coomassie brilliant blue R-250 (Bio-Rad,United States). After a thorough washing, the band of interest was cut outand subjected to Edman degradation using a Procise 492 sequencer (Ap-plied Biosystems, United States).

CO2 hydration activity assay. An electrometric method was adoptedto determine the CO2 hydration activity of the prepared samples (wholecells and cell lysates) (28). The assay was performed in a way similar to apreviously described protocol (11). First, appropriate amounts of thesamples (25, 50, or 100 �l) were added to 3 ml Tris-sulfate buffer (20 mM[pH 8.3]), and 2 ml CO2-saturated water (pH 3.8) was rapidly poured intothe mixture with vigorous stirring. All preparations and reactions wereperformed on ice. pH changes were recorded, and the time required forthe pH to drop from 8.3 to 6.3 was used for activity calculation. Based onthe Wilbur-Anderson (WA) unit, whole-cell activities per ml of unit celldensity (U/ml · OD600) were evaluated. Purified bovine carbonic anhy-drase (bCA; Sigma-Aldrich, United States) dissolved in Tris-sulfate bufferwas used as a positive control.

Sequestration of CO2 in CaCO3. The sequestration of CO2 in CaCO3

using the whole-cell catalyst was performed, and CaCO3 precipitation wasturbidimetrically monitored via A600 using UV/Vis spectrophotometer(Mecasys, South Korea). Five hundred microliters of CO2-saturated waterwas added to a reaction cuvette containing 450 �l buffer (1 M Tris, 20 mMCaCl2 [pH 8.5, 9.5, or 11]) and 50 �l of samples (or Tris-sulfate buffer [pH8.3] as a blank) and thoroughly mixed. Immediately after the cuvette wasclosed with a plastic cap to prevent CO2 leakage, the precipitation wasmonitored for an appropriate duration. The buffers and CO2-saturatedwater were prepared at ambient temperature (25 � 2°C), and the reactionwas performed at 37°C. The required time for onset of precipitation (de-fined here as the first second of the time period that showed an averagerate of increase more than 0.001 A600/s) was recorded. Once precipitationwas detected, initial slopes of absorbance curves were used to comparerelative rates.

Characterizations of CaCO3 solid crystals. The carbonate mineral wasprecipitated at pH 11 as previously described, except that the total volume wasscaled up from 1 ml to 20 ml. The reaction was carried out for 5 min, and themixture was filtered through 0.45-�m-pore membrane filters (Millipore,United States). The precipitates remaining on the membrane were dried at80°C overnight. The identities and polymorphs of the precipitates were ana-lyzed by X-ray powder diffraction (XRD) as previously described (11). Toverify the crystal morphology, imaging by scanning electron microscopy(SEM) was performed with JSM7401F (JEOL, Japan).

Thermal stability test. Purified recombinant ngCA and whole cellswith periplasmic ngCA were used for the temperature stability test. Thepurified protein was diluted to a concentration of �1 mg/ml. The sampleswere pretreated by incubation at 40 or 50°C for an appropriate time. Theywere then stored at 4°C until the activities were measured. The cells werecentrifuged and resuspended in the same buffer to eliminate the effect ofenzyme leakage from thermally induced cell lysis. Activities were esti-mated by the CO2 hydration assay, and the residual activities were calcu-lated.

RESULTS AND DISCUSSIONPeriplasmic expression of ngCA in E. coli. There are two pathwaysfor the periplasmic export of proteins in E. coli: the twin-argininetranslocation pathway (Tat) and the general secretion (Sec) pathway(29). Because cytoplasmic ngCA was strongly expressed with func-tional folding (cytCA lanes in Fig. 2a) (11), it was assumed, for thepurpose of enhancing secretion efficiency, that the Tat pathway,which transports folded proteins, would be more suitable than theSec pathway, which translocates proteins prior to folding. As one ofthe most efficient Tat signal peptides, the TorA leader sequence (30)was chosen for high-level periplasmic secretion of ngCA. As expected,the PelB signal sequence (Sec pathway) was not as effective as TorAsignal for periplasmic expression of ngCA in terms of the final expres-sion level of the mature protein (see Fig. S1 in the supplemental ma-terial). Due to the His6-tagged peptide at its C terminus, ngCA couldbe detected by Western blotting using an anti-His6 antibody evenafter signal sequence cleavage, because the signal sequence was lo-cated at the N terminus of the protein.

After we cultivated the recombinant cells and induced heterol-ogous protein expression, it was found that mature ngCA washighly expressed primarily in a soluble form, and premature poly-peptide was only observed in the insoluble fraction (periCA lanesin Fig. 2a). Surprisingly, recombinant cells harboring the periplas-mic vector pTorA-ss::NCA were poorly fractionated into periplas-mic fraction and spheroplasts by cold osmotic shock, and no bandfor ngCA was observed in the resulting periplasmic fraction(periCA lanes in Fig. 2b), while cells containing the parent vector,pET-22b(�), and the cytoplasmic vector, pET-NCA, released suf-ficient �-lactamase, a periplasmic protein marker (NC and cytCAlanes in Fig. 2b). It is unlikely that the seeming failure of periplas-mic fractionation was caused by the targeted protein stuck to thecytoplasmic membrane during the translocation process (31), be-cause most proteins were not located in the membrane fraction(Fig. 2c). N-terminal sequencing revealed that recombinant ngCAunderwent the maturation process at the correct position betweenalanine 42 and alanine 43 (Fig. 2d). Because, according to thecurrent model (29), signal cleavage occurs after target protein istranslocated, it can be concluded that the recombinant ngCA wassuccessfully expressed in the E. coli periplasm. We surmised thatthe incomplete cell fractionation and resistance of ngCA toperiplasmic release are related to a structural change in theperiplasmic region of the cell. This intriguing observation remainsto be further explored.

A minor band consistently appeared throughout the Westernblot analyses with a smaller molecular mass than that of the ma-ture form (periCA lanes in Fig. 2a). The smaller protein was iden-tified as a fragment ngCA, specifically cleaved at the C-terminalside of leucine 66 (TorA signal lane in Fig. 2d). This truncatedform of ngCA was not detected when the enzyme was producedwithout a signal sequence (cytCA lanes in Fig. 2a), nor was itdetected when it was expressed with the PelB signal sequence

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(PelB lane in Fig. S1 in the supplemental material). This impliedthat the unexpected cleavage occurred during or after the translo-cation process of the Tat pathway. The truncated form of ngCAhad no detectable activity when it was independently expressed inthe cytoplasm (see Fig. S2 in the supplemental material). This wasan expected result, because the removed region contains somehighly conserved residues, including tryptophan 52, tyrosine 54,and tryptophan 63, that have structural or functional roles fornormal CA activity (32). Thus, inhibition of the factor(s) respon-sible for this second cleavage may be needed to increase the pool ofactive periplasmic CA.

CO2 hydration ability of the constructed periplasmic whole-cell catalyst. CA catalyzes the hydration of CO2, whose product im-mediately dissociates into protons (H�) and bicarbonate (HCO3

�),and thus lowers the pH. The general electrometric or colorimetricassay for CA activity monitors the pH drop of a buffer solution di-rectly or indirectly (28, 33). We expected that these methods mightalso be applicable for measuring whole-cell CA activity because CO2,a nonpolar gaseous compound, would readily penetrate cellularmembranes (34), and the dissociated H� could be transported out ofthe cell through a specialized transport system(s) (21, 35–38). Withthese considerations, the electrometric assay was taken as an appro-priate method for measuring CA activities of whole-cell or cell lysate(see the text in the supplemental material). Purified bCA was used asa positive control, and its activity was approximately 2,600 to 3,300U/mg in our experimental system.

As shown in Fig. 3a, periplasmic expression produced a muchhigher activity level (1.77 U/ml · OD600) than cytoplasmic expres-sion (0.15 U/ml · OD600). In contrast, a cytoplasmic cell lysateshowed �1.5-fold higher hydration activity (15.82 U/ml · OD600)than a periplasmic cell lysate (10.39 U/ml · OD600) (Fig. 3b). Theseresults demonstrated that the cytoplasmic CA expression systemhas higher total CA activity, which coincides with the Western blotresults (Fig. 2a). However, the ratio of cell lysate activity to whole-

cell activity for the periplasmic CA system was an order of magni-tude lower (�6) than that of the cytoplasmic CA system (�102).This observation clearly indicates the effectiveness of periplasmictranslocation for overcoming the physical or physiological barri-ers imposed by the cytoplasmic membrane (22, 39). Again, thisindirectly demonstrated that ngCA was successfully exported intothe periplasm of E. coli. The negative-control whole cells contain-ing the parent vector showed no detectable activity (see Fig. S3a inthe supplemental material).

The pH of the cytoplasm is tightly regulated and maintained byvarious mechanisms and is not severely moved by internal or ex-ternal perturbations even under resting (nongrowing) conditions(21). If protons were rapidly generated by the action of cytoplas-mic ngCA and expelled from the cell by the various pH homeosta-sis mechanisms, the pH of the buffer would decline much fasterthan was observed in our experiments. However, this was not thecase, leaving two possible explanations: (i) many of the generatedprotons were consumed in some way (21, 35), or, as is more likely(ii) CO2 diffusion into the cytoplasm and/or H� expulsion fromthe cell was too slow to critically affect the rate of pH change in thebuffer under the experimental conditions. The hampered H� ef-flux may be explained by the fact that H� pumping out of thecytoplasm mostly requires available free energy, such as ATP orthe ion gradient (21, 35–38), because the cells of our experimentswere in the resting condition. In contrast to the cytoplasmic pH,the periplasmic pH of E. coli is in equilibrium with the external pH(40), which means that protons can freely pass in and out of theperiplasm in response to the pH gradient across the outer mem-brane. Therefore, protons formed in the periplasm can be readilytransported out of the cell and thus reduce the pH of the buffer.This explains why a whole-cell system harboring periplasmicngCA showed significant CO2 hydration activity compared to itscytoplasmic ngCA counterpart.

The ratio of periplasmic whole-cell activity to cytoplasmic

FIG 2 Analyses of ngCA expression in E. coli. (a) Fractionation of soluble and insoluble proteins. (b) Periplasmic fractionation. �-Lactamase and GroEL were used asperiplasmic and cytoplasmic protein markers, respectively. (c) Membrane fractionation. ATP synthase was chosen as a membrane protein marker. (d) (Left) Theidentities of the proteins corresponding to the Western blot bands. The gray sequence letters represent the five residues from the N termini revealed by Edmandegradation. (Right) The N-terminal partial sequence of recombinant ngCA. The TorA signal sequence, which is known to be cleaved, is underlined. The five N-terminalresidues of the cleaved protein are also indicated in gray. Lanes: M (panel a), molecular mass marker; CL, cell lysate; S, soluble fraction; IS, insoluble fraction; P,periplasmic fraction; Sp, spheroplast fraction; S*, soluble-minus-membrane fraction; M (panel c), membrane fraction. Abbreviations: NC, negative-control cellsharboring pET-22b(�); cytCA, cytoplasmic CA-expressing cells harboring pET-NCA; periCA, periplasmic CA-expressing cells harboring pTorA-ss::NCA.

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whole-cell activity was approximately 12, while the correspondingratio was only approximately 2 for the previously reported whole-cell system harboring cell-surface-anchored CA (25). Because sur-face-displayed enzymes are free to interact with any external sub-strate (41), the striking enhancement observed in our system maybe attributed to the significantly higher expression level ofperiplasmic ngCA, which even compensated for the restriction ofsubstrate diffusion by the outer membrane. This observationdemonstrates the utility of a periplasmic expression strategy forconstructing a CO2-sequestrating whole-cell biocatalyst.

Acceleration of CO2 mineralization. Although the assay forCO2 hydration activity measures H� ions, the sequestration ofCO2 in CaCO3 (or other carbonate mineral forms) requires car-bonate (CO3

2�) or bicarbonate, not protons. Because the cell is amembrane-enclosed structure and the membrane functions as aselective barrier for the passage of various substances, includingions such as HCO3

� and CO32�, CO2 hydration activity may not

directly correlate with the ability of the whole cell to precipitateCaCO3. To verify the ability of the constructed periplasmic whole-cell system to efficiently sequestrate CO2 in carbonate mineral, theconversion of CO2 to CaCO3 was examined. CaCO3 is formed bythe reaction of Ca2� and CO3

2�, the latter being formed fromHCO3

�. Because the pKa for HCO3� dissociation is quite high

(�10.3) (42), alkaline buffer with a pH of 11 was used for theconversion reaction. Although CA accelerates the rate of the CO2

hydration reaction, it does not affect the equilibrium between thedifferent species of carbonate (43). In a closed system, the finalamount of precipitated CaCO3 does not depend on whether thereaction is catalyzed by CA or not (10, 12). Therefore, we focusedon the ability of the whole-cell biocatalyst to improve the precip-itation (mineralization) rate rather than measure the quantity ofthe resulting precipitate. Turbidimetric measurement was usedhere to compare the relative mineralization rates of the whole cells(see the text in the supplemental material). The identity and mor-phology of the precipitates were confirmed by XRD (Fig. 4a) andSEM analyses (Fig. 4b). No critical differences in sizes, composi-tions, or morphologies of calcite and vaterite were observed be-

tween the examined samples, except that whole cells left rod-shaped scars indicative of cell morphology on the CaCO3 surfaces(periCA in Fig. 4b).

First, we measured the time required for precipitation to occur(44, 45). The times (means � standard deviations) required foronset of CaCO3 precipitation were as follows: blank, 108.7 � 4.2 s;negative-control cells [pET-22b(�)], 105.3 � 1.5 s; cytoplasmicCA-expressing cells (pET-NCA), 86.7 � 10.3 s; 1� (i.e., 50 �l)periplasmic CA-expressing cells (pTorA-ss::NCA), 35.7 � 2.1 s;2� (i.e., 100 �l) periplasmic CA-expressing cells (pTorA-ss::NCA), 18.0 � 1.7 s; and 0.5� (i.e., 25 �l cell lysate used instead of50 �l) periplasmic CA-expressing cell lysate (pTorA-ss::NCA),34.3 � 3.8 s. Note that the uncatalyzed reaction (blank) required�109 s to initiate precipitation, and a similar result was seen in theparent vector negative control (105.3 � 1.5 s). A significant in-crease was achieved with whole cells containing periplasmic ngCA,reducing the delay in onset of precipitation to one-third (35.7 �2.1 s) of the blank. When twice the number of the cells (2�) wasused, the required time (18.0 � 1.7 s) was reduced to half of thetime required in the 1� case (35.5 � 2.1 s). However, the perfor-mance was not significantly improved for whole cells harboringcytoplasmic ngCA. From these results, it was obvious that thetranslocation of CA out of the E. coli cytoplasm was a minimumqualification and not an option from an engineering perspective.

Next, we examined the relative initial precipitation rate, as wasdetermined from the slope of the curve. In principle, the timereduction for the onset of catalysis means that the pool of CO3

2�

required for precipitation is generated faster than in the uncata-lyzed control. In turn, this faster generation of CO3

2� entails afaster precipitation rate after the reaction has begun. Accordingly,if there are more enzymes (higher activity), there should be ahigher initial precipitation rate as well as a briefer delay of onset(Fig. 5a). We found that by satisfying this rule, the periplasmicngCA cells greatly increased the precipitation rate following onsetcompared with the other cases (Fig. 5b). Interestingly, the precip-itation rate was also slightly but meaningfully enhanced by thenegative-control cells, even though they did not have a detectable

FIG 3 CA activities assayed by CO2 hydration reactions. (a and b) Activities of whole cells (a) and cell lysates (b). The activities are expressed in WA units.Abbreviations: cytCA, cytoplasmic CA-expressing cells; periCA, periplasmic CA-expressing cells.

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CO2 hydration activity. This result is not surprising, because the E.coli cell surface carries a net negative charge (46), and this canaccelerate the rate of CaCO3 crystal growth, presenting anotheradvantage of using a whole-cell system for CO2 mineralization(26, 47). These results collectively demonstrate that our designconcept is useful in developing an efficient catalyst for acceleratingCO2 sequestration in CaCO3.

When half of the volume (0.5�) was applied, the time delay(�34 s) of the periplasmic cell lysate and the initial rate of precip-itation were very similar to those (�36 s) of the correspondingwhole-cell with the 1� volume, as described above (Fig. 5b). Thismeans that the cell lysate has an �2-fold-higher activity forCaCO3 precipitation than the whole cell. This result did not coin-cide with the result obtained in the CO2 hydration assay (�6-fold)(Fig. 3). Because the permeability of outer membrane was notincreased during the assay (see Fig. S4 in the supplemental mate-rial), this result suggests that the excretion of HCO3

� (or CO32�)

is naturally more efficient than H� expulsion through the outermembrane. Accordingly, this indicates that the actual level of theCO2 sequestration power of our periplasmic system is muchhigher than that predicted in the CO2 hydration assay. In addition,it implies that mass transfer across the outer membrane of the cellwas still a rate-limiting step in CO2 sequestration, as previouslyshown (48). The HCO3

� or CO32� produced by periplasmic CA

must be exported out of the periplasm to permit CaCO3 precipi-tation. Because these are ionic substances that cannot passthrough the lipid bilayer, transporters or channels must facilitatethe movement (Fig. 1). Porins are channel proteins in the outermembrane that are responsible for the passing of ions driven by aconcentration gradient (48, 49). Notably, homologous porinswere stably overexpressed in E. coli (50), and functional improve-ments in extracellular electron transfer were achieved as a result ofheterologously expressed porin (51). There are some reports thateven CO2 uptake can be facilitated by expression of aquaporin inplant and animal cells (52). Therefore, identification of the re-sponsible porins and increasing their number in the outer mem-brane might improve the performance of our whole-cell system.

It is not known which ion (HCO3� or CO3

2�) actually leaves theperiplasmic space. The identity of the ion is likely dependent on theextracellular pH, because the periplasmic pH follows that of the ex-tracellular environment (40), and the dissociation of HCO3

� is pHdependent (42). Because the pH value was quite high in our experi-ments, it is possible that some HCO3

� ions were further dissociatedand reacted with Ca2� to form CaCO3 within the periplasm. Thisissue needs to be further addressed to verify whether CaCO3 forma-tion in the periplasm would limit the reusability of the system bygradually destroying the periplasmic CA or would benefit the integ-

FIG 4 (a) XRD peaks and (b) SEM images of the CaCO3 precipitates obtained by the different samples. The scale bars in the SEM images are 10 �m for �1,000 (upper)and 2 �m for �4,000 (lower). The SEM images for the other whole cells were similar to the images for periCA cells. Abbreviations: Blank, uncatalyzed reaction; bCA,bovine carbonic anhydrase; NC, negative-control cells; cytCA, cytoplasmic CA-expressing cells; periCA, periplasmic CA-expressing cells; C, calcite; V, vaterite.

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rity and stability of the system by solidifying periplasmic compo-nents, including CA, into a nanocomposite (53).

CaCO3 precipitation under relatively low-pH conditions. Aspreviously mentioned, CA cannot shift the equilibrium but ratheraccelerates the reaction rate. However, it may be expected that theamount of precipitate formed before equilibrium is establishedcan be larger than that formed after the system has reached equi-librium, especially under the relatively low-pH conditions whereboth formation and dissolution of CaCO3 occur. Thus, we testedthe hypothesis with starting pHs of 8.5 and 9.5 to see how periplas-mic whole-cell catalyst would affect CaCO3 formation.

Figure 6 shows the curves for CaCO3 precipitation. With abuffer of pH 8.5, little CaCO3 formation was observed, regardless

of the presence of the catalyst. This was expected because fewCO3

2� ions exist below this pH (42). In contrast, considerableprecipitation occurred in the reactions starting at pH 9.5, and theperiplasmic ngCA cells also accelerated the precipitation rate sig-nificantly. Interestingly, the amount of CaCO3 formed in thewhole-cell CA system far exceeded the amount formed withoutCA (blank), although most of the precipitate that formed in eachexperiment dissolved within several minutes, due to the relativelylow pH (data not shown). Furthermore, the whole-cell catalystwas more stable under the low-pH condition than under thehigh-pH condition (see Fig. S4 in the supplemental material).Thus, if CaCO3 precipitate from an open, heterogeneous carbon-ate system could be harvested before its dissolution, it might bepossible to sequester CO2 more efficiently using this CA technol-ogy even at a pH below the pKa of HCO3

� or CO32�, reducing the

expense of maintaining an elevated pH.Thermal stability of the periplasmic whole-cell catalyst. Sta-

bility of CA at high temperature (40 to 60°C) is required in post-combustion CCS (7, 8). Thus, we examined the thermal stabilityof the periplasmic whole-cell system and compared it with that ofpurified enzyme as a control. Enzymes potentially leaked by celllysis during high-temperature incubation were removed beforetesting by centrifuging the cells and resuspending them in a freshbuffer. We found that the cells harboring periplasmic ngCA exhib-ited excellent stability compared with purified ngCA (Fig. 7). Dur-ing the 5-h incubation period at 40°C, periplasmic ngCA retainedall activity, while free ngCA retained less than 70% of its initialactivity (Fig. 7a). When incubated for 5 h at 50°C, the residualactivity (�60%) of the periplasmic ngCA was much greater thanthat (�25%) of the free enzyme (Fig. 7b). These results clearlyshow that the activity of periplasmic ngCA is protected by somecellular mechanism(s) in the course of high-temperature incuba-tion. Immobilization of ngCA in the periplasm, a hypothetic rea-son for the resistance to periplasmic release by osmotic shock, maybe the responsible factor contributing to the enhanced thermalstability. The mechanisms may also include protection by heat

FIG 5 Relative initial rates of CaCO3 precipitation. (a) Precipitation by different amounts of purified periplasmic ngCA. In the inset, the curves were set to the startingpoints of precipitation. Symbols represent enzyme concentrations as follows: solid circles, 2�; open circles, 3�; solid triangles, 4�; open triangles, 5�. The amount 5�corresponds to 50 �l of 1 mg/ml purified enzyme. (b) Initial precipitation rates of whole cells. The curves (showing different time delays as discussed above) were alignedat the onset points of precipitation, facilitating comparison of the initial rate differences. The intrinsic turbidities of the cells were subtracted from the curves. Abbrevi-ations: NC, negative-control cells; cytCA, cytoplasmic CA-expressing cells; periCA, periplasmic CA-expressing cells; CL, cell lysate.

FIG 6 CaCO3 precipitation patterns under relatively low-pH conditions.Changes in turbidity during the first 4 min are profiled. Buffers with pH 8.5(upper) or pH 9.5 (lower) were used. For the whole cells, the intrinsic turbid-ities were subtracted from the curves. Abbreviation: periCA, periplasmic CA-expressing cells.

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shock proteins (54) or an increase in ion permeability caused bythe elevated temperature (55). We expect that finding or engineer-ing a thermostable CA would synergistically improve the thermalstability of the periplasmic whole-cell system for practical appli-cation to postcombustion capture of industrial CO2.

Feasibility of practical application of the periplasmic whole-cell catalyst system. There are two pioneering studies on con-struction of microbial cells with recombinant CA for CO2 mitiga-tion and/or mineralization (25, 26). In both, whole cells wereconstructed using a surface display system, which resulted in quitelow CA activities. Accordingly, the periplasmic system engineeredin this work is currently the most efficient whole-cell CA catalyst,exhibiting 2 to 3 orders of magnitude higher activity than theother reported systems (Table 1).

Recently, the first detailed investigation on the economic im-pact of the biologically catalyzed system on the process cost forCO2 mineralization was reported (26). The bCA surface-display-ing yeast cells were constructed, and it was predicted that a processusing the engineered yeast is �10% more cost-effective than aprocess without biocatalytic acceleration. In addition, it was esti-mated that the cost would be further reduced (�4%) if a hypo-thetical 10� enhancement in the mineralization rate could beachieved. Our periplasmic bacterial whole-cell catalyst system hasat least �300-fold higher activity than the bCA-displaying yeast

system on the basis of cell density (Table 1). If we roughly assumethat 10� enhancement of the mineralization rate requires a 30�increase of CO2 hydration activity, with the consideration thatCO2 hydration is the rate-limiting step for CaCO3 mineralization,the periplasmic whole-cell CA system can easily attain the costreduction by �14%. Then, the remaining 10� advantage mightbe used for reducing the sizes of microbial reactor or CO2 absorbercolumn and accompanying equipment, allowing further reduc-tion of both direct and indirect costs in a significant level. Al-though the process design was optimized for the bCA-displayingyeast system, this simple calculation can show the feasibility ofenhancing the cost effectiveness of the industrial process by apply-ing our periplasmic whole-cell CA system.

ACKNOWLEDGMENTS

This work was supported the Marine Biotechnology Program and theManpower Development Program for Marine Energy funded by the Min-istry of Oceans and Fisheries, South Korea.

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