Process Biochemistry 44 (2009) 1293–1297

Short communication

Purification and characterization of an extracellular carbonic anhydrase fromPseudomonas fragi

Anjana Sharma *, Abhishek Bhattacharya, Shyamlata Singh

Bacteriology Laboratory, Department of P.G. Studies and Research in Biological Sciences, R.D. University, Jabalpur 482001 (M.P.), India

A R T I C L E I N F O

Article history:

Received 6 May 2009

Received in revised form 30 July 2009

Accepted 30 July 2009

Keywords:

Carbonic anhydrase

Pseudomonas fragi

Purification

Characterization

Alkaliphilic

Affinity chromatography

A B S T R A C T

Extracellular carbonic anhydrase was purified from Pseudomonas fragi isolated from CaCO3 enriched soil

samples. The enzyme is induced in presence of CaCO3 and is envisaged to play an important role in

bicarbonate ion transport. The 75% ammonium sulphate dialysate was purified by single step affinity

chromatography with 86% yield. It is a trimeric protein having a subunit molecular weight of 31.0 kDa

and was stable at pH 7.0–8.5 and temperature 35–45 8C. Lead, mercury and EDTA had an inhibitory effect

on CA activity, whereas zinc, iron and cadmium increased it. The presence of esterase activity along with

IC50 of sulphonamides and anionic inhibitors indicated that CA from P. fragi belonged to a-class. The CA

stability in presence of different salts, as well as in alkaline pH and high temperature makes it a potential

candidate to be exploited for biomimetic CO2 sequestration.

� 2009 Elsevier Ltd. All rights reserved.

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Process Biochemistry

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1. Introduction

The main features of the ‘modern’ biosphere were formed about2 billion years ago, when prokaryotes dominated on earth [1].Living organisms and their specific enzymes may play animportant role in the operation of dynamic ecosystems [2]. Recentstudies have demonstrated the effect of extracellular carbonicanhydrase (CA) from microbial origin on enhanced Ca2+ releasefrom limestone [3] and in assimilation of inorganic carbon (Ci), inthe form of bicarbonate from extreme ecological niche like sodalakes [1].

Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc containingmetalloenzyme catalyzing the reversible hydration of CO2

(CO2 + H2O$ HCO3�). CA is important in biological systems

because the uncatalysed interconversion between CO2 and HCO3�

is slow around neutral pH. Its high efficiency catalysis isfundamental to many biological processes, such as photosynthesis,respiration; pH homeostasis and ion transport [4]. The enzyme CAhas been located in different cellular fractions that serve variousphysiological and metabolic functions [4]. Extracellular CA hasbeen envisaged to play an important role in improving theefficiency of inorganic carbon (Ci) transport [4]. Presently CAs aredivided into three main classes, a, b and g, which have nosignificant primary sequence identity and supposedly are evolu-tionarily independent [5]. The existence of additional d [6] and e

* Corresponding author. Tel.: +91 761 2416667; fax: +91 761 2603752.

E-mail addresses: anjoo1999@gmail.com, anjoo_1999@yahoo.com (A. Sharma).

1359-5113/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2009.07.022

classes of CAs have also been reported [7]. Recent developmentshave indicated the presence of CA genes from two or even all threeknown classes, some prokaryotes even containing multiple genesfrom the same class [5]. The presence of multiple carbonicanhydrase genes suggests the importance of this enzyme inprokaryotic physiology; however the roles are still largelyunknown [5].

The enzyme has been purified from humans, other mammals,vertebrates, invertebrates, diatoms, cyanobacteria, algae, bacteriaand archaea [5,8]. Beydemir et al. [9] purified HCA I & HCA II andstudied the inhibition of two CAs with heavy metal salts of Pb(II),Co(II) and Hg(II). The nature of inhibition of both the carbonicanhydrases varied with different metal ions involved in the study.Hiser et al. [10] reported the effect of low molecular weight plasmainhibitors of rainbow trout on Sepharose 4B-L-tyrosine-sulfanyla-mide purified CA in in vitro human and in in vivo rat erythrocytes.Although the enzyme is ubiquitous in eukarya domain it hasreceived scant attention from bacteria and archaea domain, havingbeen purified from only five species [5].

Here we report the identification, purification and character-ization of a-type extracellular CA from the prokaryote Pseudomo-

nas fragi isolated from calcium carbonate enriched soil. The studyalso helps in establishing the role of CA as an efficient marker forcarbon sequestration and environmental amelioration.

2. Materials and methods

Various media, buffer components, and dialysis tubing (15 kDa cut off) were

purchased from Hi-Media, Mumbai, India. p-AMBS-agarose and bovine carbonic

anhydrase were purchased from Sigma–Aldrich, St Louis, MO, USA. Protein

A. Sharma et al. / Process Biochemistry 44 (2009) 1293–12971294

reference markers, PMSF, DTT, acrylamide and bis-acrylamide were purchased from

Bangalore Genei, Bangalore, India. All other chemicals used were of analytical grade

and were purchased from standard sources.

2.1. Strain and growth conditions

P. fragi was obtained from the Bacterial Germplasm Culture Collection,

Bacteriology laboratory, Department of Biological Sciences, Rani Durgavati

University, Jabalpur, India. The organism was originally isolated from calcium

carbonate enriched soils, around the CaCO3 kilns (Satna, Madhya Pradesh, India).

The organism was grown at pH 7.5 for 24 h at 37 8C in peptone broth containing (g/

l): glucose 1.0; beef extract 3.0; peptone 5.0; NaCl 5.0; CaCO3 6.0. [11], was

centrifuged (10,000 � g for 10 min) and the supernatant was used for enzyme assay

and purification studies.

2.2. Carbonic anhydrase assay

The electrometric method of Wilbur–Anderson, 1948 [12] was followed with

certain modifications. The samples were assayed at 4 8C by adding 0.1 ml of the

enzyme solution to 3 ml of 20 mM Tris–HCl buffer, pH 8.3. The reaction was

initiated by the addition of 2 ml ice cold CO2-saturated water. Units of activity were

calculated with the equation (tc � t)/t. The time required for the drop in one unit pH

from 8.3 to 7.3 (t) was measured, while tc (control) was the time required for the pH

change (8.3–7.3) when buffer was substituted for the test sample. Values are the

means of three replicates. Protein content was estimated colorimetrically following

the Lowry’s method [13].

2.3. Effect of calcium carbonate on extracellular CA production

The organism was grown in peptone broth (pH 7.5) supplemented with 6%

calcium carbonate and incubated for 12 h, 24 h, 36 h, and 48 h at 37 8C. Parallel

controls were established in peptone broth without calcium carbonate. To confirm

the induced nature of extracellular CA from P. fragi, seed culture was raised for 12 h

in peptone broth containing 6% calcium carbonate. This seed inoculum (1%) was

used to inoculate peptone broth without calcium carbonate. Samples were removed

at periodic intervals of 12 h, 24 h, 36 h, and 48 h and assessed for the presence of CA

under standard assay conditions following the electrometric method.

2.4. Purification of carbonic anhydrase

2.4.1. Enzyme purification

The broth supernatant was subjected to ammonium sulphate fractionations of

35%, 50%, and 75%. Enzyme activity was observed only in 75% ammonium sulphate

precipitate that was extensively dialysed (15 kDa cut off) for 16 h against 2 l of Tris–

HCl buffer (100 mM, pH 7.6). These steps and the subsequent purification steps

were performed at 4 8C [14]. Following dialysis, the soluble proteins from the

dialysate were combined with an equal volume of agarose bound p-aminomethyl-

benzenesulfonamide (p-AMBS-agarose) pre-equilibrated with 25 mM Tris–HCl/

0.1 M Na2SO4, pH 8.7. The slurry was mixed overnight at 4 8C and loaded onto a

chromatography column the following day. The column was washed with 25 mM

Tris–HCl, 22 mM Na2SO4 (pH 8.2), followed by 25 mM Tris–HCl, 300 mM NaClO4

(pH 8.7). The CA protein was eluted with 100 mM sodium acetate, 500 mM NaClO4

and 0.01 mM EDTA (pH 6.0). The protein eluted out of the column during

chromatography was detected at 280 nm [14,15,16].

2.4.2. Determination of molecular mass

The molecular mass of the purified protein was estimated by gel filtration

chromatography using sephadex G-100 that had been balanced for 24 h with buffer

(50 mM Na3PO3, 1 mM dithitreitol; pH 7.6) until no absorbance at 280 nm was

obtained. The flow rate was adjusted to 20 ml/h [16]. The subunit molecular weight

of the protein was determined by SDS-PAGE. Electrophoresis using 12.5%

polyacrylamide gels (0.8% bis-acrylamide) was performed as described by Laemmli

[17].

2.4.3. Non-denaturing PAGE and zymography

An 8% polyacrylamide concentration was optimized to perform non-denaturing

PAGE. Following electrophoresis at 10 mA the gel was cut into two halves. The first

half was stained with Coomassie brilliant blue R-250 and the second half was used

for zymography [18].

Table 1Summary of various steps involved in the purification of carbonic anhydrase from Pseu

Purification step Total activity (units) Total protein (mg)

Crude supernatant 34.5 14.1

Dialyzed (15 kDa cut off) 6.40 1.96

p-AMBS-agarose 4.47 0.405

All the steps were carried out at 4 8C. Only the 75% ammonium sulphate dialysate was

2.4.4. Esterase activity

Activity for p-nitrophenylacetate hydrolysis by CA from P. fragi was determined

at 25 8C following the method of Smith and Ferry [19].

3. Result and discussion

3.1. Induced extracellular carbonic anhydrase

CA activity (2.4 � 0.49 U/mg protein) was detected in supernatantobtained by the separation of the cells from peptone broth containingcalcium carbonate following 12 h, 24 h, 36 h and 48 h incubation.However no CA activity was detected in supernatant in absence ofcalcium carbonate irrespective to the incubation period. InterestinglyCA activity was evident only in 12 h (1.38 � 0.24 U/mg protein) and24 h (0.65 � 0.10 U/mg protein) incubated samples derived frompeptone broth (without CaCO3) containing seed culture raised inpresence of CaCO3. No activity was detected under similar conditionswhen seed culture was raised in absence of CaCO3. The results indicatedthat the extracellular CA from P. fragi is induced in presence of CaCO3.The presence of such an enzyme in P. fragi can be attributed to the site ofthe isolation of the organism. This enzyme is believed to play animportant role in the dissolution of calcium carbonate releasing Ca2+

and HCO3� ions. Carbonic acid facilitates the dissolution of lime stone;

however this conversion in nature is a slow process. CA can catalyze thehydration of CO2 to carbonic acid and may therefore acceleratelimestone dissolution [20]. Li et al. [11] demonstrated the ability ofbacterial biomass isolated from Karst areas to produce extracellular CAand its role in limestone dissolution. Similar results were obtained withbovine carbonic anhydrase [3]. Kusian et al. [21] had reported theimportance of CO2 and HCO3

� in growth of bacteria. In regions wherepH is alkaline most of the dissolved inorganic carbon is present ascarbonate and bicarbonate ions [1]. Thus extracellular CA in P. fragi isenvisaged to play an important role in improving the efficiency ofbicarbonate ion transport. The assimilation of bicarbonate in the cellcould take place by hydroxyl excretion into the outer medium [22] and/or by sodium symport [23]. Kupriyanova et al. in 2007 [1] had reporteda similar role for extracellular CA from Microcoleus cathonoplastes

isolated from the soda lakes in Russia. Similarly Puskas et al. in 2000 [4]had demonstrated that extracellular CA is essential for bicarbonateuptake in Rhodopseudomonas palustris.

3.2. Purification of the enzyme

Significant enzyme activity (8.24 U/mg protein) was observedin 75% ammonium sulphate fraction only and following dialysis1.33-fold purification with 92% enzyme yield was achieved. In thefinal step of purification the protein was passed through the p-AMBS-agarose affinity column. SDS-PAGE of the purified enzymeindicated the presence of a single band. The enzyme (11.03 U/mgprotein) was purified about 4.52-fold with a final yield of 86%(Table 1). In the purification process of CA, following dialysis, theproteins were purified directly using p-AMBS-agarose affinitycolumn. The advantages of single step affinity purification over ionexchange and hydrophobic interaction chromatography include ahigher enzyme yield and less protein loss [4,24]. An enzymerecovery of 86% during the present study validates the use of singlestep affinity purification. A similar purification strategy forDunaliella salina CA had resulted in a relative yield of 89.6% [24].

domonas fragi.

Specific activity (U/mg) Yield (%) Fold purification

2.44 100 1

3.26 92 1.33

11.03 86 4.52

loaded onto p-AMBS-agarose affinity column.

Fig. 1. (A) SDS-PAGE of purified carbonic anhydrase. Dialysate (lane 1), purified CA

(lane 2) and standard proteins of different molecular weight (97.4–29.0 kDa) (lane

3) were run in SDS-PAGE (12%) and stained with Coomassie blue. (B) Zymography

(lane 1) and native PAGE (8%) (lane 2) of purified CA.

Fig. 2. Effect of pH on stability of purified carbonic anhydrase. The pH stability of the

enzyme was determined by incubating the enzyme in different buffers for 24 h at

room temperature. The residual enzyme activity was measured according to the

standard enzyme assay. 100% CA activity was equivalent to 11.00 U/mg protein.

Fig. 3. Effect of temperature on stability of purified carbonic anhydrase. The

temperature stability of the enzyme was determined by incubating the reaction

mixture containing the purified enzyme at different temperatures between 35 8Cand 55 8C for 1 h. The residual enzyme activity was measured according to the

standard enzyme assay. 100% CA activity was equivalent to 11.00 U/mg protein.

A. Sharma et al. / Process Biochemistry 44 (2009) 1293–1297 1295

The esterase activity is present only in CA belonging to the a-class [5,19,25]. The purified CA showed high esterase activity(1465.35 � 2.34 IU), indicating that the enzyme is of a-type. Highesterase activity comparable to that of bovine carbonic anhydrasewas found in the present study. This indicates that the CA from P. fragi

belongs to the a-class. It has been assumed that a-CAs have evolvedfrom a common ancestral gene about 0.5–0.6 billion years ago [5].This is consistent with the evidence that naturally occurringecosystems are known to harbor relict microbial communities thatare considered analogues of ancient ecosystem [26].

The molecular weight of the purified CA was calculated to be�31 kDa from the log plot obtained from the relative mobility ofthe standard proteins in SDS-PAGE (Fig. 1A). However molecularmass of the intact protein was found to be 94 kDa after gel filtrationchromatography. This suggests that the enzyme is a trimer. Twoelectrophoretically separable isoforms were also detected follow-ing native page (Fig. 1B). However the zymogram indicated a singleyellow band on a purple background (Fig. 1B). HCA II is the bestcharacterized a-CA, it is a monomer with subunit molecularweight of 30 kDa. In bacterial domain a-CAs identified inHelicobacter pylori (23 kDa), Niesseria sicca (29 kDa), and Niesseria

gonorrhoeae (25 kDa) are all monomeric in nature, however a-CAdetected in R. palustris is a dimer [5]. This is the first report of atrimeric protein belonging to a-type CA in bacterial domain as wellas the purification of a-type CA from g proteobacteria. Previously,Guilloton et al. [27] had purified a b-CA from Eschericia coli. Theseresults substantiate the presence of functional as well as structuraldiversity within the CA family, endorsing the fact that CA is anexcellent example of convergent evolution. Native-Page indicatedthe presence of two electrophoretically separable isoforms,however as the two isoforms were very close, only a single largeyellow band was visible in zymography. Existence of isoformswithin the same organism has been reported in humans; 16isoforms [28], while two active external CA isozymes have beenreported in Chlamydomonas saccharophila and Chlamydomonas

reinhardtii [29].

3.3. Effect of pH and temperature on CA stability

The effect of pH on stability of purified CA was examined inthree different buffer systems, 50 mM Citrate phosphate buffer (pH5.0–6.5), Tris–HCl buffer (pH 7.0–8.5) and glycine–NaOH buffer(pH 9.0–10.0). The enzyme was found to retain 100% activity at pH8.0 while more than 80% enzyme activity was retained in the pHrange 7.0–8.5 after 24 h incubation at room temperature. Howeveronly 45% and 65% enzyme activity were retained at pH 5.0 and 10.0respectively (Fig. 2). The CA stability was measured at various

temperatures ranging from 35 8C to 55 8C for 1 h in 50 mM Tris–HCl buffer (pH 8.0). There was a progressive decline in the stabilityof CA following increase in temperature from 35 8C to 55 8C. Theenzyme was found to retain 100% activity at 35 8C while 89% and86% activity was retained at 40 8C and 45 8C respectively. Incontrast only 21.0% residual activity was observed at 55 8Cfollowing 1 h incubation at the respective temperatures (Fig. 3).The purified CA from P. fragi was found to be stable at pH range 7.0–8.5 and temperature range 35–45 8C. Similar results have beendocumented for CA from Enterobacter taylorae [30]. The human andbovine erythrocyte CA had activity in the pH range of 6.5–7.5 andtemperature range of 35–40 8C. However CA from Methanosarcina

thermophila was found to be active at 75 8C [19]. The extracellularCA from M. cathonoplastes showed two peaks of activity at pH 7.5and 10.0. In contrast CA from H. pylori showed high acid tolerance,functioning optimally in the acidic environment of humanstomach [31]. All these facts indicate to the remarkable functionaldiversity of CA and the ability of this enzyme to perform differentroles for the organisms surviving in extreme microenvironmentsand diverse ecological niche.

3.4. Effect of metal ion concentration on CA

At a concentration of 1 mM, the effect of different metal ions onthe activity of CA was examined (Table 2). CA activity wasstimulated by the addition of Zn2+, Cd2+, and Fe2+ ions. The fact thatall these three ions are part of the active metal centre in differentclasses of CA [28]; they could have a stabilization effect on CAenzyme thus enhancing the activity. In the present study Zn2+ had

Table 2Effect of metal ions on P. fragi carbonic anhydrase activity.

Metal ion (1 mm) Residual activity (1 h)

Control 100�1.89

CuCl2 99.6� 0.99

CaCl2 100�1.11

MgCl2 100� 0.67

NaCl 99� 0.76

HgCl2 40.9� 0.91

KCl 99.0� 0.67

EDTA 71.49� 0.82

PbCl2 46.6�1.13

ZnCl2 109.25�1.09

CdCl2 106.6�1.12

FeCl2 102.6� 0.87

AsCl3 37.1� 0.59

Enzyme activity measured in absence of any metal ion was

taken as 100% (11.00 U/mg protein). The remaining CA

activity was measured after preincubation of purified

enzyme with each inhibitor for 1 h at room temperature.

A. Sharma et al. / Process Biochemistry 44 (2009) 1293–12971296

maximum stimulation effect on CA activity, this holds true for a-CA as Zn(II) forms the part of the metal coordination sphere [32]. Incontrast the CA activity was strongly inhibited by Hg2+, Pb2+, Ar3+

and EDTA. Suppression of CA activity by EDTA suggests that theenzyme is a metalloprotein and/or requires certain metal ions forits activation. Kuhad et al. [33] reported that inhibition due to Hg2+

suggests the presence of thiol groups in the active site of theenzyme. The enzyme activity was not affected by the presence ofCa2+, Mg2+, K+ and Na2+ ions indicates that the enzyme couldperform optimally in presence of different salts.

3.5. Effect of inhibitors on enzyme activity

The IC50 concentration of sulfonamide and anionic for CA fromP. fragi were determined (Table 3). Both acetazolamide(IC50 = 2 � 10�5 mM) and sulfanilamide (IC50 = 7.0 � 10�3 mM)were found to strongly inhibit CA from P. fragi. The inhibitoryactivity of the anions was in the order CN� > N3

� > SCN� >I� > NO3

� > Br� > Cl� > SO42� > ClO4

� > F�. The inhibition pro-file resembled HCA II, HCA I, and other a-CAs [28]. In contrast theinhibition profile of P. fragi CA was quite different from that of wellcharacterized b-isozymes, Cab (from archeon M. thermophila) aswell as Can 2 (from Cryptococcus neoformans and Nce 103 (fromCandida albicans). This can be attributed to the different metal co-ordination spheres in these enzymes, also the fact that an overallcharge of +2 can be considered for a-CAs (three neutral Hisresidues as metal(II) ion ligands). In contrast, the charge for the

Table 3Effect of sulphonamide and anionic inhibitors on P. fragi

carbonic anhydrase.

Inhibitor IC50 (mM)

Acetazolamide 2.1�10�5

Sulfanilamide 7.0�10�3

Azide 3.0�10�3

Cyanide 8.0�10�4

Thiocyanate 1.50

Iodide 29.0

Nitrate 35.0

Bromide 69.0

Chloride 180.0

Sulphate 200.0

Chlorate 240.0

Fluoride 350.0

Enzyme activity measured in absence of any inhibitor was

taken as 100%. The percentage inhibition in terms of residual

enzyme activity was calculated. IC50 were determined by a

semi logarithmic plot of percentage inhibition vs logarithmic

concentration of inhibitor.

open active site b-CAs can be considered as zero, with the twocysteinate metal ion ligands neutralizing the +2 charge of the metalion. The difference in the charge distribution at the enzyme activesite cavity of a- and b-CAs is reflected in their quite distinctinhibition profile investigated in this study [28]. Although theinhibition profile of P. fragi CA closely resembles the HCA II, theCN� and N3

� IC50 of P. fragi CA were more closer to HCA I than HCAII. The hydrophobic contacts of the inhibitors (possessing variousionic radii) with the enzyme active site bearing specific amino acidresidues is one of the important factors controlling inhibitoryactivity. These subtle variations can be attributed to the changes inthe amino acid residues present in the active site. However suchenzyme anion adduct structures can be evidenced only byresolving with high resolution X-ray crystallography [8].

Besides the importance of CA in ion transport, this enzyme hasfound a new dimension in the field of biomimetic CO2-sequestra-tion. Application of CA in an onsite scrubber requires a robust CAactive at alkaline pH, being thermostable and minimally inhibitedby different ions [34]. All these aspects are fulfilled by the CAidentified in this study. This enzyme thus offers an alluringprospective for such a potential application. A detailed study onthis aspect is under progress in our laboratory.

4. Conclusion

Until now, the presence of external CA in the bacterial domainhas been shown only in Azotobacteriaceae colonies isolated fromKarst areas. This study for the first time reports the purification andcharacterization of an alkaline, thermostable external a-CA from gproteobacteria. The present study also emphasizes the significantrole that CA plays in improving the transport of inorganic carbonand other ions.

Acknowledgement

The authors are thankful to Department of Biotechnology, Govt.of India for providing the financial research assistance.

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