Engineered Mammalian Carbonic Anhydrases for CO2 Capture

CHAPTER 16

Engineered Mammalian Carbonic Anhydrases for CO2 Capture Christopher D. Boone, Robert McKenna

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida, USA

Contents 16.1 Atmospheric CO2 sequestration  291 16.1.1 Storage and uses of captured CO2  293 16.2 CA immobilization  294 16.2.1 Inorganic surfaces  295 16.2.2 Biopolymers  295 16.3 Biomedical CO2 capture  296 16.3.1 Confined spaces  296 16.3.2 Artificial lungs  296 16.3.3 Blood substitutes  297 16.3.4 Antidote delivery  298 16.4 CO2 capture for biofuel and biomass production  298 16.5 Directed evolution of hCA II  300 16.6 Other a-CAs  302 16.7 Conclusions  302 Acknowledgment  304 References  304

16.1  ATMOSPHERIC CO2 SEQUESTRATION The increase in atmospheric concentrations of the greenhouse gases, including CO2, methane, chloroflurocarbon, and nitrous oxide, has been associated with anthropogenic (human-induced) activities (1). Of particular concern is the rise in the most abundant greenhouse gas, CO2, since the preindustrial era (∼1850), rising from ∼280 ppm (2) to 400 ppm in 2013 (3). Measurement of the CO2 content from core extracts of Antarctic ice indicates that atmospheric concentrations are higher today than in the past 800,000 years (4–7). Other geological evidence, based on a boron isotope ratio in ancient planktonic foraminifer shells, suggests that comparable CO2 atmospheric concentrations were last seen about 20 million years ago, during the first and longest warming period of the Miocene series (8). The burning of fossil fuels has produced ∼75% of the increase in atmospheric levels of CO2 over the past 20 years, with the remainder primarily due to deforestation (9). These elevated atmospheric concentrations of CO2 since the postindustrial era (1896) Carbonic Anhydrases as Biocatalysts. DOI: 10.1016/B978-0-444-63258-6.00016-0 Copyright © 2015 Elsevier B.V. All rights reserved

291

292

Carbonic Anhydrases as Biocatalysts

have been correlated with an increase in global surface temperatures (10). Recording of the average global temperature over a 100-year span (1906–2005) revealed an average increase in temperature by 0.7 ± 0.2°C over that period, compared with the relatively stable temperature for 2000 years prior (11). Associatively, elevated surface temperatures accelerate the melting of glacier and polar ice caps, leading to a rise in sea levels, ocean acidification, and desalination, raising concerns over preservation of numerous animal and plant species, and ecological systems (12–14). Efforts to reduce and limit the emission of CO2 in 37 industrialized nations (excluding the United States) began in 1997, when the Kyoto Protocol to the United Nations Framework Convention on Climate Change was signed in an effort to reduce their emissions to 95% of their 1990 levels by 2020 (15). The first commitment period ran from 2005 to 2012 and revealed promising results as Latvia, Lithuania, and Ukraine had ∼63% reduction in their CO2 emission levels (16). Other notable decreased emissions include Estonia (∼59%), Romania (∼53%), and the United Kingdom (15%). However, some nations including Australia (∼52%), Spain, Portugal (∼36% each), and Canada (∼20%) increased CO2 emissions. The Russian Federation showed decreased emission levels (∼30%) but will not participate, along with Japan and New Zealand (increase in CO2 emissions by ∼3% and 34%, respectively), in the second commitment period scheduled to run from 2013 to 2020 (17). Selectively capturing CO2 out of a mixture of waste flue gas (typically 10–20% CO2 content) that also includes nitrogen, sulfurs, and other organic compounds can be expensive and technically challenging (18,19). Current industrial protocol employs indirect methods of CO2 capture that begins with dissolution of the relatively insoluble CO2 into an aqueous phase via amine scrubbing or mineral carbonation. An attractive alternative includes the design and incorporation of an environmentally benign, renewable, selective, and inexpensive biomimetic CO2 sequestrating agent. The hydrated CO2, expressed either as carbonic acid (H2CO3) or as the conjugate base, bicarbonate (HCO3−) (depending on pH), can then be chemically converted into calcite (CaCO3) or other mineral derivatives (aragonite, vaterite) for industrial and agricultural purposes (20) (Figure 16.1). The rate-limiting step in current industrial carbon capture methods is the hydration of CO2, warranting research into using carbonic anhydrase (CA) as a carbon sequestration catalyst (22). Human (and other mammalian) CAs offer several advantages as they are an extremely efficient and specific means for CO2 capture, are easily overexpressed in bacteria or commercially available, are reusable, and operate at ambient temperatures and under mild conditions (23,24). Human CA isoform II (hCA II) is the most common form used in these settings as it is the best studied of all the CAs and has a high catalytic rate, converting ∼106 CO2 molecules into bicarbonate per second (25–27). However, current utilization of hCA II in the industrial setting is limited by the relative instability of the enzyme in the harsh environment (mostly organic solvents at low pH and

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

Figure 16.1  Atmospheric carbon sequestration techniques utilizing CA. Flue gas produced from the burning of fossil fuels is guided into a bioreactor containing CA immobilized onto a biopolymer surface (inset). The sequestered CO2 can then be stored or chemically converted into useful by-products. Figure modified from Lee et al. (21).

high temperature), resulting in an overall reduction in cost efficiency and productivity (28–30). Additionally, hCA II is irreversibly denatured at ∼58°C (29,31) and is susceptible to inhibition by small anions including sulfate, cyanate, thiocyanate, and azide (32,33).

16.1.1  Storage and uses of captured CO2 The successful sequestration of CO2 from industrial flue gas raises concerns for its longterm storage. The sequestered gas can be either pressurized to a liquid or chemically converted to a stable compound, which can then be stored underground or in the ocean (18,34). Additionally, the production of magnesite (MgCO3) or calcite (CaCO3) with subsequent burial of the solid carbonates is actively being studied as a possible solution, but concerns over the effects of acid rain on these deposits have arisen due to the possible sudden release of CO2 (35). This potential shortcoming can be superseded if the

293

294

Carbonic Anhydrases as Biocatalysts

carbonates are stored in geographic regions that would be better suited for geosequestration such as in serpentinite or wollastonite deposits (for magnesite and calcite, respectively) or in an area where the average annual rainfall is low (35). Instead of geosequestration, the captured CO2 can be converted into various beneficial by-products including polycarbonates, acrylates, methane, stable carbonate storage polymers, and building materials (36–38). Of particular interest are the carbonates, magnesite, and calcite, which can be produced by the simple reaction of either MgCl2 or CaCl2 with bicarbonate.They have low solubility in water and are extremely durable, as evidenced by being a main constituent of shells in marine organisms (39,40). Calcite is also a common component for various pigments, acid neutralizers, and construction materials.

16.2  CA IMMOBILIZATION To improve upon the stability of CAs for use in an industrial carbon sequestration setting, researchers have studied the immobilization of CA onto various inorganic (41–45) and biopolymer surfaces (46,76), which include enriched microorganisms (48,49), in addition to adhesion onto several matrices such as chitosan, alginate, and acrylamide (50,51). The immobilization of CA onto these materials is facilitated by the number of free hydroxyl groups and surface lysine resides of the particular isoform (52) (Figure 16.2).

Figure 16.2  Immobilization of CA (shown as a cartoon) onto a biopolymer surface via an imidocarbonate linkage of surface lysine residues. The zinc metal is shown as a sphere with the coordinating His residues shown in stick view. The proton shuttle residue, His64, is shown in both the “in” and “out” conformations. The active site contains both bicarbonate and CO2, represented in stick view (PDB: 3U7C).

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

Thus, the varying surface residues found throughout the CA family can have a significant influence on the orientation and behavior of the immobilized enzyme. Overviews of some of these immobilization techniques are discussed.

16.2.1  Inorganic surfaces Several inorganic surfaces have been used as a template to covalently link bovine CA (bCA) including metal-based nanoparticles (45,53) and mesoporous silica (54,55). Ironbased nanoparticle immobilization is advantageous as these nanoparticles are highly reusable (up to 30 cycles of immobilization with replenishment via magnet separation) and retain over 80% activity over 30 days (45). Additionally, measurement of calcite precipitation revealed that the immobilized bCA efficiently captured CO2, better than that captured by the enzyme free in solution. Gold and silver nanoparticle systems have also been developed with the gold-immobilized bCA retaining ∼87% enzymatic activity after 30 days of storage. The silver-immobilized bCA stored adequately at 25°C was able to be reused 20 times while retaining full catalytic activity (45,53). Mesoporous silica, essentially a micelle-like network of silica, has been used for different immobilization techniques of bCA including covalent attachment, enzyme adsorption, and cross-linked enzyme aggregation (54). The immobilized bCAs showed similar kinetics to that free in solution, but possessed new beneficial characteristics such as enhanced stability, reusability, and durability (55). An aluminum-based derivative of the mesoporous silicate surface also showed increased stability for immobilized bCA compared with the solution form, but displayed a decreased binding affinity for the CO2 substrate (54).

16.2.2 Biopolymers In addition to the aforementioned covalent linkage and enzyme adsorption immobilization methods, protocols have been established to simply trap enzymes in a porous material such as polyurethane (PU) foam (56).This is accomplished in a very fast and efficient manner via mixture of a polyethylene glycol substituent with isocyanate end groups (named HYPOL) with the enzyme solution. Polymerization of HYPOL around the enzyme is initiated through nucelophilic attack by hydroxide on carbonyl groups, followed by subsequent release of CO2 gas, forming the spongy PU foam.The result of this mechanism is that the reacting isocyanate end groups are converted into an amine group that then rapidly cross-links with another neighboring end group, thereby cross-linking the two polymer chains. The enzyme is likewise cross-linked to HYPOL via covalent linkages among surface amine and hydroxyl groups (52).The result is a highly stable matrix that retains all catalytic activity after 45 days of storage at room temperature, whereas the enzyme free in solution has fully denatured after the same period at 4°C (52). Further research has led to the design of nontoxic and biodegradable immobilization hydrogels that are composed primarily of polysaccharides including chitosan, a linear

295

296

Carbonic Anhydrases as Biocatalysts

polysaccharide chain composed of glucosamine and N-acetylglucosamine sugars, and/or alginate, containing mannuronate and guluronate moieties (57–60). Calcite precipitation measurements of immobilized CA onto chitosan revealed more than a four-fold increase over its solution counterpart, but displayed similar esterase activities (51). Further CA immobilization studies onto the surface of various bacteria, including Bacillus pumilus, Micrococcus lylae, and Pseudomonas fragi, showed up to 50% catalytic activity after 30 days of storage (61). The successful observation of calcite precipitation and esterase activity from immobilized live P. fragi and B. pumilus cells to chitosan beads may eliminate the need for protein purification (47,49). The successful expression of Helicobacter pylori CA on the surface of E. coli (62) could lead to the advancement of immobilizing live bacterial cells with surface-expressed CA onto hydrogels for biomineralization of CO2.

16.3  BIOMEDICAL CO2 CAPTURE The successful immobilization of CA onto thin liquid membranes (TLMs) (63) has accelerated research into CO2 capture for biomedical applications. The TLMs are typically composed of polypropylene derivatives that act as a semipermeable barrier, allowing for diffusion of CO2 from a flowing gas outside the membrane into the aqueous layer located underneath. This aqueous layer contains CA that will catalyze the hydration of CO2 into a highly more soluble form, bicarbonate, which can then be subsequently desorbed by lowered CO2 partial pressures further downstream (63,64). A major benefit of TLMbased systems is that they operate very efficiently at ambient pressure and temperature, but they also have some longevity concerns that will need to be addressed, such as keeping the membranes from drying and breaking, before their practical use is feasible (65).

16.3.1  Confined spaces Management of CO2 levels in confined spaces, such as in submarines and spacecraft, is important as elevated CO2 levels in the human body can have deleterious effects such as impaired judgment, acidosis, coma, and even death (66).The life management systems in these settings employ CA-immobilized TLMs that can selectively capture CO2 from a mixture of air containing nitrogen and oxygen in ratios of 1400:1 and 900:1, respectively (51,65). These bioreactors have also been shown to outperform purely chemical methods of carbon sequestration using diethylamine, which also displays a lower selectivity for nitrogen and oxygen (400:1 and 300:1, respectively) (65).

16.3.2  Artificial lungs The same principles used in industrial TLM CO2 sequestration have been extended for implementation into extracorporeal lung assistants (artificial lungs; Figure 16.3) to facilitate blood gas exchange in respiratory failure patients, but improvements in efficacy in these devices are needed before becoming an effective alternative treatment

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

Figure 16.3  Schematic of the artificial lung system. Blood is flowed in from the inlet across the HFMs, where the dissolved bicarbonate is catalytically dehydrated into CO2 via CA. The CO2 is subsequently removed from the system via an O2 stream. Figure modified from Arazawa et al. (46).

to mechanical ventilators (67–70). Current limitations of artificial lung systems include the inefficient transfer of CO2 (from a blood inlet) across the polymetric hollow fiber membrane (HFM) where it can then be flushed out of the system by a stream of oxygen (46,71). A large surface interface (1–2 m2) is required for sufficient gas exchange, which leads to issues with hemocompatibility and biocompatibility (72–77). The transfer of CO2 across the membrane can be accelerated via immobilization of CA onto the surface of the HFM, thereby reducing the required surface area for an effective gas exchange rate (46,71,78). The “bioactive” HFM containing immobilized CA demonstrated a 75% increase in the rate of CO2 removal compared with an untreated HFM. Immobilization occurs via either isourea or N-substituted imidocarbonate covalent linkages between surface amine groups of CA and cyanate esters or cyclic imidocarbonates on the surface of the HFM. This technology has been coupled with impeller devices to increase the rate of blood mixing and CO2 transfer across the HFM, but the increased shear forces denatured the immobilized CA, leading to a loss of enzyme function (46).

16.3.3  Blood substitutes In contrast to artificial lung systems where CAs are used to capture CO2 for extraction from the blood, there have also been studies performed that utilized the same for carbon

297

298

Carbonic Anhydrases as Biocatalysts

capture in blood substitutes (79). These alternative blood supplies are primarily composed of four to five cross-linked stroma-free hemoglobin molecules (termed polySFHb) that have been shown to be advantageous over transfused whole blood because they can be autoclaved, have long storage capabilities, and contain no blood antigens (80). These polySFHb substitutes, however, displayed inadequate CO2 removal rates. As a continual source of blood is needed for surgical use and natural blood is often limited in supply, research has been devoted to improvement in these blood substitutes. The engineering of superoxide dismutase (SOD), catalase (CAT), and CA enzymes into the blood substitutes (polySFHb–SOD–CAT–CA) displayed encouraging carbon capturing capabilities and antioxidant properties (79).

16.3.4  Antidote delivery CAs have also been employed in the pharmacology field as CO2 sensors involved in antidote delivery systems used in the treatment of analgesic overdose (81). Medicines that have very potent analgesic effects include the opioids, but overdoses can cause respiratory hypoventilation that leads to elevated somatic CO2 levels and to an acidosisinduced death. Monitoring of blood opioid levels with morphine-activated enzymes that release the antidotes naltrexone and naloxone, which have been prepackaged into polymer clathrates, has shown to be a successful responsive system (82). An alternate method that does not depend on opioid blood levels is the CA treatment system, which shares similar properties to those seen with other cationic hydrogels (chitosan and alginate; see above). It is unique, however, in that it reacts to a toxicity biomarker, such as high CO2 levels or acidic pH, in an antidote feedback-regulated manner. The hydrogel is composed of N,N-dimethyaminoethyl methacrylate (DMAEMA) polymers that have been modified to have a pKa of ∼7.5, making it an adequate blood pH monitor and enabling it to be incorporated into a glucose-sensitive insulin-releasing system that included CA as a CO2 sensor (83). Further research into the hydrogel design has led to a switchable coblock polymer that undergoes a reproducible transition from gel to sol on CO2 exposure (84). This transition was utilized to trigger the CO2induced release of an encapsulated protein, extenuating future potential biomedical applications of a CA-based drug delivery system that is sensitive to changes in CO2, bicarbonate, or pH.

16.4 CO2 CAPTURE FOR BIOFUEL AND BIOMASS PRODUCTION The limited availability of fossil fuel deposits and growing concerns for the long-term global environmental effects over the burning of these products have prompted many countries, including the United States, to search for alternative fuel sources (85). In the United States alone, there are an estimated 60 billion gallons of diesel and 120 billion gallons of gasoline used for transportation every year (86). Accounting for gasoline being

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

only ∼65% as efficient as diesel, this equates to a total of ∼140 billion gallons of fuel needed every year to satisfy consumer demand. Biodiesel is preferential over conventional diesel in that it emits less gaseous pollutants, including zero CO2 and sulfate emission, into the atmosphere and is nontoxic (87). However, only 15% of the U.S. biodiesel demand could be satisfied if all of the arable land in the United States were used to grow soybean for oil production (which accounts for over half of the U.S. source for biodiesel) (85,88). The current production of biofuels also displaces croplands and has been associated with increased consumer prices (89,90). An attractive alternative to the soybean-derived production of biofuels is algaebased systems. Compared with terrestrial plants, algae (cyanobacteria) have higher oil production and carbon fixation rates (91,92). Algae are an environmentally friendly alternative as they would naturally sequester atmospheric CO2, require only sunlight and minimum micronutrients for growth, and do not compete with agricultural lands as they can be cultivated in ponds or enclosed photobioreactors located on nonarable land (85). Additional medicinal agents and by-products that can be harvested from algal cultures include proteins, fatty acids, vitamins, minerals, pigments, dietary supplements, and agents used in food production, fertilizers, and other commodity products (93–95). The effects of CA on carbon flux and fixation rates in algae have shown enhanced biomass production via addition of lysed endogenous cytoplamsic Dunaliella sp. CA to algal cultures (96,97). Ongoing research investigating the effect of adding engineered extracellular CAs to algal cultures should provide further advancement in biomass and biodiesel production (98). All photosynthetic organisms utilize CAs in carbon fixation pathways to efficiently extract CO2 from a relatively dilute atmosphere. The limiting step of biomass production for these organisms is the solubilization of CO2 into the cells as bicarbonate, and thus they have evolved carbon-concentrating mechanisms that utilize CA to deliver the soluble inorganic carbon source to ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), the first major step in carbon fixation (96,99). RuBisCO (one of the most abundant proteins on Earth) catalyzes the carboxylation of ribulose-1,5-bisphosphate (RuBP) but is also one of the slowest enzymes known, with a catalytic turnover rate of 3–10 s−1 (100). Cyanobacteria has formed a means of enhancing carbon fixation by RuBisCO via formation of a protein microcompartment, called the carboxysome, which is composed of RuBisCO, CA, active bicarbonate transporters, and structural shell proteins. On bicarbonate transportation into the cytosol of the carboxysome, it is dehydrated into CO2 via CA catalysis where it is subsequently incorporated into RuBP, which is ultimately converted into two molecules of glycerate-3-phosphate. Leakage of CO2 out of the carboxysome is prevented due to its structure and the packing arrangement of the enzymes (96). Similar to the atmospheric CO2 sequestration techniques described above, algal and cyanobacteria cultures can be used to produce calcite indirectly, as evidenced in

299

300

Carbonic Anhydrases as Biocatalysts

Chlorella and Spirulina sp., and this is an ongoing area of research (94,95,101,102). The natural precipitation of calcite in microalgae serves many proposed roles including buffering purposes and as a safeguard against active transport of bicarbonate ions (102). Studies have been performed aimed to simultaneously enhance lipid production and CO2 capture in algal cultures, with Chlorella sp. showing promising results (87,101). Along with the formation of calcite in these systems, additional evidence (101) for utilization of CA in bicarbonate production during these processes came with observed decreased CO2 capture on addition of acetazolamide, a tight-binding inhibitor of the CAs (33,103–105). The catalytic activities of extracellular and intracellular CAs in red tide dinoflagellates have shown to be pH dependent, with one species displaying increased bicarbonate uptake at or above pH 9 (106). These environmentally dependent catalytic rates can be utilized in the design of an optimized system (for pH, nutrient availability, aeration, etc.) for the simultaneous production of biofuels and calcite in a cost-efficient manner.

16.5  DIRECTED EVOLUTION OF hCA II Current limitations in the aforementioned CA carbon capturing systems include the stability and/or the catalytic activity of the enzyme. The availability of high-resolution X-ray (31,105,107) and neutron structures (104,108–110) of CAs (in particular, hCA II) has produced variants that have shown enhanced kinetics (111,112) and/or thermostability (29,113,114) that could serve as attractive candidates for these systems. The kinetically enhanced hCA II variants are of particular interest because of the inherent extreme efficiency of the enzyme, comparable only to a few other human isoforms hCA IV (115,116), hCA IX (117,118), an a-CA isolated from Sulfurihydrogenibium azorense (119,120), and other enzymes including CAT and SOD (121). Utilizing structural knowledge of the active site, the researchers were able to redistribute the water network that is involved in proton transfer out of the active site and into the bulk solvent. As the release of a proton can be a rate-limiting step during catalysis (122–124), a kinetically enhanced variant could be engineered that establishes a linear network from the zinc-bound solvent molecule and H64, the proton shuttle residue (Figure 16.4A) (110,125). Catalytic measurements using 18O-labeled mass spectrometry (25) of hCA II variants containing Y7F and/or N67Q revealed a three- to nine-fold increase in the rate in proton transfer compared with the wild-type enzyme (29,111,112). The accelerated rate is contributed to either evacuation of W3A (Figure 16.4A) (Y7F variant) from the active site or displacement of W3B (Y7F/N67Q variant), leading to an overall more linear and direct hydrogen-bonded water network through the active site. Thermostabilized variants of hCA II have been recently engineered that involve either decreasing the hydrophobicity of the enzymatic surface (29) or incorporating a

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

Figure 16.4  Structural annotation of hCA II. (A) Stick model of the active site residues involved in hydrogen bond interactions with the proton transfer network. The Zn2+ metal is shown as a sphere, and the hydrogen bonds are represented with a dashed black line. Residues and water molecules are as labeled. Hydrogens for the water molecules as determined from neutron crystallography are shown in white (PDB: 3TMJ). (B) Cartoon view of engineered thermostabilized HCA II variants with mutation sites shown in stick for L100H, L224S, and L240P (PDB: 3V3F) and a disulfide linkage between A23C and L203C (PDB: 4HBA).

disulfide linkage between residues 23 and 203 (Figure 16.4B) (113,114). The substitution of surface hydrophobic leucine residues at positions 100, 224, and 240 into hydrophilic substitutes was shown to stabilize the denaturing temperature of the variant ∼7°C higher than wild-type hCA II while also being able to retain the characteristic high catalytic efficiency of the enzyme.The enhanced thermostability of this variant was concluded from X-ray crystallographic and differential scanning calorimetric studies to be an effect of enthalpic contributions from the formation of new hydrogen bonds and a gain in entropy that occurs on releasing previously ordered water molecules around the hydrophobic interface (113,114). These stabilizing surface variants were further incorporated into the kinetically enhanced Y7F and N67Q mutants mentioned above with comparable catalytic rates but showed a slight decrease in thermostability (2–3°C), possibly due to loss of ordered hydrogen bonds between the protein and water molecules in the active site. A disulfide linkage between residues 23 and 203 (hCA II numbering) is conserved among the extracellular hCAs IV, VI, IX, XII, and XIV and several bacterial CAs (e.g., Neisseria gonorrhoeae), and has been associated with their relative stability (121). As such, engineering of this disulfide linkage into an hCA II variant (Figure 16.4B) displayed a two-fold increased resistance to denaturation by guanidine-HCl (1.7 M for the variant compared with 0.9 M for the native enzyme) (114) and ∼13°C (113) increase in thermostability while also retaining desirable active site geometry and catalytic activity.

301

302

Carbonic Anhydrases as Biocatalysts

Incorporation of a disulfide linkage on the surface of hCA II between residues 99 and 242 has also been reported to increase the resistance of hCA II denaturation up to 1.4 M guanidine-HCl (126). Further research into incorporation of the aforementioned thermostabilized and catalytically enhanced mutations into a single variant should provide encouraging results.

16.6 OTHER a-CAs Several novel a-CAs have emerged from halotolerant and thermotolerant microorganisms in the past decade that could provide templates for further rational designing of hCA II for industrial applications. CA from the marine bacterium Hahella chejuensis was identified through genome sequencing (127) and later overexpressed in E. coli (128) where it was shown to have a melting temperature ∼60°C and maximal activity at 50°C. The enzyme also displayed more favorable characteristics, however, including a very high pH and salt tolerance, activity at pH 10, and calcite precipitation. An extremely thermostable a-CA has been recently reported isolated from the chemolithotropic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) that was first identified from a hot spring in Yellowstone National Park (129). SspCA is able to retain 100% activity (comparable to that of hCA II) after 2-h incubations at 100°C. Immobilization of the bacterial enzyme onto PU foam displayed extraordinary stability, retaining activity after incubation at 100°C for 50 h, and has been extended to biomimetic CO2 capture reactors (130). The X-ray crystallographic structure of SspCA (131) revealed that the possible source for its extreme thermostability could be due to increased surface charge networks and the lack of long, flexible surface loops. With the advent of fast and cost-efficient genome sequencing, further exciting CAs with favorable biophysical properties (e.g., thermostability, enhanced kinetics, tolerance to pH, salt, and chemicals) may be anticipated.

16.7 CONCLUSIONS The favorable characteristics of the CAs, such as the efficient and selective capture of CO2, have made them attractive candidates for numerous industrial and biomedical applications ranging from atmospheric carbon sequestration to artificial lung systems (Table 16.1).The relative instability and the need for a cost-efficient, large-scale production of the enzyme are often limitations in these CO2 capture systems. Many studies have been performed to enhance the stability of CAs through either covalent immobilization onto a variety of surfaces or directed evolution techniques. Further advancements in recombinant bacterial and mammalian overexpression systems could lead to quicker and higher protein yields. The cyclic process that encompasses the carbon capturing abilities of the CAs is an extraordinary one. Incorporation of CAs into CO2 sequestrating systems has provided a

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

Table 16.1  The employment of CAs for various processes Process

Principles

CA utilization

Carbon Capture of atmospheric Immobilized onto a varisequestration CO2 produced ety of surfaces includduring the burning ing enriched microorof fossil fuels ganisms, alginates, and inorganic material Calcite Chemical converProvides bicarbonate at production sion of bicarbonate a rapid rate via the to calcite; used in catalytic hydration of construction and agCO2 that is captured as ricultural materials a result of carbon sequestration and biofuel production Artificial lungs Removal of CO2 from Immobilized onto HFMs; the bloodstream catalyzes the dehydration of bicarbonate in the blood. CO2 is then swept out of the system via an inert gas stream Cross-linked SFHb Blood Engineered into SFHbs substitutes molecules that act as along with SOD and an alternative blood CAT for enhanced supply carbon sequestration Antidote deDelivery of antidotes Release of CA from livery in the treatment of encapsulated hydrogels analgesic overdose on toxicity biomarkers such as elevated CO2 levels or acidic pH in the blood Biofuel Mass algal growth and Provides inorganic production harvesting as an alcarbon in the soluble ternative fuel source form of bicarbonate to RuBisCO; the ratelimiting step in biomass production

References

(28,41–47,49–51)

(36–38,85)

(46,67–78)

(79,80)

(81–84)

(87,94,95,101,102,106)

means to effectively prevent the emission of the pollutant into the atmosphere and convert it into a nontoxic valuable commercial product. Alternatively, the sequestered CO2 from scrubbed industrial flue gas could provide a potentially unlimited carbon source for algal cultures in the mass production of biofuels. If such a system were to be designed and optimized, the elevated CO2 concentrations of past and present societies could be erased, converting the waste gas produced during the burning of fossil fuels into a nontoxic alternate fuel source for future generations.

303

304

Carbonic Anhydrases as Biocatalysts

ACKNOWLEDGMENT This chapter has partially been funded by a NIH (GM25154) award.

REFERENCES 1. Hansen J, Sato M, Ruedy R, Lacis A, Oinas V. Global warming in the twenty-first century: an alternative scenario. Proc Natl Acad Sci U S A 2000;97:9875–80. 2. EPA. Recent climate change: atmospheric changes. Climate Change Science Program.Washington, DC: United States Environmental Protection Agency; 2007http://www.epa.gov/climatechange/science/ indicators/index.html. 3. Shukman D. Carbon dioxide passes symbolic mark. British Broadcasting Corporation. 2013. Available at: http://www.bbc.co.uk/news/science-environment-22486153. 4. Luthi D, Le Floch M, Bereiter B, Blunier T, Barnola J-M, Siegenthaler U, et al. High-resolution carbon dioxide concentration record 650,000–800,000 years before present. Nature 2008;453:379–82. 5. Petit JR, Jouzel J, Raynaud D, Barkov NI, Barnola J-M, Basile I, et al. Climate and atmospheric history of the past 420,000 years from the Vostok ice core, Antarctica. Nature 1999;399:429–36. 6. Siegenthaler U, Stocker TF, Monnin E, Luthi D, Schwander J, Stauffer B, et al. Stable carbon cycle– climate relationship during the Late Pleistocene. Science 2005;310:1313–7. 7. Spahni R, Chappellaz J, Stocker TF, Loulergue L, Hausammann G, Kawamura K, et al. Atmospheric methane and nitrous oxide of the Late Pleistocene from Antarctic ice cores. Science 2005;310:1317–21. 8. Pearson PN, Palmer MR. Atmospheric carbon dioxide concentrations over the past 60 million years. Nature 2000;406:695–9. 9. IPCC. Climate change 2001: the scientific basis. In: Houghton JT, Ding Y, Griggs DJ, Noguer M, van der Linden PJ, Dai X, editors. Contribution of Working Group I to the Third Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge/New York, NY: Cambridge University Press; 2001. p. 881. 10. Weart S. The carbon dioxide greenhouse effect. The discovery of global warming. College Park, MD: American Institute of Physics; 2011http://www.aip.org/history/climate/co2.html. 11. Jansen E, Overpeck J, Briffa KR, Duplessy J-C, Joos F, Masson-Delmotte V, et al. Palaeoclimate. In: Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, et al., editors. Climate change 2007: the physical science basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge/New York, NY: Cambridge University Press. 12. IPCC. Synthesis report summary for policymakers; observed changes in climate and their effects. 2007. Available at: https://www.ipcc.ch/pdf/assessment-report/ar4/syr/ar4_syr_spm.pdf. 13. Lindsey R. State of the climate: 2011 global sea level. ClimateWatch Magazine. NOAA Climate Services Portal. 2012. Available at: http://www.climate.gov/news-features/understanding-climate/state-climate2011-global-sea-level. 14. Morello L. Oceans turn more acidic than last 800,000 years. Scientific American. 2010. Available at: http://www.scientificamerican.com/article.cfm?id=acidic-oceans. 15. Bolin B. The Kyoto negotiations on climate change: a science perspective. Science 1998;279:330–1. 16. Van der Hoeven M. CO2 emissions from fuel combustion. Paris: International Energy Agency; 2012http://www.iea.org/co2highlights/CO2highlights.pdf. 17. Harrabin R. UN climate talks extend Kyoto Protocol, promise compensation. British Broadcasting Corporation. 2012. Available at: http://www.bbc.co.uk/news/science-environment-20653018. 18. Benson SM, Surles T. Carbon dioxide capture and storage: an overview with emphasis on capture and storage in deep geological formations. Proc IEEE 2006;94:1795–805. 19. Pierre AC. Enzymatic carbon dioxide capture. ISRN Chem Eng 2012;2012:1–22. 20. Boone CD, Gill S, Habibzadegan A, McKenna R. Carbonic anhydrases and their industrial applications. Curr Top Biochem Res 2013;14:1–10. 21. Lee SW, Park SB, Jeong SK, Lim KS, Lee SH, Trachtenberg MC. On carbon dioxide storage based on biomineralization strategies. Micron 2010;41:273–82. 22. Bond GM, Medina MG, Stringer J, Simsek EFA. CO2 capture from coal-fired utility generation plant exhausts, and sequestration by a biomimetic route based on enzymatic catalysis.Washington, DC: DOE; 2008. Available at: http://www.netl.doe.gov/publications/proceedings/01/carbon_seq/5a5.pdf.

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

23. da Costa Ores J, Sala L, Cerveira GP, Kalil SJ. Purification of carbonic anhydrase from bovine erythrocytes and its application in the enzymic capture of carbon dioxide. Chemosphere 2012;88:255–9. 24. Forsman C, Behravan G, Osterman A, Jonsson BH. Production of active human carbonic anhydrase II in E. coli. Acta Chem Scand B 1988;42:314–8. 25. Silverman DN. Carbonic anhydrase: oxygen-18 exchange catalyzed by an enzyme with rate-contributing proton-transfer steps. Methods Enzymol 1982;87:732–52. 26. Lindskog S, Coleman JE. Catalytic mechanism of carbonic-anhydrase. Proc Natl Acad Sci U S A 1973;70:2505–8. 27. Lindskog S, Silverman DN. The catalytic mechanism of mammalian carbonic anhydrases. In: Chegwidden WR, Carter ND, Edwards YH editors. The carbonic anhdyrases: new horizons. Boston: Birkhäuser Verlag; 2000. p. 175–95. 28. Bond GM, Stringer J, Brandvold DK, Simsek FA, Medina M-G, Egeland G. Development of integrated system for biomimetic CO2 sequestration using the enzyme carbonic anhydrase. Energ Fuel 2001;15:309–16. 29. Fisher Z, Boone CD, Biswas SM, Venkatakrishnan B, Aggarwal M, Tu C, et al. Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II. Protein Eng Des Sel 2012;25:347–55. 30. Kanbar B, Ozdemir E. Thermal stability of carbonic anhydrase immobilized within polyurethane foam. Biotechnol Prog 2010;26:1474–80. 31. Avvaru BS, Busby SA, Chalmers MJ, Griffin PR, Venkatakrishnan B, Agbandje-McKenna M, et al. Apo-human carbonic anhydrase II revisited: implications of the loss of a metal in protein structure, stability, and solvent network. Biochemistry 2009;48:7365–72. 32. Krishnamurthy VM, Kaufman GK, Urbach AR, Gitlin I, Gudiksen KL, Weibel DB, et al. Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein–ligand binding. Chem Rev 2008;108:946–1051. 33. Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81. 34. Zevenhoven R, Eloneva S, Teir S. Chemical fixation of CO2 in carbonates: routes to valuable products and long-term storage. Catal Today 2006;115:73–9. 35. Allen DJ, Brent GF. Sequestering CO2 by mineral carbonation: stability against acid rain exposure. Environ Sci Technol 2010;44:2735–9. 36. Arakawa H, Aresta M, Armor JN, Barteau MA, Beckman EJ, Bell AT, et al. Catalysis research of relevance to carbon management: progress, challenges, and opportunities. Chem Rev 2001;101:953–96. 37. Beckman EJ. Polymer synthesis: enhanced: making polymers from carbon dioxide. Science 1999;283: 946–7. 38. Sakakura T, Choi JC,Yasuda H. Transformation of carbon dioxide. Chem Rev 2007;107:2365–87. 39. Astachov L, Nevo Z, Brosh T, Vago R. The structural, compositional and mechanical features of the calcite shell of the barnacle Tetraclita rufotincta. J Struct Biol 2011;175:311–8. 40. Suzuki M, Saruwatari K, Kogure T, Yamamoto Y, Nishimura T, Kato T, et al. An acidic matrix protein, Pif, is a key macromolecule for nacre formation. Science 2009;325:1388–90. 41. Belzil A, Parent C. Methods of chemical immobilization of an enzyme on a solid support. Biochem Cell Biol 2005;83:70–7. 42. Bhattacharya S, Nayak A, Schiavone M, Bhattacharya SK. Solubilization and concentration of carbon dioxide: novel spray reactors with immobilized carbonic anhydrase. Biotechnol Bioeng 2004;86:37–46. 43. Hosseinkhani S, Nemat-Gorgani M. Partial unfolding of carbonic anhydrase provides a method for its immobilization on hydrophobic adsorbents and protects it against irreversible thermoinactivation. Enzyme Microb Tech 2003;33:179–84. 44. Prabhu C, Valechha A, Wanjari S, Labhsetwar N, Kotwal S, Satyanarayanan T, et al. Carbon composite beads for immobilization of carbonic anhydrase. J Mol Catal B Enzym 2011;71:71–8. 45. Vinoba M, Lim KS, Lee SH, Jeong SK, Alagar M. Immobilization of human carbonic anhydrase on gold nanoparticles assembled onto amine/thiol-functionalized mesoporous SBA-15 for biomimetic sequestration of CO2. Langmuir 2011;27:6227–34. 46. Arazawa DT, Oh H-I, Ye S-H, Johnson CA Jr, Woolley JR, Wagner WR, et al. Immobilized carbonic anhydrase on hollow fiber membranes accelerates CO2 removal from blood. J Membr Sci 2012;403– 404:25–31.

305

306

Carbonic Anhydrases as Biocatalysts

47. Sharma A, Bhattacharya A, Shrivastava A. Biomimetic CO2 sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials. Enzyme Microb Tech 2011;48:416–26. 48. Liu Z, Bartlow P, Dilmore RM, Soong Y, Pan Z, Koepsel R, et al. Production, purification, and characterization of a fusion protein of carbonic anhydrase from Neisseria gonorrhoeae and cellulose binding domain from Clostridium thermocellum. Biotechnol Prog 2009;25:68–74. 49. Prabhu C, Wanjari S, Gawande S, Das S, Labhsetwar N, Kotwal S, et al. Immobilization of carbonic anhydrase enriched microorganism on biopolymer based materials. J Mol Catal B Enzym 2009;60:13–21. 50. Bond GM, Medina MG, Stringer J, Simsek EFA. Enzymatic catalysis and CO2 sequestration.World Rev 1999;11:603–19. 51. Simsek-Ege FA, Bond GM, Stringer J. Matrix molecular weight cut-off for encapsulation of carbonic anhydrase in polyelectrolyte beads. J Biomater Sci Polym Ed 2002;13:1175–87. 52. Ozdemir E. Biomimetic CO2 sequestration: 1. Immobilization of carbonic anhydrase within polyurethane foam. Energ Fuel 2009;23:5725–30. 53. Vinoba M, Bhagiyalakshmi M, Jeong SK, Nam SC,Yoon Y. Carbonic anhydrase immobilized on encapsulated magnetic nanoparticles for CO2 sequestration. Chem Eur J 2012;18:12028–34. 54. Wanjari S, Prabhu C, Satyanarayana T,Vinu A, Rayalu S. Immobilization of carbonic anhydrase on mesoporous aluminosilicate for carbonation reaction. Micropor Mesopor Mat 2012;160:151–8. 55. Vinoba M, Bhagiyalakshmi M, Jeong SK,Yoon YI, Nam SC. Immobilization of carbonic anhydrase on spherical SBA-15 for hydration and sequestration of CO2. Colloids Surf B Biointerfaces 2012;90:91–6. 56. Wood LL, Hartdegen FJ, Hahn PA. Enzymes bound to polyurethane. US Patent US 4343834. 1982. 57. Machida-Sano I,Ogawa S,Ueda H,KimuraY,Satoh N,Namiki H.Effects of composition of iron-cross-linked alginate hydrogels for cultivation of human dermal fibroblasts. Int J Biomater 2012;2012:820513. 58. Zhai P, Chen XB, Schreyer DJ. Preparation and characterization of alginate microspheres for sustained protein delivery within tissue scaffolds. Biofabrication 2013;5:015009. 59. Yadav R, Satyanarayana T, Kotwal S, Rayalu S. Enhanced carbonation reaction using chitosan-based carbonic anhydrase nanoparticles. Curr Sci India 2011;100:520–4. 60. Hosseinkhani S, Szittner R, Nemat-Gorgani M, Meighen EA. Adsorptive immobilization of bacterial luciferases on alkyl-substituted Sepharose 4B. Enzyme Microb Tech 2003;32:186–93. 61. Prabhu C,Wanjari S, Puri A, Bhattacharya A, Pujari R,Yadav R, et al. Region-specific bacterial carbonic anhydrase for biomimetic sequestration of carbon dioxide. Energ Fuel 2011;25:1327–32. 62. Fan LH, Liu N,Yu MR,Yang ST, Chen HL. Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO2 sequestration. Biotechnol Bioeng 2011;108:2853–64. 63. Bao L, Trachtenberg MC. Facilitated transport of CO2 across a liquid membrane: comparing enzyme, amine, and alkaline. J Membr Sci 2006;280:330–4. 64. Trachtenberg MC, Tu C, Landers RA, Willson RC, McGregor ML, Laipis PJ, et al. Carbon dioxide transport by proteic and facilitated transport membranes. Life Support Biosph Sci 1999;6:293–302. 65. Cowan RM, Ge J, Qin YJ, McGregor ML, Trachtenberg MC. CO2 capture by means of an enzymebased reactor. Ann N Y Acad Sci 2003;984:453–69. 66. Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 1995;64:375–401. 67. Esteban A, Anzueto A, Frutos F, Alia I, Brochard L, Stewart TE, et al. Characteristics and outcomes in adult patients receiving mechanical ventilation: a 28-day international study. JAMA 2002;287:345–55. 68. Haft JW, Griffith BP, Hirschl RB, Bartlett RH. Results of an artificial-lung survey to lung transplant program directors. J Heart Lung Transpl 2002;21:467–73. 69. Maggiore SM, Richard JC, Brochard L. What has been learnt from P/V curves in patients with acute lung injury/acute respiratory distress syndrome. Eur Respir J 2003;22:22s–6s. 70. Ware LB, Matthay MA. The acute respiratory distress syndrome. New Engl J Med 2000;342:1334–49. 71. Kimmel JD, Arazawa DT, Ye SH, Shankarraman V, Wagner WR, Federspiel WJ. Carbonic anhydrase immobilized on hollow fiber membranes using glutaraldehyde activated chitosan for artificial lung applications. J Mater Sci Mater Med 2013;24:2611–21. 72. Beckley PD, Holt DW, Tallman RD. Oxygenators for extracorporeal circulation. In: Mora CT editor. Cardiopulmonary bypass: principles and techniques of extracorporeal circulation. New York: SpringerVerlag; 1995. p. 199–219.

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

73. Federspiel WJ, Henchir KA. Lung, artificial: basic principles and current applications. In: Wnek GE, Bowlin GL editors. Encyclopedia of biomaterials and biomedical engineering. New York: Marcel Dekker; 2004. p. 910–21. 74. Hattler BG, Federspiel WJ. Gas exchange in the venous system: support for the failing lung. In:Vaslef SN, Anderson RW editors. The artificial lung. Georgetown: Landes Bioscience; 2002. p. 133–74. 75. Okamoto T, Tashiro M, Sakanashi Y, Tanimoto H, Imaizumi T, Sugita M, et al. A new heparin-bonded dense membrane lung combined with minimal systemic heparinization prolonged extracorporeal lung assist in goats. Artif Organs 1998;22:864–72. 76. Watnabe H, Hayashi J, Ohzeki H, Moro H, Sugawara M, Eguchi S. Biocompatibility of a silicone-coated polypropylene hollow fiber oxygenator in an in vitro model. Ann Thorac Surg 1999;67:1315–9. 77. Wegner JA. Oxygenator anatomy and function. J Cardiothorac Vasc Anesth 1997;11:275–81. 78. Kaar JL, Oh H-I, Russell AJ, Federspiel WJ. Towards improved artificial lungs through biocatalysis. Biomaterials 2007;28:3131–9. 79. Bian Y, Rong Z, Chang TM. Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase: a novel biotechnology-based blood substitute that transports both oxygen and carbon dioxide and also acts as an antioxidant. Artif Cells Blood Substit Immobil Biotechnol 2012;40:28–37. 80. Gould SA, Moore EE, Hoyt DB, Ness PM, Norris EJ, Carson JL, et al. The life-sustaining capacity of human polymerized hemoglobin when red cells might be unavailable. J Am Coll Surg 2002;195: 445–52. 81. Satav SS, Bhat S,Thayumanavan S. Feedback regulated drug delivery vehicles: carbon dioxide responsive cationic hydrogels for antidote release. Biomacromolecules 2010;11:1735–40. 82. Roskos KV, Fritzinger BK, Tefft JA, Nakayama GR, Heller J. Biocompatibility and in vivo morphine diffusion into a placebo morphine-triggered naltrexone delivery device in rabbits. Biomaterials 1995;16:1235–9. 83. Hassan CM, Doyle FJ, Peppas NA. Dynamic behavior of glucose-responsive poly(methacrylic acidg-ethylene glycol) hydrogels. Macromolecules 1997;30:6166–73. 84. Han D, Boissiere O, Kumar S, Tong X, Tremblay LN, Zhao Y. Two-way CO2-switchable triblock copolymer hydrogels. Macromolecules 2012;45:7440–5. 85. Chen P, Min M, Chen Y, Wang L, Li Y, Wang C, et al. Review of the biological and engineering aspects of algae to fuels approach. Int J Agric Biol Eng 2009;2:1–30. 86. Briggs M. Widescale production from algae. New Hampshire: The University of New Hampshire, Physics Department; 2004. Available at: http://www.resilience.org/stories/2004-10-03/widescalebiodiesel-production-algae#. 87. Fulke AB, Mudliar SN,Yadav R, Shekh A, Srinivasan N, Ramanan R, et al. Bio-mitigation of CO2, calcite formation and simultaneous biodiesel precursors production using Chlorella sp. Bioresour Technol 2010;101:8473–6. 88. Monthly biodiesel production report. Washington, DC: U.S. Energy Information Administration; April 29, 2013. www.eia.gov. 89. V. M-B, Samiei H, Cheng K. Biofuel demand pushes up food prices. IMF Survey Magazine.Washington, DC: IMF Research Department; 2007. 90. Boddiger D. Boosting biofuel crops could threaten food security. Lancet 2007;370:923–4. 91. Jeong MJ, Gillis JM, Hwang J-Y. Carbon dioxide mitigation by microalgal photosynthesis. Bull Korean Chem Soc 2003;24:1763–6. 92. Johnson MB,Wen Z. Production of biodiesel fuel from the microalga Schizochytrium limacinum by direct transesterification of algal biomass. Energ Fuel 2009;23:5179–83. 93. Lopez CVG, Fernandez FGA, Sevilla JMF, Fernandez JFS, Garcia MCC, Grima EM. Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in carbon dioxide removal processes. Bioresour Technol 2009;100:5904–10. 94. Gonzalez-Fernandez C, Ballesteros M. Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation. Biotechnol Adv 2012;30:1655–61. 95. Pires JCM, Alvim-Ferraz MCM, Martins FG, Simões M. Carbon dioxide capture from flue gases using microalgae: engineering aspects and biorefinery concept. Renew Sust Energ Rev 2012;16:3043–53. 96. Badger MR. CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J Exp Bot 2003;54:609–22.

307

308

Carbonic Anhydrases as Biocatalysts

97. Bloch MR, Sasson J, Ginzburg ME, Goldman Z, Ginzburg BZ, Garti N, et al. Conversion of halophilic algae into extractable oil. US Patent US 4341038. 1982. 98. Ellis RJ. Biochemistry: tackling unintelligent design. Nature 2010;463:164–5. 99. Cannon GC, Heinhorst S, Kerfeld CA. Carboxysomal carbonic anhydrases: structure and role in microbial CO2 fixation. Biochim Biophys Acta 2010;1804:382–92. 100. Dhingra A, Portis AR Jr, Daniell H. Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants. Proc Natl Acad Sci U S A 2004;101:6315–20. 101. Ramanan R, Kannan K, Deshkar A,Yadav R, Chakrabarti T. Enhanced algal CO2 sequestration through calcite deposition by Chlorella sp. and Spirulina platensis in a mini-raceway pond. Bioresour Technol 2010;101:2616–22. 102. Shekh AY, Krishnamurthi K, Mudliar SN,Yadav RR, Fulke AB, Devi SS, et al. Recent advancements in carbonic anhydrase–driven processes for CO2 sequestration: minireview. Crit Rev Environ Sci Technol 2012;42:1419–40. 103. Aggarwal M, Boone CD, Kondeti B, Tu C, Silverman DN, McKenna R. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex. Acta Crystallogr D 2013;69:860–5. 104. Fisher SZ, Aggarwal M, Kovalesky A, Silverman DN, McKenna R. Neutron-diffraction of acetazolamide-bound human carbonic anhydrase II reveals atomic details of drug binding. J Am Chem Soc 2012;134:14726–9. 105. Sippel KH, Robbins AH, Domsic J, Genis C, Agbandje-McKenna M, McKenna R. High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design. Acta Crystallogr F 2009;65:992–5. 106. Rost B, Richter K-U, Riebesell ULF, Hansen PJ. Inorganic carbon acquisition in red tide dinoflagellates. Plant Cell Environ 2006;29:810–22. 107. Avvaru BS, Kim CU, Sippel KH, Gruner SM, Agbandje-McKenna M, Silverman DN, et al. A short, strong hydrogen bond in the active site of human carbonic anhydrase II. Biochemistry 2010;49:249–51. 108. Fisher SZ, Kovalevsky AY, Domsic J, Mustyakimov M, Silverman DN, McKenna R, et al. Enzymes for carbon sequestration: neutron crystallographic studies of carbonic anhydrase. Acta Crystallogr D 2010;66:1178–83. 109. Fisher SZ, Kovalevsky AY, Domsic JF, Mustyakimov M, McKenna R, Silverman DN, et al. Neutron structure of human carbonic anhydrase II: implications for proton transfer. Biochemistry 2010;49: 415–21. 110. Fisher Z, Kovalevsky AY, Mustyakimov M, Silverman DN, McKenna R, Langan P. Neutron structure of human carbonic anhydrase II: a hydrogen-bonded water network “switch” is observed between pH 7.8 and 10.0. Biochemistry 2011;50:9421–3. 111. Fisher SZ,Tu C, Bhatt D, Govindasamy L, Agbandje-McKenna M, McKenna R, et al. Speeding up proton transfer in a fast enzyme: kinetic and crystallographic studies on the effect of hydrophobic amino acid substitutions in the active site of human carbonic anhydrase II. Biochemistry 2007;46:3803–13. 112. Mikulski R, West D, Sippel KH, Avvaru BS, Aggarwal M, Tu C, et al. Water networks in fast proton transfer during catalysis by human carbonic anhydrase II. Biochemistry 2013;52:125–31. 113. Boone CD, Habibzadegan A, Tu C, Silverman DN, McKenna R. Structural and catalytic characterization of a thermally stable and acid-stable variant of human carbonic anhydrase II containing an engineered disulfide bond. Acta Crystallogr D 2013;69:1414–22. 114. Mårtensson L-G, Karlsson M, Carlsson U. Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond. Biochemistry 2002;41:15867–75. 115. Stams T, Nair SK, Okuyama T, Waheed A, Sly WS, Christianson DW. Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8 A resolution. Proc Natl Acad Sci U S A 1996;93:13589–94. 116. Waheed A, Okuyama T, Heyduk T, Sly WS. Carbonic anhydrase IV: purification of a secretory form of the recombinant human enzyme and identification of the positions and importance of its disulfide bonds. Arch Biochem Biophys 1996;333:432–8. 117. Alterio V, Hilvo M, Di Fiore A, Supuran CT, Pan P, Parkkila S, et al. Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX. Proc Natl Acad Sci U S A 2009;106:16233–8.

Engineered Mammalian Carbonic Anhydrases for CO2 Capture

118. Supuran CT. Inhibition of carbonic anhydrase IX as a novel anticancer mechanism.World J Clin Oncol 2012;3:98–103. 119. Akdemir A,Vullo D, De Luca V, Scozzafava A, Carginale V, Rossi M, et al. The extremo-alpha-carbonic anhydrase (CA) from Sulfurihydrogenibium azorense, the fastest CA known, is highly activated by amino acids and amines. Bioorg Med Chem Lett 2013;23:1087–90. 120. Luca VD,Vullo D, Scozzafava A, Carginale V, Rossi M, Supuran CT, et al. An alpha-carbonic anhydrase from the thermophilic bacterium Sulphurihydrogenibium azorense is the fastest enzyme known for the CO2 hydration reaction. Bioorg Med Chem 2012;21:1465–9. 121. Aggarwal M, Boone CD, Kondeti B, McKenna R. Structural annotation of human carbonic anhydrases. J Enzyme Inhib Med Chem 2013;28:267–77. 122. An H, Tu C, Ren K, Laipis PJ, Silverman DN. Proton transfer within the active-site cavity of carbonic anhydrase III. Biochim Biophys Acta 2002;1599:21–7. 123. Becker HM, Klier M, Schuler C, McKenna R, Deitmer JW. Intramolecular proton shuttle supports not only catalytic but also noncatalytic function of carbonic anhydrase II. Proc Natl Acad Sci U S A 2011;108:3071–6. 124. Duda DM, Tu C, Fisher SZ, An H, Yoshioka C, Govindasamy L, et al. Human carbonic anhydrase III: structural and kinetic study of catalysis and proton transfer. Biochemistry 2005;44:10046–53. 125. Tu CK, Silverman DN, Forsman C, Jonsson BH, Lindskog S. Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant. Biochemistry 1989;28:7913–8. 126. Karlsson M, Martensson LG, Karlsson C, Carlsson U. Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations. Biochemistry 2005;44:3487–93. 127. Jeong H,Yim JH, Lee C, Choi SH, Park YK,Yoon SH, et al. Genomic blueprint of Hahella chejuensis, a marine microbe producing an algicidal agent. Nucleic Acids Res 2005;33:7066–73. 128. Ki MR, Min K, Kanth BK, Lee J, Pack SP. Expression, reconstruction and characterization of codonoptimized carbonic anhydrase from Hahella chejuensis for CO2 sequestration application. Bioprocess Biosyst Eng 2013;36:375–81. 129. Capasso C, De Luca V, Carginale V, Cannio R, Rossi M. Biochemical properties of a novel and highly thermostable bacterial alpha-carbonic anhydrase from Sulfurihydrogenibium yellowstonense YO3AOP1. J Enzyme Inhib Med Chem 2012;27:892–7. 130. Migliardini F, de Luca V, Carginale V, Rossi M, Corbo P, Supuran C, et al. Biomimetic CO2 capture using a highly thermostable bacterial a-carbonic anhydrase immobilized on a polyurethan foam. J Enzyme Inhib Med Chem 2013;29:146–50. 131. Di Fiore A, Capasso C, De Luca V, Monti SM, Carginale V, Supuran CT, et al. X-ray structure of the first ‘extremo-alpha-carbonic anhydrase’, a dimeric enzyme from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Acta Crystallogr D 2013;69:1150–9.

309