review article carbonic anhydrase: an efficient enzyme...

Hindawi Publishing CorporationInternational Journal of Chemical EngineeringVolume 2013 Article ID 813931 6 pageshttpdxdoiorg1011552013813931

Review ArticleCarbonic Anhydrase An Efficient Enzyme with PossibleGlobal Implications

Christopher D Boone Sonika Gill Andrew Habibzadegan and Robert McKenna

Department of Biochemistry amp Molecular Biology University of Florida PO Box 100245 Gainesville FL 32610 USA

Correspondence should be addressed to Robert McKenna rmckennaufledu

Received 24 May 2013 Revised 6 August 2013 Accepted 19 August 2013

Academic Editor Francisco Jose Hernandez Fernandez

Copyright copy 2013 Christopher D Boone et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

As the global atmospheric emissions of carbon dioxide (CO2) and other greenhouse gases continue to grow to record-setting levels

so do the demands for an efficient and inexpensive carbon sequestration system Concurrently the first-world dependence oncrude oil and natural gas provokes concerns for long-term availability and emphasizes the need for alternative fuel sources At theforefront of both of these research areas are a family of enzymes known as the carbonic anhydrases (CAs) which reversibly catalyzethe hydration of CO

2into bicarbonate CAs are among the fastest enzymes known which have a maximum catalytic efficiency

approaching the diffusion limit of 108Mminus1sminus1 As such CAs are being utilized in various industrial and research settings to helplower CO

2atmospheric emissions and promote biofuel productionThis review will highlight some of the recent accomplishments

in these areas along with a discussion on their current limitations

1 Introduction

The atmospheric concentrations of greenhouse gases suchas carbon dioxide (CO

2) methane chlorofluorocarbons

and nitrous oxides have been increasing accordingly dueto human-induced activities [1] CO

2is the most abundant

greenhouse gas being produced primarily by the burning offossil fuels such as coal oil and natural gas The atmosphericconcentration of CO

2has increased since preindustrial era

from sim280 ppm [2] to 400 ppm in 2013 [3] According toAntarctic ice core extractions these levels are significantlyhigher than those at any time during the past 800000years [4ndash7] Less direct geological evidence based on boron-isotope ratios in ancient planktonic foraminifer shells sug-gests that such high CO

2atmospheric concentrations were

last seen about 20 million years ago [8]Since 1896 a trend has been associated with elevated CO

2

levels in the atmosphere and an increase in the average globaltemperature [9] In sim100 years (1906ndash2005) the averageglobal temperature increased by 07 plusmn 02∘C compared to arelatively constant average over the previous two thousandyears [10]This rise in CO

2levels is linked not only to surface

temperature increases (with rising sea levels melting of

the glacier and polar ice caps) but also to increased oceanacidity [11ndash13] Fossil fuel burning accounts for 75 ofthe elevated atmospheric CO

2levels from human activity

over the past 20 years with the remainder being associatedprimarily with deforestation [14]

2 Carbonic Anhydrase

The need for development of an efficient and inexpensivecarbon sequestration system and the drive for an alternativefuel supply have both been led by a group of enzymes calledthe carbonic anhydrases (CAs EC 4211) The CAs areubiquitously found in all kingdoms of lifeand are mostly zincmetalloenzymes that catalyze the interconversion betweencarbon dioxide and bicarbonate [15ndash17] CAs exist in threestructurally distinct and evolutionarily unrelated classes120572 120573 and 120574 The 120572-class is found throughout the animalkingdom and in the periplasmic function of Chlamydomonasreinhardtii which is a type of green alga [18] In addition120572-CAs can be found in Neisseria gonorrhoeae and otherNeisseria species [19ndash21] The 120573-CAs are found in plantswith C3 and C4 metabolism as well as monocotyledons anddicotyledons arthropods and bacteria [17 22ndash27]The 120574-CA

2 International Journal of Chemical Engineering

was first isolated and characterized in the Methanosarcinathermophila a methanogenic archebacterium [28] Homol-ogous sequences of 120574-CAs have been found in eubacteriahowever it is not known whether these sequences representfunctional CAs [17]

Despite their structural differences all CAs utilize thesame catalytic mechanism a two-step ping-pong mechanismthat catalyzes the reversible hydrationdehydration of CO

2

into bicarbonate and a proton [17] as

E Zn-OHminus + CO2

H2OlArr997904997904rArr E Zn-H

2O +HCO

3

minus (1)

E Zn-H2O + BlArr997904997904rArr E ZnOHminus + BH+ (2)

In the first reaction the zinc-bound hydroxide (ZnOH)nucleophilically attacks the CO

2to form bicarbonateThen a

molecule of water coordinates directly to the zinc ion whichpromotes diffusion of the bicarbonate out of the active site(Reaction (1)) The zinc-bound water molecule (ZnH

2O) is

deprotonated in the next reaction to regenerate the ZnOH(Reaction (2)) The proton is transferred from the ZnH

2O

to the bulk solvent (B) via a highly ordered array of watermolecules in the active site [29ndash32] The amino acid residuethat acts as the proton shuttle residue (ie the link betweenthe buried ZnH

2O in the active site and bulk solvent) is His64

in the case of human CA isoform II (HCA II) [33] and severalother HCAs [15] HCA II is a highly efficient catalyst witha turnover rate (119896cat) on the order of one per microsecondand an overall catalytic efficiency (119896catK119872) approaching thediffusion rate limit of 108Mminus1 sminus1 [15 29 34ndash37] Due to thishighly efficient CO

2hydration there is considerable interest

in using CAs in carbon sequestration systems as well as inbiofuel and calcite production (Figure 1 and Table 1) Thisreview will outline the current status advantages and limitsof these applications

3 Carbon Sequestration

The typical industrial flue gas contains 10minus20 CO2

which by current processes is cost-inefficient and requiresharsh chemical environments at elevated temperatures [60]Selectively capturing CO

2out of a mixture of waste gas

that may also include nitrogen sulfur and other compoundscan be technically challenging and expensive [61] Currentindustrial protocol separates postcombustion CO

2via amine

scrubbing mineral carbonation pressure storage or absorp-tion into solids or into liquids An attractive alternativeto these methods includes the use of an environmentallybenign renewable selective and inexpensive biomimeticCO2sequestering agent

The CAs are the leading candidate for this application asthey are fairly inexpensive to be produced are reuseable andcan work at ambient temperatures and undermild conditions[38] Adequate bovine CA can be purified from simplechloroform extraction or ammonium sulfate precipitationof slaughterhouse blood samples [62] Human (and othermammalian) CAs are easily overexpressed in bacteria [63]and are commercially available for purchase While bothhuman and bovine CAs have been used in the industrial

setting there is a growing need for improved CAs withhigher stability andor catalytic rates (to improve reusabilityand cost-efficiency resp) [38 64] A technique currentlyemployed in the industrial setting to improve the stability ofCAs is via immobilization onto a variety of inorganic [39ndash43] and biopolymer surfaces [44 45] including enrichedmicroorganisms [21 46] as well as onto matrices containingacrylamide alginate and chitosan-alginate [47 48] Othertechniques include site-directed mutagenesis to rationallydesign faster [65] andor more stabile [64 66] variants ofHCA II Research on stable CAs isolated from halo- andthermotolerant microorganisms could provide further CO

2

sequestration candidates in the industrial settingOnce CO

2has been scrubbed from the flue gas it can be

chemically converted into stable compounds such as variouscarbonates or it can be pressurized to a liquid state for masstransport for geosequestration (storage either undergroundor in the ocean) [67 68] However there are concerns overthe cost stability and the long-term biological impacts (egthe release of CO

2upon contact with acidic rain) associated

with geosequestration [68] Chemical conversion of CO2into

ecologically friendly products such as calcite (CaCO3) has

gained recent interest Calcite is the main constituent ofshells in marine organisms [69 70] and is readily preparedby reacting CaCl

2with bicarbonate the product of CA

catalysis As such calcite is routinely used in the makingof cement and other building materials but it can alsobe used as a pigment for paint formulation or as an acidneutralizer Sequestered CO

2can also be converted into other

useful products such as polycarbonates acrylates methanecarbonate storage polymers and other constructionmaterials[56ndash58]

4 Biofuel Production

The potential long-term global environmental effects andthe limited availability of oil and natural gas sources haveprompted many countries including the US to initiatemethods to find an environment friendly alternate fuel source[59] There are an estimated 60 billion gallons of diesel and120 billion gallons of gasoline used in the US per year [71]This equates sim140 billion gallons of biodiesel needed for totaltransportation fuel in the US each year Biodiesel is preferredover conventional diesel as it does not contribute to CO

2or

sulfate levels in the atmosphere emits less gaseous pollutantsand is nontoxic [49] Soybean oil accounts for over half of thesource of US biodiesel production [72] However only sim15of the biodiesel demand could be met if all the arable land inthe US were used to grow soybean for oil production [59]Additionally the current production of biofuels displacescroplands previously used for food and has been associatedwith increased consumer prices [73 74]

An alternative to the soybean-derived biodiesel is algae-based systems Algae are attractive candidates as they havehigher oil production and carbon fixation rates compared toterrestrial plants [75 76] and do not compete with traditionalagriculture as they can be cultivated in ponds or in closedphotobioreactors located on nonarable land [59] Continuous

International Journal of Chemical Engineering 3

CO2 sequestration Biofuel production

Carbonic anhydrase

Calcite production Algal raceway pond

Diesel

Figure 1 Schematic of the centralized role for CA in converting CO2into beneficial products The catalytic conversion of CO

2produced

during the combustion of fossil fuels into bicarbonate (HCO3

minus) via CA yields a valuable source of inorganic carbon for algal cultures grownin raceway ponds The lipids and oils from the algae cultures are excellent sources for biofuels whereas the ldquowasterdquo product yields additionalbeneficial proteins vitamins minerals and dietary supplementsThe algal CA can also serve as a great source for calcite (CaCO

3) production

being critical in many construction agricultural and industrial materials

Table 1 CA usage in industrial settings

Technique Principles CA utilization References

Carbon sequestration Capture of atmospheric CO2 produced duringthe burning of fossil fuels

Immobilized onto a variety of surfaces includingenriched microorganisms alginates and inorganicmaterial

[38ndash48]

Biofuel production Mass algal growth and harvesting as analternative fuel source

Provides inorganic carbon in the soluble form ofbicarbonate to Rubisco the rate-limiting step inbiomass production

[49ndash55]

Calcite production Chemical conversion of bicarbonate to calciteused in construction and agricultural materials

Provides bicarbonate at a rapid rate via the catalytichydration of CO2 that is captured as a result ofcarbon sequestration and biofuel production

[56ndash59]

cultivation of algae would also yield beneficial medicinalagents such as proteins fatty acids vitamin A mineralspigments dietary supplements and other biomolecules [77]

The CAs play an important role in the carbon fixationpathways in photosynthetic organisms (plants algae andcyanobacteria) The rate-limiting step of biomass produc-tion in these organisms is the uptake of CO

2into cells

as bicarbonate A carbon-concentrating mechanism thatincludes CA delivery of inorganic carbon to RuBisCo wasevolved to counterattack this limitation [59 78] The CO

2

is fixated into phosphoglyceric acid enters the Calvin cycleand ultimately results in sugar Numerous research effortsare being conducted to improve the efficiency of carbonfixation pathways aiming to improve food crop cultivationand biomass production [79]

Endogenous algal and cyanobacteria CA can also beused indirectly in calcite deposition as evidenced by theenhanced CO

2capture and sequestration in the presence of

the algal species Chlorella and Spirulina [50ndash53] These algalspecies also provide the extra benefit of producing calciteduring cell with Chlorella produced the greatest yield of lipidbiomass in photobioreactors [50] Other studies in simulated

raceway ponds showed similar results with the additionalobservation of decreased CO

2capture with increasing levels

of acetazolamide a commonly used CA inhibitor [50 80]The role of CA in this calcite precipitation is proposed tobe a vital one as it provides inorganic carbon in a solubleform as bicarbonate the preferred source for a variety ofalgal strains (Figure 1 and Table 1) [54 55] However muchwork is needed in determining the right conditions (pHsubstratenutrient availability aeration etc) to optimize thesimultaneous production of biofuels and calcite with algalspecies

5 Conclusion

Manrsquos dependence on fossil fuels and other natural gasproducts has brought forth an era when CO

2atmospheric

levels are higher than those at any other time in recordedhistory The long-term environmental consequences of thispandemic caused by humanities need for energy are bleakbut also unpredictable The global reduction of greenhousegas emissions is the first critical step in the reversing process


as an alternative source of fuel is found The need for asolution has prompted much research into studying theviability of utilizing CAs for both of these challenges Ideallyan optimized system would include a cyclic productionof biofuels via algal andor microalgal cultures that wouldsubstitute in fossil fuel combustionThe flue gas could then bescrubbed forCO

2by theCAs in the same algal cultures which

would also promote the formation of bicarbonate inducingfurther biomass production and increasing the rate of calciteprecipitation (Figure 1) As such this system would providethe benefits of reducedCO

2emissionswhile also providing an

essentially self-enclosed fuel and calcite generator providedother essential ingredients and nutrients are available Muchresearch is also needed however in designing a highly activeand stable variant of CA that can be easily overexpressed insuch system

References

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[2] EPA ldquoRecent Climate Change Atmospheric Changesrdquo Cli-mate Change Science Program United States EnvironmentalProtection Agency 2007 httpwwwepagovclimatechangescienceindicatorsindexhtml

[3] D Shukman ldquoCarbon dioxide passes symbolic mark BritishBroadcasting Corporationrdquo 2013 httpwwwbbccouknewsscience-environment-22486153

[4] R Spahni J Chappellaz T F Stocker et al ldquoAtmosphericscience atmospheric methane and nitrous oxide of the latepleistocene fromAntarctic Ice Coresrdquo Science vol 310 no 5752pp 1317ndash1321 2005

[5] U Siegenthaler T F Stocker E Monnin et al ldquoAtmosphericscience stable carbon cycle-climate relationship during the latepleistocenerdquo Science vol 310 no 5752 pp 1313ndash1317 2005

[6] J R Petit J Jouzel D Raynaud et al ldquoClimate and atmospherichistory of the past 420000 years from the Vostok ice coreAntarcticardquo Nature vol 399 no 6735 pp 429ndash436 1999

[7] D Luthi M le Floch B Bereiter et al ldquoHigh-resolution carbondioxide concentration record 650000-800000 years beforepresentrdquo Nature vol 453 no 7193 pp 379ndash382 2008

[8] P N Pearson and M R Palmer ldquoAtmospheric carbon dioxideconcentrations over the past 60 million yearsrdquoNature vol 406no 6797 pp 695ndash699 2000

[9] S Weart ldquoThe carbon dioxide greenhouse effectrdquo in TheDiscovery of Global Warming American Institue of Physics2011 httpwwwaiporghistoryclimateco2htm

[10] K Jansen ldquoWhat Do Reconstructions Based on PalaeoclimaticProxies Showrdquo pp 466ndash478 2007

[11] C Kennedy ldquoState of the Climate 2011 Global Sea LevelrdquoClimateWatch Magazine NOAA Climate Services Portal 2012

[12] IPCC ldquoSynthesis Report Summary for Policymakers Observedchanges in climate and their effectsrdquo 2007

[13] LMorello ldquoOceansTurnMoreAcidicThanLast 800000YearsrdquoScientific American 2010 httpwwwscientificamericancomarticlecfmid=acidic-oceans

[14] J THoughton Y Ding D J Griggs et al IPCC Climate Change2001 The Scientific Basis Cambridge University Press Cam-bridge UK 2001 Working Group I to the Third AssessmentReport of the Intergovernmental Panel on Climate Change

[15] M Aggarwal C D Boone B Kondeti and R McKennaldquoStructural annotation of human carbonic anhydrasesrdquo Journalof Enzyme Inhibition and Medicinal Chemistry vol 28 pp 267ndash277 2013

[16] V M Krishnamurthy G K Kaufman A R Urbach et alldquoCarbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand bindingrdquo Chem-ical Reviews vol 108 no 3 pp 946ndash1051 2008

[17] S Lindskog ldquoStructure and mechanism of Carbonic Anhy-draserdquo Pharmacology and Therapeutics vol 74 no 1 pp 1ndash201997

[18] D Hewett-Emmett and R E Tashian ldquoFunctional diversityconservation and convergence in the evolution of the 120572- 120573-and 120574-carbonic anhydrase gene familiesrdquoMolecular Phylogenet-ics and Evolution vol 5 no 1 pp 50ndash77 1996

[19] B Elleby L C Chirica C Tu M Zeppezauer and S LindskogldquoCharacterization of carbonic anhydrase from Neisseria gonor-rhoeaerdquo European Journal of Biochemistry vol 268 no 6 pp1613ndash1619 2001

[20] S Huang Y Xue E Sauer-Eriksson L Chirica S Lindskogand B-H Jonsson ldquoCrystal structure of carbonic anhydrasefrom Neisseria gonorrhoeae and its complex with the inhibitoracetazolamiderdquo Journal of Molecular Biology vol 283 no 1 pp301ndash310 1998

[21] Z Liu P Bartlow R M Dilmore et al ldquoProduction purifi-cation and characterization of a fusion protein of carbonicanhydrase from Neisseria gonorrhoeae and cellulose bind-ing domain from Clostridium thermocellumrdquo BiotechnologyProgress vol 25 no 1 pp 68ndash74 2009

[22] A Maresca D Vullo A Scozzafava and C T SupuranldquoInhibition of the alpha- and beta-carbonic anhydrases fromthe gastric pathogen Helycobacter pylori with anionsrdquo Journalof Enzyme Inhibition andMedicinal Chemistry vol 28 no 2 pp388ndash391 2013

[23] I Nishimori T Minakuchi T Kohsaki et al ldquoCarbonic anhy-drase inhibitors the 120573-carbonic anhydrase from Helicobacterpylori is a new target for sulfonamide and sulfamate inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 17 no 13 pp3585ndash3594 2007

[24] R S Rowlett ldquoStructure and catalytic mechanism of the 120573-carbonic anhydrasesrdquo Biochimica et Biophysica Acta vol 1804no 2 pp 362ndash373 2010

[25] R S Rowlett C Tu M M McKay et al ldquoKinetic character-ization of wild-type and proton transfer-impaired variants of120573-carbonic anhydrase from Arabidopsis thalianardquo Archives ofBiochemistry and Biophysics vol 404 no 2 pp 197ndash209 2002

[26] L Syrjanen M Tolvanen M Hilvo et al ldquoCharacterizationof the first beta-class carbonic anhydrase from an arthropod(Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebratesrdquo BMC Biochemistryvol 11 no 1 article 28 2010

[27] S A Zimmerman and J G Ferry ldquoThe 120573 and 120574 classes ofcarbonic anhydraserdquoCurrent Pharmaceutical Design vol 14 no7 pp 716ndash721 2008

[28] T M Iverson B E Alber C Kisker J G Ferry and D C ReesldquoA closer look at the active site of 120574-class carbonic anhydrases


high-resolution crystallographic studies of the carbonic anhy-drase frommethanosarcina thermophilardquo Biochemistry vol 39no 31 pp 9222ndash9231 2000

[29] D N Silverman ldquoCarbonic anhydrase oxygen-18 exchangecatalyzed by an enzyme with rate-contributing Proton-transferstepsrdquoMethods in Enzymology vol 87 pp 732ndash752 1982

[30] D N Silverman and S Lindskog ldquoThe catalytic mechanism ofcarbonic anhydrase implications of a rate-limiting protolysis ofwaterrdquo Accounts of Chemical Research vol 21 no 1 pp 30ndash361988

[31] D N Silverman and R Mckenna ldquoSolvent-mediated protontransfer in catalysis by carbonic anhydraserdquo Accounts of Chem-ical Research vol 40 no 8 pp 669ndash675 2007

[32] D N Silverman C Tu X Chen S M Tanhauser A J Kresgeand P J Laipis ldquoRate-equilibria relationships in intramolecularproton transfer in human carbonic anhydrase IIIrdquoBiochemistryvol 32 no 40 pp 10757ndash10762 1993

[33] C K Tu D N Silverman C Forsman B-H Jonsson andS Lindskog ldquoRole of histidine 64 in the catalytic mechanismof human carbonic anhydrase II studied with a site-specificmutantrdquo Biochemistry vol 28 no 19 pp 7913ndash7918 1989

[34] S Lindskog and J E Coleman ldquoThe catalytic mechanism ofcarbonic anhydraserdquo Proceedings of the National Academy ofSciences of the United States of America vol 70 no 9 pp 2505ndash2508 1973

[35] S Lindskog and D N Silverman ldquoThe catalytic mechanism ofmammalian carbonic anhydrasesrdquo inTheCarbonic AnhdyrasesNew Horizons W R Chegwidden N D Carter and Y HEdwards Eds pp 175ndash195 Birkhauser Boston Mass USA2000

[36] THMaren ldquoCarbonic anhydrase inhibition in ophthalmologyaqueous humour secretion and development of sulphonamideinhibitorsrdquo inTheCarbonic Anhydrases NewHorizons W R CChegwidden N D Carter and Y H Edwards Eds pp 425ndash436 2000

[37] C T Supuran ldquoCarbonic anhydrasesmdashan overviewrdquo CurrentPharmaceutical Design vol 14 no 7 pp 603ndash614 2008

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2sequestration using the enzyme carbonic

anhydraserdquo Energy and Fuels vol 15 no 2 pp 309ndash316 2001[39] A Belzil and C Parent ldquoQualification methods of chemical

immobilizations of an enzyme on solid supportrdquo Biochemistryand Cell Biology vol 83 no 1 pp 70ndash77 2005

[40] S Bhattacharya A Nayak M Schiavone and S K Bhat-tacharya ldquoSolubilization and concentration of carbon dioxidenovel spray reactors with immobilized carbonic anhydraserdquoBiotechnology and Bioengineering vol 86 no 1 pp 37ndash46 2004

[41] S Hosseinkhani and M Nemat-Gorgani ldquoPartial unfolding ofcarbonic anhydrase provides a method for its immobilizationon hydrophobic adsorbents and protects it against irreversiblethermoinactivationrdquo Enzyme and Microbial Technology vol 33no 2-3 pp 179ndash184 2003

[42] C Prabhu A Valechha S Wanjari et al ldquoCarbon compositebeads for immobilization of carbonic anhydraserdquo Journal ofMolecular Catalysis B vol 71 no 1-2 pp 71ndash78 2011

[43] M Vinoba K S Lim S K Lee S K Jeong and MAlagar ldquoImmobilization of human carbonic anhydrase ongold nanoparticles assembled onto aminethiol-functionalizedmesoporous SBA-15 for biomimetic sequestration of CO

2rdquo

Langmuir vol 27 no 10 pp 6227ndash6234 2011

[44] D T Arazawa H-I Oh S-H Ye et al ldquoImmobilized carbonicanhydrase on hollow fiber membranes accelerates CO

2removal

from bloodrdquo Journal ofMembrane Science vol 403-404 pp 25ndash31 2012

[45] A Sharma A Bhattacharya and A Shrivastava ldquoBiomimeticCO2sequestration using purified carbonic anhydrase from

indigenous bacterial strains immobilized on biopolymericmaterialsrdquo Enzyme and Microbial Technology vol 48 no 4-5pp 416ndash426 2011

[46] C Prabhu S Wanjari S Gawande et al ldquoImmobilization ofcarbonic anhydrase enriched microorganism on biopolymerbased materialsrdquo Journal of Molecular Catalysis B vol 60 no1-2 pp 13ndash21 2009

[47] G M Bond M G Medina J Stringer and E F A SimsekldquoEnzymatic Catalysis and CO

2Sequestrationrdquo World Resource

Review vol 11 pp 603ndash619 1999[48] F A Simsek-Ege GM Bond and J Stringer ldquoMatrixmolecular

weight cut-off for encapsulation of carbonic anhydrase inpolyelectrolyte beadsrdquo Journal of Biomaterials Science PolymerEdition vol 13 no 11 pp 1175ndash1187 2002

[49] A B Fulke S N Mudliar R Yadav et al ldquoBio-mitigation ofCO2 calcite formation and simultaneous biodiesel precursors

production using Chlorella sprdquo Bioresource Technology vol 101no 21 pp 8473ndash8476 2010

[50] R Ramanan K Kannan A Deshkar R Yadav and TChakrabarti ldquoEnhanced algal CO

2sequestration through cal-

cite deposition by Chlorella sp and Spirulina platensis in amini-raceway pondrdquo Bioresource Technology vol 101 no 8 pp2616ndash2622 2010

[51] J C M Pires M C M Alvim-Ferraz F G Martins and MSimoes ldquoCarbon dioxide capture from flue gases usingmicroal-gae engineering aspects and biorefinery conceptrdquo Renewableand Sustainable Energy Reviews vol 16 no 5 pp 3043ndash30532012

[52] C Gonzalez-Fernandez and M Ballesteros ldquoLinking microal-gae and cyanobacteria culture conditions and key-enzymes forcarbohydrate accumulationrdquo Biotechnology Advances vol 30pp 1655ndash1661 2012

[53] A Y Shekh K Krishnamurthi S N Mudliar et al ldquoRecentadvancements in carbonic anhydrase-driven processes for CO

2

sequestration minireviewrdquo Critical Reviews in EnvironmentalScience and Technology vol 42 pp 1419ndash1440 2012

[54] Z Chi J V OrsquoFallon and S Chen ldquoBicarbonate produced fromcarbon capture for algae culturerdquo Trends in Biotechnology vol29 no 11 pp 537ndash541 2011

[55] B Rost K-U Richter U Riebesell and P J Hansen ldquoInorganiccarbon acquisition in red tide dinoflagellatesrdquo Plant Cell andEnvironment vol 29 no 5 pp 810ndash822 2006

[56] T Sakakura J-C Choi and H Yasuda ldquoTransformation ofcarbon dioxiderdquo Chemical Reviews vol 107 no 6 pp 2365ndash2387 2007

[57] H Arakawa M Aresta J N Armor et al ldquoCatalysis researchof relevance to carbon management progress challenges andopportunitiesrdquo Chemical Reviews vol 101 no 4 pp 953ndash9962001

[58] E J Beckman ldquoMaking polymers fromcarbondioxiderdquo Sciencevol 283 no 5404 pp 946ndash947 1999

[59] P Chen M Min Y Chen et al ldquoReview of the biological andengineering aspects of algae to fuels approachrdquo InternationalJournal of Agricultural and Biological Engineering vol 2 pp 1ndash30 2009


[60] S M Benson and T Surles ldquoCarbon dioxide capture andstorage an overview with emphasis on capture and storage indeep geological formationsrdquo Proceedings of the IEEE vol 94 no10 pp 1795ndash1805 2006

[61] A C Pierre ldquoEnzymatic carbon dioxide capturerdquo ISRN Chem-ical Engineering vol 2012 Article ID 753687 22 pages 2012

[62] J da Costa Ores L Sala G P Cerveira and S J KalilldquoPurification of carbonic anhydrase from bovine erythrocytesand its application in the enzymic capture of carbon dioxiderdquoChemosphere vol 88 no 2 pp 255ndash259 2012

[63] C Forsman G Behravan A Osterman and B H JonssonldquoProduction of active human carbonic anhydrase II in E colirdquoActa chemica Scandinavica B vol 42 no 5 pp 314ndash318 1988

[64] Z Fisher C D Boone S M Biswas et al ldquoKinetic and struc-tural characterization of thermostabilized mutants of humancarbonic anhydrase IIrdquo Protein Engineering Design amp Selectionvol 25 no 7 pp 347ndash355 2012

[65] S Z Fisher C Tu D Bhatt et al ldquoSpeeding up proton transferin a fast enzyme kinetic and crystallographic studies on theeffect of hydrophobic amino acid substitutions in the active siteof human carbonic anhydrase IIrdquo Biochemistry vol 46 no 12pp 3803ndash3813 2007

[66] C D Boone A Habibzadegan C Tu D N Silverman andR McKenna ldquoStructural and catalytic characterization of athermally stable and acid-stable variant of human carbonicanhydrase II containing an engineered disulfide bondrdquo ActaCrystallographica D vol 69 pp 1414ndash1422 2013

[67] R Zevenhoven S Eloneva and S Teir ldquoChemical fixation ofCO2in carbonates routes to valuable products and long-term

storagerdquo Catalysis Today vol 115 no 1ndash4 pp 73ndash79 2006[68] D J Allen and G F Brent ldquoSequestering CO

2by mineral car-

bonation stability against acid rain exposurerdquo EnvironmentalScience and Technology vol 44 no 7 pp 2735ndash2739 2010

[69] M Suzuki K Saruwatari T Kogure et al ldquoAn acidic matrixprotein Pif is a key macromolecule for nacre formationrdquoScience vol 325 no 5946 pp 1388ndash1390 2009

[70] L Astachov Z Nevo T Brosh and R Vago ldquoThe structuralcompositional andmechanical features of the calcite shell of thebarnacle Tetraclita rufotinctardquo Journal of Structural Biology vol175 no 3 pp 311ndash318 2011

[71] M Briggs Widescale Production From Algae Physics Depart-ment The University of New Hampshire 2004

[72] Monthly Biodiesel Production Report US Energy InformationAdministration 2013 httpwwweiagov

[73] D Boddiger ldquoBoosting biofuel crops could threaten foodsecurityrdquoThe Lancet vol 370 no 9591 pp 923ndash924 2007

[74] V Mercer-Blackman H Samiei and K Cheng ldquoBiofueldemand pushes up food pricesrdquo IMF Research DepartmentIMF Survey Magazine 2007

[75] M L Jeong J M Gillis and J-Y Hwang ldquoCarbon DioxideMitigation byMicroalgal PhotosynthesisrdquoBulletin of the KoreanChemical Society vol 24 no 12 pp 1763ndash1766 2003

[76] M B Johnson and Z Wen ldquoProduction of biodiesel fuel fromthe microalga schizochytrium limacinum by direct transesteri-fication of algal biomassrdquo Energy and Fuels vol 23 no 10 pp5179ndash5183 2009

[77] C V Gonzalez Lopez F G Acien Fernandez J M FernandezSevilla J F Sanchez Fernandez M C Ceron Garcıa and EMolina Grima ldquoUtilization of the cyanobacteria Anabaena spATCC 33047 in CO

2removal processesrdquo Bioresource Technol-

ogy vol 100 no 23 pp 5904ndash5910 2009

[78] G C Cannon S Heinhorst and C A Kerfeld ldquoCarboxysomalcarbonic anhydrases structure and role in microbial CO

2

fixationrdquoBiochimica et Biophysica Acta vol 1804 no 2 pp 382ndash392 2010

[79] R J Ellis ldquoBiochemistry tackling unintelligent designrdquoNaturevol 463 no 7278 pp 164ndash165 2010

[80] S Z Fisher M Aggarwal A Y Kovalevsky D N Silvermanand RMcKenna ldquoNeutron diffraction of acetazolamide-boundhuman carbonic anhydrase II reveals atomic details of drugbindingrdquo Journal of the American Chemical Society vol 134 pp14726ndash14729 2012

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DistributedSensor Networks



was first isolated and characterized in the Methanosarcinathermophila a methanogenic archebacterium [28] Homol-ogous sequences of 120574-CAs have been found in eubacteriahowever it is not known whether these sequences representfunctional CAs [17]

Despite their structural differences all CAs utilize thesame catalytic mechanism a two-step ping-pong mechanismthat catalyzes the reversible hydrationdehydration of CO

2

into bicarbonate and a proton [17] as

E Zn-OHminus + CO2

H2OlArr997904997904rArr E Zn-H

2O +HCO

3

minus (1)

E Zn-H2O + BlArr997904997904rArr E ZnOHminus + BH+ (2)

In the first reaction the zinc-bound hydroxide (ZnOH)nucleophilically attacks the CO

2to form bicarbonateThen a

molecule of water coordinates directly to the zinc ion whichpromotes diffusion of the bicarbonate out of the active site(Reaction (1)) The zinc-bound water molecule (ZnH

2O) is

deprotonated in the next reaction to regenerate the ZnOH(Reaction (2)) The proton is transferred from the ZnH

2O

to the bulk solvent (B) via a highly ordered array of watermolecules in the active site [29ndash32] The amino acid residuethat acts as the proton shuttle residue (ie the link betweenthe buried ZnH

2O in the active site and bulk solvent) is His64

in the case of human CA isoform II (HCA II) [33] and severalother HCAs [15] HCA II is a highly efficient catalyst witha turnover rate (119896cat) on the order of one per microsecondand an overall catalytic efficiency (119896catK119872) approaching thediffusion rate limit of 108Mminus1 sminus1 [15 29 34ndash37] Due to thishighly efficient CO

2hydration there is considerable interest

in using CAs in carbon sequestration systems as well as inbiofuel and calcite production (Figure 1 and Table 1) Thisreview will outline the current status advantages and limitsof these applications

3 Carbon Sequestration

The typical industrial flue gas contains 10minus20 CO2

which by current processes is cost-inefficient and requiresharsh chemical environments at elevated temperatures [60]Selectively capturing CO

2out of a mixture of waste gas

that may also include nitrogen sulfur and other compoundscan be technically challenging and expensive [61] Currentindustrial protocol separates postcombustion CO

2via amine

scrubbing mineral carbonation pressure storage or absorp-tion into solids or into liquids An attractive alternativeto these methods includes the use of an environmentallybenign renewable selective and inexpensive biomimeticCO2sequestering agent

The CAs are the leading candidate for this application asthey are fairly inexpensive to be produced are reuseable andcan work at ambient temperatures and undermild conditions[38] Adequate bovine CA can be purified from simplechloroform extraction or ammonium sulfate precipitationof slaughterhouse blood samples [62] Human (and othermammalian) CAs are easily overexpressed in bacteria [63]and are commercially available for purchase While bothhuman and bovine CAs have been used in the industrial

setting there is a growing need for improved CAs withhigher stability andor catalytic rates (to improve reusabilityand cost-efficiency resp) [38 64] A technique currentlyemployed in the industrial setting to improve the stability ofCAs is via immobilization onto a variety of inorganic [39ndash43] and biopolymer surfaces [44 45] including enrichedmicroorganisms [21 46] as well as onto matrices containingacrylamide alginate and chitosan-alginate [47 48] Othertechniques include site-directed mutagenesis to rationallydesign faster [65] andor more stabile [64 66] variants ofHCA II Research on stable CAs isolated from halo- andthermotolerant microorganisms could provide further CO

2

sequestration candidates in the industrial settingOnce CO

2has been scrubbed from the flue gas it can be

chemically converted into stable compounds such as variouscarbonates or it can be pressurized to a liquid state for masstransport for geosequestration (storage either undergroundor in the ocean) [67 68] However there are concerns overthe cost stability and the long-term biological impacts (egthe release of CO

2upon contact with acidic rain) associated

with geosequestration [68] Chemical conversion of CO2into

ecologically friendly products such as calcite (CaCO3) has

gained recent interest Calcite is the main constituent ofshells in marine organisms [69 70] and is readily preparedby reacting CaCl

2with bicarbonate the product of CA

catalysis As such calcite is routinely used in the makingof cement and other building materials but it can alsobe used as a pigment for paint formulation or as an acidneutralizer Sequestered CO

2can also be converted into other

useful products such as polycarbonates acrylates methanecarbonate storage polymers and other constructionmaterials[56ndash58]

4 Biofuel Production

The potential long-term global environmental effects andthe limited availability of oil and natural gas sources haveprompted many countries including the US to initiatemethods to find an environment friendly alternate fuel source[59] There are an estimated 60 billion gallons of diesel and120 billion gallons of gasoline used in the US per year [71]This equates sim140 billion gallons of biodiesel needed for totaltransportation fuel in the US each year Biodiesel is preferredover conventional diesel as it does not contribute to CO

2or

sulfate levels in the atmosphere emits less gaseous pollutantsand is nontoxic [49] Soybean oil accounts for over half of thesource of US biodiesel production [72] However only sim15of the biodiesel demand could be met if all the arable land inthe US were used to grow soybean for oil production [59]Additionally the current production of biofuels displacescroplands previously used for food and has been associatedwith increased consumer prices [73 74]

An alternative to the soybean-derived biodiesel is algae-based systems Algae are attractive candidates as they havehigher oil production and carbon fixation rates compared toterrestrial plants [75 76] and do not compete with traditionalagriculture as they can be cultivated in ponds or in closedphotobioreactors located on nonarable land [59] Continuous



Carbonic anhydrase


Diesel


2produced



3) production






[38ndash48]



[49ndash55]



[56ndash59]



2into cells


2








5 Conclusion


2atmospheric








References
















































2rdquo



2removal

















2


















2by mineral car-














2






RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation











Carbonic anhydrase


Diesel


2produced



3) production






[38ndash48]



[49ndash55]



[56ndash59]



2into cells


2








5 Conclusion


2atmospheric








References
















































2rdquo



2removal

















2


















2by mineral car-














2






RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation















References
















































2rdquo



2removal

















2


















2by mineral car-














2






RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation




























2rdquo



2removal

















2


















2by mineral car-














2






RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation



















2by mineral car-














2






RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation











RoboticsJournal of





Journal of



RotatingMachinery





VLSI Design



Shock and Vibration







Journal of



Volume 2014


SensorsJournal of





Propagation









review article carbonic anhydrase: an efficient enzyme...

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