Int. J. Mol. Sci. 2015, 16, 15456-15480; doi:10.3390/ijms160715456 OPEN ACCESS
International Journal of
Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review
Thermostable Carbonic Anhydrases in Biotechnological Applications Anna Di Fiore *, Vincenzo Alterio, Simona M. Monti, Giuseppina De Simone and Katia D’Ambrosio * Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Napoli, Italy; E-Mails: [email protected] (V.A.); [email protected] (S.M.M.); [email protected] (G.D.S.) * Authors to whom correspondence should be addressed; E-Mails: [email protected] (A.D.F.); [email protected] (K.D.); Tel.: +39-081-2532215 (A.D.F.); +39-081-2532044 (K.D.). Academic Editor: Charles A. Collyer Received: 26 May 2015 / Accepted: 2 July 2015 / Published: 8 July 2015
Abstract: Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application. Keywords: CO2 capture protein engineering
process;
thermostable
enzyme;
carbonic
anhydrases;
1. Introduction Increased atmospheric carbon dioxide levels have been correlated with global warming. CO2 can be emitted from various sources, but mostly from the burning of fossil fuels [1–3]. To reduce the levels of atmospheric CO2, the use of renewable source of energy and the shift toward activities requiring less
Int. J. Mol. Sci. 2015, 16
15457
energy have been encouraged all around the world. However, considering the present energy demand, fossil fuels are difficult to completely substitute in the near future. Therefore, in the transition period, it is necessary to develop methods to reduce the concentration of CO2 in the atmosphere. One practical and effective approach is to capture CO2 from flue gas. Once that CO2 is sequestered, it can be either pressurized to a liquid or chemically converted to a stable compound, which can be subsequently stored in the ocean or underground [4,5]. It is also possible to convert the captured CO2 into various beneficial by-products which include acrylates, polycarbonates, stable carbonate storage polymers, methane and building materials [6–8]. Different techniques have been developed to perform CO2 capture [9]; however, the majority of these methods are too expensive and of limited efficiency. Thus, some biological methodologies, also called “bio-mimetic” CO2 capture systems, have been implemented as more economic and more sustainable technologies. They are based on the use of enzymes involved in CO2 biological processes, occurring naturally in living organisms. Carbonic anhydrases (CAs) (EC 4.2.1.1), which catalyze the reversible hydration of the CO2 molecule (CO2 + H2O ↔ HCO3− + H+), can be efficiently used in these processes [10]. CAs are ubiquitous metallo-enzymes present in prokaryotes and eukaryotes, being encoded by six distinct, evolutionarily unrelated gene families: the α-CAs (present in vertebrates, eubacteria, algae and cytoplasm of green plants), β-CAs (predominantly in eubacteria, algae and chloroplasts of both monoas well as dicotyledons), γ-CAs (mainly in Archaea and some eubacteria), δ- and ζ-CAs (both discovered in marine diatoms), and the recently identified η-CAs (present in different Plasmodium spp.) [10–12]. To perform catalytic activity, CAs need the presence in their active site of a metal ion (generally Zn2+) coordinated by three residues, which can be hystidines, cysteines or glutamine, depending on the enzyme class. The catalytic mechanism for the CO2 hydration reaction, studied in detail for the α-, β- and γ-class, consists of two steps [13,14]. In the first step a zinc-bound hydroxide leads the nucleophilic attack on a CO2 molecule with formation of bicarbonate bound to the zinc ion, which is then substituted by a water molecule (Equation (1)). The second step, the rate limiting one, consists of the regeneration of the enzyme reactive species, the zinc-bound hydroxide, via a proton transfer reaction, which occurs from the zinc-bound water molecule to the external buffer (Equation (2)). This process is generally assisted by an enzyme residue which acts as proton shuttle; in most human isoforms this residue is a histidine [15,16]. HO CO2 + EZnOH− ↔ EZnHCO3− ←⎯⎯ → EZnH2O + HCO3−
(1)
EZnH2O + B ↔ EZnOH− + BH+
(2)
2
The last years have seen an increasing interest in using CAs in CO2 capture and storage processes [17–19]; however, this use is greatly limited by the harsh conditions required in these processes, i.e., high temperatures and high concentrations of organic ions and metals [20–22]. In this context the employment of thermostable enzymes may represent an interesting possibility. These enzymes can be obtained through either isolation from microorganisms living at high temperature, i.e., thermophiles, or protein engineering techniques applied to mesophilic proteins. In this review an extensive overview of the thermostable CAs so far reported and their biotechnological applications will be provided. In particular, the first part of the paper will be dedicated to the description of the processes in which the involvement of these enzymes has been described, highlighting the most recent advances in such field. The second part will be instead focused on the
Int. J. Mol. Sci. 2015, 16
15458
biochemical and structural data currently available in literature on thermostable CAs, both those isolated from thermophilic organisms and those obtained by protein engineering techniques. 2. Thermostable CAs in CO2 Capture and Storage Processes The term “carbon capture and storage (CCS)” is generally used to describe the set of technologies aimed at capturing CO2 produced by major emitters and its long term storage. Different techniques have been developed in order to capture CO2, such as absorption into a liquid, adsorption on a solid, gas phase separation, and membrane systems [9], and among these the use of thermostable CAs has been described for chemical absorption [23]. This process involves the reaction of CO2 with a chemical solvent to form an intermediate compound, which is then treated with heat, regenerating the original solvent and a CO2 stream. In particular, in a typical chemical absorption process (Figure 1), the flue gas containing CO2 enters from the bottom into a packed absorber column at low temperature (30–50 °C) and then contacts a CO2 absorber; after absorption, the CO2-rich solvent is sent to a stripper operating at high temperatures (120–140 °C) to recover the absorber and CO2. After regeneration, the CO2-lean solvent is pumped back to the absorber for cyclic use, while the pure CO2 is compressed for the subsequent transportation and storage [23,24]. The main energy cost associated to this process is due to the heat required for the desorption and the pumping of the absorbing solution around the system [25].
Figure 1. Schematic representation of a chemical absorption process. Red lines indicate the absorber regeneration path. Alkanolamines are among the most widely used absorbers for CO2 capture [26]; they include primary, secondary and tertiary amines, such as monoethanolamine (MEA), diethanolamine (DEA) and N-methyldiethanolamine (MDEA) [27]. The proposed mechanism of CO2 absorption by alkanolamines is described by the Equations (3)–(6): R1R2NH + CO2 ↔ R1R2NH+COO−
(3)
R1R2NH+COO− + R1R2NH ↔ R1R2NCOO− + R1R2NH2+
(4)
R1R2NCOO− + H2O ↔ R1R2NH + HCO3−
(5)
R1R2R3N + H2O + CO2 ↔ R1R2R3NH+ + HCO3−
(6)
Int. J. Mol. Sci. 2015, 16
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where R1 is always an alkyl group, R2 is a hydrogen atom for primary amines and an alkyl group for secondary and tertiary amines and R3 is an alkyl group. In the case of primary and secondary amines the reaction with CO2 proceeds via a zwitterion mechanism. Initially, one amine group interacts with CO2 producing a zwitterion intermediate (Equation (3)), which reacts with a base, i.e., a second amine group, generating a carbamate (Equation (4)). Finally, the interaction of the latter compound with a water molecule leads to bicarbonate (Equation (5)). On the other side, the mechanism involving tertiary amines is essentially a base-catalyzed hydration of CO2 and implies the formation of bicarbonate directly, without passing through the carbamate (Equation (6)) [28–31]. Several studies demonstrated that the use of tertiary amines in chemical absorption processes is more advantageous with respect to the use of primary and secondary amines, since they have a lower heat of regeneration [32] and a higher capture capacity. Indeed, in tertiary amines 1 mole of amine is required to absorb 1 mole of CO2, while primary and secondary amines load 0.5 mole of CO2 per mole of amine [33]. However, the reaction of CO2 with tertiary amines is significantly slower than that with primary and secondary amines [34]. Thus, the use of CAs has been proposed to increase the CO2 absorption rate [35], facilitating the intermolecular transfer of protons [36]. Considering the high temperature used in this process, several heat-stable CAs have been employed, such as the CAs isolated from Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) [37,38] and from Caminibacter mediatlanticus (CmCA), both belonging to the α class [39,40]. Moreover, two highly stable variants of β-CAs from Desulfovibrio vulgaris (DvCA) [41] and Methanobacterium thermoautotrophicum (Cab) [42], obtained by protein engineering techniques, have also been tested for their capability to accelerate CO2 absorption in alkaline solvents. Transportation of the pure CO2 for subsequent storage also requires expensive technology. In this context the CA-catalyzed CO2 capture and its direct storage as CaCO3, which can be safely returned to the environment, is a very promising approach [20]. This method involves a number of steps, which are summarized below: CO2(g) ↔ CO2(aq)
(7)
CO2(aq) + H2O ↔ H2CO3
(8)
H2CO3 ↔ H+ + HCO3−
(9)
HCO3− ↔ H+ + CO32−
(10)
CO32− + Ca2+ ↔ CaCO3↓
(11)
The first step involves the dissolution of gaseous CO2 (Equation (7)) and its consequent reaction with water forming carbonic acid (Equation (8)), which then ionizes in bicarbonate (Equation (9)) and carbonate ions (Equation (10)). Finally, in the presence of Ca2+ ions calcium carbonate is formed and precipitates (Equation (11)). Since hydration of CO2 (Equation (8)) constitutes the rate-limiting step of this process, starting from 2001 CAs have been used to accelerate this reaction [20]. The overall process of is schematically depicted in Figure 2. Even if several studies reported the use in this process of CAs isolated from mesophilic microorganisms, such as Serratia sp. ISTD04 [43] and Citrobacter freundii [44], only recently two CAs
Int. J. Mol. Sci. 2015, 16
15460
isolated from thermophilic bacteria, Persephonella marina and Thermovibrio ammonificans, have been utilized [45–49].
Figure 2. Schematic representation of CA-based CO2 capture system and its storage as CaCO3. CO2 was sequestered in the scrubber column and the obtained carbonate ions were transformed in CaCO3 in presence of CaCl2 in the precipitation column. In both CCS technologies above described, the utilization of free CAs in solution has some disadvantages, such as a low enzyme stability, a limited repeatable usage, and the impossibility of enzyme recovery from the reaction environment. These disadvantages can be eliminated by immobilizing the enzyme within solid supports. Immobilization can be achieved both by physical or chemical methods. In the first case it involves the absorption of the enzyme onto a water insoluble matrix or supporting material, while in the second case it consists of the formation of covalent bonds between a water insoluble matrix and the enzyme [50]. So far a number of matrices have been used, such as metal-based nanoparticles [51], chitosan beads [52] and polyurethane [53], but only few thermostable CAs have been immobilized. Among these, SspCA has been immobilized on a polyurethane foam (HYPOL2060) [54] and CmCA on a hollow fibers membrane [40], generating in both cases highly stable and active enzymes. Following immobilization unwanted release of enzyme from reactor surface can happen; thus, to develop more efficient immobilization procedures, a recent patent reports the utilization of two thermostable γ-CAs isolated from Methanosarcina thermofila and Pyrococcus horikoshii, respectively [55]. In particular, this study describes the formation of γ-CA nanoassemblies, where individual enzymes are connected each other and make multiple linked interactions with the reactor surface. This can be achieved by mutating specific enzyme residues to cysteines, in order to introduce sites for biotinylation, thus allowing the subsequent formation of stable nanostructures by cross-linking of biotinylated-γ-CAs with streptavidin tetramers [55]. Further addition of an immobilization sequence at amino- or carboxy-terminus also allows for a controlled and reversible immobilization of the γ-CA to a functionalized surface.
Int. J. Mol. Sci. 2015, 16
15461
3. Carbonic Anhydrases Isolated from Thermophilic Bacteria 3.1. α-Carbonic Anhydrases Although in the past only a few studies have reported on thermostable α-CAs, and most of them has been described in patents [40,47,56], recently numerous data on these enzymes have been published in open scientific literature. In particular, four highly thermostable α-CAs, isolated from Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) [57,58], Sulphurihydrogenibium azorense (SazCA) [59–61], Thermovibrio ammonificans (TaCA) [62], and Persephonella marina EX-H1 (PmCA) [46], have been extensively studied both biochemically and kinetically. SspCA, the first of these enzymes to be kinetically characterized, showed a very high catalytic activity for the CO2 hydration reaction (kcat = 9.35 × 105 s−1; kcat/KM = 1.1 × 108 M−1·s−1 at 20 °C and pH 7.5), comparable to that of human carbonic anhydrase II (hCA II), the most active human CA isoform (see Table 1) [57,58]. This enzyme also presented esterase activity, and a thermoactivity analysis revealed an optimum working temperature of 95 °C [57]. Table 1. Kinetic parameters for the CO2 hydration reaction catalyzed by thermostable CAs. Data on hCA II have been added for comparison. Enzyme hCA II SspCA SazCA TaCA PmCA Cab MtCam (expressed in E. coli and purified aerobically) MtCam (expressed in E. coli and purified anaerobically) MtCam (expressed in M. acetivorans and purified anaerobically)
Class α α α α α β
kcat (s−1) 1.40 × 106 9.35 × 105 4.40 × 106 1.60 × 106 3.2 × 105 1.7 × 104
kcat/KM (M−1·s−1) 1.5 × 108 1.1 × 108 3.5 × 108 1.6 × 108 3.0 × 107 5.9 × 106
Ref. [63] [57] [61] [62] [46] [64]
γ
6.8 × 104
3.1 × 106
[65]
γ
24.3 × 104
5.4 × 106
[65]
γ
23.1 × 104
3.9 × 106
[66]
Thermal stability studies highlighted for SspCA an extraordinary resistance to high temperatures; indeed, it is able to retain CO2 hydration activity after incubation at 100 °C for 3 h, while its human homologues, hCA I and hCA II, are inactivated at temperature higher than 60 °C for each incubation time tested [67]. Moreover, long-term stability studies showed that SspCA has a half-life of 53 and 8 days at 40 and 70 °C, respectively (Table 2), and that after incubation at these temperatures for 28 days the enzyme still presents a significant residual activity (74% at 40 °C and 10% at 70 °C) [68]. Finally, the effect of the immobilization within a polyurethane (PU) foam on SspCA kinetic properties and its long-term stability has also been evaluated, showing that after immobilization the enzyme is still active and stable up to 50 h at 100 °C [54].
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Table 2. Long-term stability of some thermostable α-CAs at different temperatures. Data on bovine CA II (bCA II) have also been reported for comparison [45]. Temperature (°C) 40 60 70
bCA II 6 n.d.
International Journal of
Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review
Thermostable Carbonic Anhydrases in Biotechnological Applications Anna Di Fiore *, Vincenzo Alterio, Simona M. Monti, Giuseppina De Simone and Katia D’Ambrosio * Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Napoli, Italy; E-Mails: [email protected] (V.A.); [email protected] (S.M.M.); [email protected] (G.D.S.) * Authors to whom correspondence should be addressed; E-Mails: [email protected] (A.D.F.); [email protected] (K.D.); Tel.: +39-081-2532215 (A.D.F.); +39-081-2532044 (K.D.). Academic Editor: Charles A. Collyer Received: 26 May 2015 / Accepted: 2 July 2015 / Published: 8 July 2015
Abstract: Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application. Keywords: CO2 capture protein engineering
process;
thermostable
enzyme;
carbonic
anhydrases;
1. Introduction Increased atmospheric carbon dioxide levels have been correlated with global warming. CO2 can be emitted from various sources, but mostly from the burning of fossil fuels [1–3]. To reduce the levels of atmospheric CO2, the use of renewable source of energy and the shift toward activities requiring less
Int. J. Mol. Sci. 2015, 16
15457
energy have been encouraged all around the world. However, considering the present energy demand, fossil fuels are difficult to completely substitute in the near future. Therefore, in the transition period, it is necessary to develop methods to reduce the concentration of CO2 in the atmosphere. One practical and effective approach is to capture CO2 from flue gas. Once that CO2 is sequestered, it can be either pressurized to a liquid or chemically converted to a stable compound, which can be subsequently stored in the ocean or underground [4,5]. It is also possible to convert the captured CO2 into various beneficial by-products which include acrylates, polycarbonates, stable carbonate storage polymers, methane and building materials [6–8]. Different techniques have been developed to perform CO2 capture [9]; however, the majority of these methods are too expensive and of limited efficiency. Thus, some biological methodologies, also called “bio-mimetic” CO2 capture systems, have been implemented as more economic and more sustainable technologies. They are based on the use of enzymes involved in CO2 biological processes, occurring naturally in living organisms. Carbonic anhydrases (CAs) (EC 4.2.1.1), which catalyze the reversible hydration of the CO2 molecule (CO2 + H2O ↔ HCO3− + H+), can be efficiently used in these processes [10]. CAs are ubiquitous metallo-enzymes present in prokaryotes and eukaryotes, being encoded by six distinct, evolutionarily unrelated gene families: the α-CAs (present in vertebrates, eubacteria, algae and cytoplasm of green plants), β-CAs (predominantly in eubacteria, algae and chloroplasts of both monoas well as dicotyledons), γ-CAs (mainly in Archaea and some eubacteria), δ- and ζ-CAs (both discovered in marine diatoms), and the recently identified η-CAs (present in different Plasmodium spp.) [10–12]. To perform catalytic activity, CAs need the presence in their active site of a metal ion (generally Zn2+) coordinated by three residues, which can be hystidines, cysteines or glutamine, depending on the enzyme class. The catalytic mechanism for the CO2 hydration reaction, studied in detail for the α-, β- and γ-class, consists of two steps [13,14]. In the first step a zinc-bound hydroxide leads the nucleophilic attack on a CO2 molecule with formation of bicarbonate bound to the zinc ion, which is then substituted by a water molecule (Equation (1)). The second step, the rate limiting one, consists of the regeneration of the enzyme reactive species, the zinc-bound hydroxide, via a proton transfer reaction, which occurs from the zinc-bound water molecule to the external buffer (Equation (2)). This process is generally assisted by an enzyme residue which acts as proton shuttle; in most human isoforms this residue is a histidine [15,16]. HO CO2 + EZnOH− ↔ EZnHCO3− ←⎯⎯ → EZnH2O + HCO3−
(1)
EZnH2O + B ↔ EZnOH− + BH+
(2)
2
The last years have seen an increasing interest in using CAs in CO2 capture and storage processes [17–19]; however, this use is greatly limited by the harsh conditions required in these processes, i.e., high temperatures and high concentrations of organic ions and metals [20–22]. In this context the employment of thermostable enzymes may represent an interesting possibility. These enzymes can be obtained through either isolation from microorganisms living at high temperature, i.e., thermophiles, or protein engineering techniques applied to mesophilic proteins. In this review an extensive overview of the thermostable CAs so far reported and their biotechnological applications will be provided. In particular, the first part of the paper will be dedicated to the description of the processes in which the involvement of these enzymes has been described, highlighting the most recent advances in such field. The second part will be instead focused on the
Int. J. Mol. Sci. 2015, 16
15458
biochemical and structural data currently available in literature on thermostable CAs, both those isolated from thermophilic organisms and those obtained by protein engineering techniques. 2. Thermostable CAs in CO2 Capture and Storage Processes The term “carbon capture and storage (CCS)” is generally used to describe the set of technologies aimed at capturing CO2 produced by major emitters and its long term storage. Different techniques have been developed in order to capture CO2, such as absorption into a liquid, adsorption on a solid, gas phase separation, and membrane systems [9], and among these the use of thermostable CAs has been described for chemical absorption [23]. This process involves the reaction of CO2 with a chemical solvent to form an intermediate compound, which is then treated with heat, regenerating the original solvent and a CO2 stream. In particular, in a typical chemical absorption process (Figure 1), the flue gas containing CO2 enters from the bottom into a packed absorber column at low temperature (30–50 °C) and then contacts a CO2 absorber; after absorption, the CO2-rich solvent is sent to a stripper operating at high temperatures (120–140 °C) to recover the absorber and CO2. After regeneration, the CO2-lean solvent is pumped back to the absorber for cyclic use, while the pure CO2 is compressed for the subsequent transportation and storage [23,24]. The main energy cost associated to this process is due to the heat required for the desorption and the pumping of the absorbing solution around the system [25].
Figure 1. Schematic representation of a chemical absorption process. Red lines indicate the absorber regeneration path. Alkanolamines are among the most widely used absorbers for CO2 capture [26]; they include primary, secondary and tertiary amines, such as monoethanolamine (MEA), diethanolamine (DEA) and N-methyldiethanolamine (MDEA) [27]. The proposed mechanism of CO2 absorption by alkanolamines is described by the Equations (3)–(6): R1R2NH + CO2 ↔ R1R2NH+COO−
(3)
R1R2NH+COO− + R1R2NH ↔ R1R2NCOO− + R1R2NH2+
(4)
R1R2NCOO− + H2O ↔ R1R2NH + HCO3−
(5)
R1R2R3N + H2O + CO2 ↔ R1R2R3NH+ + HCO3−
(6)
Int. J. Mol. Sci. 2015, 16
15459
where R1 is always an alkyl group, R2 is a hydrogen atom for primary amines and an alkyl group for secondary and tertiary amines and R3 is an alkyl group. In the case of primary and secondary amines the reaction with CO2 proceeds via a zwitterion mechanism. Initially, one amine group interacts with CO2 producing a zwitterion intermediate (Equation (3)), which reacts with a base, i.e., a second amine group, generating a carbamate (Equation (4)). Finally, the interaction of the latter compound with a water molecule leads to bicarbonate (Equation (5)). On the other side, the mechanism involving tertiary amines is essentially a base-catalyzed hydration of CO2 and implies the formation of bicarbonate directly, without passing through the carbamate (Equation (6)) [28–31]. Several studies demonstrated that the use of tertiary amines in chemical absorption processes is more advantageous with respect to the use of primary and secondary amines, since they have a lower heat of regeneration [32] and a higher capture capacity. Indeed, in tertiary amines 1 mole of amine is required to absorb 1 mole of CO2, while primary and secondary amines load 0.5 mole of CO2 per mole of amine [33]. However, the reaction of CO2 with tertiary amines is significantly slower than that with primary and secondary amines [34]. Thus, the use of CAs has been proposed to increase the CO2 absorption rate [35], facilitating the intermolecular transfer of protons [36]. Considering the high temperature used in this process, several heat-stable CAs have been employed, such as the CAs isolated from Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) [37,38] and from Caminibacter mediatlanticus (CmCA), both belonging to the α class [39,40]. Moreover, two highly stable variants of β-CAs from Desulfovibrio vulgaris (DvCA) [41] and Methanobacterium thermoautotrophicum (Cab) [42], obtained by protein engineering techniques, have also been tested for their capability to accelerate CO2 absorption in alkaline solvents. Transportation of the pure CO2 for subsequent storage also requires expensive technology. In this context the CA-catalyzed CO2 capture and its direct storage as CaCO3, which can be safely returned to the environment, is a very promising approach [20]. This method involves a number of steps, which are summarized below: CO2(g) ↔ CO2(aq)
(7)
CO2(aq) + H2O ↔ H2CO3
(8)
H2CO3 ↔ H+ + HCO3−
(9)
HCO3− ↔ H+ + CO32−
(10)
CO32− + Ca2+ ↔ CaCO3↓
(11)
The first step involves the dissolution of gaseous CO2 (Equation (7)) and its consequent reaction with water forming carbonic acid (Equation (8)), which then ionizes in bicarbonate (Equation (9)) and carbonate ions (Equation (10)). Finally, in the presence of Ca2+ ions calcium carbonate is formed and precipitates (Equation (11)). Since hydration of CO2 (Equation (8)) constitutes the rate-limiting step of this process, starting from 2001 CAs have been used to accelerate this reaction [20]. The overall process of is schematically depicted in Figure 2. Even if several studies reported the use in this process of CAs isolated from mesophilic microorganisms, such as Serratia sp. ISTD04 [43] and Citrobacter freundii [44], only recently two CAs
Int. J. Mol. Sci. 2015, 16
15460
isolated from thermophilic bacteria, Persephonella marina and Thermovibrio ammonificans, have been utilized [45–49].
Figure 2. Schematic representation of CA-based CO2 capture system and its storage as CaCO3. CO2 was sequestered in the scrubber column and the obtained carbonate ions were transformed in CaCO3 in presence of CaCl2 in the precipitation column. In both CCS technologies above described, the utilization of free CAs in solution has some disadvantages, such as a low enzyme stability, a limited repeatable usage, and the impossibility of enzyme recovery from the reaction environment. These disadvantages can be eliminated by immobilizing the enzyme within solid supports. Immobilization can be achieved both by physical or chemical methods. In the first case it involves the absorption of the enzyme onto a water insoluble matrix or supporting material, while in the second case it consists of the formation of covalent bonds between a water insoluble matrix and the enzyme [50]. So far a number of matrices have been used, such as metal-based nanoparticles [51], chitosan beads [52] and polyurethane [53], but only few thermostable CAs have been immobilized. Among these, SspCA has been immobilized on a polyurethane foam (HYPOL2060) [54] and CmCA on a hollow fibers membrane [40], generating in both cases highly stable and active enzymes. Following immobilization unwanted release of enzyme from reactor surface can happen; thus, to develop more efficient immobilization procedures, a recent patent reports the utilization of two thermostable γ-CAs isolated from Methanosarcina thermofila and Pyrococcus horikoshii, respectively [55]. In particular, this study describes the formation of γ-CA nanoassemblies, where individual enzymes are connected each other and make multiple linked interactions with the reactor surface. This can be achieved by mutating specific enzyme residues to cysteines, in order to introduce sites for biotinylation, thus allowing the subsequent formation of stable nanostructures by cross-linking of biotinylated-γ-CAs with streptavidin tetramers [55]. Further addition of an immobilization sequence at amino- or carboxy-terminus also allows for a controlled and reversible immobilization of the γ-CA to a functionalized surface.
Int. J. Mol. Sci. 2015, 16
15461
3. Carbonic Anhydrases Isolated from Thermophilic Bacteria 3.1. α-Carbonic Anhydrases Although in the past only a few studies have reported on thermostable α-CAs, and most of them has been described in patents [40,47,56], recently numerous data on these enzymes have been published in open scientific literature. In particular, four highly thermostable α-CAs, isolated from Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) [57,58], Sulphurihydrogenibium azorense (SazCA) [59–61], Thermovibrio ammonificans (TaCA) [62], and Persephonella marina EX-H1 (PmCA) [46], have been extensively studied both biochemically and kinetically. SspCA, the first of these enzymes to be kinetically characterized, showed a very high catalytic activity for the CO2 hydration reaction (kcat = 9.35 × 105 s−1; kcat/KM = 1.1 × 108 M−1·s−1 at 20 °C and pH 7.5), comparable to that of human carbonic anhydrase II (hCA II), the most active human CA isoform (see Table 1) [57,58]. This enzyme also presented esterase activity, and a thermoactivity analysis revealed an optimum working temperature of 95 °C [57]. Table 1. Kinetic parameters for the CO2 hydration reaction catalyzed by thermostable CAs. Data on hCA II have been added for comparison. Enzyme hCA II SspCA SazCA TaCA PmCA Cab MtCam (expressed in E. coli and purified aerobically) MtCam (expressed in E. coli and purified anaerobically) MtCam (expressed in M. acetivorans and purified anaerobically)
Class α α α α α β
kcat (s−1) 1.40 × 106 9.35 × 105 4.40 × 106 1.60 × 106 3.2 × 105 1.7 × 104
kcat/KM (M−1·s−1) 1.5 × 108 1.1 × 108 3.5 × 108 1.6 × 108 3.0 × 107 5.9 × 106
Ref. [63] [57] [61] [62] [46] [64]
γ
6.8 × 104
3.1 × 106
[65]
γ
24.3 × 104
5.4 × 106
[65]
γ
23.1 × 104
3.9 × 106
[66]
Thermal stability studies highlighted for SspCA an extraordinary resistance to high temperatures; indeed, it is able to retain CO2 hydration activity after incubation at 100 °C for 3 h, while its human homologues, hCA I and hCA II, are inactivated at temperature higher than 60 °C for each incubation time tested [67]. Moreover, long-term stability studies showed that SspCA has a half-life of 53 and 8 days at 40 and 70 °C, respectively (Table 2), and that after incubation at these temperatures for 28 days the enzyme still presents a significant residual activity (74% at 40 °C and 10% at 70 °C) [68]. Finally, the effect of the immobilization within a polyurethane (PU) foam on SspCA kinetic properties and its long-term stability has also been evaluated, showing that after immobilization the enzyme is still active and stable up to 50 h at 100 °C [54].
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Table 2. Long-term stability of some thermostable α-CAs at different temperatures. Data on bovine CA II (bCA II) have also been reported for comparison [45]. Temperature (°C) 40 60 70
bCA II 6 n.d.
