Viviana De Luca

Biomimetic CO2 capture using a highly thermostable bacterial α-carbonic anhydrase immobilized on a polyurethane foam

Abstract The biomimetic approach represents an interesting strategy for carbon dioxide (CO2) capture, offering advantages over other methods, due to its specificity for CO2 and its eco-compatibility, as it allows concentration of CO2 from other gases, and its conversion to water soluble ions. This approach uses microorganisms capable of fixing CO2 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 α-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 CO2 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 CO2 capture capability in conditions close to those of a power plant application. In this bioreactor, a gas phase, containing CO2, was put into contact with a liquid phase under conditions, where CO2 contained in the gas phase was absorbed and efficiently converted into bicarbonate by the extremo-α-CA.

Biochemical properties of a new α-carbonic anhydrase from the human pathogenic bacterium, Vibrio cholerae

Abstract 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 α-CA of V. cholerae (VchCA), which has been identified by translated genome inspection. The α-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 CO2 to bicarbonate.

DNA cloning, characterization, and inhibition studies of an α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

We have cloned, purified, and characterized an α-carbonic anhydrase (CA, EC 4.2.1.1) from the human pathogenic bacterium Vibrio cholerae, VchCA. The new enzyme has significant catalytic activity, and an inhibition study with sulfonamides and sulfamates led to the detection of a large number of low nanomolar inhibitors, among which are methazolamide, acetazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, and indisulam (KI values in the range 0.69-8.1 nM). As bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit the in vivo virulence, we propose that VchCA may be a target for antibiotic development, exploiting a mechanism of action rarely considered until now.

Anion inhibition studies of an α-carbonic anhydrase from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1.

The newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 encodes an α-carbonic anhydrases (CAs, EC 4.2.1.1) which is highly catalytically active and thermostable. Here we report the inhibition of this enzyme, denominated SspCA, with inorganic and complex anions and other molecules interacting with zinc proteins. SspCA was inhibited in the micromolar range by diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic and phenylarsonic acid, trithiocarbonate and selenocyanide (K(I)s of 4-70 μM) and in the submillimolar one by iodide, cyanide, (thio)cyanate, hydrogen sulfide, azide, nitrate, nitrite, many complex anions incorporating heavy metal ions and iminodisulfonate (K(I)s of 0.48-0.86 mM). SspCA was not substantially inhibited by bicarbonate and carbonate, hydrogensulfite and peroxidisulfate (K(I)s in the range of 21.1-84.6mM). The exceptional thermostability and lack of strong affinity for hydrogensulfide, bicarbonate, and carbonate make this enzyme an interesting candidate for biotechnological applications of enzymatic CO(2) fixation.

An α-carbonic anhydrase from the thermophilic bacterium Sulphurihydrogenibium azorense is the fastest enzyme known for the CO2 hydration reaction

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 first activation study of a bacterial carbonic anhydrase (CA). The thermostable α-CA from Sulfurihydrogenibium yellowstonense YO3AOP1 is highly activated by amino acids and amines.

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) was investigated for its activation with a series of amino acids and amines. D-His, L-Phe, L-Tyr, L- and D-Trp were the most effective SspCA activators, with activation constants in the range of 1-12 nM, whereas L-His, L/D-DOPA, D-Tyr, and several biogenic amines/catecholamines were slightly less effective activators (K(A) in the range of 37 nM-0.97 μM). The least effective SspCA activator was d-Phe (K(A) of 5.13 μM). The thermal stability, robustness and very high catalytic activity of SspCA make this enzyme an ideal candidate for biomimetic CO(2) capture processes.

Biochemical characterization of recombinant β-carbonic anhydrase (PgiCAb) identified in the genome of the oral pathogenic bacterium Porphyromonas gingivalis

Abstract Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β-, γ-, δ- and ζ-CAs are ubiquitous metalloenzymes present in prokaryotes and eukaryotes. CAs started to be investigated in detail only recently in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many such organisms they are essential for the life cycle of the organism. CA inhibition leads to growth impairment or growth defects in several pathogenic bacteria. The microbiota of the human oral mucosa consists of a myriad of bacterial species, Porphyromonas gingivalis being one of them and the major pathogen responsible for the development of chronic periodontitis. The genome of P. gingivalis encodes for a β- and a γ-CAs. Recently, our group purified the recombinant γ-CA (named PgiCA) which was shown to possess a significant catalytic activity for the reaction that converts CO2 to bicarbonate and protons, with a kcat of 4.1 × 105 s−1 and a kcat/Km of 5.4 × 107 M−1 × s−1. We have also investigated its inhibition profile with a range of inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate. Here, we describe the cloning, purification and kinetic parameters of the other class of CA identified in the genome of P. gingivalis, the β-CA, named PgiCAb. This enzyme has a good catalytic activity, with a kcat of 2.8 × 105 s−1 and a kcat/Km of 1.5 × 107 M−1 × s−1. PgiCAb was also inhibited by the clinically used sulfonamide acetazolamide, with an inhibition constant of 214 nM. The role of CAs as possible virulence factors of P. gingivalis is poorly understood at the moment but their good catalytic activity and the fact that they might be inhibited by a large number of compounds, which may pave the way for finding inhibitors with antibacterial activity that may elucidate these phenomena and lead to novel antibiotics.

Biochemical characterization of the γ-carbonic anhydrase from the oral pathogen Porphyromonas gingivalis, PgiCA

Abstract Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. CAs are present in many pathogenic species and are involved in the bicarbonate metabolism/biosynthetic reactions involving this ion. Ubiquity of these enzymes suggests a pivotal role in microbial virulence and pathogenicity. Porphyromonas gingivalis is an anaerobic bacterium, which colonizes the oral cavity, being involved in the pathogenesis of periodontitis, an inflammatory disease leading to tooth loss. Recently, we reported an anion inhibitory study on the γ-CA (denominated PgiCA) identified in the genome of this Gram-negative bacterium. In this paper we continue our research on PgiCA, and describe the biochemical characterization of the recombinant protein, its thermal stability, the oligomeric state and the enzyme kinetics. PgiCA is a polypeptide chain formed of 192 amino acids and displays an identity of 30–33% when compared with the prototypical γ-CAs, CAM or CAMH (from Methanosarcina thermophila) or CcmM (from Thermosynechococcus elongatus). A subunit molecular mass of 21 kDa was estimated by SDS-PAGE, while HPLC size exclusion chromatography under native conditions gave an estimated molecular mass of 65 kDa suggesting that the recombinant enzyme self-associate in a homotrimer, as all other γ-CAs studied so far. Enzyme kinetic analysis showed that PgiCA is 62 times more effective as a catalyst compared to CAM, the only other γ-CA characterized in detail kinetically. All these features represent an interesting attractive for the drug design of inhibitors/activators of this new enzyme.

Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense YO3AOP1

A new carbonic anhydrase (CA, EC 4.2.1.1) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 was identified and characterized. The bacterial carbonic anhydrase gene was expressed in Escherichia coli yielding an active enzyme, which was purified in large amounts. The recombinant protein (SspCA) was found to belong to the α-CA class and displays esterase activity. The kinetic parameters were determined by using CO2 and p-nitrophenylacetate (p-NpA) as substrates. The bacterial enzyme presented specific activity comparable to that of bovine carbonic anhydrase (bCA II) but it showed biochemical properties never observed for the mammalian enzyme. The thermophilic enzyme, in fact, was endowed with high thermostability and with unaltered residual activity after prolonged exposure to heat up to 100°C. SspCA and the bovine carbonic anhydrase (bCA II) were immobilized within a polyurethane (PU) foam. The immobilized bacterial enzyme was found to be active and stable at 100°C up to 50 h.

Associations of selenium status with cardiometabolic risk factors: An 8-year follow-up analysis of the Olivetti Heart Study

OBJECTIVE High selenium status has been associated with adverse cardiometabolic outcomes in selenium-replete populations such as the US. In populations with lower selenium status such as in Italy, there is little epidemiological evidence about the association of selenium with cardiometabolic risk factors. We therefore examined cross-sectional and prospective relationships of serum selenium concentrations with cardiometabolic risk factors including blood pressure, diabetes and blood lipids in the Olivetti Heart Study. METHODS The study population consisted of 445 adult male individuals for whom baseline serum selenium measurement and cardiometabolic risk factors at baseline (1994-1995) and follow-up examination (2002-2004: average follow-up=8 years) were available. Serum selenium was measured by atomic absorption spectrophotometry. RESULTS Average serum selenium concentration at baseline was 77.5 ± 18.4 μg/L. In cross-sectional analyses, serum selenium levels were positively associated with serum total cholesterol (p for trend <0.0001) and prevalent diabetes (p for trend <0.05). In prospective analysis, serum selenium at baseline was likewise a strong predictor of serum total cholesterol (p=0.002) and LDL-cholesterol (p=0.001) at follow-up, after adjustment for age, BMI, cigarette smoking, physical activity, and lipid-lowering medication. These associations, however, were no longer significant after additional adjustment for baseline blood lipids. Selenium at baseline did not predict changes in total cholesterol levels between the baseline and follow-up examinations [β-coefficient (± SE)= 0.09 ± 0.12 (p=0.46)]. CONCLUSION These findings corroborate previous cross-sectional associations of high selenium status with adverse blood lipid profile and diabetes. However, prospective analyses do not support the causality of these relations. Randomized and experimental evidence is necessary to clarify the mechanisms underlying the observed cross-sectional associations.