Heat-stable and alkalistable carbonic anhydrase

Abstract

A carbonic anhydrase having high heat stability and alkalistability, use of the carbonic anhydrase in the in the extraction of carbon dioxide from a carbon dioxide-containing medium.

Description

[0001] HEAT-STABLE AND ALKALISTABLE CARBONIC ANHYDRASE

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to a carbonic anhydrase enzyme and its use in the extraction of carbon dioxide (CO2).

[0004] BACKROUND OF THE INVENTION

[0005] The European Union committed to reducing greenhouse gas emissions by at least 40% by 2030 under the 2016 Paris Agreement. To achieve timely decarbonization of the industrial sector, experts, including the Center for Climate and Energy Solutions, advocate for the adoption of carbon capture, utilization, and storage technologies (CCUS) as the most viable approach. Various CO2 capture technologies, such as chemical and physical absorption, membrane separation, and cryogenic distillation, have been explored. Notably, chemical absorption, especially in post-combustion capture, has gained prominence due to its multiple advantages. The prevailing technology relies on amines, like monoethanolamine (MEA), known for their exceptional CO2 removal capabilities. However, amine-based strategies have drawbacks, including energy-intensive operation, high maintenance costs, and environmental concerns related to potential toxic emissions. An alternative eco-friendly approach involves utilizing enzymes called carbonic anhydrases (CAs), which catalyze the reversible hydration of CO2 to bicarbonate (HCCh' ) and hydrogen ions (H+). CAs, found in all living organisms, exhibit remarkable reaction rates, significantly accelerating CO2 dissolution. Carbonic anhydrases are known to be used in combination with conventional amine-based solvents in CO2 capture technologies and enhancing drastically the overall CO2 capture efficiency. On the other hand, there is a great interest in utilizing carbonic anhydrases in novel green CO2 capture technologies that include the use of aqueous carbonate solutions, which are known to be eco-friendly, unlike amines. Such technologies can overall overcome the techno economical bottleneck of the conventional approaches by requiring less energy for regeneration and at the same time achieving very high CO2 capture efficiency. Despite their abundance in nature, CAs face challenges in industrial pipelines due to the harsh conditions that are employed in industrial settings.

[0006] There are several characterized thermostable carbonic anhydrases (CAs). The beta-class CA (Cab) from Methanobacterium thermoautotrophicum AH, which has been reported to be heat stable to up to 75 °C and the gamma-class carbonic anhydrase (Cam) from Methanosarcina thermophila TM-1. Cam was isolated for the first time in 1994, and in 1996 it was shown to be stable to heating at 55 °C for 15 min. Cam is the only isolated enzyme of the gamma-class and has been subject to a lot of characterization studies since its discovery. WO 2010 / 081007 describes heat stable variants of Cam. US 2006 / 0257990 describes variants of human carbonic anhydrase II where the most stable variant shows activity up to 65 °C. US 2004 / 0259231 discloses the use of Cab as well as the non-thermostable human CA isoform IV in a CO2 solubilization and concentration process. WO 2008 / 095057 describes heat-stable alpha-carbonic anhydrases from Bacillus clausii and Bacillus halodurans and their use for the extraction of CO2. WO 2010 / 151787 (application no. PCT / US2010 / 040022 ) describes heat-stable alphacarbonic anhydrases from Caminibacter and their use for the extraction of CO2. US 11 ,773,386 B2 describes heat-stable metagenomic carbonic anhydrases derived from the Logatchev Hydrothermal and their use in CO2 capture pipelines.

[0007] SUMMARY OF THE INVENTION

[0008] The present invention provides a novel carbonic anhydrase, which can be used in the extraction of CO2 from a CO2-containing medium.

[0009] The carbonic anhydrase according to the present invention maintains high residual activity at an elevated temperature and maintains high residual solubility after incubation in aqueous 20 % (w / v) K2CO3.

[0010] The stability of the carbonic anhydrase according to the present invention allows for its use in bioreactors where flue gasses are distributed in aqueous medium and CO2 is extracted enzymatically. For this process high temperatures can be utilized for efficient mass transfer but the process can also be performed in ambient temperatures. Heat stability due to enzyme use may include situations where the carbonic anhydrase carries out catalysis in one temperature (e.g. 20- 65 °C) and then, due to different stages in the process, is exposed to higher temperatures (e.g. 70 - 120 °C). In this setup, the enzyme has to withstand repeated exposure to low and high temperatures during the process cycle. Furthermore, the carbonic anhydrase can be used in immobilization matrixes.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : pET28 plasmid map. The map shows the codon optimized gene of carbonic anhydrase having the sequence SEQ ID NO: 3 as cloned in the pET28a (+) vector along with the in frame 6X Histidine-tag and other plasmid features like KanRantibiotic resistance gene and the origin of replication (or / ). Figure 2: Thermostability of carbonic anhydrase having the sequence SEQ ID NO: 4. The thermostability was evaluated by measurements of residual carbonic anhydrase activity after high-temperature exposure at 80 °C and 90 °C for up to 24 h. The presented data represents the mean value of minimum three independent experiments, each conducted in technical triplicates. The error bars indicate the standard deviation of the mean values.

[0013] Figure 3: Stability in 20% (w / v) K2CO3. The alkalistability of carbonic anhydrase having the sequence SEQ I D NO: 4 was evaluated through the measurement of residual solubility after incubation of the enzyme in aqueous 20% (w / v) K2CO3 aqueous solution for different time periods spanning up to 30 days. The incubated enzyme preparation was centrifuged to remove denatured protein and the remaining soluble protein concentration was measured photometrically. The measurements of residual soluble protein were performed in triplicates and expressed as the mean average with standard deviation.

[0014] DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a novel carbonic anhydrase which can be used in the extraction of CO2 from CO2-containing media, such as gas, liquid or multiphase media. The carbonic anhydrase of the present invention in particularly useful in applications where the temperature of the CO2-containing medium is above 70 °C and above the optimum temperature of the carbonic anhydrases of the prior art. Also, the carbonic anhydrase of the present invention is especially useful for use in carbon-capture media of high salinity, especially those that consist of high concentration of K2CO3, a very commonly used CO2-capture medium.

[0016] The term "carbonic anhydrase activity" or "CA activity" or “CO2 hydratase activity” is defined herein as an EC (Enzyme Commission) 4.2.1.1 CO2 hydration activity which catalyzes the conversion between carbon dioxide and bicarbonate [CO2 + H2O HCOs' + H+], CA activity can be determined according to a standard procedure known in the art, as also described in Example 3 hereinbelow. One unit of CA activity is defined after Wilbur [1 WAU = (tO - 1) / 1] where WAU is the activity in WA units and to and t represent the time in seconds for the catalyzed and uncatalyzed reaction. A polypeptide according to the present invention is considered to have CA activity if it has at least 20%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, and most preferably at least 100% of the CA activity of the polypeptide consisting of the amino acid sequence corresponding to amino acid residues 1 to 168 of SEQ ID NO: 2.

[0017] The term "CO2-containing medium” is used to describe any material which contains at least 0.001 % (v / v) CO2, preferably at least 0.01% (v / v), more preferably at least 1% (v / v), even more preferably at least 20% (v / v), and most preferably at least 50% (v / v) CO2. Preferably the CCh-containing medium has a temperature between 5 °C and 110 °C, more preferably between 30 °C and 90 °C, even more preferably between 40 °C and 90 °C, and most preferably between 60 °C and 90 °C at any pressure. CCh-containing media are in particular gaseous phases (including gas mixtures), liquids or multiphase mixtures, but may also be solid. A CCh-containing gaseous phase is for example raw natural gas obtainable from oil wells, gas wells, and condensate wells, syngas) generated by the gasification of a carbon containing fuel (e.g., methane) to a gaseous product comprising CO and H2, or emission streams from combustion processes, e.g., from carbon based electric generation power plants, or from flue gas stacks from such plants, industrial furnaces, stoves, ovens, or fireplaces or from airplane or car exhausts. A CO2-containing gaseous phase may alternatively be ambient air (including hot (above 40 °C) air, e.g., desert air), or from respiratory processes in mammals (such as the CCh-containing gas phase in an artificial lung), living plants and other CO2 emitting species, in particular from green-houses. A CCh-containing gas phase may also be off-gas, from aerobic or anaerobic fermentation, such as brewing, fermentation to produce useful products such as ethanol, or the production of biogas. Such fermentation processes can occur at elevated temperatures if they are facilitated by thermophilic microorganisms, which are for example encountered in the production of biogas. A CCh-containing gaseous phase may alternatively be a gaseous phase enriched in CO2 for the purpose of use or storage. The above-described gaseous phases may also occur as multiphase mixtures, where the gas co-exists with a certain degree of fluids (e.g., water or other solvents) and / or solid materials (e.g., ash or other particles). CCh-containing liquids are any solution or fluid, in particular aqueous liquids, containing measurable amounts of CO2, preferably at one of the levels mentioned above at any pressure. CCh-containing liquids may be obtained by passing a CCh-containing gas or solid (e.g., dry ice or soluble carbonate containing salt) into the liquid. CCh-containing fluids may also be compressed CO2 liquid (that contains contaminants, such as dry-cleaning fluid), supercritical CO2, or CO2 solvent liquids, like ionic liquids. A CCh-containing liquid may also be referred to as a “carrier liquid”. A CO2- containing liquid may also include compounds capable of improving the CCh-containing capacity of the liquid, such as HCCh' (KHCO3 or NaHCCh), COs-2(Na2COs or K2CO3), HPO42‘ (K2HPO4 or Na2HPC>4) or MDEA or any amine or Tris.

[0018] The term "CO2 extraction” is to be understood as a reduction of carbon from a CO2- containing medium. Such an extraction may be performed from one medium to another, e.g., gas to liquid, liquid to gas, gas to liquid to gas, liquid to liquid or liquid to solid, but the extraction may also be the conversion of CO2 to bicarbonate, carbonate or carbonic acid within the same medium or the conversion of bicarbonate to CO2 within the same medium. The term CO2 capture is also used to indicate extraction of CO2 from one medium to another or conversion of CO2 to bicarbonate / carbonate or conversion of bicarbonate / carbonate to CO2.

[0019] When used herein the term “coding sequence” means a polynucleotide sequence, which directly specifies the amino acid sequence of product polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG and TGA. The coding sequence may be a DNA, cDNA, mRNA, synthetic or recombinant polynucleotide.

[0020] The term "functional fragment of a polypeptide" or "a polypeptide fragment” having carbonic anhydrase activity” is used to describe a polypeptide which is derived from a longer polypeptide (parent polypeptide), e.g., a mature polypeptide, and which has been truncated either in the N-terminal region or the C-terminal region or in both regions to generate a fragment of the parent polypeptide. To be a functional polypeptide the fragment must maintain at least 20%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90%, and most preferably at least 100% of the CA activity of the parent polypeptide.

[0021] The term "identity" is used to describe the relatedness between two amino acid sequences or two nucleic acid sequences. For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package, preferably version 3.0.0 or later.

[0022] The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0023] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0024] For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package, preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0025] (Identical DeoxyribonucleotidesxlOO) I (Length of Alignment-Total Number of Gaps in Alignment)

[0026] The term “heat-stable” or “thermostable” as used in reference to an enzyme, such as a carbonic anhydrase, indicates that the enzyme is functional or active (i.e., can perform catalysis) at an elevated temperature, i.e., above 45 °C, preferably above 60 °C, more preferably above 80 °C, even more preferably above 90 °C, and most preferably above 100 °C. For a carbonic anhydrase to be considered as heat-stable it remains active after at least 15 minutes, preferably for at least 24 hours, more preferably for at least 7 days, even more preferably for at least 30 days, and most preferably for at least 50 days at the elevated temperature. The level of activity can be measured using an assay known to the art, as also described in Example 3 hereinbelow, after incubation for the given time at each temperature. The activity may be compared with the enzyme activity prior to the temperature elevation, thereby obtaining the residual activity of the enzyme after the heat treatment. Preferably, the residual activity is at least 30% after the given time at the elevated temperature, more preferably at least 60%, even more preferably at least 80%, and most preferably the level of residual activity is at least equal to or unchanged after the given time at the elevated temperature.

[0027] The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0028] The term “isolated polynucleotide” means a polynucleotide isolated from the metagenomic DNA material of an environment or the genomic material of an organism. Preferably, the isolated polynucleotide is at least 1% more preferably at least 20% pure, even more preferably at least 80% pure and most preferably at least 95% pure, as determined by agarose electrophoresis. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

[0029] The term "isolated polypeptide" as used herein refers to a polypeptide which is at least 20% pure, preferably at least 40% pure, more preferably at least 80% pure, even more preferably at least 90% pure, and most preferably at least 95% pure, as determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The term "operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0030] The term “substantially pure polypeptide” denotes herein a polypeptide preparation which contains at most 10%, preferably at most 5%, more preferably at most 4%, even more preferably at most 1%, and most preferably at most 0.5% by weight of other polypeptide material with which it is natively associated. It is, therefore, preferred that the substantially pure polypeptide is at least 95% pure, more preferably at least 96% even more preferably at least 99%, and most preferably 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polypeptides are in “essentially pure form”, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively associated. This can be accomplished, for example, by preparing the polypeptide by means of well-known recombinant methods or by classical purification methods.

[0031] The term “syngas” or “synthesis gas” is used to describe a gas mixture that contains varying amounts of carbon monoxide and hydrogen generated by the gasification of a carbon containing fuel (e.g., methane or natural gas) to a gaseous product with a heating value. CO2 is produced in the syngas reaction and must be removed to increase the heating value.

[0032] The term “thermophilic” in relation to an organism, describes an organism which thrives at relatively high temperatures, i.e., above 45 °C. Hyperthermophile organisms thrive in extremely hot environments, that is, hotter than around 60 °C with an optimal temperature above 80 °C.

[0033] The present invention provides a carbonic anhydrase which is a) a polypeptide comprising or having the amino acid sequence SEQ ID NO: 2; or b) a polypeptide comprising or having an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to amino acid residues of SEQ ID NO: 2 or c) a peptide of (a) or (b) or having carbonic anhydrase activity; or d) a polypeptide encoded by a nucleic acid sequence with: i) a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 2; or ii) a polynucleotide sequence of SEQ ID NO: 1.; or iii) a subsequence of (i) or (ii), of at least 100 contiguous nucleotides, or iv) a complementary strand of (i) or e) a polypeptide encoded by a nucleic acid sequence which, because of the degeneracy of the genetic code, does not hybridize with the polynucleotide sequence of SEQ ID NO: 1 , but which codes for a polypeptide having an amino acid sequence according to a) or b); or f) a polypeptide encoded by a nucleic acid sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. The polypeptide in b), d) or g) can be synthetic if the polypeptide falls within the claimed identities and maintain carbonic anhydrase activity.

[0034] Polynucleotide sequences with a given % identity to SEQ ID NO: 1 DNA sequence or polypeptide sequences with a given % identity to amino acid sequence SEQ ID NO: 2, may be obtained from naturally occurring sources such as bacterial strains. Alternatively, the polypeptide or polynucleotide sequences may be obtained by substitution, deletion, and / or insertion of one or more amino acids or nucleic acids in the parent sequence (for example the sequence SEQ ID NO: 1 for polynucleotides, or sequence SEQ ID NO: 2 for polypeptides). Preferably the number of amino acids which is changed in the parent sequence, or the polypeptide encoded by the parent polynucleotide is between 1 to 5, 1 to 10, 1 to 20, 1 to 30 or 1 to 40 amino acids. The amino acid changes are, preferably, of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an aminoterminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine tag or a polyhistidine-glutamine tag, an antigenic epitope or a binding domain. Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter specific activity are known in the art and are described. The most commonly occurring exchanges are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly.

[0035] Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. In 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. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. A large number of these analyses have already been performed on carbonic anhydrases. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides which are related to a polypeptide according to the invention.

[0036] Beta-carbonic anhydrases are identified by their consensus sequence motifs: CxDxR and HxxC. The respective consensus residues correspond to positions 41 to 45 for the motif CxDxR and the positions 94 to 97 for the motif HxxC both in SEQ ID NO: 2. In a preferred embodiment all consensus positions are present in the carbonic anhydrase.

[0037] The following amino acid residues C41 , H94, and C97 (numbering according to SEQ ID NO: 2) are predicted to form a catalytic triad which is important for CO2 hydration reaction. In a preferred embodiment of the present invention the carbonic anhydrase contains a cysteine in position 41 , a histidine in position 94 and a cysteine in position 97 (SEQ ID NO: 2 numbering).

[0038] The following cysteine residues C2, C36, C139 and C165 (using SEQ ID NO: 2 numbering) are predicted to engage in a cysteine bridge and may therefore be important for the stability of the carbonic anhydrase. In a preferred embodiment of the present invention the carbonic anhydrase contains a cysteine in position 2, 36, 139 and 165 (using SEQ ID NO: 2 numbering).

[0039] Single or multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure. Other methods that can be used include error-prone PCR, phage display, and region-directed mutagenesis.

[0040] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules 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.

[0041] Preferably, the carbonic anhydrase according to the present invention maintains residual activity of at least 30%, more preferably at least 60%, even more preferably at least 90%, and most preferably 100% after incubation for 30 min at a temperature of 60 °C. The thermostability of the carbonic anhydrase according to the present invention is its tolerance at high temperatures which is evaluated by measuring its residual activity.

[0042] Preferably, the carbonic anhydrase according to the present invention maintains residual concentration of at least 30%, more preferably above 60%, even more preferably above 90%, and most preferably 100% after incubation for 30 min in 20% (w / v) K2CO3 buffer at pH 11.5.

[0043] The stability in K2CO3of the carbonic anhydrase according to the present invention is its tolerance in the presence of K2CO3which is evaluated by measuring its residual concentration.

[0044] The carbonic anhydrase according to the present invention is useful in a series of applications, such as those described hereinbelow.

[0045] The carbonic anhydrase according to the present invention can be used in the extraction of CO2 from a CCh-containing medium, such as a gas, a liquid, or multiphase mixture. Preferably, the CO2 is extracted from one medium, such as a gas, to a second medium such as a liquid involving the conversion of CO2 to bicarbonate within the second medium, this is also termed absorption of CO2. The reverse extraction process where bicarbonate in the CCh-containing medium is converted to CO2 which can then be released from the first medium to a second medium, such as a gas, is also a desirable process where the carbonic anhydrase of the present invention can be applied. This process is also termed desorption of CO2. The present invention is in particular useful where the temperature of the CC>2-containing medium and / or the temperature of certain stages of the extraction process where carbonic anhydrase is present is above the temperature optimum for commercially available carbonic anhydrases, such as CA-I or CA-II isolated from human or bovine erythrocytes, which have temperature optimums at approximately 37 °C. One example of a process stage where elevated temperatures may occur is when the hot flue gas is brought into contact with the carbonic anhydrase containing liquid used to absorb the CO2 from the flue gas. Another example is the current CO2 scrubbing technologies, such as chemical absorption with carbonates (e.g., hot potassium carbonate process), alkanolamines (e.g., monoethanolamine, methyldiethanolamine, etc.) or other amines (e.g., ammonia), which use elevated temperatures (up to about 120 to 130 °C) in the desorption process.

[0046] The carbonic anhydrase according to the present invention may be used for carbon dioxide extraction from CO2 emission streams, e.g., from carbon-based or hydrocarbonbased 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 carbonic anhydrase may also be used to remove CO2 in the preparation of industrial gases such as acetylene (C2H2), carbon monoxide (CO), chlorine (CI2), hydrogen (H2), methane (CH4), nitrous oxide (N2O), propane (CsHs), sulfur dioxide (SO2), argon (Ar), nitrogen (N2), and oxygen (O2). The carbonic anhydrase can also be used to remove CO2 from raw natural gas during the processing to natural gas. Removal of CO2 from the raw natural gas will serve to enrich the methane (CH4) content in the natural gas, thereby increasing the thermal units / m3. Raw natural gas is generally obtained from oil wells, gas wells, and condensate wells. Natural gas contains between 1% (v / v) to 10% (v / v) CO2 when obtained from geological natural gas reservoirs by conventional methods, but depending on the natural source or recovery method used, may contain up to 50% (v / v) CO2 or even higher. The carbonic anhydrase can also be used to purify natural gas such that it is substantially free of CO2, e.g., such that the CO2 content is below 1 % (v / v), preferably below 0.5% (v / v), more preferably below 0.1 % (v / v), and most preferably below 0.02% (v / v). In resemblance to the methane enrichment of natural gases, the carbonic anhydrase can also be used to enrich the methane content in biogases. Biogases will always contain a considerable degree of CO2, since the bacteria used in the fermentation process produce methane (60-70% v / v) and CO2 (30-40% v / v). Biogas production may be performed using mesophilic or thermophilic microorganisms. Thermophilic strains allow the fermentation to occur at elevated temperatures, e.g., from 40 °C to 80 °C, or from 50 °C to 70 °C, or from 55 °C to 60 °C. In such processes a heat-stable carbonic anhydrase is particularly useful to remove CO2 from the methane. The carbonic anhydrase of the present invention can be used to reduce the carbon dioxide content in a biogas. Preferably the CO2 content is reduced such that it constitutes less than 25% (v / v), more preferably less than 5% (v / v), even more preferably less than 1 % (v / v), and most preferably less than 0.1 % (v / v). Furthermore, the carbonic anhydrase may be used in the production of syngas by removing the CO2 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 heatstable carbonic anhydrase is an advantage. The present invention provides for the use of a carbonic anhydrase to reduce the carbon dioxide content in syngas production. Preferably, the CO2 content is reduced such that it constitutes less than 25% (v / v), more preferably less than 10% (v / v), even more preferably less than 2% (v / v), and most preferably less than 0.1 % (v / v). The temperature stability and / or longevity of the carbonic anhydrase can be increased to some extent by formulation, e.g., by immobilization of the enzyme. In a preferred embodiment, the carbonic anhydrase of the invention maintains at least 75% activity when incubated for 6 hours in 90 °C and 50% activity when incubated for 24 hours at 80 °C. According to this preferred embodiment, the carbonic anhydrase remains soluble and active for at least 10 days of incubation in 20% (w / v) K2CO3 (at pH 11 .5).

[0047] CO2 extraction from a CCh-containing medium can, for example, can be performed in enzyme-based bioreactors. Before the carbon dioxide-containing medium is processed in a bioreactor, it may be purified to free it from contaminants which may disturb the enzymatic reaction or interfere with bioreactor functionality in other ways, e.g., by clotting outlets or membranes. Gasses / multiphase mixtures emitted from combustion processes, e.g., flue gases or exhausts, are preferably cleared of ash, particles, NOx and / or SO2, before the gas / multiphase mixture is passed into the bioreactor. The raw natural gas from different regions may have different compositions and separation requirements. Preferably, oil, condensate, water, and natural gas liquids, if present in the raw natural gas, are removed prior to the extraction of CO2 in an enzyme-based bioreactor. The CO2 emitted from combustion processes or present in the raw natural gas may be extracted in the same process as the sulfur removal, or it may be extracted in a completely separate process. If the gas at this point exceeds the temperature optimum of the carbonic anhydrase of the present invention, some degree of cooling may be needed. The temperature to which carbonic anhydrase is exposed during CO2 extraction process whether it is the process temperature in the bioreactor or the feed gas temperature may be, for example, between 0 °C and 120 °C. Preferably the process temperature is between 45 °C and 110 °C, more preferably between 50 °C and 100 °C, even more preferably between 60 °C and 80 °C, and most preferably between 65 °C and 75 °C.

[0048] Reactors and processes for gas separation, including CO2 extraction, are well known in the art and are used commercially for various purposes. There are several types of reactors which may be combined with the carbonic anhydrase of the present invention to generate a bioreactor (a reactor comprising biological material such as an enzyme) for extracting CO2 from gases, such as combustion gases or respiration gases. Because carbonic anhydrase improves the rate of CO2 extraction, combining carbonic anhydrase with the CO2 extraction reactor enables reactor and process improvements such as smaller size and less expensive absorption modules (e.g., shorter absorption column) and use of low energy consuming and low volatility carrier liquids, as well as overall lower operating temperatures compared to the conventional approaches.

[0049] CO2 extraction in a direct gas-liquid contact reactor may involve a first absorption stage followed by optionally a subsequent desorption, precipitation, utilization, collection, regeneration or release stage. A general description of the absorption stage is as follows. When the absorption reactor is in operation, a water-containing liquid enters the reactor at one end, preferably the top, and flows to the other end, preferably the bottom, and the CC>2-containing gas stream (feed gas) enters the reactor at one end, preferably at the opposite end (the bottom" ("countercurrent") from the liquid and the gas passes through the liquid and exits, minus the CO2 extracted into the liquid, through a gas outlet at the opposite end (preferably, the top of the reactor). The liquid that exits the absorption reactor is enriched in bicarbonate / carbonate (CCh-rich liquid) and the exit gas is reduced in the CO2 content compared to the feed gas. The CCh-rich liquid may be processed in subsequent reactions, for example to generate pure CO2 by passing through a desorption reactor or produce carbonate precipitates such as CaCCh. The CCh-rich liquid from the absorption reactor can also be utilized, e.g., to enhance algae growth, collected, e.g., by pumping the CCh-rich liquid into a contained geological formation, released, e.g., by pumping the CCh-rich liquid into the environment, such as release of bicarbonate liquid into seawater from a submarine life support system, evaporated or desalinated. The CO2- rich liquid containing bicarbonate anion can be used in industrial processes, such as in the manufacturing processes for ammonium carbonate and ammonium bicarbonate, which are useful as fertilizer, or in processes for the removal and neutralization of acid gases such as sulfur dioxide.

[0050] In the CO2 capture processes described above the carbonic anhydrase according to the present invention may be combined with one or more other carbonic anhydrases. The different process steps in the whole CO2 capture process may require different operating conditions, e.g., temperature, pH, carrier liquid compositions, pressure and so forth. The carbonic anhydrase of the present invention may be combined with other carbonic anhydrases operating at different optimal conditions which are needed in the CO2 capture process. For example, one carbonic anhydrase could circulate in the carrier liquid and a different carbonic anhydrase could be immobilized at one or more locations in the reactor.

[0051] The carbonic anhydrase of the present invention can also find more unconventional applications such as in pilot cockpits, submarine vessels, aquatic gear, safety and firefighting gear and astronauts' space suits and artificial lung devices to keep breathing air free of toxic CO2 levels. Other applications are to remove CO2 from confined spaces, such as to reduce hazardous CO2 levels from inside breweries and enclosed buildings carrying out fermentation, and from CO2 sensitive environments like museums and libraries, to prevent excessive CO2 from causing acid damage to books and artwork. A further alternative application is to remove CO2 from hot ambient air, e.g., in a desert. In this case the carbonic anhydrase could for example be used in a reactor suitable for extracting CO2 from ambient air, such a reactor could for example take the form of an "artificial tree" or a windmill.

[0052] The carbonic anhydrase according to the present invention” can be used as an independent CO2 extraction catalyst, or it may alternatively be combined with conventional CO2 extraction technologies such as chemical absorption via amine-based solvents or aqueous ammonia or physical solvents such as Selexol™ (Union Carbide) or polyethylene glycol ethers.

[0053] The carbonic anhydrase of the present invention can also be combined with a carbon dioxide absorbing compound such as amine-based compounds, for example, aqueous alkanolamines, including monoethanolamine (MEA), diethanolamine (DEA), methyldiethanolamine (MDEA), 2-amino-2-hydroxymethyl-1 ,3-propanediol (Tris), diglycolamine (DGA), 2-amino-2-methyl-1 -propanol (AMP), 2-amino-2-hydroxymethyl- 1 ,3-propanediol (AHPD), diisopropanol amine (DIPA), aqueous soluble salt (e.g. sodium or potassium salts) of N-methylaminopropionic acid or N,N-dimethylaminoacetic acid or N-methylalanine, N-methylglycine, beta-alanine (3-aminopropanoic acid) or other natural or modified amino acids (e.g. N-substituted amino acid derivatives), 2-(2- aminoethylamino)ethanol (AEE), triethanolamine (TEA) or another primary, secondary, tertiary or hindered amine-based solvents, or aqueous soluble salts of glycine (e.g. sodium or potassium glycinate) and taurine or other liquid absorbers such as aqueous NaOH, KOH, LiOH, carbonate salt (e.g. sodium, potassium, or ammonium) or bicarbonate salt solutions such as K2CO3 or Na2COs at different ionic strengths, molar concentrations (ranging from dilute solutions to highly concentrated solutions, up to the solubility limit of the salts, which may vary based on the temperature) or aqueous electrolyte solutions and promoters such as piperazine, or polyethylene glycol ethers, or a blend of them or analogs or blends thereof. The aqueous soluble salts and solvents may be combined with pH buffering and mineral sequestering compounds, such as phosphate salts, polyphosphate salts and borate salts, to provide mixed salt solutions, such as potassium bicarbonate with potassium phosphate. The aqueous soluble salts and solvents may be combined with simple electrolytes (e.g. alkali halides, such as NaCI, KCI, and metal halides, such as ZnCh). The combination may either be applied in the bioreactors described above or it may be applied to already existing CO2 scrubbing facilities based on conventional techniques. In conventional bioreactors, the concentration of alkanolamines is typically 15-30 weight percent. In an embodiment of the present invention the concentration of alkanolamines could be in the conventional range or preferably at a lower concentration such as below 15% (v / v), more preferably below 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.2% (v / v) and most preferably below 0.1 % (v / v).

[0054] In conventional processes, corrosion and oxidation inhibitors, such as contained in Fluor Daniel's proprietary EconAmine FG solvent, are added to provide for increasing the amine concentration while reducing the risk of corrosion. Inorganic corrosion inhibitors include vanadium (e.g., sodium metavanadate), antimony, copper, cobalt, tin, and sulfur compounds. Organic corrosion inhibitors include thiourea and salicylic acid.

[0055] Other auxiliary carrier liquid components can include wetting agents, chelating agents (e.g. ethylenediamine tetra acetic acid, polyphosphate salts), and viscosity reducers, and other compounds capable of increasing the flux of CO2 into or out of the carrier liquid.

[0056] In conventional processes, techniques to reduce and / or avoid foam formation are commonly employed. These include removal of foam-causing impurities prior to CO2 extraction and use of antifoaming agents and foam inhibitors such as silicone compounds or high-boiling alcohols such as oleyl alcohol or octyl phenoxyethanol.

[0057] Another aspect of the present invention relates to biogas production where the CO2 extraction is performed directly in the biogas fermentation broth, as an alternative to passing the biogas through a bioreactor as described above by adding the carbonic anhydrase of the present invention to the anaerobic broth

[0058] Thus, the present invention further provides the use of the carbonic anhydrase of the present invention as an additive in a biogas fermentation broth.

[0059] The present invention further provides the use of the carbonic anhydrase of the present invention to enhance growth of algae and other aquatic plants that utilize bicarbonate as a carbon source by catalyzing the conversion of CO2 to bicarbonate in or for delivery to the aquatic plant environment. This approach can, for example, be used to simultaneously remove CO2 from a combustion exhaust gas, such as a flue gas, and provide CO2 for conversion to bicarbonate by contacting the exhaust gas with liquid from a cultivation pond. Certain approaches to cultivating algae and aquatic plants involve use of enclosed tubes or shallow troughs or ponds in which heat from sunlight raises the water temperature. Hence a heat stable carbonic anhydrase is particularly useful at elevated cultivation temperatures.

[0060] Polynucleotides

[0061] The present invention also relates to isolated or synthetic polynucleotides encoding a polypeptide with carbonic anhydrase activity as defined above. In a preferred embodiment the polynucleotides of the present invention are synthetic which have been codon optimized to increase the expression in a selected host cell.

[0062] The present invention also relates to an isolated or synthetic polynucleotide encoding a polypeptide having carbonic anhydrase activity, where the polynucleotide is selected from the group consisting of: a) a polynucleotide obtained by codon optimization of SEQ ID NO: 1 where the nucleotide sequence of the codon optimized polynucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1 b) having a nucleotide sequence corresponding to SEQ ID NO: 3.; or c) a polynucleotide which is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3; or d) a fragment of (a) or (b) encoding a polypeptide having carbonic anhydrase activity.

[0063] Nucleic Acid Constructs

[0064] The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0065] A polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0066] The control sequence may be a promoter sequence, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a carbonic anhydrase of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the carbonic anhydrase. The promoter may be any polynucleotide that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0067] The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked t’ the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention. The control sequence may also be a suitable leader sequence, when transcribed is a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked t’ the 5'-terminus of the polynucleotide encoding the carbonic anhydrase. Any leader sequence that is functional in the host cell of choice may be used.

[0068] Expression Vectors

[0069] The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the carbonic anhydrase at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0070] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0071] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0072] The vector preferably contains one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for antibiotic, biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. The vector preferably contains an element(s) that permits integration of the vector into the host’s cell genome or autonomous replication of the vector in the cell independent of the genome.

[0073] For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the carbonic anhydrase or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0074] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0075] Examples of bacterial origins of replication are the origins of replication of plasmids pET28, pbr322, Puc19, pACYC177, and pACYC184 permitting replication in E. coli, and pub110, Pe194, pTA1060, and pAMB1 permitting replication in Bacillus.

[0076] Examples of origins of replication for use in a yeast host cell are the 2micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0077] Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI . Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished.

[0078] More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0079] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art.

[0080] Host Cells

[0081] The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the production of a carbonic anhydrase of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will largely depend upon the gene encoding the carbonic anhydrase and its source.

[0082] The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.

[0083] The prokaryotic host cell may be any gram-positive or gram-negative bacterium. Grampositive bacteria include, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0084] The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0085] The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells. The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0086] The introduction of DNA into an E. coli cell may, for instance, be affected by protoplast transformation or electroporation. The introduction of DNA into a Streptomyces cell may, for instance, be affected by protoplast transformation and electroporation, by conjugation, or by transduction. The introduction of DNA into a Pseudomonas cell may, for instance, be affected by electroporation or by conjugation. The introduction of DNA into a Streptococcus cell may, for instance, be affected by natural competence, by protoplast transformation, by electroporation or by conjugation. However, any method known in the art for introducing DNA into a host cell can be used.

[0087] Methods of Production

[0088] The present invention also relates to methods of producing the carbonic anhydrase of the present invention, comprising: (a) cultivating a cell, which in its wild-type form produces the carbonic anhydrase, under conditions conducive for production of the carbonic anhydrase; and (b) recovering the carbonic anhydrase.

[0089] The present invention also relates to methods of producing the carbonic anhydrase of the present invention, comprising: (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the carbonic anhydrase; and (b) recovering the carbonic anhydrase.

[0090] The host cells are cultivated in a nutrient medium suitable for production of the carbonic anhydrase using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the carbonic anhydrase to be expressed and / or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the carbonic anhydrase is secreted into the nutrient medium, the carbonic anhydrase can be recovered directly from the medium. If the carbonic anhydrase is not secreted, it can be recovered from cell lysates.

[0091] The carbonic anhydrase may be detected using methods known in the art that are specific for polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the carbonic anhydrase.

[0092] The carbonic anhydrase may be recovered using methods known in the art. For example, the carbonic anhydrase may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0093] The carbonic anhydrase may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS- PAGE, or extraction to obtain substantially pure polypeptides.

[0094] In an alternative aspect, the carbonic anhydrase of the present invention can be produced from the host cell together with other polypeptides and / or fermentation products to provide a non-purified or minimally purified mixture that may be less expensive to produce than substantially pure polypeptides while still providing the desired carbonic anhydrase performance.

[0095] In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the present invention expressing a polypeptide is used as a source of the polypeptide.

[0096] The present invention further provides a composition comprising the carbonic anhydrase of the present invention and preferably an excipient and a method for preparing such a composition comprising admixing the carbonic anhydrase of the invention with an excipient.

[0097] In a particular embodiment the carbonic anhydrase of the present invention is the major (polypeptide) component of the composition, e.g., a mono-component composition. In a mono-component composition the carbonic anhydrase of the present invention preferably constitutes at least 80% of the carbonic anhydrase activity, more preferably at least 90%, even more preferably at least 95% and most preferably 100% of the carbonic anhydrase activity. The excipient in this context is to be understood as any auxiliary agent or compound used to formulate the composition and includes solvent (e.g., water, inorganic salts, fillers, pigments, waxes), carriers, stabilizers, cross-linking agents, adhesives, preservatives, buffers and the like. The composition may further comprise one or more additional enzymes, such as one or more additional carbonic anhydrases, a decarboxylase, laccase, or oxidase.

[0098] The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a solid composition. For instance, the enzyme composition may be formulated using methods known to the art of formulating technical enzymes and / or pharmaceutical products, e.g., into coated or uncoated granules or micro-granules. The carbonic anhydrase of the invention may thus be provided in the form of a granule, preferably a non-dusting granule, a liquid, in particular a stabilized liquid, a slurry or a protected polypeptide.

[0099] For certain applications, immobilization of the carbonic anhydrase may be preferred. An immobilized enzyme comprises two essential functions, namely the non-catalytic functions that are designed to aid separation (e.g., isolation of catalysts from the application environment, reuse of the catalysts and control of the process) and the catalytic functions that are designed to convert the target compounds (or substrates) within the time and space desired. When an enzyme is immobilized, it is made insoluble to the target compounds (e.g., substrates) it aids converting and to the solvents used. An immobilized enzyme product can be separated from the application environment in order to facilitate its reuse, or to reduce the amount of enzyme needed, or to use the enzyme in a process where substrate is continuously delivered and product is continuously removed from proximity to the enzyme, which, e.g., reduces enzyme cost. Furthermore, enzymes are often stabilized by immobilization. A process involving immobilized enzymes is often continuous, which facilitates easy process control. The immobilized enzyme can be retained as a heterogeneous catalyst by mechanical means, or by inclusion in a definite space. The latter can be done by microencapsulation, e.g., in semi permeable membranes or by inclusion in UF systems using, e.g., hollow fiber modules, etc. Immobilization on porous carriers is also commonly used. This includes binding of the enzyme to the carrier, e.g., by adsorption, complex / ionic / covalent binding, or just simple absorption of soluble enzyme on the carrier and subsequent removal of solvent. Crosslinking of the enzyme can also be used as a means of immobilization. Immobilization of enzyme by inclusion into a carrier is also industrially applied. Specific methods of immobilizing enzymes such as carbonic anhydrase include, but are not limited to, spraying of the enzyme together with a liquid medium comprising a polyfunctional amine and a liquid medium comprising a cross-linking agent onto a particulate porous carrier, linking of carbonic anhydrase with a cross-linking agent (e.g., glutaraldehyde) to an ovalbumin layer which in turn adhere to an adhesive layer on a polymeric support, or coupling of carbonic anhydrase to a silica carrier or to a silane, or a CNBr activated carrier surface such as glass, co-polymerization of carbonic anhydrase with methacrylate on polymer beads or using globular protein and adhesive. The carbonic anhydrase may also be immobilized using tags such as histidine-like tags (e.g., 6x His tag or HQ tag) or a cellulose binding module (CBM).

[0100] In a further embodiment of the present invention the carbonic anhydrase is immobilized on a matrix. The matrix may for example be selected from the group beads, fabrics, fibers, hollow fibers, membranes, particulates, porous surfaces, rods, structured packing, and tubes. Specific examples of suitable matrices include alumina, bentonite, biopolymers, calcium carbonate, calcium phosphate gel, carbon, cellulose, ceramic supports, clay, collagen, glass, hydroxyapatite, ion-exchange resins, kaolin, nylon, phenolic polymers, polyaminostyrene, polyacrylamide, polypropylene, polymerhydrogels, sephadex, sepharose, silica gel, precipitated silica, and TEFLON-brand PTFE. In an embodiment of the present invention carbonic anhydrase is immobilized on a nylon matrix. The carbonic anhydrase to be included in the composition may be stabilized in accordance with methods known in the art e.g., by stabilizing the carbonic anhydrase in the composition by adding an antioxidant or reducing agent to limit oxidation of the carbonic anhydrase or it may be stabilized by adding polymers such as PVP, PVA, PEG, sugars, oligomers, polysaccharides or other suitable polymers known to be beneficial to the stability of polypeptides in solid or liquid compositions or it may be stabilized by adding stabilizing ions, such as zinc (e.g. zinc chloride or zinc sulphate) which is present in the enzyme active site. A preservative, such as Proxel™, or penicillin, can be added to extend shelf life or performance in application.

[0101] EXAMPLES

[0102] In the examples hereinbelow the name CA-KR1 is used for the carbonic anhydrase of the present invention having the sequence SEQ ID NO: 4 and the name ca-kr1 is used for the gene that encodes CA-KR1. SEQ ID NO: 4 contains the amino acids of SEQ ID NO: 2 plus an epitope having a leucine, a glutamic acid and six histidine residues.

[0103] EXAMPLE 1

[0104] Cloning, overexpression and purification of CA-KR1 carbonic anhydrase in E. coli

[0105] A synthetic gene based on the protein sequence CA-KR1 carbonic anhydrase was designed and the gene codon usage was optimized for E. coli The codon optimization process designed a gene using synonymous codons using method is known in the art.

[0106] A 6X-His tag encoding polynucleotide, was cloned in frame to the DNA encoding the codon optimized carbonic anhydrase gene. Also, the nucleotide sequences: ccatgg and ctcgag (which correspond to Ncol and Xhol restriction sites) were inserted at the start 5’ and end 3’ of the aforementioned optimized carbonic anhydrase-6HIS-tag gene. The nucleotide sequence of the fusion product corresponds to SEQ ID NO: 3.

[0107] The synthetic carbonic anhydrase gene (CA gene SEQ ID NO: 3 was Gblock synthesized and purchased. The synthetic gene was cloned in a pET28a (+) E. coli vector resulting in plasmid pET28 CA-KR1.

[0108] A plasmid MAP of pET28 CA-KR1 containing the optimized CA gene ca-kr1 (SEQ ID NO: 3) is shown in Fig 1 . The CA gene was expressed by control a T7 promoter and a terminator (term). Also, the plasmid pET28 CA-KR1 contained a gene coding for Aminoglycoside 3'-phosphotransferase (kanR) which was used as cloning selection for E. coli growth. The plasmid also contained an E. coli origin of replication.

[0109] E. coli DH5a competent cells were transformed with plasmid pET28 CA-KR1 and one clone was selected using methods known in the art. Competent E. coli Origami 2(DE3) (Novagen) cells were transformed with the plasmid isolated from the selected E. coil clone.

[0110] Kanamycin resistant E. coli clones were analyzed by DNA sequencing to verify the correct DNA sequence of the construct. The SEQ ID NO: 3 translated protein sequence corresponds to SEQ ID NO: 4, where amino acids leucine and glutamic acid at the positions 169 and 170 correspond to the Xhol restriction site, the amino acid sequence at 171-176 correspond to the 6-HIS tag and amino acids 1 to 168 correspond to the predicted carbonic anhydrase.

[0111] One expression clone was selected and was cultivated on a rotary shaking table in 2 L culture flasks each containing 500 ml Luria Broth (LB) media supplemented with 50 pg / ml kanamycin at final concentration. The clone was cultivated for approximately 2 hours at 37 °C until the optical density (OD) of the culture reached the value of 0.5. Then the culture was induced by supplementing Isopropyl p-D-1 -thiogalactopyranoside (IPTG) AT 0.2 mM and Zink Chloride (ZnCh) AT 0.5 mM final concertation. It was later determined that there was carbonic anhydrase activity in the IMAC purified protein sample as described in Example 2. To purify the heat-stable CA-KR1 from the E. coli cells, the cell pellet was initially collected via centrifugation at 6.000 x g for 10 min and was resuspended in 25 mM T ris-HCL / 100 mM NaCI / 10 mM imidazole, pH 8.3 buffer by mild manual stirring on ice. The cells were lysed with the use of a cell disruptor (Constant Systems, CF2) at 30 KPSI. The resulting lysate was subsequently fractionated by centrifugation at 47.000 x g, 4 °C for 45 min and the supernatant was loaded on an immobilized metal affinity chromatography (IMAC) purification column. The buffers used were 25 mM Tris-HCL / 100 mM NaCI / 10 mM imidazole, pH 8.3 (for column equilibration), 25 mM Tris-HCL / 100 mM NaCI / 25 mM imidazole, pH 8.3 (for washing) and 25 mM Tris-HCL / 100 mM NaCI / 250 mM imidazole, pH 8.3 (for the elution of the protein). The purity of the eluted protein was evaluated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) along with Coomassie Blue gel staining. CA-KR1 was further purified by size exclusion chromatography (SEC), using an AKTA pure 25 system (GE Healthcare Lifesciences) with a HiLoad 16 / 600 Superdex 75 pg column and 25 mM Tris-HCL, 100 mM NaCI, pH 8.3 buffer.

[0112] EXAMPLE 2

[0113] Detection of Carbonic Anhydrase Activity

[0114] After the successful purification of the CA-KR1 protein, its potential CA catalytic activity was assayed by employing a modification of the Wilbur-Anderson assay. During the CO2 hydration reaction gaseous carbon dioxide is converted to bicarbonate anion as given in equation 1 :

[0115] [CO2+ H2O HCOT + H+]

[0116] For the preparation of the reaction, fully saturated CO2 water (60-70 mM) was prepared by bubbling pure CO2 gas IN 200 mL of distilled, ice-cold H2O for at least 60 min. The reaction was initiated by the addition of 10 pL of approximately 0.6 mg / ml purified CA- KR1 in 100 pL of 100 mM Tris-HCL, 0.2 mM Phenol red pH 8.3 buffer, followed by immediate addition of 200 pL of CO2 saturated water and rapid mixing. For measuring of the control reaction, 10 pL of the same buffer, without the addition of the enzyme, were used instead. All reagents used were ice cold and multiple measurements were performed in parallel in 96-well plates, using a multi-channel pipette for simultaneous mixing of all samples due to the time-sensitivity of the reaction. As the reaction goes on more protons are produced resulting in lowering the pH of the reaction. The time required for the pH to drop from value 8.3 to 6.3 was measured through the change in color of phenol red (transition point of the dye) from red to yellow respectively for both catalyzed and uncatalyzed reactions. Typically, the reaction time for the uncatalyzed samples was in the range of 80 to 100 sec while the time required in the presence of the enzyme was in between 30 to 45 seconds. This finding supports that the CA-KR1 enzyme is indeed an enzyme carrying carbonic anhydrase activity.

[0117] In order to cross-verify the CA activity of CA-KR1 , the protein was subjected to protonography with a few modifications of the original protocol. The protein sample was mixed with native loading dye (without SDS, mercaptoethanol, or Dichlorodiphenyltrichloroethane (DTT)), loaded in duplicates on a 15% (w / v) polyacrylamide SDS gel next to a protein standard sample, and run at 180 V for 45 min. Afterwards, the gel was vertically split into two parts: the first one contained the protein standard alongside one of the two identical CA-KR1 lanes and stained with Coomassie blue stain while the other half containing the other CA-KR1 lain was subjected to Protonography to identify CA activity. CA activity was visualized in-gel by observation of the topical color change of the pH indicator bromothymol blue from blue to yellow, indicating a localized drop of pH due to the release of protons in the CO2 hydration reaction after the immersion of the gel in CCh-saturated water. The protonography result clearly indicated two CA activity zones in the gel. The low M.W band corresponds to the monomer of the enzyme at 19 kDa while the high M.W band corresponds to the dimer at 38 kDa. The fact that those bands do not totally agree with the protein standards could be due to the semi-denaturing conditions used for this assay, actively changing the total amount of negative charges in each protein conformation and subsequently interfering with the electrophoretic mobility. These results verify that CA-KR1 is indeed an active, dimeric CA.

[0118] EXAMPLE 3

[0119] Thermostability of CA-KR1 carbonic anhydrase

[0120] The thermostability of CA-KR1 was studied by measuring its residual (%) CO2 hydratase activity after prolonged incubation at different temperatures and for varying time periods. It is important to note that assays involving gaseous substrates, such as CO2, tend to produce significant variabilities in measurements, especially when sampling time points are selected by observation, a practice that introduces the bias in the perception of color. Consequently, the method selected to eliminate such deviations was stopped-flow spectrophotometry (SFA-20 Rapid Kinetics Accessory (TgK Scientific) equipped with two 2.5 mL Kloehn Drive Syringes). For each measurement, 75 pL of CA-KR1 of concentration ranging from 1-3 mg / mL were initially mixed with 3 mL of 50 mM Tris-SCL, 0.1 mM phenol red pH 8.3, and loaded in one of the reservoir syringes. The other syringe was filled with CC>2-saturated water (continues bubbling on ice for 1 hour) and the contents of the two reservoirs were mixed instantaneously at a 1 :1 volume ratio in the stopped-flow cell. The time required for the absorbance at 570 nm to drop from the maximum (pH 8.3) to the value corresponding to pH 6.3 was recorded photometrically (SPECTROstar Nano, BMG LABTECH) for both catalyzed and uncatalyzed reaction and the activity was calculated in WA units: 1 WA Unit= (T0-T) / T, were TO is the time required for the uncatalyzed reaction to finish and T is the time required for the catalyzed reaction to finish, as described previously. CA-KR1 aliquots were incubated at 80 °C and 90 °C for time periods ranging from 1-24 h and the residual activity was calculated. All measurements were performed as part of at least 3 independent experiments (starting from protein overexpression) each including a minimum of 3 technical replicates. As shown in Fig. 2, CA-KR1 has a half-life of 24 h when incubated at 80 °C and retains approximately 75% of its initial activity after 6 h of incubation at 90 °C. Interestingly, CA-KR1 also presents thermal activation at 80 °C at 2 h of incubation, thus indicating the probable near-optimum temperature of action under the specific conditions.

[0121] SspCA is a widely studied CA, which has been reported as a benchmark enzyme for industrial CO2 capture as it exhibits high thermostability in industrial application-relevant temperatures. Specifically, SspCA retains -76% and -71% of its initial activity after 3 h of incubation at 80 °C and 90 °C, respectively. In comparison, CA-KR1 exhibits 100% and 77% of residual activity when tested in the same conditions. In the case of TaCA, the oxidized enzyme oTaCA is reported to be the most thermostable form. oTaCA has been reported to maintain -65% and - 35% of its activity after 1 h of incubation at 80 °C and 90 °C respectively, while CA-KR1 exhibits 87% and 89% of residual activity under the same conditions, respectively. Furthermore, TaCA has been recently claimed as the most thermostable CA ever discovered based on the fact that it exhibits a half-life of 77 days at 60 °C although this temperature is not optimum for CO2 capture. Numerous studies have applied protein engineering strategies to optimize CAs and enhance their thermostability. In fact, the most recently reported thermostable CA is a mutant of SspCA, named K100G, acquired through rational design. K100G retains 30% of its activity after 1 h of incubation at 85 °C, while CA-KR1 shows superior thermostability as it exhibits 89% of residual activity after 1 h of incubation at the higher temperature of 90 °C.

[0122] EXAMPLE 4 of CA-KR1 in K2CO3 solvent The enzyme-assisted CO2 capture technologies which have been proposed use carbonate salts like K2CO3 at high concentrations to allow for sustainable and efficient carbon conversion rates. The high salinity and pH of the 20% (w / v) K2CO3 aqueous solution that circulates between the absorption and desorption column, challenges the stability of biocatalysts, which, as all proteins, tend to denature in strong alkaline media. The al kalistability of CA-KR1 was studied under conditions resembling an industrial CO2 process (20% w / v K2CO3, pH 11.5). For this purpose, 1.4-3.4 mg / mL of pure CA-KR1 was mixed 1 :1 with 40% (w / v) K2CO3 aqueous solution and incubated at room temperature for time periods varying from 1 to 30 d. At the end of each incubation period, the solution was centrifuged at 22,000 g, 4 °C for 15 min to remove the denatured protein. The stability of the CA-KR1 was evaluated by measuring the residual concentration of soluble enzyme after incubation. Upon measurements, all samples were also subjected to native PAGE followed by Coomassie staining and native protonography (see Example 2) to relate residual solubility to CA activity. All concentration measurements were performed as part of at least 3 independent experiments each including a minimum of 3 technical replicates and the values are reported as the mean average with standard deviation. Strikingly, the residual concentration of CA-KR1 , as well as the protonography activity tests showed that CA-KR1 retains 90% of its initial concentration even after 30 d of incubation, while remaining catalytically active. These findings, combined with the thermostability data of CA-KR1 , demonstrate the potential of the carbonic anhydrase of the present invention to perform multiple absorption-desorption cycles of CO2 in the proposed carbon capture setups at realistic conditions.

Claims

CLAIMS1. A carbonic anhydrase comprising or having an amino acid sequence which is at least 95% identical to SEQ ID NO: 2.

2. The carbonic anhydrase according to claim 1 , comprising or having an amino acid sequence which is at least 96% identical to SEQ ID NO: 2.

3. The carbonic anhydrase according to claim 1 or 2, comprising or having an amino acid sequence which is at least 97% identical to SEQ ID NO: 2.

4. The carbonic anhydrase according to any one of the preceding claims, comprising or having an amino acid sequence which is at least 98% identical to SEQ ID NO: 2.

5. The carbonic anhydrase according to any one of the preceding claims, comprising or having an amino acid sequence which is at least 99% identical to SEQ ID NO: 2.

6. The carbonic anhydrase according to any one of the preceding claims, comprising the amino acid sequence SEQ ID NO: 2.

7. The carbonic anhydrase according to any one of the preceding claims, having the amino acid sequence SEQ ID NO: 2.

8. The carbonic anhydrase according to any one of the preceding claims, wherein the carbonic anhydrase maintains residual activity of at least 60% after incubation for 30 min at a temperature of 60 °C.

9. The carbonic anhydrase according to any one of the preceding claims, wherein the carbonic anhydrase maintains residual activity of at least 90% after incubation for 30 min at a temperature of 60 °C.

10. The carbonic anhydrase according to any one of the preceding claims, wherein the carbonic anhydrase maintains residual activity of 100% after incubation for 30 min at a temperature of 60 °C.11 . The carbonic anhydrase according to any one of the preceding claims, wherein the carbonic anhydrase maintains residual concentration of at least 60% after incubation for 30 min in 20% (w / v) K2CO3 buffer at pH 11 .5.

12. The carbonic anhydrase according to any one of the preceding claims, wherein the carbonic anhydrase maintains residual concentration of at least 90% after incubation for 30 min in 20% (w / v) K2CO3 buffer at pH 11 .5.

13. The carbonic anhydrase according to any one of the preceding claims, wherein the carbonic anhydrase maintains residual concentration of 100% after incubation for 30 min in 20% (w / v) K2CO3 buffer at pH 11 .5.

14. Use of the carbonic anhydrase according to any one of the preceding claims for the extraction of carbon dioxide containing medium.

15. Use according to claim 14, wherein the carbon dioxide containing medium is a gas, a liquid, or a multiphase mixture.

16. Use according to claim 14 or 15, wherein the extraction of carbon dioxide is performed at temperatures between 45 °C and 110 °C.

17. An expression vector comprising a nucleic acid sequence encoding a carbonic anhydrase according to any one of claims 1 to 13, operably linked to a promoter.

18. A host cell genetically modified to produce recombinantly a carbonic anhydrase according to any one of claims 1 to 13, or to carry an expression vector according to claim 17.

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