WO2013064195A1 - A new heat-stable carbonic anhydrase and uses thereof - Google Patents

Abstract

The present invention relates to an isolated alpha class carbonic anhydrase (CA) enzyme that is heat-active and heat-stable for at least 2 hours up to near 100°C and functional derivatives thereof wherein said derivatives maintain the carbonic anhydrase activities of the enzyme herein described. In addition the present invention relates to use of said heat-stable carbonic anhydrase or said derivative thereof at temperatures higher than 65° in C0₂ extraction (in the range from 70°C to 1 10°C) as well as to isolated polypeptides coding for said anhydrase or derivatives thereof and to having said carbonic anhydrase activity and to isolated polynucleotides encoding said polypeptides.. The invention also relates to nucleic acid constructs or vectors comprising said polynucleotides, and host cells comprising said constructs or vectors.

Description

A NEW HEAT-STABLE CARBONIC ANHYDRASE AND USES THEREOF The present invention relates to an isolated alpha class carbonic anhydrase (CA) enzyme that is heat-active and heat-stable for at least 2 hours up to near 100°C and functional derivatives thereof wherein said derivatives maintain the carbonic anhydrase activities of the enzyme herein described.

The enzyme or derivatives thereof herein disclosed have a maximum enzymatic activity at temperatures in the range from 65°C to 100°C and even to about 110°C.

In addition the present invention relates to use of said heat-stable carbonic anhydrase or said derivative thereof at temperatures higher than 65°C in C0₂ extraction (e.g. in the range from 70°C to 100°C and even to about 1 10°C), e.g. , from carbon dioxide (C02) containing media. Furthermore, the invention relates to isolated polypeptides coding for said anhydrase or derivatives thereof and to having said carbonic anhydrase activity and to isolated polynucleotides encoding said polypeptides. The invention also relates to nucleic acid constructs or vectors comprising said polynucleotides, and host cells comprising said constructs or vectors.

STATE OF THE ART

In addition to energy conservation and use of low-carbon renewable energy sources, the separation or "capture" and sequestration of carbon dioxide (C0₂) from power generation and industrial m ixed gas streams, especially combustion emissions, is an increasingly important option for decreasing the levels of this greenhouse gas released to the atmosphere. Carbon dioxide capture and storage (CCS) is a multi-stage process in which C0₂ is separated from mixed gas streams, transported, for example through pipelines, to a storage location, such as deep geological formations, and isolated ("sequestered") from the atmosphere by trapping C0₂ in the storage site. The capture part of the process is the most energy intensive stage. C0₂ capture using chemical absorption and desorption of C0₂ in amine based solvents, which are currently used for natural gas processing and ammonia manufacture, is projected to consume on the order of 30% of the net power generation of a coal burning power plant, causing significant economic burdens as well as increasing the need for petroleum-based amine chemicals.

Therefore, alternative technologies that can decrease the cost of C0₂ capture and minimize environmental impacts are urgently needed.

Carbon dioxide (C0₂) is a key metabolite in all living organisms, and organisms produce enzymes specific for C0₂ related process.

Carbonic anhydrases are enzymes distributed in all the domains of life, they are zinc-containing enzymes that catalyse the reversible reaction between carbon dioxide hydration and bicarbonate dehydration and have been found in all kingdoms of life.

Carbonic anhydrase catalyses the following reaction:

H₂0 + C0₂ H⁺ + HC0₃

Carbonic anhydrases exist in various distinct classes named alpha, beta, gamma and delta. These classes evolved from independent origin (Bacteria, Archaea, Eukarya) and have no significant sequence or structural identity except for the presence of a single zinc atom at the catalytic site (Tripp et all J. Biol. Chem. 276:486115-48618-2001). Alpha classes CA are present in mammals where more than 1 1 isozymes have been identified, beta classes in algae and plants, gamma classes in prokariotes and Archaea, (Alber and Ferry, Proc. Natl. Acad. Science 91 :6909-6913,1994; Parisi et all, Plant Mol. Boil. 55: 193-207,2004).

Many prokaryotes contain carbonic anhydrase genes from more than one class or more than one gene of a certain class.

Mammalian carbonic anhydrases are between the most active enzymes described with a turnover number of aboutI O⁶ molecules of C0₂ per second. The use of these enzymes, either free or immobilized, has been reported in several technical approach for capturing C0₂, from combustion and other gases mixtures, for its subsequent utilization or disposal. (WO2006/089423, WO2004/007058, WO2004/028667; US2004/0029257, US7, 132,090, WO2005/1 14417, US6, 143,556, WO2004/1 04160, US2005/214636, WO2008/095057, US2010/00447866 A1 , US7,699,910 B2).

The conversion of C0₂ by CA to bicarbonate and /or carbonate ions, opens different possibilities such as: the precipitation as carbonate salts, its utilization to favour the growth of algae and other microorganism, for example, for the production of methane, and many other applications.

Generally, the carbonic anhydrases, isolated from mammals or prokaryotes and other mesophilic sources, are active at physiological temperatures (37°C) and are, like many enzymes, quite unstable under operative conditions.

US Patent 7,892,814 discloses anhydrases defined as heat-stable. The patent, although claiming anhydrases used in reactions up to 100°C, discloses in tables 2 and 4 anhydrases having a maximum activity between 37 and 60°C and a residual activity of max 16.9% at 80°C.

The anhydrases disclosed in US Patent 7,892,814, (also WO2008095057), although being an improvement over the prior art, do not allow in practice to carry out effective enzymatic reactions at a temperature higher than 45°C and is few cases up to 60°C due to the medium heat stability of the anhydrases disclosed therein.

WO2008095057, that represents the closest prior art of the present application, teaches that the more heat stable carbonic anhydrases show a decrease in stability at about 60°C and that said anhydrases are substantially inactive (max activity reported about 16.9%) at 80°C.

The same patent indicates that carbonic anhydrase, especially heat-stable carbonic anhydrase, may be used for carbon dioxide extraction from C0₂ emission streams, e.g. , from carbon-based or hydrocarbon-based combustion in electric generation power plants, or from flue gas stacks from such plants, industrial furnaces, stoves, ovens, or fireplaces or from airplane or car exhausts. The prior art teaches that heat-stable carbonic anhydrases, could be used to remove C0₂ in the preparation of industrial gases such as acetylene (C₂H₂), carbon monoxide (CO), chlorine (Cl₂), hydrogen (H₂), methane (CH₄), nitrous oxide (N₂0), propane (C₃H₈), sulphur dioxide (S0₂), argon (Ar), nitrogen (N₂), and oxygen (0₂) and that the enzyme can also be used to remove C0₂ from a raw natural gas during the processing to natural gas. Raw natural gas is generally obtained from oil wells, gas wells, and condensate wells. Natural gas contains between 3 to 10% C0₂ when obtained from geological natural gas reservoirs by conventional methods. In particular, carbonic anhydrases can be used to enrich the methane content in biogases. Biogases always contain a discrete amount of C0₂. The prior art indicates, that a carbonic anhydrase that tolerates higher temperatures would offer improved robustness in actual use and storage related to biogas processes utilizing mesophilic strains. Thermophilic strains allow the fermentation to occur at elevated temperatures; in such processes a heat-stable carbonic anhydrase would be particularly useful to remove C0₂ from the C0₂ containing medium. Furthermore, carbonic anhydrase may be applied in the production of syngas by removing the C0₂ generated by the gasification of a carbon containing fuel (e.g., methane or natural gas) thereby enriching the CO, H₂ content of the syngas. Where syngas production occurs at elevated temperatures the use of a heat-stable carbonic anhydrase is an advantage.

Thermophile microorganisms, living at temperatures from 70°C to 1 10°C, are known in the art. In certain cases it has been possible to isolate from said organisms heat-stable enzymes showing activities at the temperatures of survival of said microorganisms. The characteristics and the properties of biocatalysts deriving from said organisms would enlarge the limits of biotechnological operative conditions both for traditional processes and for designing new processes for innovative products.

Notwithstanding the fact that heat-stable microorganisms are known in the art and notwithstanding the interest for heat-stable (or thermo-stable) alpha carbonic anhydrases, no alpha carbonic anhydrases having a range of activity from about 70°C to about 110°C have never been described in the art.

Hence, an alpha CA heat-stable (or heat-stable) and thermo-active at temperature from about 70°C to about 1 10°C would provide a real advantage for all the technologies already known in the art for C0₂ capture and utilization.

DESCRIPTION

In the present invention, a gene encoding a heat-stable and heat-active CA enzyme from Y03AOP1 (Topt 72°C), a strain isolated by Reyenbach A.I. et al. .J.Bacteriol (2009 191 :1992-1993), was identified, isolated, cloned and expressed in E.coli and the recombinant enzyme obtained, was purified and characterized.

The enzyme isolated from the species Aquificales sulfurihydrogenibium showed extraordi nary properties such an extremely h ig h heat-activity at temperatures in the range from about 65°C to about 100°C and even to about 1 10°C and a heat-stability at similar temperatures that reaches at least 2 hours without significant losses of activity. Furthermore, the enzyme has shown an extremely high stability at alkaline pH also in the presence of 30% mono-ethanolamine. Such properties allow the use of this enzyme at temperatures and experimental conditions never used for the capture and isolation of C0₂ in the harshest environments.

To date, no alpha CA with such or similar properties have been disclosed. The present invention discloses for the first time an isolated, heat-stable, alpha carbonic anhydrase (also indicated as CA) having the maximum carbonic anhydrase enzymatic activity in the range from 65°C to 100°C and even to about 1 10°C in standard CA activity tests as defined below in the detailed description. By maximum CA activity it is intended that the highest peak of activity of the enzyme is in the range indicated.

The anhydrase has been the sole anhydrase having these features isolated form thermophilic microorganisms notwithstanding the wide screening and analysis carried out by the inventors.

The inventors have screened various thermophilic strains belonging to the Archaea and Bacteria kingdom for sequences similar or partially similar to two sequences of alha class CAs, namely from Rhodopseudomonas palustris and from Bacillus clausii KSM-K16.

Only the genus Aquificales sulfurihydrogenibium showed the presence, in the genomes of the strain Y03AOP1 of a gene encoding a putative alpha class CA with an identity of about 17% and 43% to the sequences of reference. The putative enzyme showed an identity with bovine alpha CA of 33,2% and with human alpha CA of 29,5%,

Due to the known and documented independent evolution of these classes of genes, the author could not a priori be sure to find putative matches to the sequences of reference. In fact, the data obtained, demonstrate that the highest identity found was extremely low and that it was not possible to predict a priori whether a match would have been found and whether the match would have, indeed, lead to the identification of a heat-stable anhydrase.

The author has hence isolated a heat-stable class alpha CA, said anhydrase being characterised, as demonstrated by the figures and examples below, by an extremely high heat-stability and heat-activity.

In fact, the enzyme disclosed herein has the peak of CA enzymatic activity in the temperature range from 65°C to 100°C and even to about 1 10°C whereas all the class alpha CA disclosed so far show a decrease in their CA enzymatic activity from 60°C and higher.

The invention hence encompasses an isolated heat stable alpha carbonic anhydrase having the highest CA activity at a temperature range from about 65°C to about 100°C and even to about 1 10°C, enzymatically active fragments thereof said fragments maintaining said peak of carbonic anhydrase enzymatic activity; an isolated polynucleotide coding for said carbonic anhydrase or fragment thereof ; an isolated polynucleotide construct comprising said polynucleotide, said construct of being a vector; a host cell transformed with said vector, a method for the production of said isolated heat-stable alpha carbonic anhydrase having the maximum carbonic anhydrase enzymatic activity in the range from 65°C to 100°C and even to about 1 10°C or enzymatically active fragments thereof said fragments maintaining said peak of carbonic anhydrase enzymatic activity comprising the steps of

a. colturing a host cell transformed with the vector of claim 7

b. extracting the carbonic anhydrase produced thereby;

a method for the extraction of carbon dioxide from a medium wherein

a. said extraction is carried out at temperatures in the range from 50°C to 100°C or to about 1 10°C ;

b. said carbonic anhydrase or fragments is used and

a reactor for extracting carbon dioxide wherein said reactor comprises a bicarbonate buffer with a pH of at least 8 and a heat-stable alpha carbonic anhydrase having the maximum carbonic anhydrase enzymatic activity in the range from 65°C to 100°C or to 110°C or enzymatically active fragments thereof said fragments maintaining said peak of carbonic anhydrase enzymatic activity

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 represents. Gene/protein sequences of carbonic anhydrases from the Sulfurihydrogenibium specie identified by the present inventor, respectively SEQ IDs 1 and 2 of strain Y03AOP1

Figure 2 represents the stability of the enzyme at various temperatures after a 30, 60, 120 and 180 minutes incubation. The data of figure 2 are summarised in Table 1.

Units were calculated according to the description,

Figure 3 represents the results of experiments comparing the activity of bovine CA and the CA of the invention at increasing temperatures from 0 to 100°C.

The figure clearly shows that the CA of the invention has an activity of more than 2 folds of the maximum bovine CA activity already at 50°C and that at temperatures between 80 and 100°C the activity of the CA of the invention is of about 4 folds the maximum activity of bovine CA, with a peak at about 90°C.

Units were calculated according to the description.

DETAILED DESCRIPTION OF THE SEQUENCES SEQ ID 1 Sulfurihydrogenibium sp. Y03AOP1 alpha carbonic anhydrase nucleotide sequence

atg aga aaa ata eta att tct gca gtt tta gta tta tea age att tct ata tct ttt get gag cat gaa tgg agt tat gaa ggt gaa aag gga ccg gag cat tgg gcg cag tta aaa cct gaa ttc ttt tgg tgt aaa tta aaa aat caa tct cct ata aac att gat aaa aaa tat aaa gtt aaa gca aac ctg cca aag tta aac ttg tac tac aaa act gca aaa gaa tea gaa gta gta aac aat ggt cat act att cag ata aat ata aaa gaa gat aac act tta aac tac ctt gga gaa aag tat cag ctt aaa cag ttt cat ttc cac aca cca agt gaa cat aca ata gag aaa aaa tct tat ccg ttg gaa att cac ttt gtt cat aaa aca gaa gat ggt aag att ttg gtc gtt ggt gta atg get aaa ctt ggg aaa act aat aaa gag tta gat aaa att tta aac gtt get cct get gaa gaa gga gaa aaa att ctt gat aag aat tta aat tta aac aat ctt ata cca aaa gat aag aga tat atg aca tac tea ggc tea tta ace act cca cca tgc act gaa ggt gta aga tgg att gtc ttg aaa aaa cct att tct ata tct aag cag caa ctt gaa aag tta aaa tct gtt atg gtg aat cct aac aac aga cct gtt caa gag att aat tea aga tgg ata att gaa gga ttt taa

SEQ ID 2 Sulfurihydrogenibium sp. Y03AOP1 alpha carbonic anhydrase amino acid sequence

Met Arg Lys lie Leu lie Ser Ala Val Leu Val Leu Ser Ser lie Ser lie Ser Phe Ala Glu His Glu Trp Ser Tyr Glu Gly Glu Lys Gly Pro Glu His Trp Ala Gin Leu Lys Pro Glu Phe Phe Trp Cys Lys Leu Lys Asn Gin Ser Pro lie Asn lie Asp Lys Lys Tyr Lys Val Lys Ala Asn Leu Pro Lys Leu Asn Leu Tyr Tyr Lys Thr Ala Lys Glu Ser Glu Val Val Asn Asn Gly His Thr lie Gin lie Asn lie Lys Glu Asp Asn Thr Leu Asn Tyr Leu Gly Glu Lys Tyr Gin Leu Lys Gin Phe His Phe His Thr Pro Ser Glu His Thr lie Glu Lys Lys Ser Tyr Pro Leu Glu lie His Phe Val His Lys Thr Glu Asp Gly Lys lie Leu Val Val Gly Val Met Ala Lys Leu Gly Lys Thr Asn Lys Glu Leu Asp Lys lie Leu Asn Val Ala Pro Ala Glu Glu Gly Glu Lys lie Leu Asp Lys Asn Leu Asn Leu Asn Asn Leu lie Pro Lys Asp Lys Arg Tyr Met Thr Tyr Ser Gly Ser Leu Thr Thr Pro Pro Cys Thr Glu Gly Val Arg Trp lie Val Leu Lys Lys Pro lie Ser lie Ser Lys Gin Gin Leu Glu Lys Leu Lys Ser Val Met Val Asn Pro Asn Asn Arg Pro Val Gin Glu lie Asn Ser Arg Trp lie lie Glu Gly Phe

SEQ ID 3, a sequence coding for SEQ I D 3 was in vector pETCA according to example 2 in replacement of nucleotides 1-60 of SEQ ID NO 1

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Leu

DETAILED DESCRIPTION OF THE INVENTION

As already mentioned, the isolated heat-stable alpha carbonic anhydrase of the invention, is an alpha CA, having a maximum carbonic anhydrase enzymatic activity in the range from 65°C to 100°C and even to about 1 10°C or enzymatically active fragments thereof said fragments maintaining said peak of carbonic anhydrase enzymatic activity.

As disclosed in the examples below, in the present description, the CA activity was assayed using the Wilburn assay (Wilburn , 1948, J . Biol . Chem. 176: 147-154) as modified by Chirica (Chirica et all, 2001 , Biochim. Biophys. Acta 1544:55-63) that is the conventional assay for measuring CA activity in the art.

A more detailed definition of enzymatically active fragment is provided in the glossary below.

The alpha CA or the active fragments thereof can be also referred to as "the active CA enzymes" or "the CA" or "SpCA" in the present description.

In an embodiment of the invention, the alpha CA or the active fragments thereof have a maximum carbonic anhydrase enzymatic activity in the range from about 70°C to about 110°C or to about 100°C, from about 80°C to about 1 10°C or to about 100°C.

In another embodiment, the alpha CA or the active fragments thereof have an optimum temperature activity at about 90°C.

As clearly disclosed in the examples of this description, the CA of the invention has a unique feature with respect for alpha CA disclosed in the art.

In fact (see figure 3 and example 3), when the activity of the CA is compared to the CA activity of Carbonic Anhydrase from bovine erythrocytes (C5024 Sigma- Aldrich) (used as a control in all experiments herein reported) by incubating an equal amount in weight of enzyme in 15 mM Tris-sulphate buffer at pH 7.5 with 3mM of nitrophenilacetate, the optimum activity of alpha bovine CA is of about 60°C (with a decrease in said activity immediately over 60°C) whereas the CA activity of the alpha CA of the invention, shows an increase of activity up to 90°C with a small decrease at about 100°C.

Moreover, as clear from figure 3, the activity of the alpha CA of the invention is more than twice the activity of the same amount in weight, of the alpha bovine CA used as control, i.e. , x ng of bovine CA corresponded to about 1 unit of enzyme at 60°C whereas the same amount of the alpha CA of the invention correspond to about 3 units of enzyme at the same temperature.

The active CA enzymes of the invention, hence, not only are stable and active at temperatures well above the temperatures of stability and activity of alpha Carbonic Anhydrases disclosed in the art, but, even at temperatures that are optimum temperature activity of other known alpha CAs such as the bovine alpha CA, show an activity that is more than twice than the one of the known alpha CA. Hence the enzymes of the invention have a wider activity range and a higher activity with respect to the state of the art.

As already indicated, in the present description, the CA activity was assayed using the Wilburn assay (Wilburn ,1948, J.Biol. Chem. 176: 147-154) as modified by Chirica ( Chirica et all, 2001 , Biochim.Biophys. Acta 1544:55-63).

The arbitrary unit of the enzyme is defined as the quantity of the enzyme needed to bring the pH of 2 ml_ solution from 8,3 to 6,3 in 1 minute in the condition described.

Using p-nitrophenyl acetate as substrate the enzyme Unit is calculated at

0°C and pH 7.6 according the formula: 1 U = (Abs 348nm/min - Abs blank/min)/ 0.05 x (mg enzyme/ml reaction); where 0.05 is the μηιο^Γ extinction coefficient of p- NpA.

In an embodiment of the invention, the active CA enzymes of the invention retain at least 90% of the carbonic anhydrase activity they show at 40, 50 or 60°C, after incubation at 90°C for 20 minutes.

In a further embodiment, they retain at least 90% their carbonic anhydrase activity at 40, 50 or 60°C, after incubation at 80°C for a time period up to 180 minutes.

Since the presence of CA increases the power of monoethanolammine

(MEA) and diethanolammine (DEA) to capture C0₂, the active CA enzymes of the invention are conveniently stable in the presence of different concentrations of the two amines at 25 and 80°C for a time period up to 180 minutes as reported in the examples below. He n ce , th e active CA e nzy m es of th e i nve nti o n a re advantageously stable in these harsh conditions with a very alkaline pH. The activity can be assayed using the Wilburn method.

The CA of the invention can be a CA isolated from Aqificales sulfurihydrogenibium species, in particular from strain Y03AOP1.

I n an embodiment the active CA enzymes of the invention are isolated polypeptides of SEQ ID NO 2 or have at least 90%, 95%, 98% or 99% of sequence identity to SEQ I D NO 2. With relation to the enzymatically active fragments or derivatives of the CA of the invention, it is to be noted that a sequence of a CA of the invention is provided (SEQ ID NO 2) and that enzymatically active fragments can readily be derived by the skilled person following teachings known in the art.

In fact, essential amino acids in the isolated polypeptide of SEQ ID NO 2 can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). I n the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e., carbonic anhydrase activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.

The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, e.g. by nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutation of putative contact site amino acids (de Vos et al. , 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wodaver et al., 1992, FEBS Lett. 309: 59-64).

The skilled person can refer to similar analyses performed on carbonic anhydrases, as reviewed in Tripp et al., 2001 , J. Biol. Chem. 276: 48615-48618 and Lindskog, 1997, Pharmacol. Ther. 74: 1-20. The invention also encompasses single or multiple amino acid substitutions in SEQ I D NO 2 that can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, and that can be screened as described in Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.

The sequence of the CA of the invention can be also subjected to error- prone PCR, phage display (e.g., Lowman et al., 1991 , Biochem. 30: 10832-10837; U . S. Patent No. 5,223,409; WO 92/06204) , and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a/., 1988, DNA 7: 127).

The derivatives and or fragments obtained with anyone of the methods above or other can be com bined with high-throughput, automated screening methods to detect activity of cloned, mutagenised polypeptides expressed by host cells. Mutagenised D NA m o lecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

The above identified methods for designing enzymatically active fragments or derivatives would allow the skilled person to carry out exactly the steps to follow in order to obtain the desired products without use of inventive skill and without undue burden.

The invention further relates to compositions comprising the isolated heat- stable alpha carbonic anhydrase or enzymatically active fragments thereof as described above or mixtures thereof and one or more excipient where the excipient can be a solvent such as water, inorganic salts, fillers, pigments, waxes, buffers, carriers, stabilizers, cross linking agents, adhesives, preservatives, glycerol and the like or mixture thereof.

The composition may be in the form of a liquid, a solid, granules coated or uncoated, micro-granules, semi solid, slurry.

The composition may comprise also one or more additional enzyme.

The present invention also relates to an isolated polynucleotide coding for the carbonic anhydrase of the invention or its CA enzymatically active fragments thereof. In particular, the active CA enzymes of the invention can be coded by SEQ I D NO 1 or fragments thereof, taking into account the degeneration of the genetic code, hence all polynucleotide sequences coding for the same amino acid sequence coded by SEQ ID NO 1 are intended as encompassed by the present invention.

In fact, the skilled person would perfectly know that SEQ I D 1 , coding for SEQ ID NO 2, could be replaced by other sequences still coding for SEQ I D NO 2 merely by taking into account the degeneration of the genetic code. Most textbook of genetics or molecular genetics provide the well known table of correspondence between nucleotide triplets and amino acids.

The same obviously applies to fragments of SEQ I D NO 1 coding for enzymatically active fragments of the enzyme coded by SEQ ID NO 1.

A nucleotide sequence encoding a polypeptide of the present invention can be modified in order to synthesise a polypeptide having an amino acid sequence that has at least one substitution, deletion and/or insertion as compared to the amino acid sequence of SEQ ID NO: 2.

Only modifications preserving the maximum carbonic anhydrase enzymatic activity in the range from 65°C to 100°C and even to about 1 10°C are part of the present invention, by way of example, modification outside regions critical to the function of the enzyme can be carried out. Amino acid residues which are essential to the function are therefore preferably not subject to modification.

The skilled person can identify amino acid residues essential to the heat stable CA activity according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g. , Cunningham and Wells, 1989, Science 244: 1081-1085). According to the known techniques, the sites of substrate-enzyme i nteraction can be determ i ned by analysis of the three- dimensional structure e.g. by nuclear magnetic resonance analysis, crystallography or photo affinity labelling (see, e.g. , de Vos et al. , 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The invention relates also to an isolated polynucleotide construct comprising the polynucleotide as defined above. The construct can be, by way of example, a construct comprising, operatively linked to the polynucleotide as defined above (SEQ ID NO 1 , fragments thereof or corresponding nucleotide sequences coding for the same amino acids of SEQ ID NO 1 or its fragments), one or more of: an origin of replication, a promoter, a termination sequence, an enhancer, a tag for purification, a selection marker, (such as a sequence coding for a his tag or other tags known in the art that facilitate the purification of the enzyme), restriction enzyme sites and other components commonly used in the art for the preparation of a cloning or of an expression vector.

In a particular embodiment of the invention, the polynucleotide construct is a vector, said vector being any suitable vector for cloning and/or expression in bacterial, fungi, yeast, insect, mammalian and plant cells as well as phages and bacteriophages.

The vector can be any commonly used and available cloning or expression vector and the skilled person will merely need to follow the manufacturer instructions or the published instructions concerning said vector.

Molecular cloning is common knowledge for the skilled person and the sequence coding for the enzyme of interest can be easily cloned into any known suitable vector without need of inventive skill and without undue burden.

An example is provided in this description but any cloning or expression system and vector will be suitable for the scope of the present invention.

I n a particular embodiment the vector will be a recombinant expression vector so to allow direct production of the active CA enzyme in the cell or organism of interest.

In an embodiment of the invention the expression vector can comprise an isolated polynucleotide of the present invention that can be operatively linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

Several vector and hosts systems are known in the art.

As used herein, the phrase "expression vector" is used to refer to a vector that is suitable for the production of an encoded product (e.g., a protein of interest). The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operatively links the nucleic acid sequence to regulatory sequences in the vector that enable the transcription and translation of the nucleic acid sequence within the recombinant host cell.

According to the present invention, the phrase "operatively linked" refers to linking a nucleic acid molecule to an expression control sequence (e. g. , a transcription control sequence and/or a translation control sequence) in a manner such that the molecule can be expressed when transfected (i.e., transformed, transduced, transfected, conjugated or conduced) into a host cell.

Alternatively, a polynucleotide sequence of the present invention may be inserted into the polylinker of an appropriate vector for expression.

The vector or the construct may further comprise nucleotide sequences coding for tags which may aid purification or immobilization of the polypeptide. A non limiting example of tags useful in this invention is given by a polyhistidine tag (His tag), however other purification tags known in the art may be used. Preferably, the tag will be positioned in the N-terminal or C-terminal of the polypeptide, and may be encoded directly by the vector. Alternatively, the tag may be located internally to the polypeptide, as long as it does not affect the functionality of the polypeptide.

The recombinant expression vector may be any vector (e.g. , a plasmid, phagemid, cosmid, artificial chromosome, transposon, phage or virus) that can be conveniently subjected to recombinant DNA procedures and that will allow the expression of the nucleotide sequence in the host cell of choice. The skilled person will select suitable vector-host systems known in the art.

The vector may be an autonomously replicating vector and may contain any means for assuring self-replication. Alternatively, the vector may be introduced into the host cell and integrated into the genome and replicated together with the chromosome(s) into which it has been integrated (e.g. transposon).

The vectors of the present invention may further contain one or more selectable markers for the selection of the transformed cells.

The invention also provides a host cell transformed with the vector above described, i.e. comprising the polynucleotide construct as defined above.

The cell can be a prokaryotic cell such as a bacterial or an archaea cell, or an eukaryotic cell such as a yeast cell, a fungal cell, a plant cell, an insect cell or a mammalian cell. The introduction of the vector into the host cell can be carried out by any commonly known technique e.g. using competent cells, by means of electroporation, conjugation and other transformation techniques commonly known by those skilled in the art.

A non limiting example of suitable vector construction and suitable host-cells is provided in this description in the example section.

The present invention also relates to the production of an isolated heat- stable alpha carbonic anhydrase having the maximum carbonic anhydrase enzymatic activity in the range from 65°C to 100°C or to 1 10°C or enzymatically active fragments thereof said fragments maintaining said peak of carbonic anhydrase enzymatic activity comprising the steps of

a. culturing a host cell transformed with the vector defined above

b. extracting the carbonic anhydrase produced thereby.

According to the vector and host cell selected, the alpha carbonic anhydrase or the active fragments thereof of the invention can be directly secreted into the culture media or extracted by the transformed cells according to standard procedures.

In an embodiment, said anhydrase or active fragments thereof retain at least

95%, or at least 90% or at least 80% of the carbonic anhydrase activity after incubation at 90°C for 20 minutes.

In another embodiment said anhydrase is of SEQ I D NO 2 or has at least

90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO 2.

The present invention also provides a method for the extraction of carbon dioxide from a medium wherein

a. said extraction is carried out at temperatures in the range from 50°C to 100°C or to 110°C ;

b. the carbonic anhydrase or fragments thereof of anyone of claims 1 to 4 is used.

As an example, the extraction may be carried out at temperatures in the range from about 65°C or 70°C or 75°C or 80°C to about 100°C or 1 10°C, from about 75°C to about 80°C, or 90°C, or 95°C or 100°C or 1 10°C.

In a particular embodiment the extraction will be carried out in a temperature range from about 80°C to about 100°C or from about 80°C to 90° about °C.

The state of the art teaches various methods for C0₂ extraction that make use of alpha carbonic anhydrase, in principle, any known method using this class of enzymes can be carried out with the active CA enzymes of the invention and the enzymes can be used in any known reactor for C0₂ extraction in processes wherein the extraction is based on the use of alpha carbonic anhydrases.

The CA of the invention may be used for carbon dioxide extraction from C0₂ emission streams such as carbon-based or hydrocarbon-based combustion in electric generation power plants, or from flue gas stacks from such plants, industrial furnaces, stoves, ovens, or fireplaces or from airplane or car exhausts. The enzymes of the invention can be used to remove C0₂ in the preparation of industrial gases such as acetylene (C₂H₂), carbon monoxide (CO), chlorine (Cl₂), hydrogen (H₂), methane (CH₄), nitrous oxide (N₂0), propane (C₃H₈), sulphur dioxide (S0₂), argon (Ar), nitrogen (N₂), and oxygen (0₂). Carbonic anhydrase can also be used to remove C0₂ from a raw natural gas during the processing to natural gas. When co₂ is removed from a raw natural gas or biogas, the result is the final "enrichment" in other components (such as methane by way of example). Furthermore, the CA of the invention can be used in a method for reducing the carbon dioxide content in a biogas, in a syngas thus enriching the biogas or syngas in other gases content. According to the invention the carbon dioxide containing method may be a fluid, a biogas, a natural gas, a syngas, a multiphase mixture.

In an embodiment of the invention the extraction can be carried out at a pH greater than 8. Furthermore, the extraction can be carried out in the presence of amines such as monoethanolammine (MEA) and diethanolammine (DEA).

The data provided in the example section (example 4) demonstrate the particular suitability of the CA of the invention for this embodiment. Accordingly, the present invention also relates to a reactor for extracting carbon dioxide wherein said reactor comprises a bicarbonate buffer with a pH of at least 8 and a the heat-stable alpha carbonic anhydrase or enzymatically active fragments thereof as defined above, the extraction being optionally carried out in the presence of amines such as monoethanolammine (MEA) and diethanolammine (DEA).

The C0₂ extraction from a C0₂-containing medium can be carried out in enzyme based reactors (such as bioreactors). The carbon dioxide-containing medium can be purified from contaminants e.g., by dotting outlets or membranes before processing into the reactor. By way of example, the C0₂-containing media (such as gasses/ multiphase mixtures emitted from combustion processes, e.g., flue gases or exhausts), can be cleared of ash, particles, NOx and/or S0₂, before the gas/ multiphase mixture is passed into the reactor. Raw natural gas from different regions may have different compositions and separation requirements; oil, condensate, water and natural gas liquids, if present in the raw natural gas, can be removed prior to the extraction of C0₂ in the reactor according to the invention.

The C0₂ from the raw natural gas may be extracted in the same process as the sulphur removal, or may be extracted in a completely separate process. According to the invention the reaction temperature of the C0₂ extraction process can be between 65°C and 110°C, such as between 70°C and 1 10°C, or between 75°C and 1 10°C, or between 80°C and 1 10°C, or between 85°C and 110°C, or between 90°C and 110°C, or between 95°C and 110°C, or between 65°C and 100°C, or between 70°C and 100°C, or between 75°C and 100°C, or between 80°C and 100°C, or between 85°C and 100°C, or between 90°C and 100°C, or between 95°C and 100°C, or between 65°C and 95°C, or between 70°C and 95°C, or between 75°C and 95°C, or between 80°C and 95°C, or between 85°C and 95°C, or between 90°C and 95°C, or between 65°C and 90°C, or between 70°C and 90°C, or between 75°C and 90°C, or between 80°C and 90°C, or between 85°C and 90°C, or between 65°C and 850°C, or between 70°C and 85°C, or between 75°C and 85°C, or between 80°C and 85°C, or between 65°C and 80°C, or between 70°C and 80°C, or between 75°C and 80°C, or between 65°C and 75°C, or between 70°C and 75°C.

Bioreactors suitable for the extraction process of the invention can be bioreactors in which a mixed gas stream (e.g. , containing oxygen, nitrogen and carbon dioxide) contacts the enzyme of the invention, at a gas-liquid interface to catalyze the conversion of carbon dioxide contained in the gas to bicarbonate or carbonate. The gas-liquid interface in such a bioreactor can for example be provided by an enzyme based hollow fiber membrane bioreactor (HFM B). An example of H FM B is a hollow fiber contained liquid membrane (H FCLM) as described by Majumdar et al. , 1988, AIChE 1 135-1 145. CLMs are made by sandwiching a core liquid between two polymer membranes. According to the art, the core liquid is preferably continuously re-supplied through a reservoir of liquid membrane solvent. An alternative type of enzyme based CLM permeator useful in a bioreactor is described in Cowan et al., 2003, Ann. NY Acad. ScL 984: 453-469 (hereby incorporated by reference).

In summary, the bioreactor of the invention can comprise a liquid membrane constructed by sandwiching phosphate buffered solution containing the carbonic anhydrase of the invention between two hydrophobic, microporous, polypropylene membranes (e.g. , Celgard PP-2400). The CA concentration can be, by way of example between 100-166 micro-M, and the buffer suitably can have a phosphate concentration between 50-75 mM and a pH between 6.4 and 8.0. The skilled person is aware that the concentrations of CA and buffer depend on the C02 concentration in the feed. Accordingly, the pH optimum is a function of the C0₂ concentration and the buffer strength. According to the art, the thickness of the aqueous phase can be of about 330 micro-m, and can also be varied from 70 micro-m to 670 micro-m using annular spacers. The liquid membrane fluid volume is maintained by hydrostatic fluid addition from a reservoir, so to provide a constant liquid membrane thickness and to prevent the separation between the polymer membrane and the metal support. One side of the CLM (the feed membrane) is contacted with a C0₂- containing feed gas stream, and the other side of the CLM (the sweep membrane) is in contact with a C0₂-free sweep gas stream, for example argon. In this bioreactor C0₂ from the feed gas stream is converted to bicarbonate in the liquid phase and then returned as C0₂ to the sweep gas stream from where it can be stored in the form of compressed C0₂. The entire process is catalysed by the carbonic anhydrase. The CLM permeator described above is capable of capturing C0₂ from feed gas streams with down to 0.1 % C0₂. Alternative CLM permeators are known in the art and the skilled person will know how to adapt CA and buffer concentration and pH selection to said permeators without use of inventive skill.

Glossary in the meaning of the invention

The term "carbonic anhydrase activity" or "CA activity" is defined herein as an EC 4.2.1.1 activity which catalyzes the inter-conversion between carbon dioxide and bicarbonate [C0₂ + H₂0→ HC0₃ ^" + H⁺]. For purposes of the present invention, CA activity is determined according to the procedure described in Example 3.

One unit of CA activity is defined after Wilbur [1 U = (1/tc)-(1/tu) x 1000] where U is units and tc and tu represent the time in seconds for the catalyzed and uncatalyzed reaction, respectively (Wilbur, 1948, J. Biol. Chem. 176: 147-154).

The arbitrary unit of the enzyme is defined as the quantity of the enzyme needed to bring the pH of 2 mL solution from 8,3 to 6,3 in 1 minute in the condition described.

Using p-nitrophenyl acetate as substrate the enzyme Unit is calculated at 0°C and pH 7.6 according the formula: 1 U = (Abs 348nm/min - Abs blank/min)/ 0.05 x (mg enzyme/ ml reaction); where 0.05 is the μηιο^Γ extinction coefficient of p- NpA.

The term "isolated" with reference to the CA of the invention indicates a CA that has been isolated from its natural environment or a recombinant CA, expressed in an organism or in a cell that is not Aqificales sulfurihydrogenibium strain Y03AOP1.

The term "C0₂-containing medium" is used to describe any material which may contain at least 0.001 % C0₂, up to about 50%, the medium can be in particular in the form gaseous phases, liquids or multiphase mixtures, but may also be solid.

The term "C0₂ extraction" (or capture) is intended as a reduction of C0₂ from a C0₂-containing medium. Such an extraction may be performed from one medium to another and may also be the conversion of C0₂ to bicarbonate or carbonate within the same medium.

The term "functional fragment" when referred to the enzyme of the invention is used to describe a polypeptide which is derived from SEQ ID NO 2, and which has been truncated either in the N-terminal region or the C-terminal region or in both regions or that has mutations or deletions within the amino acid sequence so to generate a fragment or a mutant of the parent polypeptide. To be a functional fragment the resulting polypeptide shall maintain at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the CA activity of the parent polypeptide of SEQ ID NO 2 and the maximum of said CA activity has to be in the range between 65°C and 110°C.

EXAMPLES

Example 1

Identification of the thermophilic microrganism containing the gene of the alpha class CA.

A search in non-redundant protein database using two sequences of alha class CAs, namely from Rhodopseudomonas palustris and from Bacillus clausii KSM-K16 as queries, was performed using the blastp algorithm available at the site http://blast.ncbi.nlm.nih.gov/Blast.cgi. The databases were inspected by individual genus groups belonging to both Archaea and Bacteria (hyper)thermophilic kingdoms. Although various putative CA were found, belonging to different CA families, only the genus Sulfurihydrogenibium showed the presence, in the genome of the Y03AOP1 strain of a gene encoding a putative alpha class CA that subsequently showed a CA activity. In fact, all other putative CA enzymes found with the query did not show esterase activity.

Example 2.

Expression of the genes, purification and characterization of the recombinant proteins The gene from Sulfuri hydrogeni bi um WAS synthesised (Mr. Gene, Regensburg, Germany) with optimized codon usage for E. coli. The nucleotide sequence is shown in Fig. 2.

The codon CTC was inserted at position 60 of SEQ ID NO 1 to generate the Xhol restriction site and an additional Xhol site was added immediately downstream of the stop codon (Xhol sites underlined on the sequences). The Xhol restriction fragment was inserted in the expression vector pET15b (Novagen), producing the vector pETCA that comprised SEQ ID NO 1 deprived of nucleotides 1-60 coding for aa 1-20 of SEQ I D NO 2, that were replaced by nucleotides coding for the aa of S EQ I D N O 3. Therefore a sequence coding for aa of SEQ ID NO 3 (i.e. a nucleotide stretch in the pET15b plasmid) was fused in frame with nucleotides 61- 741 of SEQ ID NO 1 in order to code for a fusion protein having 6X histidine tag at the N-terminus consisting of the aa of SEQ I D NO 3 attached to the aa 21-246 of SEQ ID NO 2.

The pETCA plasmid was transferred into E.coli cells treated with CaCI₂ according to the procedures described by Sambrook and Russel (2001 Molecular Cloning. A Laboratory Manual Vol. 1 page 116,1 18.

The transformed cells were grown at 37°C in LB in the presence of 50 micrograms per litre of kanamycin. At an optical density of 0.6 at 600 nm, it was added IPTG at a concentration of 1 mM and ZnS0₄ at a concentration of 0.5 mM. After 5 hours the cells were harvested by centrifugation, suspended in the buffer Tris-HCI 10 mM pH 8.3 and lysed by sonication. After centrifugation at 10000 rpm for 45 min the enzyme was purified by thermoprecipitation keeping the solution for 30 min at 80°C. After centrifugation, the supernatant containing the enzyme was absorbed by a DE52 exchange chromatography, in the buffer Tris-HCI 10 mM pH 8.3, and eluted with a saline gradient from 0 to 0.5 M NaCI. The analysis of the peak containing the enzyme revealed the presence of a single band.

The CA activity was assayed using the Wilburn assay (Wilburn , 1948, J.Biol. Chem. 176: 147-154) as modified by Chirica (Chirica et all, 2001 , Biochim.Biophys. Acta 1544:55-63).

The arbitrary unit of the enzyme is defined as the quantity of the enzyme needed to bring the pH of 2 mL solution from 8,3 to 6,3 in 1 minute in the condition described.

Using p-nitrophenyl acetate as substrate the enzyme Unit is calculated at 0°C and pH 7.6 according the formula: 1 U = (Abs 348nm/min - Abs bianco/min)/ 0.05 x (mg of enzyme/ ml of reaction); where 0.05 is the μηιο^Γ extinction coefficient of p-NpA. MW determinations of S:sp CA by native and SDS electrophoresis showed a molecular mass of 26.3 KD indicating that the enzyme was a monomer.

The V max and Km of S.sp CA at 0°C, using C02 as substrate with the Wilburn assay, were comparable to that of bovine CA. In fact a Km of 13.5 mM and a Vmax of 1 1.2 Units / μg of enzyme were determined. Using p-nitro-phenyl acetate as substrate a value of Km of 2.51 mM and a Vmax of 3,76 units/ μg of enzyme were obtained.

Example 3

Thermoactivity and thermostability of the S.sp CA enzyme.

The optimum temperature activity for bovine and S. sp CA activity was determined by incubating enzymes in 15 mM Tris-sulphate buffer, pH 7.6 at 25°C, 40°C, 50°C, 60°C, 70°C and 80°C with 3mM of p-nitrophenilacetate (Figure 3). After 5min, the reaction was monitored following the absorbance at 348nm. The optimum temperature activity for bovine enzyme was found to be 60°C, while the activity S.sp CA increased constantly up to a temperature of 90°C. Using the same quantity of the two enzymes, the value of the activity of SpCA at 60°C was more than the double than that of bovine CA.

In Figure 2 is reported the results of experiments comparing the stability at different temperatures of the bovine and the S sp CA.

The experiments showed that after 20 minutes of incubation of the enzymes at the indicated temperatures, the activity of the bacterial CA was stable while the bovine enzyme was inactivated at temperature above 50°C. We also investigated the stability of S.sp CA at 90°C in 10 mM Tris-HCI, pH 8.3. Aliquots were removed at appropriate times (10, 30 and 90 and 180 minutes) and the residual activity was measured at 0°C using C02 as substrate with Wilburn assay. The S.sp CA retained 100% of activity after 2 hours at 90°C.

Figure 2 shows the comparison of the stability of the enzyme with different incubation times (30, 60, 120, and 180 minutes), the same are summarised in table 1 below.

The experiment demonstrates that the enzyme shows stability at extremely high temperatures also after 2 hours incubation. Table 1

Thermostability of SpCA.

The thermostability of SpCA has been assessed by incubating the enzyme at 25°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C e 100°C in 0.01 mM Tris-HCI pH 8.3 (2 ml_). Aliquots were assayed at different time of incubation (30, 60, 120 e 180 min). The activity was determined with the Wilburn assay using 30 ng of enzyme.

Example 4

Stability of S.sp CA in the presence of MEA and DEA

Since the presence of CA increases the power of monoethanolammine (MEA) and diethanolammine (DEA) to capture C02, it has been evaluated the stability of S.sp CA in the presence of different concentrations of the two amines at 25 and 80°C. The enzyme was incubated for 180 min. at the two temperatures with MEA or DEA, at concentrations form 1 to 30%, as reported in the Table2. After 180 min. the samples were dialysed and activity determined. The results show that the S.sp CaA is stable in these harsh conditions since the pH is very alkaline. The activity was assayed using the Wilburn method. No differences were found on the effect of the two amines. Table 2

Effect of MEA and DEA on the enzyme stability

U/MQ of enzyme (25°C) ΙΙ/μς of enzyme (80°C)

No solvent 7.7 7.9

MEA o DEA 1 % 6.7 6.5

MEA o DEA 5% 6.5 6.6

MEA o DEA 10% 6.4 6.6

MEA o DEA 20% 6.6 6.5

MEA o DEA 30% 6.6 6.4

Claims

1 . An isolated heat-stable alpha carbonic anhydrase, having maximum carbonic anhydrase enzymatic activity in the range from 65° C to 1 1 0° C or enzymatically active fragments thereof said fragments maintaining said peak of carbonic anhydrase enzymatic activity.

2. The alpha carbonic anhydrase or fragments thereof of claim 1 wherein said anhydrase or fragments thereof retains at least 90% of its carbonic anhydrase activity after incubation at 90°C for 20 minutes.

3. The alpha carbonic anhydrase or fragments thereof of claim 1 or 2 wherein said alpha carbonic anhydrase derives from Aquificales sulfurihydrogenibium species.

4. The alpha carbonic anhydrase of anyone of claims 1 to 3 wherein said anhydrase is of SEQ ID NO 2 or has at least 95% sequence identity to SEQ ID NO 2.

5. The alpha carbonic anhydrase or fragment thereof of anyone of claims 1 to 4 having the maximum carbonic anhydrase enzymatic activity at about 90°C.

6. An isolated polynucleotide coding for the carbonic anhydrase or fragment thereof.

7. The isolated polynucleotide of claim 6 wherein said polynucleotide is of SEQ ID NO 1.

8. An isolated polynucleotide construct comprising the polynucleotide of claims 6 or 7.

9. The construct of claim 8 wherein said construct is a vector.

10. The vector of claim 9 that is an expression vector.

1 1. A host cell transformed with the vector of claims 9 or 10.

12. A method for the production of an isolated heat-stable alpha carbonic anhydrase having the maximum carbonic anhydrase enzymatic activity in the range from 65°C to 1 10°C or enzymatically active fragments thereof said fragments maintaining said peak of carbonic anhydrase enzymatic activity comprising the steps of

a. culturing a host cell transformed with the vector of claim 8

b. extracting the carbonic anhydrase produced thereby.

13. The method of claim 12 wherein said anhydrase or fragments thereof retains at least 90% of its carbonic anhydrase activity after incubation at 90°C for 20 minutes.

14. The method of claim 12 or 13 wherein said anhydrase is of SEQ ID NO 2 or has at least 95% sequence identity to SEQ ID NO 2.

15. A method for the extraction of carbon dioxide from a medium wherein a. said extraction is carried out at temperatures in the range from 50°C to

1 10°C;

b. the carbonic anhydrase or fragments thereof of anyone of claims 1 to 5 is used.

16. The method of claim 15 wherein the carbon dioxide containing medium is a fluid, a biogas, a natural gas, a syngas, a multiphase mixture.

17. The method of claim 15 or 16 wherein the extraction of carbon dioxide is carried out at temperatures in the range from 65°C to 1 10°C.

18. The method of claims 15 or 16 wherein the extraction of carbon dioxide is carried out at temperatures in the range from 75°C to 100°C.

19. The method of any one of claims 15 to 18 wherein the extraction is carried out at a ph greater than 8.

20. A reactor for extracting carbon dioxide wherein said reactor comprises a bicarbonate buffer with a pH of at least 8 and a the heat-stable alpha carbonic anhydrase having the maximum carbonic anhydrase enzymatic activity in the range from 65°C to 1 1 0°C or enzymatically active fragments thereof said fragments maintaining said peak of carbonic anhydrase enzymatic activity.

21. The reactor of claim 20 wherein said anhydrase or fragments thereof retains at least 90% of its carbonic anhydrase activity after incubation at 90°C for 20 minutes.

22. The reactor of claim 20 or 21 wherein said anhydrase is of SEQ ID NO 2 or has at least 95% sequence identity to SEQ ID NO 2.

23. The reactor of anyone of claims 20 to 22 wherein said reactor is a bioreactor.

24. Compositions comprising the isolated heat-stable alpha carbonic anhydrase or enzymatically active fragments thereof according to claims 1-5 or mixtures thereof and one or more excipient.