Investigating the Application of Enzyme Carbonic Anhydrase for CO2

Jan 5, 2007 - Biological sequestration of carbon dioxide (CO2) in geological formations is one of the proposed methods to reduce the carbon dioxide re...
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Ind. Eng. Chem. Res. 2007, 46, 921-926

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GENERAL RESEARCH Investigating the Application of Enzyme Carbonic Anhydrase for CO2 Sequestration Purposes Parissa Mirjafari, Koorosh Asghari, and Nader Mahinpey* Faculty of Engineering, UniVersity of Regina, 3737 Wascana Parkway, Regina, Saskatchewan, Canada S4S 0A2

Biological sequestration of carbon dioxide (CO2) in geological formations is one of the proposed methods to reduce the carbon dioxide released into the atmosphere. In this method, an enzyme is used to enhance the hydration and subsequent precipitation of CO2. In the present work, the effect of bovine carbonic anhydrase on the hydration of CO2, and its precipitation in the form of calcium carbonate, was studied. The enzyme enhanced the hydration reaction. The rate of hydration reaction increased with both the enzyme concentration and temperature. The precipitation of calcium carbonate was promoted in the presence of the enzyme. The concentration of the enzyme did not affect the precipitation; however, temperature impacted the precipitation of calcium carbonate. At higher temperatures, less calcium carbonate was formed. Also, in the presence of the enzyme, calcium carbonate settled more quickly. The enzyme activity was not influenced by the pH of the reaction mixture. In contrast, the formation of calcium carbonate was affected by the pH of the solution. A kinetic analysis was performed for the bovine carbonic anhydrase. Based on the experimental results, the activation energy and catalytic rate constant are estimated as 700.91 cal/mol and 0.65 s-1, respectively. Introduction Carbon dioxide (CO2), as well as other greenhouse gases, has been emitted into the atmosphere by anthropogenic activities since the industrial revolution. Carbon dioxide is the most abundant greenhouse gas. It is emitted from industries that utilize fossil fuels, such as power plants that burn coal. The concentration of CO2 has increased by 40% from the preindustrial level, which is equivalent to a concentration rise from 280 to 360 ppm.1 It is believed that the increase in the concentration of CO2 is responsible for global warming, as the temperature of the earth has increased 0.3 °C per decade.1,2 This is considered to have a significant impact on the earth’s climate. Hence, it is essential to find ways to reduce the emission of CO2 to the atmosphere. There are various methods of reducing the concentration of CO2 in the atmosphere. These methods are normally classified as (1) reduction of formation of CO2 and (2) reduction of emission of CO2. Formation of CO2 can be reduced by a shift of consumption patterns toward activities requiring less energy, by developing energy-efficient technologies, by replacing fossil fuels with clean fuels, such as hydrogen, and by utilization of biomass as a renewable source of energy.2 However, due to both the abundance and the comparatively reasonable price associated with the use of fossil fuels, it is unlikely that they will be phased out in the near future. Therefore, in the transition period, before an energy supply and demand system with acceptable CO2 emissions is reached, it is necessary to develop methods to reduce the concentration of CO2 in the atmosphere. One approach is to recover CO2 from industrial flue gases and transport it to suitable locations for storage. Various locations have been proposed for CO2 storage, including the oceans, deep aquifers, and depleted oil and gas reservoirs.3 Storing CO2 in the oceans may affect aquatic life by decreasing * To whom correspondence should be addressed. Tel.: (306) 5854490. Fax: (306) 585-4855. E-mail: [email protected].

the pH of water. Storage of CO2 in depleted oil and gas reservoirs poses problems in terms of ensuring the safety of long-term CO2 disposal and the potential environmental impact and danger to human life in the event of leakage of CO2.3 Another alternative for CO2 disposal is sequestration based on the chemical fixation of CO2 in the form of carbonate minerals such as calcite, magnesite, and dolomite. This is a safe and permanent method of disposing of CO2. These carbonate minerals are abundant in nature and are environmentally benign and stable. Mineralization of CO2 can be achieved by direct contact of gaseous CO2 with mineral sources of calcium or magnesium or by dissolving CO2 in water and then bringing the solution into contact with the minerals. Either way will produce calcium or magnesium carbonate, which are solids and will precipitate.4,5 Reactions in the Indirect Method In the indirect method of mineralization of CO2, calcium carbonate is produced through a reaction between calcium ions and aqueous CO2. The following reactions take place in this process: (1) First, gaseous CO2 dissolves in water to form aqueous CO2 (reaction 1).

CO2(g) T O2(aq)

(1)

(2) Then, aqueous CO2 reacts with water to form carbonic acid: k2,-2

CO2(aq) + H2O 798 H2CO3 k2 ) 6.2 × 10-2 s-1 and k-2 ) 23.7 s-1 6,7 K2 )

k2 6.2 × 10-2 ) ) 2.6 × 10-3 k-2 23.7

10.1021/ie060287u CCC: $37.00 © 2007 American Chemical Society Published on Web 01/05/2007

(2)

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(3) In the next step, carbonic acid dissociates to bicarbonate and carbonate ions: k3,-3

H2CO3798 H+ + HCO3k4,-4

HCO3- 798 H+ + CO32-

(3) (4)

The equilibrium constant of reaction 3 is equal to 1.7 × 10-4:

K3 )

k3 8 × 106 (s-1) ) ) 1.7 × 10-4 k-3 4.7 × 1010 (s-1)

Reaction 3 is very rapid and is virtually diffusion controlled. (4) At the end, in the presence of calcium cations, calcium carbonate forms and precipitates:

Ca2+ + CO32- f CaCO3V

(5)

Among reactions 1-5, reaction 2 is the slowest, and it is the rate-limiting step. It is proposed that a biological catalyst be used to increase the rate of this reaction.8-10 The biological catalyst for this reaction is enzyme carbonic anhydrase. Carbonic anhydrases are very well-known enzymes that are ubiquitous in nature. They can be found in animals and plants and even in the human erythrocyte. They exist in different forms, with different structures and molecular weights, and their activities vary from one to another.11 They are among the fastest enzymes known. For instance, each molecule of isozyme C from the human body can catalyze 1.4 × 106 molecules of CO2 in 1 s.12 In the presence of an anhydrase enzyme, the mechanism of hydration of CO2 changes completely. The evidence suggests that the catalysis of CO2 hydration is initiated by the nucleophilic attack on the carbon atom of CO2, by zinc-bound OH-, to produce bicarbonate, which is then displaced from zinc by a water molecule:11

E‚ZnH2O T EZnOH- + H+

(6)

E‚ZnOH- + CO2 T EZnHCO3-

(7)

E‚ZnHCO3- + H2O T EZnH2O + HCO3-

(8)

In this new mechanism, reaction 2, which has the slowest reaction rate, is eliminated, and, therefore, the overall reaction rate is enhanced dramatically. In the present work, the feasibility of using this enzyme as a catalyst for hydration of CO2, as well as its precipitation in the form of calcium carbonate, was studied in a batch system. The effects of enzyme concentration, temperature, and buffer on the hydration of CO2 and formation of calcium carbonate were investigated. In addition, a kinetic analysis was performed on the experimental results to determine the activation energy of bovine carbonic anhydrase. Materials and Methods Enzymatic Hydration of CO2. Bovine carbonic anhydrase (Sigma-Aldrich Co.) was used for the experiments in this study. Carbonic anhydrase catalyzes the hydration reaction of CO2, and consequently, hydrogen ions are transferred between the active site of the enzyme and the surrounding buffer. This results in a change in pH. Therefore, measuring pH via the delta pH method is a viable method to monitor the progress of this

enzymatic reaction.13 The pH electrodes used for this study are Beetrode electrodes manufactured by World Precision Instruments Incorporation (WPI). These are specialized electrodes with a solid-state pH sensor with ideal characteristics over a wide pH range, which exhibits a larger Eo than conventional glass electrodes. The proper application of these electrodes requires a separate reference electrode such as WPI’s Dri-Ref Series. Therefore, the pH meter should be capable of connecting both Beetrode and reference electrodes. The tip diameter for a Beetrode electrode is 0.1 mm, and the pH measurement response time for this electrode is less than 1 s. The effect of the enzyme on the hydration of CO2 was studied using a reaction mixture containing 15 mL of phosphate buffer (potassium dehydrate phosphate/sodium hydroxide buffer with a pH 6.86, Fisher Scientific) and 5 mL of the enzyme solution at concentrations of 0.2, 0.4, 0.8, 2, and 6 µM. Each reaction mixture containing a particular concentration of the enzyme was transferred to a beaker, and its temperature was maintained at 0 °C. The mixture was stirred with a magnetic stirrer. Carbon dioxide solution was prepared by bubbling deionized water with gaseous CO2, and then, at the start of the experiments, 20 mL of this solution was introduced to the mixture of the enzyme and buffer. The progress of the reaction was monitored by measuring the pH of the mixture. The experiment was terminated when there was no further change in pH. The effect of temperature on the enzymatic hydration of CO2 was studied by changing the temperature of the reaction mixture from 0 to 30 °C. The presence of buffer is important for the precipitation of calcium carbonate. In the absence of buffer, the presence of CO2 drives pH to low values, which decelerates the dissociation of bicarbonate (eq 4) in the forward direction to produce carbonate ions (CO32-). Therefore, in order to avoid the condition of low pH, buffer solution should be added to the reaction mixture. To observe the effect of buffer on the reaction, all the above experiments were repeated in the absence of the phosphate buffer. In these experiments, 15 mL of phosphate buffer was replaced by 15 mL of deionized water in the reaction mixture. A control reaction mixture was prepared by replacing the enzyme solution with 5 mL of deionized water. This was used to compare the reaction progress with and without the enzyme. Enzymatic Precipitation of Calcium Carbonate. The influence of bovine enzyme on the precipitation of CO2 in the form of calcium carbonate (CaCO3) was studied using a reaction mixture containing 15 mL of the enzyme solution (with concentrations of 3 and 6 µM), 0.9 g of CaCl2‚2H2O, and the buffer solution. The buffer solution was prepared by dissolving 2.52 g of Tris buffer (tris(hydroxymethyl)aminomethane provided by EM Science)) in 15 mL of deionized water. To start the reaction, 60 mL of CO2 solution was added to this mixture. The reaction mixture was maintained at approximately 0 °C. In the original experiments, after CO2 was added, the mixture was left for 2 h and then filtered and dried to measure the weight of CaCO3 that was precipitated. The effect of temperature on the enzymatic formation of CaCO3 was studied by changing the temperature from 0 to 30 °C and to 50 °C. To compare the rate of enzymatic formation of CaCO3 with the rate of the nonenzymatic reaction, three samples of the original reaction mixture with the enzyme concentration of 6 µM were prepared (samples 1, 2, and 3). Also, three other samples (4, 5, and 6) were prepared by replacing 15 mL of the enzyme with 15 mL of deionized water (nonenzymatic precipitation). For each series of samples, the experiment was initiated by adding CO2 solution

Ind. Eng. Chem. Res., Vol. 46, No. 3, 2007 923 Table 1. Summary of Precipitation Experiments set

temp (°C)

1 2 3 4 5 6 7 8 9

0 0 0 0 30 30 50 50 50

enzyme concn (µM) 3 6 6 no enzyme 3 6 6 no enzyme 6

buffer

wt of precipitate (g)

no. of runs

error (%)

yes yes no yes yes yes yes yes no

0.2098 0.2106 0 0.19 0.1280 0.1283 0.096 0.0941 0

2 4 3 2 2 4 3 2 2

0.1 4.2 N/A 1 0.16 1.8 1.7 1.5 N/A

to the first sample of that series and then, after 5 min, the sample was filtered. Then, CO2 was added to the second sample. After 10 min, the second sample was filtered. The third sample was filtered after 15 min. This gave the change in the amount of CaCO3 in 5-min intervals. Table 1 summarizes all of the precipitation experiments. Turbidity experiments were conducted to observe the rate of formation and settlement of CaCO3 at different conditions. To observe the effect of the enzyme on the precipitation, 6 mL of the original reaction mixture with the enzyme concentration of 6 µM was transferred to a test cell, and then 13.5 mL of CO2 was injected into the cell. The change in the turbidity of the sample was monitored using a Orbeco-Hellige digital turbidimeter (Model 965-10A). To investigate the precipitation in the absence of the enzyme, the enzyme was replaced by the deionized water, and to observe the precipitation in the absence of buffer, Tris buffer was eliminated from the reaction mixture. Table 1 summarizes the experiments on the precipitation of calcium carbonate.

concentration of the enzyme. At higher enzyme concentrations, pH drops more quickly. Also, the results show that in the absence of the enzyme (control run) the rate of pH decrease is much slower compared to the rate of pH drop in the presence of the enzyme, even at low concentration of the enzyme (e.g., 0.2 µM). These results demonstrate that enzyme carbonic anhydrase increases the rate of hydration of CO2, even at low concentrations. The results of the experiments at 30 °C and in the presence of buffer are shown in Figure 3. The same trend can be seen in this figure, but in this case the rate of change in pH was higher than that at 0 °C. The comparison of rates at different concentrations at 30 °C is illustrated in Figure 4. These results suggest that, at high temperatures, the concentration of the enzyme is not as effective in the promotion of hydration of CO2 as it is in lower temperatures. This is beneficial because, in the

Results and Discussion Enzymatic Hydration of CO2. Figure 1 demonstrates the pH change at 0 °C when the buffer was in the system. Addition of CO2 to the reaction mixture at time zero tended to drop the pH of the mixture to the low values, but the presence of the buffer balanced this effect, and the pH leveled off at a value slightly lower than the pH of the buffered solution. Since the initial rate of pH drop (rA) is equal to the slope of the curves at the first few seconds of the reaction, to compare the rate of pH drop at different concentrations, the slope of the curves in Figure 1 were calculated for the early part of the reaction and -1/slope, which is equivalent to 1/rA for each enzyme concentration, as shown in Figure 2. It is clearly observed in this figure that the pH drop depends on the

Figure 2. Comparison of the rate of pH drop for different enzyme concentrations at 0 °C.

Figure 1. pH experiments at T ) 0 °C and in the presence of buffer.

Figure 3. pH experiments at T ) 0 °C and in the presence of the buffer.

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Figure 4. Comparison of the rate of pH drop for different enzyme concentrations at 30 °C.

Figure 5. Results of pH experiment at T ) 0 °C, no buffer.

Figure 6. Results of pH experiment at T ) 30 °C, no buffer.

warm environments, a low concentration of the enzyme will result in a higher rate of hydration. The same experiments at 0 and 30 °C were repeated, but in this case no phosphate buffer was added to the reaction mixture. Figures 5 and 6 illustrate these results. Since there was no buffer present in these experiments, there was no control on pH. In Figures 5 and 6, it is clearly seen that pH decreased to low values around 4. The pH values were slightly lower at 30 °C, which is due to the effect of temperature on the pH of a solution. Enzymatic Precipitation of Calcium Carbonate. The results of precipitation experiments showed that the enzymatic pre-

Figure 7. Comparison of precipitation with and without enzyme.

cipitation of CaCO3 was not dependent on the concentration of bovine enzyme. With bovine enzyme at concentrations of 3 and 6 µM, the weight of CaCO3 was 0.2098 and 0.2106 g, respectively, at 0 °C and 0.1280 and 0.1283 g, respectively, at 30 °C. These numbers are very close, and the difference is negligible. These results are tabulated in Table 1. The amount of CaCO3 decreased as the temperature increased. The weight of CaCO3 at 0, 30, and 50 °C was 0.2098, 0.1283, and 0.096 g, respectively. This is because the solubility of CO2 in water decreases with increasing temperature.14 In the experiments in which the Tris buffer was eliminated from the reaction mixture, there was no precipitation at all. This result did not depend on the temperature or concentration of the enzyme. When the buffer is absent from the reaction mixture, addition of CO2 to the mixture drives the pH down to low values (near 4). The chemistry of CO2 hydration and bicarbonate dissociation shows that, in low pH, there is not enough carbonate ion present.15 As a result, the solution does not become saturated with CaCO3. This is the reason precipitation was not observed in this condition. In the absence of the enzyme, precipitation was observed after 2 h. The amount of CaCO3 was almost the same as its amount in the presence of the enzyme (Table 1). However, Figure 7 indicates that precipitation of calcium carbonate was much faster in the presence of bovine enzyme. This figure presents a comparison of the precipitation in the presence and absence of the enzyme. It is clearly seen that, in the presence of the enzyme, CaCO3 reached its maximum value in less than 10 min; however, when no enzyme was added to the reaction mixture, the formation of calcium carbonate took place very slowly. The results from the precipitation experiments were in very good agreement with the turbidity experiments. The turbidity measurements showed that, in the presence of the enzyme, the precipitation started momentarily after injection of CO2 and rapidly jumped to its highest extent. This result confirms the results in Figure 7. Also, the turbidity measurements showed that CaCO3 settled much faster with the enzyme (Figure 8). Figure 8 shows very well that although CaCO3 was formed in the absence of the enzyme, the rate of formation was slow and it settled slowly. Also, this figure shows that precipitation of CaCO3 did not take place when the buffer was not added to the mixture. The effect of enzyme bovine carbonic anhydrase on the precipitation of calcium carbonate was also studied by Liu et al.16 The experimental solutions in their study are ionic solutions with high concentrations of calcium and magnesium. The effect of different parameters such as the presence of enzyme,

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Figure 8. Results of the turbidity experiment.

temperature, and concentration of calcium and magnesium are studied. A direct comparison of the results of the Liu study with the results obtained from this study might be rather misleading due to the ionic nature of their study. Kinetic Analysis. Since the activation energy for the bovine carbonic anhydrase was not reported previously, our attempt was to find this activation energy. It is known from previous studies that the rate of reaction 8, like many other enzymatic reactions, can be expressed by the Michaelis-Menten equation. Also, at the initial moments of the reaction, the rate of reaction 8 is equivalent to the rate of production of hydrogen. Based on this fact, a reaction rate was developed for the hydrogen production, considering reactions 6, 7, and 4. A kinetic analysis was established, using the developed equation and also the results for the pH change. The rate statement for the production of hydrogen was derived using reactions 6, 7, and 4. All of these reactions are in the steady-state condition. Based on these facts and assumptions, and by writing the mass balance for all the intermediates and components in reactions 6, 7, and 4, the final rate for the hydrogen production was as follows:

r H+ )

kcat[CO2][E0] Km + [CO2]

(9)

where kcat is the catalytic rate constant for reaction 8 and Km is the Michaelis-Menten constant. Applying the Arrhenius equation17 to eq 9 yields

d[H+] K0(exp(-Ea/RT))[CO2][E0] ) dt Km + [CO2]

(10)

In this equation, K0 is the Arrhenius constant and Ea is the activation energy. During the experiments, the change in pH was measured at different enzyme concentrations and temperatures. These results are shown in Figures 1 and 3. In order to obtain the change in the concentration of hydrogen (d[H+]/dt), the pH data on these figures were first converted to the hydrogen concentration and then the hydrogen concentration was plotted against time. Since the rates were calculated at the initial moments of the reactions, the concentration of CO2 was assumed to be equal to its initial concentration (solubility of CO2 in water). A nonlinear regression was performed to solve the equations for kcat, Km, and Ea. The NLREG program was used for the nonlinear regression. The results are as follows: kcat ) 0.65 s-1, Km ) 0.0179 mol/ L, and Ea ) 700.9115 cal/mol.

Implications and Future Work. The study presented here is only the beginning of the efforts of using enzyme as a means for CO2 sequestration. Additional studies on addressing various issues related to scaling up and also other operating conditions, such as higher pressure, are underway. In order for this process to work for CO2 sequestration, it is necessary to have calcium ions in the reaction environment. This creates a limitation on the number of geological formations where this process can be implemented. It is expected that before implementing this technique for CO2 sequestration in aquifers and/or depleted oil and gas reservoirs the locations under study will be tested for the presence and extent of calcium ions available. The presence of calcium ions is a screening criterion for applicability of this enzymatic process in underground formations. On the other hand, if this process is going to be used for removing CO2 from smaller emitters, such as smaller plants, then sources of calcium ion should be brought in as part of the process. With respect to economic feasibility, although the enzyme is relatively expensive, since it acts as a very active catalyst, it will not be lost during reactions and, hence, a large amount of enzyme does not seem to be necessary. Enzyme carbonic anhydrase has shown stability up to a temperature of 70 °C18,19 and within a pH range of 5-10.20 Therefore, although additional economic studies are needed for a more detailed evaluation, the cost of enzyme does not seem to be a strong prohibitive factor. In addition, it is envisioned that the flue gas generated from power plants and other large CO2 emitters goes through a CO2 capture facility before injection into underground formations. Therefore, the stream of CO2 will be of high concentration and less volume. The current work is, to our knowledge, the first application of enzyme for CO2 abatement and climate change technology. There is obvious room for further effort and refinement. Conclusions Mineralization of carbon dioxide is investigated as a method of converting CO2 to mineral carbonates. The slow rate of hydration of CO2 has been a limiting factor to make this method widely accepted. Different enzymes, including enzyme carbonic anhydrase, have been shown to be credible as biological catalysts to overcome this shortcoming. The primary objective of this research was to study the hydration reaction and also precipitation of CaCO3 in the presence of the enzyme bovine carbonic anhydrase. The effects of enzyme concentration, temperature, and buffer presence on the hydration reaction and on precipitation were studied. The results showed that this enzyme was a very effective catalyst. It promoted the hydration of CO2 and, consequently, the precipitation of CaCO3. The rate of the hydration reaction increased with the temperature. At a constant temperature, a higher rate was obtained with higher enzyme concentrations, but the concentration effect faded at higher temperatures. Enzyme carbonic anhydrase not only enhanced the hydration reaction of CO2, it also promoted the formation of CaCO3. The rate of precipitation of CaCO3 was higher in the presence of the enzyme. Although a higher concentration of the enzyme increased the rate of hydration, it did not affect the precipitation of CaCO3. On the contrary, the pH of a solution was shown to influence precipitation of CaCO3. Acknowledgment Financial support from the Petroleum Technology Research Center (PTRC) and Faculty of Graduate Studies and Research (FGSR) at the University of Regina is gratefully acknowledged.

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Literature Cited (1) Drake, F. Global warming, the Science of climate change; Oxford University Press: New York, 2000. (2) Hendriks, C. Carbon dioxide remoVal from coal-fired power plants; Kluwer Academic: Dordrecht, The Netherlands, 1994. (3) Bachu, S. Sequestration of CO2 in geological media: criteria and approach for site selection in response to climate change. Energy ConVers. Manage. 2000, 41, 953-970. (4) Lackner, K. S.; Butt, D. P.; Wendt, C. H.; Sharp, D. H. Carbon dioxide disposal in solid form. Proceedings of the 21st International Conference on Coal Utilization and Fuel Systems, Clearwater, FL, March 18-21, 1996. (5) Lackner, K. S.; Wendt, C. H.; Butt, D. P.; Joyce, E. L.; Sharp, D. H. Carbon dioxide disposal in carbonate minerals. Energy 1995, 20 (11), 1153-1170. (6) Sullivan, B. P.; Krist, K.; Guard, H. E. Electrical and electrocatalytic reactions of carbon dioxide; Elsevier: New York, 1993. (7) Ho, C.; Strurevant, J. M. The kinetics of hydration of carbon dioxide at 25 °C. J. Biol. Chem. 1963, 238 (10), 1-18. (8) Bond, G. M.; Egeland, G.; Brandvold, D. K.; Medina, M. G.; Simsek, F. A.; Stringer, J. Enzymatic catalysis and CO2 sequestration. World Resour. ReV. 1999, 11 (4), 603-618. (9) Bond, G. M.; Egeland, G.; Brandvold, D. K.; Medina, M. G.; Stringer, J. CO2 sequestration via a biomimetic approach. EPD Congress: Proceedings of sessions and symposia; 1999; pp 763-781. (10) Bond, G. M.; Stringer, J.; Brandvold, D. K.; Simsek, F. A.; Medina, M. G.; Egeland, G. Development of integrated system for biomimetic CO2 sequestration using the enzyme carbonic anhydrase. Energy Fuels 2001, 15, 309-316.

(11) Chegwidden, W. R.; Carter, N. D.; Edwards, Y. H. The carbonic anhydrase new horizons; Birkha¨user Verlag: Basel, Switzerland, 2000. (12) Khalifah, R. C. The carbon dioxide hydration activity of carbonic anhydrase. J. Biol. Chem. 1971, 246 (8), 2561-2573. (13) Henry, R. P. The carbonic anhydrase: cellular physiology and molecular genetics; Plenum Press: New York, 1991. (14) Green, P. Perry’s Chemical Engineering Handbook, 7th ed.; McGraw-Hill: New York, 1997. (15) Morgan, S. Aquatic chemistry, Chemical Equilibria and Rates in Natural Waters; Wiley-Interscience: New York, 1996. (16) Liu, N.; Bond, G. M.; Abel, A.; McPherson, B. J.; Stringer, J. Biomimetic sequestration of CO2 in carbonate form: role of produced waters and other forms. Fuel Process. Technol. 2005, 86, 1615-1625. (17) Levenspiel, O. Chemical reaction engineering, 3rd ed.; John Wiley & Sons: New York, 1999; p 72. (18) Lavecchia, R.; Zugaro, M. Thermal denaturation of erythrocyte carbonic anhydrase. FEBS Lett. 1991, 292 (1, 2), 162-164. (19) Garett, R. H.; Grisham, C. M. Biochemistry; Saunders College Publishing: Orlando, FL, 1995. (20) Kernohan, J. C. The pH-activity curve of bovine carbonic anhydrase and its relationship to the inhibition of the enzyme by anions. Biochem. Biophys. Acta 1965, 96, 304-317.

ReceiVed for reView March 10, 2006 ReVised manuscript receiVed November 21, 2006 Accepted November 22, 2006 IE060287U