SspCA, a novel `extremo-α-carbonic anhydrase' isolated from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1, is an efficient catalyst for the hydration of CO2 and presents exceptional thermostability. Indeed, SspCA retains a high catalytic activity even after being heated to 343-373 K for several hours. Here, the crystallographic structure of this α-carbonic anhydrase (α-CA) is reported and the factors responsible for its function at high temperature are elucidated. In particular, the study suggests that increased structural compactness, together with an increased number of charged residues on the protein surface and a greater number of ionic networks, seem to be the key factors involved in the higher thermostability of this enzyme with respect to its mesophilic homologues. These findings are of extreme importance, since they provide a structural basis for the understanding of the mechanisms responsible for thermal stability in the α-CA family for the first time. The data obtained offer a tool that can be exploited to engineer α-CAs in order to obtain enzymes with enhanced thermostability for use in the harsh conditions of the CO2 capture and sequestration processes.

No abstract

We report the biochemical characterization of a new carbonic anhydrase (CA, EC 4.2.1.1), named SazCA, identified by translated genome inspection in Sulfurihydrogenibium azorense, a thermophilic bacterium from terrestrial hot springs of the Azores. SazCA is an α-CA showing kinetic parameters that make it the fastest enzyme of the CA family described so far. The biochemical properties, thermostability and inhibition of SazCA were compared with those of the thermophilic and mesophilic counterparts, demonstrating the special features of this unique enzyme.

The biomimetic approach represents an interesting strategy for carbon dioxide (CO 2 ) capture, offering advantages over other methods, due to its specificity for CO 2 and its eco-compatibility, as it allows concentration of CO 2 from other gases, and its conversion to water soluble ions. This approach uses microorganisms capable of fixing CO 2 through metabolic pathways or via the use of an enzyme, such as carbonic anhydrase (CA, EC 4.2.1.1). Recently, our group cloned and purified a novel bacterial a-CA, named SspCA, from the thermophilic bacteria, Sulfurihydrogenibium yellowstonense YO3AOP1 living in hot springs at temperatures of up to 110 C. This enzyme showed an exceptional thermal stability, retaining its high catalytic activity for the CO 2 hydration reaction even after being heated at 70 C for several hours. In the present paper, the SspCA was immobilized within a polyurethane (PU) foam. The immobilized enzyme was found to be catalytically active and showed a long-term stability. A bioreactor containing the ''PU-immobilized enzyme'' (PU-SspCA) as shredded foam was used for experimental tests aimed to verify the CO 2 capture capability in conditions close to those of a power plant application. In this bioreactor, a gas phase, containing CO 2 , was put into contact with a liquid phase under conditions, where CO 2 contained in the gas phase was absorbed and efficiently converted into bicarbonate by the extremo-a-CA.

Vibrio cholerae, a Gram-negative bacterium, is the causative agent of cholera and colonizes the upper small intestine where sodium bicarbonate is present at a high concentration. Sodium bicarbonate is a potential inducer of virulence gene expression. Bacteria can increase cytosolic bicarbonate levels through the existence of transporter family proteins or through the action of metalloenzymes, called carbonic anhydrases (CAs, EC 4.2.1.1). Vibrio cholerae, lacking of transporter proteins in its genome, utilizes the CA system to accumulate bicarbonate into the cell suggesting a pivotal role of this metalloenzymes in the microbial virulence. Here, we report for the first time the characterization of the a-CA of V. cholerae (VchCA), which has been identified by translated genome inspection. The a-CA encoding gene was cloned and expressed in Escherichia coli and the recombinant protein purified to homogeneity. This investigation aimed to study the biochemical properties of VchCA and to provide preliminary insights in the field of this pathogen virulence. VchCA has a low esterase activity with 4-nitrophenyl acetate as substrate, and a high activity for the hydration of CO 2 to bicarbonate.