Thermostable Carbonic Anhydrase Research Articles

Improved Solubility and Stability of a Thermostable Carbonic Anhydrase via Fusion with Marine-Derived Intrinsically Disordered Solubility Enhancers.

Carbonic anhydrase (CA), an enzyme catalyzing the reversible hydration reaction of carbon dioxide (CO2), is considered a promising biocatalyst for CO2 reduction. The α-CA of Thermovibrio ammonificans (taCA) has emerged as a compelling candidate due to its high thermostability, a critical factor for industrial applications. However, the low-level expression and poor in vitro solubility have hampered further utilization of taCA. Recently, these limitations have been addressed through the fusion of the NEXT tag, a marine-derived, intrinsically disordered small peptide that enhances protein expression and solubility. In this study, the solubility and stability of NEXT-taCA were further investigated. When the linker length between the NEXT tag and the taCA was shortened, the expression level decreased without compromising solubility-enhancing performance. A comparison between the NEXT tag and the NT11 tag demonstrated the NEXT tag's superiority in improving both the expression and solubility of taCA. While the thermostability of taCA was lower than that of the extensively engineered DvCA10, the NEXT-tagged taCA exhibited a 30% improvement in long-term thermostability compared to the untagged taCA, suggesting that enhanced solubility can contribute to enzyme thermostability. Furthermore, the bioprospecting of two intrinsically disordered peptides (Hcr and Hku tags) as novel solubility-enhancing fusion tags was explored, demonstrating their performance in improving the expression and solubility of taCA. These efforts will advance the practical application of taCA and provide tools and insights for enzyme biochemistry and bioengineering.

Carbonic Anhydrase Robustness for Use in Nanoscale CO2 Capture Technologies.

Continued dependence on crude oil and natural gas resources for fossil fuels has caused global atmospheric carbon dioxide (CO2) emissions to increase to record-setting proportions. There is an urgent need for efficient and inexpensive carbon sequestration systems to mitigate large-scale emissions of CO2 from industrial flue gas. Carbonic anhydrase (CA) has shown high potential for enhanced CO2 capture applications compared to conventional absorption-based methods currently utilized in various industrial settings. This study aims to understand structural aspects that contribute to the stability of CA enzymes critical for their applications in industrial processes, which require the ability to withstand conditions different from those in their native environments. Here, we evaluated the thermostability and enzyme activity of mesophilic and thermophilic CA variants at different temperature conditions and in the presence of atmospheric gas pollutants like nitrogen oxides and sulfur oxides. Based on our enzyme activity assays and molecular dynamics simulations, we see increased conformational stability and CA activity levels in thermostable CA variants incubated week-long at different temperature conditions. The thermostable CA variants also retained high levels of CA activity despite changes in solution pH due to increasing NO and SO2 concentrations. A loss of CA activity was observed only at high concentrations of NO/SO2 that possibly can be minimized with the appropriate buffered solutions.

Rational design engineering of a more thermostable Sulfurihydrogenibium yellowstonense carbonic anhydrase for potential application in carbon dioxide capture technologies

Implementing hyperthermostable carbonic anhydrases into CO2 capture and storage technologies in order to increase the rate of CO2 absorption from the industrial flue gases is of great importance from technical and economical points of view. The present study employed a combination of in silico tools to further improve thermostability of a known thermostable carbonic anhydrase from Sulfurihydrogenibium yellowstonense. Experimental results showed that our rationally engineered K100G mutant not only retained the overall structure and catalytic efficiency but also showed a 3 °C increase in the melting temperature and a two-fold improvement in the enzyme half-life at 85 °C. Based on the molecular dynamics simulation results, rearrangement of salt bridges and hydrogen interactions network causes a reduction in local flexibility of the K100G variant. In conclusion, our study demonstrated that thermostability can be improved through imposing local structural rigidity by engineering a single-point mutation on the surface of the enzyme.

Enhanced Production of a Thermostable Carbonic Anhydrase in Escherichia coli by Using a Modified NEXT Tag.

Carbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO2) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO2 capture and utilization. In particular, the CA of Thermovibrio ammonificans (taCA) has attracted increasing attention as a highly stable enzyme. However, the poor solubility and the low expression level in Escherichia coli have hampered further utilization of taCA. In a recent study, these limitations were partly resolved by using a small solubility-enhancing fusion tag named NEXT, which originates from the N-terminal extension of Hydrogenovibrio marinus CA. In this study, the NEXT tag was engineered by adding small peptides to the N terminus to further increase the production yield of NEXT-tagged taCA. The addition of ng3 peptide (His-Gly-Asn) originating from the N-terminal sequence of Neisseria gonorrhoeae CA improved the expression of NEXT-taCA, while the previously developed translation-enhancing element (TEE) and Ser-Lys-Ile-Lys (SKIK) tag were not effective. The expression test with all 16 codon combinations for the ng3 sequence revealed that the change in translation initiation rate brought about by the change in nucleotide sequence was not the primary determinant for the change in expression level. The modified ng3-NEXT tag may be applied to increase the production yields of various recombinant proteins.

Molecular dynamics simulations identify the regions of compromised thermostability in SazCA.

The present study examined the structure and dynamics of the most active and thermostable carbonic anhydrase, SazCA, probed using molecular dynamics simulations. The molecular system was described by widely used biological force-fields (AMBER, CHARMM22, CHARMM36, and OPLS-AA) in conjunction with TIP3P water model. The comparison of molecular dynamics simulation results suggested AMBER to be a suitable choice to describe the structure and dynamics of SazCA. In addition to this, we also addressed the effect of temperature on the stability of SazCA. We performed molecular dynamics simulations at 313, 333, 353, 373, and 393 K to study the relationship between thermostability and flexibility in SazCA. The amino acid residues VAL98, ASN99, GLY100, LYS101, GLU145, and HIS207 were identified as the most flexible residues from root-mean-square fluctuations. The salt bridge analysis showed that ion-pairs ASP113-LYS81, ASP115-LYS81, ASP115-LYS114, GLU144-LYS143, and GLU144-LYS206, were responsible for the compromised thermal stability of SazCA.

High-level production in a plant system of a thermostable carbonic anhydrase and its immobilization on microcrystalline cellulose beads for CO2 capture

Plant-produced SazCA and its application to CO2 capture. Technologies that rely on chemical absorption or physical adsorption have been developed to capture CO2 from industrial flue gases and sequester it at storage sites. Carbonic anhydrases (CAs), metalloenzymes, that catalyze the reversible hydration of CO2 have recently received attention as biocatalysts in the capture of CO2 from flue gases, but their cost presents a major obstacle for use at an industrial scale. This cost, however, can be reduced either by producing a long-lasting enzyme suitable for CO2 capture or by lowering production costs. High-level expression, easy purification, and immobilization of CAs from Sulfurihydrogenibium azorense (SazCA) were investigated in a plant system. Fusion of the 60-amino acid-long ectodomain (M-domain) of the human receptor-type tyrosine-protein phosphatase C increased the levels of SazCA accumulation. Fusion of the cellulose-binding module (CBM3) from Clostridium thermocellum resulted in tight binding of recombinant protein to microcrystalline cellulose beads, enabling easy purification. The chimeric fusion protein, BMC-SazCA, which consisted of SazCA with the M and CBM3 domains, was expressed in tobacco (Nicotiana benthamiana), giving a recombinant protein yield in leaf extracts of 350mg/kg fresh weight. BMC-SazCA produced in planta was active in the presence of various chemicals used in CO2 capture. Immobilization of BMC-SazCA on the surface of microcrystalline cellulose beads extended its heat stability, allowing its reuse in multiple rounds of the CO2 hydration reaction. These results suggest that production of SazCA in plants has great potential for CA-based CO2 sequestration and mineralization.

Use of an immobilised thermostable α-CA (SspCA) for enhancing the metabolic efficiency of the freshwater green microalga Chlorella sorokiniana

There is significant interest in increasing the microalgal efficiency for producing high-quality products that are commonly used as food additives in nutraceuticals. Some natural substances that can be extracted from algae include lipids, carbohydrates, proteins, carotenoids, long-chain polyunsaturated fatty acids, and vitamins. Generally, microalgal photoautotrophic growth can be maximised by optimising CO2 biofixation, and by adding sodium bicarbonate and specific bacteria to the microalgal culture. Recently, to enhance CO2 biofixation, a thermostable carbonic anhydrase (SspCA) encoded by the genome of the bacterium Sulfurihydrogenibium yellowstonense has been heterologously expressed and immobilised on the surfaces of bacteria. Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyse the physiologically reversible reaction of carbon dioxide hydration to bicarbonate and protons: CO2 + H2O ⇄ HCO3 − + H+. Herein, we demonstrate for the first time that the fragments of bacterial membranes containing immobilised SspCA (M-SspCA) on their surfaces can be doped into the microalgal culture of the green unicellular alga, Chlorella sorokiniana, to significantly enhance the biomass, photosynthetic activity, carotenoids production, and CA activity by this alga. These results are of biotechnological interest because C. sorokiniana is widely used in many different areas, including photosynthesis research, human pharmaceutical production, aquaculture-based food production, and wastewater treatment.

Characterization and High-Level Periplasmic Expression of Thermostable α-Carbonic Anhydrase from Thermosulfurimonas Dismutans in Escherichia Coli for CO2 Capture and Utilization.

Carbonic anhydrase (CA) is a diffusion-controlled enzyme that rapidly catalyzes carbon dioxide (CO2) hydration. CA has been considered as a powerful and green catalyst for bioinspired CO2 capture and utilization (CCU). For successful industrial applications, it is necessary to expand the pool of thermostable CAs to meet the stability requirement under various operational conditions. In addition, high-level expression of thermostable CA is desirable for the economical production of the enzyme. In this study, a thermostable CA (tdCA) of Thermosulfurimonas dismutans isolated from a deep-sea hydrothermal vent was expressed in Escherichia coli and characterized in terms of expression level, solubility, activity and stability. tdCA showed higher solubility, activity, and stability compared to those of CA from Thermovibrio ammonificans, one of the most thermostable CAs, under low-salt aqueous conditions. tdCA was engineered for high-level expression by the introduction of a point mutation and periplasmic expression via the Sec-dependent pathway. The combined strategy resulted in a variant showing at least an 8.3-fold higher expression level compared to that of wild-type tdCA. The E. coli cells with the periplasmic tdCA variant were also investigated as an ultra-efficient whole-cell biocatalyst. The engineered bacterium displayed an 11.9-fold higher activity compared to that of the recently reported system with a halophilic CA. Collectively these results demonstrate that the highly expressed periplasmic tdCA variant, either in an isolated form or within a whole-cell platform, is a promising biocatalyst with high activity and stability for CCU applications.

ART (ARduino-based pH Tracker), a new tool designed for characterization of a highly thermostable carbonic anhydrase

ART (ARduino-based pH Tracker), a new tool designed for characterization of a highly thermostable carbonic anhydrase

Rational Design of Thermostable Carbonic Anhydrase Mutants Using Molecular Dynamics Simulations.

The stability of enzymes is critical for their application in industrial processes, which generally require different conditions from the natural enzyme environment. Both rational and random protein engineering approaches have been used to increase stability, with the latter requiring extensive experimental effort for the screening of variants. Moreover, some general rules addressing the molecular origin of protein thermostability have been established. Herein, we demonstrate the use of molecular dynamics simulations to gain molecular level understanding of protein thermostability and to engineer stabilizing mutations. Carbonic anhydrase (CA) is an enzyme with a high potential for biotechnological carbon capture applications, provided it can be engineered to withstand the high temperature process environments, inevitable in most gas treatment units. In this study, we used molecular dynamics simulations at 343, 353, and 363 K to study the relationship between structure flexibility and thermostability in bacterial α-CAs and applied this knowledge to the design of mutants with increased stability. The most thermostable α-CA known, TaCA from Thermovibrio ammonificans, had the most rigid structure during molecular dynamics simulations, but also showed regions with high flexibility. The most flexible amino acids in these regions were identified from root mean square fluctuation (RMSF) studies, and stabilizing point mutations were predicted based on their capacity to improve the calculated free energy of unfolding. Disulfide bonds were also designed at sites with suitable geometries and selected based on their location at flexible sites, assessed by B-factor calculation. Molecular dynamics simulations allowed the identification of five mutants with lower RMSF of the overall structure at 400 K, compared to wild-type TaCA. Comparison of free-energy landscapes between wild-type TaCA and the most promising mutants, Pro165Cys-Gln170Cys and Asn140Gly, showed an increased conformational stability of the mutants at 400 K.

Halotolerant carbonic anhydrase with unusual N-terminal extension from marine Hydrogenovibrio marinus as novel biocatalyst for carbon sequestration under high-salt environments

Carbonic anhydrase (CA), an enzyme that catalyzes the carbon dioxide (CO2) hydration, has been suggested as a potentially powerful agent for CO2 capture and utilization. For successful application, CA should withstand the harsh environment presented by CO2-capturing facilities. While there have been intensive efforts to identify and engineer thermostable CAs, other required conditions such as the high salt concentration of CO2 absorbents have often been ignored. Herein, we expressed, purified, and characterized a novel α-type CA (hmCA) possessing an unusual N-terminal extension from the halophilic marine bacterium Hydrogenovibrio marinus. We found that the N-terminal extension strongly influenced the enzyme solubility. Recombinant hmCA showed catalytic efficiency comparable to other bacterial α-type CAs. hmCA was less inhibited by anionic inhibitors showing 1.6- (NO3−), 3.1- (NO2−), and 3.7-fold (Cl−) higher inhibition constants than those of mesophilic bovine CA (bCA), suggesting halotolerance. Recombinant hmCA was markedly stabilized using most of the alkali metal salts tested, showing 19 °C higher melting temperature at 1 M NaCl compared to bCA that was significantly destabilized. The region of N-terminal extension seemed not to be involved in halotolerance. The remarkable halotolerance may be attributed to the uneven distribution of electrostatic potential and the localized negative charge on the hmCA surface. hmCA displayed ∼29-fold longer half-life than that of bCA at 40 °C in potassium carbonate as a practical absorbent, suggesting that halotolerance should be considered another key characteristic in the development of biocatalysts for CO2 capture using high-salt-containing CO2 absorbents.

Laboratory to bench-scale evaluation of an integrated CO2 capture system using a thermostable carbonic anhydrase promoted K2CO3 solvent with low temperature vacuum stripping

An advanced post-combustion CO2 capture process with combined attributes of a thermostable carbonic anhydrase (CA) enzyme catalyst, low-enthalpy K2CO3 solvent, and vacuum stripping utilizing low exergy steam was evaluated from laboratory concept to application performance testing in an integrated 30 standard liters per minute gas flow bench-scale system operated for 500 h. Laboratory concept studies were performed using a wetted wall column to characterize solvent CO2 absorption kinetics and using a recirculating temperature loop to evaluate CA thermo-stability. Wetted wall column tests showed a dramatic 5-fold increase in CO2 mass transfer coefficient when combining 2 g/L CA with aqueous 23.5 wt% K2CO3 solvent. Further increasing the CA concentration resulted in a gradual increase in mass transfer coefficient until a performance plateau was observed beyond a 4 g/L CA dose. Operating temperature had limited impact on CO2 capture over the range 30–50 °C. Surface tension measurements of 23.5 wt% K2CO3 solvent exhibited a gradual decrease with increasing CA concentration. Thermo-stability tests in a temperature cycling loop designed to mimic the temperature swings between absorption and desorption showed that CA longevity could be extended by decreasing the total cycle time spent at high temperature. Parametric tests in the bench-scale unit resulted in a CO2 capture efficiency increase of 4.6-fold when increasing the CA concentration from zero to 2.5 g/L. Capture efficiency increased with higher reboiler duty (i.e. reboiler temperature) and lower absorber temperature. Tests with a 30 °C absorber temperature delivered >90% capture. Variation in solvent flow rate had little impact on capture efficiency because the reaction closely reached equilibrium at the top of the absorber. The integrated bench-scale system operated successfully for an accumulated 500 h under conditions of 40 °C absorber temperature and stripper at 35 kPa pressure with an approximate 77 °C stripper bottom temperature, delivering an average 84% CO2 capture with 23.5 wt% K2CO3-based solvent containing 2.5 g/L CA. Dissolved CA replenishment and conventional process controls were demonstrated as straightforward approaches to maintain system performance of this benign, low-temperature, CA-promoted process for CO2 capture.

Biocatalyzed Accelerated Post-combustion CO2 Capture and Stripping in Monoethanolamine

The existing solvent mediated CO2 capture process is energy intensive due to high temperature requirement for solvent regeneration. Therefore, improvement of solvent systems for optimized and low energy intensive CO2 capture and desorption is of enormous interest. In this regard, we report a novel matrix composed of ZnO/Fe2O3 encapsulated mesoporous silica tethered thermostable carbonic anhydrase (CA) (designated as CA@fn-Zn/Fe/MS). The enzyme coupled matrix was used for efficient post combustion CO2 capture and low energy intensive desorption. It was observed that the use of CA@fn-Zn/Fe/MS (0.5 wt%) in 30% Monoethanolamine (MEA) enhances the CO2 uptake to 0.82 mol/mol of solvent and the regeneration efficiency by 2.6 times as compared to the neat MEA at 30 wt% . The enzyme coupled matrix is stable over a broad range of pH and can be used for several cycles. The development adds values to improve conventional amine based CO2 scrubbing technology.

Accelerated CO2 capture in hybrid solvent using co-immobilized enzyme/complex on a hetero-functionalized support

There has been increasing interest in the development of solvent systems for efficient and less energy intensive CO2 capture and desorption. In this work, we have developed a hybrid solvent system (30wt.% MEA+7.5wt% PZ+15wt%K2CO3) containing a thermo-stable carbonic anhydrase (CA) and Zn complex (Zn-Im) together on a hetero-functional support (HFSCaCO3/H3BO3/SiO2) (termed as CA/Zn-Im: HFS). The hybrid solvent containing CA/Zn-Im: HFS exhibit excellent gravimetric CO2 uptake (21.65wt%) over the neat hybrid solvent (13.75wt%). Gravimetric and pressure drop experiments confirmed that HFS and Zn-Im plays key role in improving the CO2 capture activities of CA in hybrid solvent. Low temperature desorption (90°C) suggested that the hybrid solvent containing CA/Zn-Im: HFS has 1.57 times higher desorption kinetics compared to the neat hybrid solvent. Moreover, CA was highly stable in the hybrid solvent medium and could be reused for 15 consecutive cycles with retention of 90% of its initial activity. The development in various key aspects of CO2 capture suggested that the hybrid solvent with CA/Zn-Im: HFS is an efficient solvent system to improve conventional amine based scrubbing technology.

A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9.35 × 105 s−1 and kcat/Km = 1.1 × 108 M−1 s−1) which was maintained after heating the enzyme at 80 °C for 3 h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe3O4 nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.

Highly thermostable carbonic anhydrase from Persephonella marina EX-H1: Its expression and characterization for CO2-sequestration applications

The codon-optimized carbonic anhydrase gene of Persephonella marina EX-H1 (PMCA) was expressed and characterized. The gene with the signal peptide removed, PMCA(sp−), resulted in the production of approximately five times more purified protein than from the intact gene PMCA using an Escherichia coli expression system. PMCA(sp−) is formed as homo-dimer complex. PMCA(sp−) has a wide pH tolerance (optimum pH 7.5) and a high thermostability even at 100°C (88min of thermal deactivation half-life). The melting temperature for PMCA(sp−) was 84.5°C. The apparent kcat and Km values for CO2 hydration were 3.2×105s−1 and 10.8mM. The activity of the PMCA(sp−) enzyme was enhanced by Zn2+, Co2+, and Mg2+, but was strongly inhibited by Cu2+, Fe3+, Al3+, Pb2+, Ag+, and Hg2+. PMCA(sp−) readily catalyzed the hydration of CO2, precipitating CaCO3 as calcite in the presence of Ca2+.

Bacterial extremo-α-carbonic anhydrases from deep-sea hydrothermal vents as potential biocatalysts for CO2 sequestration

a b s t r a c t Carbonic anhydrase (CA) is an enzyme that rapidly catalyzes the reversible hydration of carbon dioxide (CO2) in aqueous solutions. Recent progresses in CA research suggest that this biocatalyst can be used as an alternative or an additive for chemical CO2 capture, which is considered an essential green route for the reduction of anthropogenic CO2 emissions. However, the facilities in which CA will most likely be used usually operate under high temperature conditions, requiring a highly thermostable CA. Here, we attempted to find and characterize -CAs originated from Perse- phonella marina and Thermovibrio ammonificans, thermophilic Gram-negative bacteria in deep-sea hydrothermal vents. The recombinant -CAs expressed in Escherichia coli showed catalytic efficien- cies better than the previously characterized thermophilic - and -CAs from archaea, and their activities were significantly increased at higher temperatures. Remarkably, these enzymes exhibited outstanding thermostability (stable up to at least 80 ◦C). Through long-term stability tests at 40 ◦C and 60 ◦ C, we discovered that the novel -CA of T. ammonificans might be the most thermostable CA ever characterized. Also, the recombinant CAs successfully accelerated CO2 sequestration in CaCO3, more efficiently than mesophilic bovine CA under high temperature condition. Collectively, these results demonstrate that the -CAs of thermophilic bacteria from deep-sea hydrothermal vents are promising biocatalysts for practical industrial CO2 capture in terms of both activity and stability.

Characterization and Properties of a New Thermoactive and Thermostable Carbonic Anhydrase

A new carbonic anhydrase was isolated and characterized from the thermophilic bacterium Sulfurihydrogenibium sp. YO3AOP1. The encoding gene was cloned and expressed in Escherichia coli and the recombinant protein purified to homogeneity. This enzyme (SspCA) belongs to the ?? class of the carbonic anhydrase family, is a monomer of 26.1 kDa and shows esterase activity. The kinetic parameters were determined by using CO2 and p-nitrophenylacetate (p-NpA) as substrates. Thermoactivity and thermostability studies showed that SspCA is active in the temperature range from 0 to 100 °C and retains full activity after 2 h incubation at 100 °C. SspCA was immobilized within a polyurethane foam and was found to be unalterably active and stable up to 50 h at 100 °C.

Kinetic Assessment of Thermostable Carbonic Anhydrase for CO2 Capture Processes

The most recent challenge to reduce greenhouse gas emissions is the development of Carbon Capture and Sequestration (CCS) processes for CO2 removal from flue gases. The biomimetic strategy is based on the adoption of Carbonic Anhydrase (CA) as an industrial biocatalyst as an alternative to conventional additives (e.g. amines) to increase CO2 absorption rate in aq. solutions.The present contribution concerns the kinetic assessment of a recombinant CA (SspCA) identified and characterized in the thermophile bacterium Sulfurihydrogenibium sp YO3AOP1. The CA characterization - long term stability included - was carried out under operating conditions close to those typically adopted in CCS plants. The absorption rate of pure CO2 into aq. solutions was assessed by working out time-resolved measurements of gas pressure decay in a batch stirred reactor. The first order enzyme kinetics for SspCA was assessed at 25 °C in buffer at pH 9.6. Long term stability of SspCA at 40 and 70 °C was promising compared with that of CA from bovine erythrocytes.