Carbonic anhydrase (CA) is an enzyme that catalyzes reversible hydration of carbon dioxide into bicarbonate and proton. It plays various physiological functions in virtually all of the living organisms including mammals, plants, and bacteria. It has been recently suggested that this remarkably fast ...
Carbonic anhydrase (CA) is an enzyme that catalyzes reversible hydration of carbon dioxide into bicarbonate and proton. It plays various physiological functions in virtually all of the living organisms including mammals, plants, and bacteria. It has been recently suggested that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. For its practical application, however, there are two major limitations that should be intensively dealt with; i) the enzyme should be economically produced in a sufficient amount, and ii) once produced, the enzyme should retain its activity under harsh conditions such as high temperature. To address the issues, we developed efficient and economic biocatalysts with high stabilization, based on large production of a recombinant CA from Neisseria gonorrhoeae (ngCA) in Escherichia coli. In addition, we characterized new CAs from thermophilic bacteria that possess high thermostability to approach more practical application of CA. First, some properties of the recombinant ngCA produced in E. coli were examined in relevance to its application to CO2 sequestration. ngCA without N-terminal signal sequence was highly expressed in E. coli. The production yield was ~3 g of soluble enzyme per L-culture in 150-L pilot scale fermentation. The activity of ngCA was slightly inhibited by several metal ions, particularly by Ni2+ although it was not significant. Interesting result was observed by Co2+ ion treatment where the ion even stimulated the catalytic activity. Co2+ is known to substitute the active site Zn2+, and the stimulation might be exploited to produce high-activity version of ngCA. The pH dependence of esterase activity was similar to the previously reported profile of CO2 hydration. Temperature optimum was near 50 °C, a temperature frequently found in CO2 capturing facilities. The thermal stability of ngCA seems to be the major limitation for its practical application; it was rapidly deactivated under high temperature conditions. If the thermostability can be enhanced by immobilization in an efficient and economical manner, ngCA will serve as a promising biocatalyst for CO2 sequestration. Second, we engineered ngCA in the periplasm of E. coli to promote the economical use of enzymes, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. Third, we developed and characterized bioinspired silica nanoparticle with auto-encapsulated recombinant ngCA. The silica formation was mediated by the silica-condensing R5 peptide fused to the ngCA. The encapsulation efficiency was increased with a hyperbolic relation as the enzyme concentration was raised. The strong silica-R5 interaction prevented enzyme leakage after encapsulation. The catalytic efficiency of esterase activity was over 55% compared to free enzyme, while that of CO2 hydration was only ~10% of the free enzyme activity. This discrepancy did not seem to be related to the surface charges of silica, which may differently affect the accessibility to the encapsulated enzyme of different substrates. The encapsulated enzyme retained the full activity after incubation for 30 min at 60 °C, while the free enzyme lost half of the initial activity showing great enhancement of thermostability. The biosilica had amorphous, spherical structure with a diameter of 300 to 500 nm that were fused to each other displaying average particle size of ~2,000 nm. About half of the total surface area in the silica nanoparticle was exposed to external environment, implying that the outermost surface was catalytically of great importance. This bioinspired silica nanoparticle with CA can be efficiently applied to CO2 sequestration with the outstanding entrapment, catalytic performance, and stability. Finally, we attempted to find and characterize α-CAs originated from Persephonella marina and Thermovibrio ammonificans, thermophilic Gram-negative bacteria in deep-sea hydrothermal vents. The recombinant α-CAs expressed in E. coli showed catalytic efficiencies better than thermophilic β- and γ-CAs from archaea, and their activities were significantly increased at higher temperatures. Remarkably, these enzymes exhibited outstanding thermostability (stable to at least 80 °C). Through long-term stability tests at 40°C and 60°C, we discovered that the novel α-CA of T. ammonificans is the most thermostable CA ever characterized. These results demonstrate that the α-CAs of thermophilic bacteria from deep-sea hydrothermal vents are promising biocatalysts for practical industrial CO2 capture in terms of both activity and stability. Collectively, in this thesis, we developed efficient biocatalysts for CO2 sequestration, with green and economical strategies based on biological and bioinspired ways. We constructed whole-cell biocatalyst with periplasmic CA, which can be produced more economically. The recombinant CA was encapsulated in nanosilica particles that exhibited excellent catalytic performance and thermostability. Novel thermophilic CAs were cloned and characteri
Carbonic anhydrase (CA) is an enzyme that catalyzes reversible hydration of carbon dioxide into bicarbonate and proton. It plays various physiological functions in virtually all of the living organisms including mammals, plants, and bacteria. It has been recently suggested that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. For its practical application, however, there are two major limitations that should be intensively dealt with; i) the enzyme should be economically produced in a sufficient amount, and ii) once produced, the enzyme should retain its activity under harsh conditions such as high temperature. To address the issues, we developed efficient and economic biocatalysts with high stabilization, based on large production of a recombinant CA from Neisseria gonorrhoeae (ngCA) in Escherichia coli. In addition, we characterized new CAs from thermophilic bacteria that possess high thermostability to approach more practical application of CA. First, some properties of the recombinant ngCA produced in E. coli were examined in relevance to its application to CO2 sequestration. ngCA without N-terminal signal sequence was highly expressed in E. coli. The production yield was ~3 g of soluble enzyme per L-culture in 150-L pilot scale fermentation. The activity of ngCA was slightly inhibited by several metal ions, particularly by Ni2+ although it was not significant. Interesting result was observed by Co2+ ion treatment where the ion even stimulated the catalytic activity. Co2+ is known to substitute the active site Zn2+, and the stimulation might be exploited to produce high-activity version of ngCA. The pH dependence of esterase activity was similar to the previously reported profile of CO2 hydration. Temperature optimum was near 50 °C, a temperature frequently found in CO2 capturing facilities. The thermal stability of ngCA seems to be the major limitation for its practical application; it was rapidly deactivated under high temperature conditions. If the thermostability can be enhanced by immobilization in an efficient and economical manner, ngCA will serve as a promising biocatalyst for CO2 sequestration. Second, we engineered ngCA in the periplasm of E. coli to promote the economical use of enzymes, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. Third, we developed and characterized bioinspired silica nanoparticle with auto-encapsulated recombinant ngCA. The silica formation was mediated by the silica-condensing R5 peptide fused to the ngCA. The encapsulation efficiency was increased with a hyperbolic relation as the enzyme concentration was raised. The strong silica-R5 interaction prevented enzyme leakage after encapsulation. The catalytic efficiency of esterase activity was over 55% compared to free enzyme, while that of CO2 hydration was only ~10% of the free enzyme activity. This discrepancy did not seem to be related to the surface charges of silica, which may differently affect the accessibility to the encapsulated enzyme of different substrates. The encapsulated enzyme retained the full activity after incubation for 30 min at 60 °C, while the free enzyme lost half of the initial activity showing great enhancement of thermostability. The biosilica had amorphous, spherical structure with a diameter of 300 to 500 nm that were fused to each other displaying average particle size of ~2,000 nm. About half of the total surface area in the silica nanoparticle was exposed to external environment, implying that the outermost surface was catalytically of great importance. This bioinspired silica nanoparticle with CA can be efficiently applied to CO2 sequestration with the outstanding entrapment, catalytic performance, and stability. Finally, we attempted to find and characterize α-CAs originated from Persephonella marina and Thermovibrio ammonificans, thermophilic Gram-negative bacteria in deep-sea hydrothermal vents. The recombinant α-CAs expressed in E. coli showed catalytic efficiencies better than thermophilic β- and γ-CAs from archaea, and their activities were significantly increased at higher temperatures. Remarkably, these enzymes exhibited outstanding thermostability (stable to at least 80 °C). Through long-term stability tests at 40°C and 60°C, we discovered that the novel α-CA of T. ammonificans is the most thermostable CA ever characterized. These results demonstrate that the α-CAs of thermophilic bacteria from deep-sea hydrothermal vents are promising biocatalysts for practical industrial CO2 capture in terms of both activity and stability. Collectively, in this thesis, we developed efficient biocatalysts for CO2 sequestration, with green and economical strategies based on biological and bioinspired ways. We constructed whole-cell biocatalyst with periplasmic CA, which can be produced more economically. The recombinant CA was encapsulated in nanosilica particles that exhibited excellent catalytic performance and thermostability. Novel thermophilic CAs were cloned and characteri
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