WO2014090327A1 - Novel enzymes for enhanced gas absorption - Google Patents

Abstract

The present invention relates to isolated natural polypeptides having carbonic anhydrase activity at elevated temperature and enhanced absorbing/desorbing abilities in regard to acidic components e.g. C0₂ from/to a gas mixture e.g. flue gas, natural gas or biogas. The present invention relates further to use of the isolated polypeptides.

Description

Novel enzymes for enhanced gas absorption

FIELD OF THE INVENTION

The present invention relates to new isolated polypeptides having carbonic anhydrase activity at elevated temperature and enhanced absorbing/desorbing abilities in regard to acidic components e.g. C0₂ from/to a gas mixture e.g. flue gas, natural gas or biogas. The present invention relates further to use of the isolated polypeptides.

BACKGROUND OF THE INVENTION

Carbonic anhydrases (KEGG: EC 4.2.1.1) are widely spread in nature and catalyze the interconversion of carbon dioxide and water to bicarbonate and protons (or vice versa).

Carbonic anhydrase is one of the fastest enzymes known, with one molecule able to turnover a million molecules of bicarbonate per second. The enzyme will increase the kinetics in a reaction towards equilibrium but will not change equilibrium values.

Carbonic anhydrases are categorized in five distinct classes (alpha, beta, gamma, delta and epsilon) evolving from different origins, which may explain why between members of different families, no significant sequence similarity are found on the amino acid level. One common feature among most known carbonic anhydrases is, however, a zinc ion in the catalytic site, by which the enzyme binds its substrate. The pKa is then lowered and allows for nucleophilic attack on the carbon dioxide group. Co-factors other than zinc ions have been described for individual carbonic anhydrase enzymes. However, zinc ions are most preferred among the known carbonic anhydrase enzymes.

Carbon dioxide capture is an important step in both energy production and

environmental preservation and is of importance to control carbon dioxide emission to the atmosphere. Both chemical and enzymatic absorptions have been described.

The main problems with the existing technology are a low overall efficiency, a slow absorption rate of the gas (C0₂) and that most catalysts used up to date to increase absorption kinetics are toxic. Further, the most effective absorbents, such as amines, could harm the environment. Amines will decompose over time and generate a waste problem. Carbonates, for instance K₂C0₃ and /or Na₂C0₃, are non toxic and could be used as absorbents instead of amines, but have much slower absorption rate than amines. NH₃ could, however, be used instead of amines.

The existing technology has also low energy efficiency, i.e. it consumes large amounts of energy, for heating, cooling and operating pumps, compressors, blowers etc. There is therefore a need for non-toxic effective absorbents/desorbents and/or catalysts. One category of catalysts that has been suggested is enzymes. Enzymes are widely distributed in nature, and some are active in catalyzing C0₂ absorption/desorption during respiration. Enzymes may therefore prove to be both effective and non toxic catalysts. If enzymes are to be used as catalysts with the existing technology, flue gas from power plants has to be cooled down in a heat exchanger before it can be treated with enzymes.

The higher the operating temperature of the enzymatic process, the smaller the heat exchanger can be. The use of thermostable enzymes will reduce the need to cool the flue gas and temperatures above 60°C will reduce the risk of microbial growth in the reactor(s).

The carbonic anhydrase^'s capability to capture C0₂ to control C0₂ emission by capturing C0₂ gas before emission to the environment has been suggested and investigated for some years and has been described in:

WO 2010/014774 describes the extraction of C0₂ gas from a gas flow as catalyzed by the use of enzymes (carbonic anhydrase). Enzymes are also used in the desorption step. The reactor may contain two or more different enzymes.

WO 2008/095057 and WO 2010/151787 refer to heat-stable carbonic anhydrases and their use in C0₂ extraction.

US 2009/0155889 describes a system and a method for absorbing C0₂ from a gas flow, where absorbent solution includes amines and the catalyst includes one or more enzymes.

US 2010/0086983 refers to a procedure to remove carbon dioxide from a gas flow using immobilized enzymes.

WO 2009/000025 shows a method to absorb C0₂ from a gas flow, whereby the absorption is catalyzed by the use of enzymes on a solid carrier.

US 6,143,556 refers to the use of enzymes to isolate specific gases from a gas flow. For this purpose, it is described using a bioreactor containing beads coated with enzymes. One or more different enzymes may be used and the carrier material may also include various types of material.

US 2008/0003662 refers to a method for separating carbon dioxide from a gas flow through the use of an enzyme (carbonic anhydrase). WO 98/55210 discloses an apparatus and process for extraction of carbon dioxide from a gas flow. The bioreactor contains an immobilized enzyme (carbonic anhydrase) that catalyses the process.

US 3,896,212 refers to the absorption of C0₂ in a gas flow by using different concentrations of catalyst, and that the amount is varied from the inlet to the outlet of absorbent, but does not show the use of enzymes.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide isolated polypeptides having a more efficient carbonic anhydrase activity at elevated temperatures, which may at least partly overcome the above mentioned problems. This and other objects will be apparent from the following description, and is achieved by the isolated polypeptides of the present invention, having carbonic anhydrase activity and the use of said polypeptides, according to the appended independent claims. Embodiments are set forth in the dependent claims.

According to a first aspect, the present invention relates to an isolated polypeptide having carbonic anhydrase activity as defined in any one of a) through e) or any combinations thereof: a) a polypeptide having an amino acid sequence corresponding to amino acid

residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22 , SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or

a polypeptide which is at least 60% identical to amino acid residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or a fragment of a) to b) having carbonic anhydrase activity, or a polypeptide encoded by nucleic acid sequence which hybridizes under medium stringency conditions with a polynucleotide sequence encoding a polypeptide of: i) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or ii) polynucleotide sequence of: SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23 SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31; iii) or a complementary strand of i) or ii); or iv) a subsequence of i); or e) a polypeptide encoded by the nucleic acid sequence which due to the degeneracy of the genetic code does not hybridize with the polynucleotide sequence of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 or SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31; but which codes for a polypeptide having an amino acid sequence according to a) or b).

A further aspect of the invention relates to an isolated polypeptide or a mixture of polypeptides comprising at least one of the polypeptides according to the first aspect or embodiments thereof of the present invention.

Another aspect of the present invention relates to a composition comprising the isolated polypeptide or the mixture of polypeptides and an immobilizing agent.

Yet another aspect of the present invention relates to an isolated polynucleotide having the nucleotide sequence encoding the isolated polypeptide of the first aspect of the invention.

A further aspect relates to a nucleic acid construct comprising an operable linked control sequence directing the expression of the polynucleotide.

Yet a further aspect relates to a vector comprising the polynucleotide or the nucleic acid construct.

Another aspect relates to a host cell comprising the polynucleotide, the nucleic acid construct or the vector.

Yet another aspect relates to use of the isolated polypeptide or the composition for absorbing/desorbing an acidic component from/to an absorbing medium. Further aspects relates to use of the isolated polynucleotide, use of the nucleic acid construct, use of the vector and use of the host cell.

Preferred embodiments of the present invention is set forth in the dependent claims and in the detailed description of the invention

DESCRIPTION OF THE FIGURES

Figure 1 Illustrates gene synthesis and cloning of

SCA01/SCA02/SCA03/SCA04/SCA06b/SCA09/SCA10/SCAl l

carbonic anhydrase encoding genes into pET16b based expression vectors for recombinant production in E. coli. xx in pSINxx-I and pSINxx-II indicates the two-digit number of the respective SCAxx encoding gene, i.e. SCA04/SCA06b/SCA09/SCAl l .

Figure 2 Illustrates SDS-PAGE analysis of recombinant production of

SCA04/SCA06b/SCA09/SCAl l in E. coli. pET16b, negative control derived from a culture containing the empty expression plasmid pET16b; SI, crude extract; S2, heat- treated extract (20 min at 65 °C). Arrows indicate the expected target size of protein SCA04/SCA06b/SCA09/SCAl 1.

Figure 3 Illustrates carbonic anhydrase activity measurements using crude extracts from recombinant production of SCA04/SCA06b/SCA09/SCAl 1 in E. coli. P buffer, phosphate buffer A (reference); pET16b, negative control derived from a culture containing the empty expression plasmid pET16b. Dilutions 1 : 10 and 1 :20 in buffer A prior to measurement are given behind the protein name where applicable.

Figure 4 Illustrates carbonic anhydrase activity measurements using crude extracts from recombinant production of SCA04/SCA06b/SCA09/SCAl 1 in E. coli after incubation at 23 °C (RT), 65 °C or 80 °C for 1 h or 5 h, as indicated. Blue bars (left) represent measurements diluted with ion free water containing 1 μΜ ZnS0₄, red bars (right) represent measurements diluted to 20 % (w/v) K₂CO₃, 1 μΜ ZnS0₄ final concentration.

Figure 5A Illustrates specific activity of SCA04 as a function of substrate concentration. Data series 1 and 2 are shown in open squares and open diamonds respectively. All data were included in the calculations. Lines are calculated from the K_m and V_max found from non-linear fitting of the Michaelis-Menten equation.

Figure 5B. Illustrates specific activity of SCA09 as a function of substrate concentration. Data series 1 and 2 are shown in open squares and open diamonds, respectively. All data were included in the calculations. Lines are calculated from the K_m and V_max found from non-linear fitting of the Michaelis-Menten equation.

Figure 5C. Illustrates specific activity of SCA11 as a function of substrate concentration. Data series 1 and 2 are shown in open squares and open diamonds, respectively. All data were included in the calculations. Lines are calculated from the K_m and V_max found from non-linear fitting of the Michaelis-Menten equation.

Figure 6 Illustrates the relative C0₂ absorption rate as a function of C0₂ loading at different enzyme concentrations;

Figure 7 Illustrates the impact of the absorbent concentration on the reaction kinetics. Figure 8 Illustrates temperature stability of SCA04 at 80 °C

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to isolate and identify novel heat stable carbonic anhydrases having the ability to more efficiently increase the reaction rate, i.e. the absorption/desorption of an acidic component from/to a gas mixture e.g flue gas, natural gas or biogas. The acidic component might be, but is not limited to C0₂. Also other areas of use as improved oil recovery, biomass production, C0₂ storage or artificial lung support are suggested.

The isolated carbonic anhydrases according to the present invention have been isolated from microorganisms having high temperature oil and gas reservoirs as their natural habitat, also isolates from other habitats are however, possible. The isolated carbonic anhydrases are able to perform their catalytic activity under elevated temperatures resulting in a reaction process with high energy efficiency. Using the enzymes of the present invention in an absorber /desorber system may increase the efficiency at least 10 times with regard to the kinetic rate than existing technology, preferentially even higher. Making the technical process more efficient will have a significant impact on reducing the operational costs. Beneficial environmental advantages will also be achieved, in that the use of toxic absorbents/desorbents and catalysts may be avoided.

It is suggested to use at least one carbonic anhydrase enzyme to increase absorption/ desorption in an absorption/desorption unit to make the absorption/ desorption system work more effectively. Utilizing the carbonic anhydrase enzyme(s) according to the present invention, the C0₂ capture will be improved. Both flue gas capture systems (atmospheric pressure) and C0₂ capture from natural gas at elevated or high pressure will benefit from the present invention. In existing technology, gas from power plants has to be cooled down in a heat exchanger before it can be treated with enzymes. The higher the operating temperature of the enzymatic process, the smaller the heat exchanger can be. The use of

thermostable enzymes will reduce the need to cool the flue gas and temperatures above 60°C will reduce the risk of microbial growth in the reactor(s).

The catalyzed reaction depends on the C0₂ concentration in the gas. In a C0₂- absorption process the C0₂ concentration in the gas will decrease from the inlet to the outlet of the absorber. This will have an impact on how efficient the enzyme, or the mixture of enzymes, should be to catalyze the process. In a capture system where the concentration varies considerably in the gas phase from inlet to outlet, it will be beneficial to operate an absorber/desorber system where the enzyme or mixture of enzymes are optimized for each section.

The inventors surprisingly detected that using an enzyme or a mixture of enzymes with different K_m values at different sections in the absorber/desorber system, made the absorption/desorption process more efficient. At the inflow of an absorber system, the C0₂ concentration will be high, an enzyme or a mixture of enzymes with high K_m value(s) is more efficient at high C0₂ content. At the outlet part, with lower C0₂ concentration, an enzyme or a mixture of enzymes with low K_m value(s) should be the catalyst, as it is more efficient at low C0₂ content.

A further optimization of the reaction may be reached by using an immobilizing agent.

The enzyme for carbonic anhydrase based C0₂ capture should have a long life-time in the process, be very efficient, and have a suitable K_m value for C0₂ and a suitable K_m value for HCO₃ ^" which is different from the K_m value for C0₂.

The inventors identified the carbonic anhydrase enzymes with the above characterstics based on the protein sequence of the carbonic anhydrase from a putative micororganism selected from the group or any combination thereof: Methanocaldococcus

sp./Psychromonas sp./Desulfovibrio sp./Sulfurospirillum sp./Methanococcus

sp./Pelobacter sp./Thermococcus sp. /Sulfur ovum sp.. A synthetic gene was designed, and codon optimized for recombinant expression in E. coli. The gene was further cloned by standard methods into a pUC vector and the desired coding sequence was confirmed by sequencing. Subsequently the gene was excised and cloned into an expression vector. Heat shock competent cells of E. coli were transformed with the carbonic anhydrase gene, and an expression clone was picked and cultivated. The bacterial cells were disrupted and insoluble cell debris was pelleted by centrifugation, resulting in a crude extract containing the soluble protein. Further purification was performed, and finally a carbonic anhydrase enriched supernatant was achieved. Methods describing the cloning of the carbonic anhydrase gene and recombinant production of carbonic anhydrase are further described in the example section.

The isolated carbonic anhydrases according to the present invention and their amino acid sequences are identified as follows: SCAOl (SEQ ID NO:26), SCA02 (SEQ ID NO:28), SCA03 (SEQ ID NO:30) SCA04 (SEQ ID NO:2), SCA05 (SEQ ID NO:6), SCA06b, (SEQ ID NO: 10) SCA07 (SEQ ID NO: 14), SCA09 (SEQ ID NO: 18), SCA10 (SEQ ID NO:32) and SCAl l (SEQ ID NO:22), and were identified in an oil reservoir metagenome derived DNA sequence database assembled from read sequences obtained by 454 pyrosequencing of the metagenomic DNA. Sequences were found similar to sequences from: Methanocaldococcus FS406-22, Thermococcus AM4, Sulfurovum NB C3 '-\,Methanocaldococcus fervens AG86, Psychromonas ingrahamii 37, Desulfovibrio sp, ND132, Sulfur o spirillum deleyianum DSM 6946, Methanococcus aeolicus Nankai- 3, Methanococcus aeolicus Nankai-3 and Pelobacter carbinolicus DSM 2380, respectively.

Said carbonic anhydrases were codon optimized for expression in E.coli and have the following sequence identity: SCA04 (SEQ ID NO:4), SCA05 (SEQ ID NO:8), SCA06b (SEQ ID NO: 12), SCA07 (SEQ ID NO: 16), SCA09 (SEQ ID NO:20) and SCAl l (SEQ ID NO:24)

The DNA sequences encoding the isolated carbonic anhydrases and the codon optimized anhydrases have the following identity: SCA04 (SEQ ID NOs: l and 3), SCA05 (SEQ ID NOs:5 and 7), SCA06b (SEQ ID NOs:9 and 11), SCA07 (SEQ ID NOs: 13 and 15), SCA09 (SEQ ID NOs: 17 and 19), SCAl l (SEQ ID NOs:21 and 23) respectively. Further DNA sequences encoding the isolated carbonic anhydrases have the following identity: SCAOl (SEQ ID NO:25), SCA02 (SEQ ID NO:27), SCA03 (SEQ ID NO:29) and SCA10 (SEQ ID NO:31).

Accordingly, in a first aspect the present invention relates to an isolated polypeptide having carbonic anhydrase activity as defined in any one of a) through e) or any combinations thereof: a) a polypeptide having an amino acid sequence corresponding to amino

residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or

a polypeptide which is at least 60% identical to amino acid residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID N0: 16, SEQ ID N0: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or c) a fragment of a) or b) having carbonic anhydrase activity; or d) a polypeptide encoded by nucleic acid sequence which hybridizes under medium stringency conditions with a polynucleotide sequence encoding a polypeptide of: i) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or

or; ii) polynucleotide sequence of: SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31; iii) or a complementary strand of i) or ii); or iv) a subsequence of i); or e) a polypeptide encoded by the nucleic acid sequence which due to the

degeneracy of the genetic code does not hybridize with the polynucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:3; but which codes for a polypeptide having an amino acid sequence according to a) or b).

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least65%, identical to the amino acid residues selected from the group or any

combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32. In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 70%, identical to the amino acid residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 75%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 80%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 85%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 90%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 95%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32. In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 96%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID

NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 97%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 98%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16,

SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In one embodiment of this aspect the polypeptide as defined in b) is preferably at least 99%, identical to the amino acid residues selected from the group or any combinations thereof of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.

In a further embodiment the hybridization conditions in d) may be high stringency.

The present isolated polypeptides may further comprise putative metal ion binding sites comprising the following sites:

CI 6, D18, H71, C74 of SEQ ID NOs:2 and 4;

C38, D40, H91, C94 of SEQ ID NOs:6 and 8;

C42, D44, H103, C106 of SEQ ID NO: 10 and C43, D45, H104, C107 of SEQ ID NO: 12;

C42, D44, H103, C106 of SEQ ID NO: 14 and C43, D45, H104, C107 of SEQ ID NO: 16;

H71, H88, H93 of SEQ ID NO: 18 and H72, H89, H94 of SEQ ID NO:20;

H72, HI 07 and HI 12 of SEQ ID NOs:22 and 24:

H72, H89, H94 of SEQ ID NO:26;

H65, H82, H87 of SEQ ID NO:28; H64, H89, H94 of SEQ ID NO:30;

H139, H156, H161 of SEQ ID NO:32.

The binding sites are believed to bind Zn²⁺ but also other active binding sites involving e.g. Cd ions and Fe ions are possible.

In a preferred embodiment the isolated polypeptide may further comprise a high K_m value or a low K_m value, for instance may the low K_m value be chosen from the following range: from about 1 to about 25mM and the high K_m value may be chosen from the following range: from about 25 to about 60mM.

In one or more embodiments the carbonic anhydrase activity may be maintained at a temperature above 65°C for at least one hour. The activity may also be maintained for at least five hours in the temperature range of about 65°C to about 100°C, showing that the enzyme may perform its activity at high temperatures, i.e. temperatures of: above 40°C, preferably above 50°C, more preferably above 55°C, more preferably above 60°C, even more preferably above 65°C most preferably above 70°C, most preferably above 80°C, most preferably above 85°C, most preferably above 90°C and even most preferably above 100°C.

In one embodiment the carbonic anhydrase activity is maintained at a temperature of at least 80°C for at least two hours at a level corresponding to at least 90% of the initial activity.

Further the carbonic anhydrase activity may be maintained at a temperature of above 65° C for at least 5 hours at a K₂CO₃ concentration of 20% (w/v) even 80°C may be tolerated.

The K₂CO₃ concentration of 20% (w/v) may have a stabilizing effect on the enzyme.

Another aspect of the invention relates to an isolated polypeptide or a mixture of isolated polypeptides comprising at least one of the isolated polypeptide identified above. Also its use in absorbing/desorbing an acidic component from/to an absorbing medium, wherein the acidic component may be C0_2, is an aspect of the present invention.

According to another aspect of the present invention, a composition is provided comprising an immobilizing agent and the isolated polypeptide(s) as defined above. In one more embodiments of this aspect the immobilizing agent may comprise a matrix, surface or substrates as for instance beads, fabrics, fibers, porous materials, CLEAs, structured or random packing, crystals such as monoliths or any combinations thereof. The isolated polypeptides may be in an aqueous phase or immobilized on particles. More specifically, the enzyme(s) may (a) be but is not limited to dissolve in the absorbing liquid and flowing through the appropriate section of the absorber, (b) it may be immobilized on the respective section of the absorber, or (c) may be immobilized on particles floating inside the absorbing liquid.

Further, the composition may be used for absorbing/desorbing an acidic component from/to a gas mixture e.g. flue gas, natural gas or biogas, wherein the acidic component may be C0₂.

Yet another aspect of the present invention relates to an isolated polynucleotide having the nucleotide sequence encoding the polypeptide of the present invention.

Further a nucleic acid construct may be operable linked to a control sequence directing the expression of the polynucleotide.

According to another aspect of the present invention, a vector is provided, comprising the polynucleotide or the nucleic acid construct.

In another aspect of the present invention, a host cell comprising the said isolated polynucleotide is provided, the nucleic acid construct or the vector comprising the polynucleotide.

A further aspect of the present invention relates to the use of an isolated polypeptide according to the present invention or a composition as described above for

absorbing/desorbing an acidic component from/to a gas mixture, wherein the the acidic component may be C0₂

Yet another aspect of the present invention relates to the use of an isolated

polynucleotide having a nucleotide sequence encoding the polypeptide of the present invention. Further, a nucleic acid construct may be operable linked to a control sequence directing the expression of the polynucleotide.

According to another aspect of the present invention relates to the use of a vector comprising the polynucleotide or the nucleic acid construct.

Another aspect of the present invention relates to the use of a host cell comprising the said isolated polynucleic acid, the nucleic acid construct or said vector. DEFINITIONS

The term "isolated polypeptide" as used herein refers to a 20-100% pure polypeptide determined by SDS-PAGE.

The term "carbonic anhydrase activity" as used herein is defined as an activity which catalyzes the conversion between carbon dioxide and bicarbonate.

The term "heat stable" or "thermostable" is used herein to describe an enzyme that maintains activity over an elongated period of time at elevated temperatures. The thermostability of the enzyme can be increased or enhanced in some way by immobilization, chemical modification (e.g. cross-linking) or use of stabilizing chemicals.

The term "operably linked" as used herein is defined as a configuration wherein a control sequence is placed in a position relative to the coding sequence of a

polynucleotide sequence and is thereby able to control the expression of the coding sequence.

The term "sequence identity" as used herein refers to a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. The two sequences to be compared must be aligned to the best possible fit with the insertion of gaps or alternatively, truncation at the ends of the protein sequences. Sequence identity has been calculated by the BLASTP program (Altschul et al, 1990).

The term "gas mixture" as used herein refers to the C0₂ containing gas stream. Such a gas stream can be and is not limited to raw natural gas from oil or gas wells, syngas from the gasification of a carbon containing fuel, emission stream from combustion processes, flue gas from e.g. electric generation power plants, catalytic crackers, boilers etc, or biogas.

The terms "absorbent", "absorbing liquid", "solvent" are used in the present invention to describe a reacting liquid compound that has the ability to absorb C0₂. It may comprise carbonates, and/or primary, and/or secondary and/or tertiary amines and/or blends thereof, and/or alkanolamines, and/or amino acid salts.

The term "K_m" is defined in the present invention as the Michaelis-Menten constant. Michaelis-Menten kinetics is a model of enzyme kinetics in the form of an equation describing the rate of enzymatic reactions by relating the reaction rate to the concentration of a substrate. The Michaelis-Menten constant, K_m, is the substrate concentration at which the reaction rate is half of the maximum rate achieved by the system, at maximum (saturating) substrate concentrations.

The Michaelis-Menten equation, the reaction rate equation for a one-substrate enzyme catalyzed reaction is:

_ Vmax [S]

° K_m + [S] wherein

Vo is the initial reaction rate,

V_max is the maximum reaction rate,

[S] is the substrate concentration

K_m is the Michaelis-Menten constant.

The terms "absorbent", "absorbing liquid", "solvent" are used in the present invention to describe a reacting liquid compound that has the ability to absorb C0₂.

The term "catalyst" is defined herein as any chemical entity that catalyses the hydration of carbon dioxide to bicarbonate. For the purposes of the present invention, it is closely related but not limited to the carbonic anhydrase family of enzymes.

The term "immobilizing agent" as used herein refers to an agent having the ability to stabilize enzyme(s) on e.g. on a matrix, surface or substrate. It can be at least partially composed of e.g. beads, fabrics, fibers, porous materials, structured or random packing, crystals or combinations thereof.

Having now fully described the present invention in some detail by way of illustration and example for purpose of clarity of understanding, it will be obvious to one skilled in the art that same can be performed by modifying or changing the invention by a wide and equivalent range of conditions, formulations and other parameters thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. EXAMPLES

Example 1

Cloning of a carbonic anhydrase gene

(SCA04/SCA05/SCA06b/SCA07/SCA09/SCAll, respectively) from a putative Methanocaldococcus

s )JMethanococcus s )JPelobacter sp. respectively in E. coli

Based on the predicted protein sequence of the carbonic anhydrase

SCA04/SCA05/SCA06b/SCA07/SCA09/SCAl l respectively, from a putative

Methanocaldococcus sp.Psychromonas sp./Desulfovibrio sp./Sulfurospirillum sp./Methanococcus sp./Pelobacter sp., respectively, a synthetic gene including the coding sequence for a Factor Xa protease cleavable 8x His-tag was designed, and the gene was codon optimized for recombinant expression in E. coli. For alternative cloning with and without the His-tag coding region, a Pcil restriction site was introduced immediately upstream of the CA coding region. The synthetic gene to which protective bases and appropriate restriction sites (5'-Xbal, 3'-BamRT) were added was synthesized by GenScript (Piscataway, NJ, USA) and cloned blunt-ended into EcoRV digested plasmid pUC57. Correct cloning was confirmed by restriction digest analysis, and the desired coding sequence was confirmed by sequencing (GenScript). Subsequently, the gene was excised from the pUC57 derivative and simultaneously cloned in two alternative ways into expression vector pET16b (Novagen). For recombinant expression including the His-tag coding region, the pUC57 resident carbonic anhydrase gene was excised from the pUC57 derivative using Ncol and BamHl and cloned into the similarly digested pET16b expression vector to give plasmid pSIN04-I/pSIN05-I/pSIN06b- I/pSIN07-I/pSIN09-I/pSINl l-I, respectively . Alternatively, for recombinant expression without a His-tag coding region, the carbonic anhydrase gene was excised from the pUC57 derivative using Pcil and BamHl and cloned into the compatible ends of an Ncol and BamHl digested pET16b vector fragment to give plasmid pSIN04-II/pSIN05- II/pSIN06b-II/pSIN07-II/pSIN09-II/pSINl l-II, respectively.

Correct cloning was confirmed by restriction analysis, and the desired coding sequence was confirmed by sequencing (GenScript). A schematic representation of the cloning experiments is given in Figure 1. Example 2

Recombinant production of carbonic anhydrase SCA04/SCA06b/SCA09/SCAll, respectively, from a putative Methanocaldococcus

s )JMethanococcus s )JPelobacter sp., respectively, in E. coli

Heat-shock competent cells of E. coli BL21(DE3) were transformed with the carbonic anhydrase gene carrying pET16b derivative pSIN04-II/pSIN06b-II/pSIN09-II/pSINl 1- II, respectively. One single clone was selected to inoculate 5 ml LB Amp¹⁰⁰ liquid medium. The culture was incubated at 37 °C and 200 rpm overnight on a shaking incubator. 3 ml of this pre-culture was then used to inoculate 1000 ml LB medium supplemented with 100 μg/ml ampicillin in a baffled 3 1 shake flask. The culture was incubated at 37 °C and 150 rpm while following culture growth by OD₆oo measurement. At an OD₆oo of approximately 1.0, gene expression from the T7 promoter was induced by addition of 0.5 mM final concentration inducer isopropyl β-D-l- thiogalactopyranoside (IPTG) from a sterile stock solution. Simultaneously, also 0.5 mM final concentration zinc sulphate was added from a sterile stock solution. The gene expression induced culture was incubated for another 5 h at 16 °C and 150 rpm. Subsequently, cells were harvested by centrifugation (9000 xg, 4 °C, 15 min). The wet weight of the cell paste was determined. The pellet was re-suspended in a small volume of supernatant, transferred to 50 ml tubes and centrifuged again (6800 xg, 4 °C, 15 min). The supernatant was discarded, and the cell paste was stored at -80 °C prior to extraction. For extraction, the biomass was thawed on ice, and cells were re-suspended in 25 ml buffer A (50 mM potassium phosphate, pH 6.8, 1 μΜ zinc sulphate) for each 5 g of wet weight. Cells were disrupted while held on ice by 5x1 min sonication using a Branson Sonifier (duty cycle 50 %, output control 3) with intermediate mixing. The sonicated cell suspension was centrifuged (6800 xg, 4 °C, 15 min), and the supernatant was transferred to a fresh tube. An aliquot of this crude extract was incubated for 20 min at 65 °C on a water bath. The heated extract was centrifuged (6800^xg, 4 °C, 20 min), and the supernatant (heat treated crude extract) was transferred to a fresh tube. The resulting target enzyme enriched supernatant and the original crude extract were analysed for the presence of the target enzyme monomer using SDS-PAGE under reducing conditions. A similarly produced extract of E. coli carrying the empty expression vector pET16b was used as a negative control. The result of SDS-PAGE analysis is given in Figure 2.

SDS-PAGE revealed a strong band of the expected molecular size in the crude extract for all of the four proteins SCA04/SCA06b/SCA09/SCAl 1 which was absent in the negative control (pET16b), indicating an efficient recombinant production of all four proteins (Figure 2). By heating for 20 min at 65 °C, a strong enrichment of target protein relative to the protein background was observed for SCA04/SCA09/SCA11, an indication for thermostability of these proteins at 65 °C. SCA06b had earlier been shown to be quantitatively denatured by incubation at 65 °C. Example 3

Determination of carbonic anhydrase activity at room temperature (~23 °C) of recombinantly produced SCA04/SCA06b/SCA09/SCAll

The following procedure was used to determine carbonic anhydrase activity, representing a modified version of the carbonic anhydrase activity assay as described by Wilbur, 1948, J. Biol. Chem. 176, 147-154: On a magnet stirrer at room temperature, a 50 ml plastic beaker (reaction vessel) containing a small stirrer magnet was filled with 12 ml 20 mM Tris-S0₄ buffer pH 8,3, 1 μΜ ZnS0₄. A calibrated pH electrode connected to a PHM210 pH meter (Radiometer Analytical) was installed in the buffer solution to allow for both continuous pH measurement as well as thorough mixing of the vessel's content at 750 rpm. 100 μΐ culture crude extract prepared in buffer A

(50 mM potassium phosphate, pH 6.8, 1 μΜ zinc sulfate) prepared according to

Example 2 was added to the Tris buffer while mixing. After pH logging was started (WaveScan 2.0 controlling an Advantec USB-4711 A multifuntion module connected to the pH-meter's recorder output; channel range: +/-1.25mV), 9 ml C0₂ saturated ion- free water was added to the reaction vessel. C0₂ saturated water was prepared by bubbling C0₂ gas from dry ice in an isolation bottle through 0.5 L ion free water while stirring overnight. The decrease in pH was recorded at a resolution of 50 ms until a constant pH was obtained (25-80 s). The time needed for the pH to drop from pH 7.8 to pH 7.0 (dt) was determined and used as a measure for carbonic anhydrase enzymatic activity. Three independent measurements were regularly performed in order to define the standard deviation between individual measurements, regularly leading to a variation of less than 5 %. 100 μΐ pure buffer A or 100 μΐ of a reference crude extract (prepared from a similarly treated culture containing the empty expression vector pET16b) was used as a negative control. A spontaneous pH change from pH 7.8 to pH 7.0 in the absence of enzymatic activity was regularly obtained within 18-35 s. The result is given in Figure 3 A. Activity units were calculated using the formula U=(dt_PH7.8-_PH7-o, buffer A-dt_pH7.8-_PH7-o, extract)/dt_PH7.8-pH7-o, extract * factor of dilution with buffer A and correlated to 1 ml sample volume (Figure 3B).

All four proteins SCA04/SCA06b/SCA09/SCAl l could be shown to exhibit carbonic anhydrase activity (Figure 3B). Measuring at room temperature (23 °C), SCAl 1 showed the highest activity, followed by SCA04. SCA06b and SCA09 activity could be detected, though at very low levels. No activity was observed in the negative control extract (Figure 3B).

Example 4

Assessment of thermostability and high salt tolerance of recombinantly produced SCA04/SCA06b/SCA09/SCAll, respectively, in crude extract

In order to evaluate the stability of recombinantly produced

SCA04/SCA06b/SCA09/SCAl 1, respectively, at 65 °C and/or 80 °C and/or at high salt concentration [20 % (w/v) K₂C0₃], 175 μΙ_, of crude extract prepared as described in Example 2 were diluted with 325 μΐ. 30.8 % (w/v) K₂C0₃, 1 μΜ ZnS0₄, pH8.3 to give a final concentration of 20 % (w/v) K₂C0₃ in the diluted extract. In parallel, 175 crude extract were diluted with 325 μΐ_^ ion free water containing 1 μΜ ZnS0₄.

Dilutions were incubated at room temperature (~23 °C), 65 °C or 80 °C for 1 h or 5 h. After incubation, samples were centrifuged in a microliter centrifuge (14,000 rpm, 4 °C, 5 min), and 285 μΐ, of the cleared supernatant, corresponding to 100 μΐ of undiluted crude extract, was used for activity measurement as described in Example 3. For the ion free water/ 1 μΜ ZnS0₄ diluted samples, the time for the pH to drop from pH 7.8 to pH 7.0 (dt_PH7.8-pH7.o) was determined, while for the respective high salt samples, due to a higher initial pH, the drop time from pH 8.2 to pH 7.4 (dt_pH8.2-_PH7.4) was determined, both corresponding to an almost linear decrease of pH over time. Dilutions (1 : 10, 1 : 100 or 1 :200) were applied where necessary in order to obtain dt values between 5 and 20 seconds. Activity units were calculated using the formula U=(dt_PH8.2-_PH7.4, ref dt_pH8.₂-pH7.4, extract )/dt_pH8.2-pH7.4, extract * factor of dilution with 20 % (w/v) K₂C0₃, 1 μΜ ZnS0₄, pH8.3 for the samples containing 20 % (w/v) K₂C0₃ and U=(dt_PH7.8-_PH7.o, ref dt_pH7.8-pH7.o, extract)/dt_PH7.8-pH7.o, extract * factor of dilution with ion free water containing 1 μΜ ZnS0₄ for the samples diluted with ion free water containing 1 μΜ ZnS0₄. The reference measurements for the two different conditions (ref) were obtained from measuring similarly diluted crude extract from a culture containing the empty expression vector pET16b incubated for 1 h at 65 °C. The calculated activity units were correlated to 1 ml extract volume. The results are presented in Figure 4. The four enzymes

SCA04/SCA06b/SCA09/SCAl 1 exhibited very different characteristics with respect to stability at high temperature and/or high salt concentration (Figure 4). SCA04 was found to be very stable under all condition tested. Even after incubation for 5 h in 20 % (w/v) K₂C0₃, more than 50 % of the original activity was retained. SCA11 was found to be relatively stable when incubated at 65 °C, though a clear decrease of activity over time was observed at this temperature. The combination of high temperature (65 °C) and 20 % (w/v) K₂C0₃ was not tolerated, leading to a rapid loss of functionality. It had been shown before that SCA11 was in general quickly degraded at 80 °C (data not shown). SCA06b was found to be stable at room temperature, but quantitatively degraded already after 1 h incubation at 65 °C. Interestingly, higher carbonic anhydrase activity was observed for SCA06b in the presence of 20 % (w/v) K₂C0₃. The high salt concentrations obviously had a stabilizing effect on the enzyme. This effect was also observed when SCA06b was incubated at 65 °C. SCA09 showed a relatively high stability when incubated at 65 °C or 80 °C, though some loss of activity was observed especially after 5 h of incubation. The additional presence of 20 % (w/v) K₂C0₃ had no additional destabilizing effect at 65 °C, while after 5 h incubation at 80 °C, most of the activity was lost. Example 5

Determination of K_m values of SCA04, SCA09 and SCA11

(i) Recombinant production in E. coli and preparation of heat enriched crude extracts containing SCA04, SCA09 and SC All

For the recombinant production of the enzymes SCA04, SCA09 and SCA11 in E. coli, high cell density (HCD) fed-batch fermentation experiments in bench-top scale fermentors were performed. E. coli strains generated based on strain BL21(DE3) and carrying the respective CA encoding gene on a pET16b derived plasmid were pre- cultivated in 100 ml LB(g) medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 10 g/L glucose*H₂0) containing 100 mg/1 ampicillin in baffled 500 ml shake flasks at 30 °C and 200 rpm on a shaking incubator. After approx. 10 h of incubation, 125 μΐ of the grown cultures on this medium were used to inoculate 100 ml Hi. l inoculation medium (8.6 g/L Na₂HP0₄*2H₂0, 3 g/L KH₂P0₄, 1 g/L NH₄C1, 0.5 g/L NaCl, 0.06 g/L Fe(III) citrate hydrate, 0.003 g/L H₃B0₄, 0.015 g/L MnCl₂*4H₂0, 0.0084 g/L EDTA*2H₂0, 0.0015 g/L CuCl₂*2H₂0, 0.0025 g/L Na₂Mo₄0₄*2H₂0, 0.0025 g/L CoCl₂*6H₂0, 0.008 g/L Zn(CH₃COO)₂*2H₂0, 10 g/L glucose, 0.6 g/L MgS0₄*7H₂0) containing 100 mg/1 ampicillin in baffled 500 ml shake flasks which was then incubated at 30 °C and 200 rpm to an OD600 of approx. 6 (after approx. 18 h). 750 ml Hf.l medium (16.6 g/L KH₂P0₄, 4 g/L (NH₄)₂HP0₄, 2.1 g/L citric acid, 0.075 g/L Fe(III) citrate hydrate, 0.0038 g/L H₃B0₄, 0.0188 g/L MnCl₂*4H₂0, 0.0105 g/L EDTA*2H₂0, 0.0019 g/L CuCl₂*2H₂0, 0.0031 g/L Na₂Mo0₄*2H₂0, 0.0031 g/L CoCl₂*6H₂0, 0.01 g/L Zn(CH₃COO)₂*2H₂0, 25 g/L glucose, 1.5 g/L MgS0₄*7H₂0) containing 100 mg/1 ampicillin were then inoculated to a calculated final OD₆oo of 0.05. Fermentations were performed at 30 °C and pH 6.8, automatically adjusted with 12.5 % NH₃ solution. Using an aeration rate of 0.35 to 1.5 wm, a minimum level of dissolved oxygen (DO) of 0.2 was maintained by automatic adjustment of the stirrer speed. After approx. 12 h of batch cultivation, exponential feeding was started using a 50 % glucose/MgS0₄ solution at an initial rate of 10 g/(L culture volume*h) [i.e. 7.5 g/(750 ml*h)] up to approx. 35 g/h. After that, constant feeding at 35 g/h was applied. Glucose levels were monitored manually when necessary and held limiting throughout the fed-batch phase. CA gene expression was induced at an OD₆oo of approx. 70 by addition of 0.75 ml 1 M IPTG and 1.5 ml 500 mM ZnS0₄ from sterile stock solutions. Sample P0 was withdrawn immediately before induction, and subsequent samples [usually 5 ml in total for OD₆oo measurement and biomass + supernatant (3x1 g)] were taken every 2-3 h. All samples were stored at -20 °C until analysis/extraction. Approx. 6 h after induction of CA gene expression, fermentation was stopped, and biomass was harvested by centrifugation in two centrifugation bags each. The supernatant was disposed, and the biomass was frozen and stored at -80 °C until further processing. Based on the time series samples withdrawn after induction of gene expression, recombinant production of active enzyme was confirmed by SDS- PAGE and using the carbonic anhydrase pH assay. HCD fermentation derived biomass (48.3 g for SCA04, 77.6 g/L for SCA09, 61.6 g for SCA11) was processed using the following procedure for the extraction of soluble CA enzyme (result: crude extract) and the subsequent enrichment of the thermostable enzymes by heat treatment (result: heat enriched crude extract): each 10 g wet weight of biomass was re-suspended in 20 ml buffer A (50 mM potassium phosphate, 1 μΜ ZnSC"4, pH 6,8) by pipetting and/or whirl-mixing (e.g. in a 50 ml tube). While placed on ice, the biomass suspension was sonicated (Branson Sonifier, flat tip, duty cycle 50 %, output control 4) for 10 x 1 min with thorough mixing after each minute of sonication. The treated sample was centrifuged (20 min, 20000 xg, 4 °C), and the supernatant was transferred to a fresh reaction tube. The supernatant (crude extract) was stored at -80 °C (alternatively short term storage at 4 °C). To produce a crude extract enriched for the thermostable CA enzyme, an aliquot of the respective crude extract was heated for 25 min at 65 °C in a water bath while inverting the tube several times during incubation to ensure thorough heating. After incubation, the sample was centrifuged (15 min, 20000 xg, 4 °C), and the supernatant (=heat enriched crude extract) was stored in aliquots a 1-1.5 ml at -80 °C.

(ii) Determination of enzyme concentrations and purities of SCA04, SCA09 and SCA11 in heat enriched crude extracts

The quantification of enzymes SCA04, SCA09 and SCA11 in heat enriched crude extracts was performed by a combination of (i) the determination of total protein concentrations using the Bradford protein assay and bovine serum albumin (BSA) as a standard, and (ii) SDS-PAGE based band intensity quantification.

The Bradford assay was performed as follows: from a commercial stock solution of BSA (NEB, lO mg/ml) and a derived lOOx dilution (100 μg/ml), 800 μΐ each of the dilutions of 0, 1, 5, 7.5 and 10 μg/ml BSA in ion free water were prepared and used as standards. Enriched crude extract samples were diluted 1 :10000, 1 :2000 and 1 : 1000 in 800 μΐ final volume in order to fit the results to the linear OD595 detection range of the Spectramax microtiter plate reader. To each 800 μΐ diluted samples and standards, 200 μΐ Bradford color solution (Bio-Rad protein assay concentrate) was added and mixed thoroughly. 3x 200 μΐ of each reaction were transferred to a 96-well plate (three parallels of each concentration of sample and standards), and absorbance was measured at 595 nm wave length in the Spectramax reader. The OD595 results for the standard dilutions were plotted against the BSA concentration to produce a standard curve, and sample results were correlated to this standard curve to determine and calculate the total protein concentration in the sample dilution and the original sample. The determined total protein concentrations were 11.4 mg/ml (SCA04), 17.6 mg/ml (SCA11) and 12.8 mg/ml (SCA09).

SDS-PAGE and quantification of the CA enzyme monomers of SCA04, SCA09 and SCA11 were performed as follows: six dilutions each of the respective enriched crude extracts were generated in a final volume of 20 μΐ. This 20 μΐ sample dilution and 10 μΐ gel loading dye were mixed and boiled for 3 min. 25 μΐ of the heated mixtures was then applied on 12 % Clare Page SDS-PA gels. The protein standards used were the BioRad Dual color and Broad range standards. Lysozyme and BSA were used in dilution as further references. The gel images were analyzed using the ChemDoc software, and Image Reports were generated. Based on this analysis and on visual inspections of the protein gels, it was concluded that the portions of carbonic anhydrase monomeric protein in the respective enriched crude extracts were approximately 60 % for SCA04, 95 % for SCA09 and 90 % for SCA11. From this result and the total protein concentrations, the concentrations of the CA enzymes in the enriched crude extract were calculated to be 6.84 mg/ml (SCA04), 12.16 mg/ml (SCA09) and 15.84 mg/ml (SCA11).

(Hi) Production of C0₂-saturated water

To produce C0₂-saturated water as substrate stock solution for subsequent CA activity measurements for K_m determination, a thermos bottle was filled with dry ice, and the developing gas was bubbled through a flask containing 500 ml ion free water while stirring. The system was left overnight to reach saturation, before the bottle was tightly closed and stored for at least one hour to overnight to equilibrate. The C0₂ concentration in the substrate stock solution was determined by titration with 0.01 M NaOH in the presence of the pH indicator phenolphthalein and continued until the indicator turned pale pink (typically 33-36 ml for 10 ml C0₂-saturated water).

(iv) Measuring enzymatic activity and data conversion

Enzymatic activity was monitored by following the pH decrease after the addition of substrate solution and enzyme solution and subtracting the respective results from a control reaction where no enzyme was added. This decrease was linear between pH 8.3 and pH 7.3, and only values in this range were included in the calculation of the kinetic parameters. The reaction mixture consisted of 12 ml buffer (20 mM Tris-S0₄, 1 μΜ ZnS0₄, pH 8.3), 0.5-9 ml substrate stock solution (C0₂-saturated water), 8.5-0 ml ion free water, and 0.1 ml enzyme solution or buffer (control). The total reaction volume in all cases was 21.1 ml. Buffer and ion free water were mixed, and the pH electrode was inserted in the reaction vessel. The mixture was stirred at maximum stirrer speed, and the measurement/logging was started. After ~5 seconds, the substrate solution was added, and immediately afterwards, the enzyme was added. The decrease in pH was then monitored and logged at a resolution of 50 ms for about one minute.

The datasets obtained report the pH decrease in pH/10 as a function of time, and to convert pH/10-units into C0₂ concentrations in mM, the change in pH/10 was plotted as a function of 'C0₂-concentration' added to the reaction. This plot was semi-linear for values up to ~7 mM. Correlation curves were made for each individual experiment. The addition of 1 mM C0₂ was found to correlate to a signal change of -0.0143 (SCA04 measurements), -0.0158 (SCA09 measurements) and -0.0143 (SCA11 measurements) in "pH/10" units.

(v) K_m determination ofSCA04

The SCA04 enzyme was assayed using a 5-fold diluted enriched crude extract sample. For each substrate concentration, the activities were determined as the difference in slope values of the curve with enzyme added and the respective reference curve without enzyme added (buffer only). These values (in units of pH/10 per second) were then divided by the slope value -0.0143 pH/10 per mM, obtaining activities in the units of mM/s. By dividing these values by the concentration of functional enzyme in the system (correcting for dilutions and enzyme purity), activities in mmol/s per mg protein were calculated. Enzyme units (U) are often referred to as the amount of enzyme needed to produce 1 Mol product per minute (or second). Here, it is defined as the amount of enzyme needed to consume 1 Mol C0₂ per second, and specific activities (U/mg) were found by multiplying the mmol/s per mg protein- values with 1000.

In Figure 5A, the specific activities of SCA04 are plotted as a function of the substrate concentration. Two individual series were included, plotted as open squares and open diamonds, respectively, while the sum of the two is presented as closed diamonds. Values for the Michaelis-Menten kinetic parameters K_m and V_max were determined by using the Microsoft Excel solver tool to minimize the sum of squared deviations between measured and values calculated from the Michaelis-Menten equation including all measurements.

(vi) K_m determination ofSCA09

The SCA09 enzyme was assayed using an undiluted enriched crude extract sample. For each substrate concentration, the activities were determined as the difference in slope values of the curve with enzyme added and the respective reference curve without enzyme added (buffer only). These values (in units of pH/10 per second) were then divided by the slope value -0.0158 pH/10 per mM, obtaining activities in the units of mM/s. By dividing these values by the concentration of functional enzyme in the system (correcting for dilutions and enzyme purity), activities in mmol/s per mg protein were calculated. Here, units are defined as the amount of enzyme needed to consume 1 Mol C0₂ per second, and specific activities (U/mg) were found by multiplying the mmol/s per mg protein- values with 1000.

In Figure 5B, the specific activities are plotted as a function of substrate concentration. Two individual series were included, plotted in open squares and open diamonds, respectively, while the sum of the two is seen in closed diamonds. Values for the Michaelis-Menten kinetic parameters K_m and V_max were determined by using the Microsoft Excel solver tool to minimize the sum of squared deviations between measured and values calculated from the Michaelis-Menten equation including all measurements.

(vii) K_m determination ofSCAll

The SCA11 enzyme was assayed using a 100-fold diluted enriched crude extract sample. For each substrate concentration, the activities were determined as the difference in slope values of the curve with enzyme added and the respective reference curve without enzyme added (buffer only). These values (in units of pH/10 per second) were then divided by the slope value -0.0143 pH/10 per mM, obtaining activities in the units of mM/s. By dividing these values by the concentration of functional enzyme in the system (correcting for dilutions and enzyme purity), activities in mmol/s per mg protein were calculated. Here, units are defined as the amount of enzyme needed to consume 1 Mol C0₂ per second, and specific activities (U/mg) were found by multiplying the mmol/s per mg protein- values with 1000.

In Figure 5C, the specific activities are plotted as a function of substrate concentration. Two individual series were included, plotted in open squares and open diamonds, respectively, while the sum of the two is seen in closed diamonds. Values for the Michaelis-Menten kinetic parameters KM and Vmax were determined by using the Microsoft Excel solver tool to minimize the sum of squared deviations between measured and values calculated from the Michaelis-Menten equation including all measurements.

(viii) Summary

The kinetic parameters determined for the three CA enzymes SCA04, SCA09 and SCA11 are listed in Table 1.

Table 1. Kinetics parameters for SCA04, SCA09 and SCA11

SCA04 SCA09 SCA11

mM U/mg mM U/mg mM U/mg

9.1 205977 6.1 15752 48.8 8295138

Example 6

Effect of the amount of enzyme added

To demonstrate the impact of free carbonic anhydrase on the reaction kinetics, tests were conducted in a stirred cell at enzyme concentrations of 1 , 2 and 4 wt% in a 20 wt% K₂C0₃ solution. The initial loading of the solution was adjusted to 0, 0.1 and 0.2 mole C0₂/mole K₂C0₃. The enzyme used is a variant of carbonic anhydrase from human erythrocyte cells type II (A known amount of absorption solution was placed in the cell, and let equilibrate with pure C0₂ under constant atmospheric pressure. The volume of C0₂ absorbed is logged as a function of time and is used to calculate the rate of absorption. The results were compared to tests conducted without the presence of enzyme (points presented as 0 wt%) and are expressed as relative C0₂ absorption rate, i.e. as the ratio of absorption rate with enzyme to absorption rate in the absence of enzyme. The results indicate that the enzyme enhances the absorption rate for all tested K₂C0₃ solutions. As the initial loading of the solution increases, there is a decrease in the relative rate of absorption. The enzymes succeed to increase the kinetics by a noticeable and important factor, but it is expected that as the loading increases further, the enhancement will be reduced mainly due to the fact that as C0₂ is absorbed in the solution, accumulation of HC0₃ ^~ species reduces the rate of absorption. Figure 6 illustrates the relative C0₂ absorption rate as a function of C0₂ loading at different enzyme concentrations.

Example 7

Effect of the solvent concentration

To demonstrate the impact of the solvent concentration on the reaction kinetics, tests were conducted in a stirred cell with a fixed amount of enzyme (4 wt%) at different K₂C0₃ concentrations. The results are shown in Figure 7, which illustrates the relative C0₂ absorption rate as a function of the K₂C0₃ concentration (wt%) at an amount of 4 wt% enzyme.

Example 8

Temperature stability of SCA04 at 80° C

Crude extract of E. coli containing recombinantly produced SCA04 was prepared as described in Example 2. Fractions of 500 μΐ crude extract in 1.5 ml reaction vials were incubated on a heating block at 80 °C for 0, 15, 30, 60, 120, 180, 240 and 300 min, respectively. After incubation, samples were centrifuged in a microliter centrifuge (13,200 rpm, 22 °C, 10 min), and the cleared supernatant was used for carbonic anhydrase activity measurements at room temperature in triplicate as described in Example 3. The time for the pH to drop from pH 7.8 to pH 7.0 (dt_pH7.8-_PH7.o) was determined, and activity units were calculated using the formula U=(dt_PH7.8-_PH7.o, ref dt_PH7.8-pH7.o, extract)/dtpH7.8-_PH7.o, extract- The reference data (ref) were obtained by measuring in triplicate extraction buffer A (50 mM potassium phosphate, pH 6.8, 1 μΜ zinc sulphate) instead of cell extract. Calculated activity units for three parallel measurements were averaged and standard deviations were determined to be below 5 %. The mean result for the measurements of the sample incubated at 80 °C for 15 min was set as 100 % (0 min data excluded as an outlier) and the results for the other time-series samples were correlated to this. The percentage of residual activity was plotted as a function of incubation time at 80 °C as presented in Figure 8. From the resulting curve, it can be derived that SCA04 tolerates incubation at 80 °C over extended periods of time with only minor loss of activity. After three hours of incubation, still more than 90 % of the initial carbonic anhydrase activity could be detected.

Claims

WE CLAIM

1.

An isolated polypeptide having carbonic anhydrase activity as defined in any one of a) through e) or any combinations thereof: a) a polypeptide having an amino acid sequence corresponding to amino residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32;

a polypeptide which is at least 60% identical to amino acid residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22 or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32;

a fragment of a) or b) having carbonic anhydrase activity, or a polypeptide encoded by nucleic acid sequence which hybridizes under medium stringency conditions with a polynucleotide sequence encoding a polypeptide of: i) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID

NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24„ SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or ii) polynucleotide sequence of: SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 or SEQ ID NO:23„SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31;

iii) or a complementary strand of i) or ii); or iv) a subsequence of i); or e) a polypeptide encoded by the nucleic acid sequence which due to the degeneracy of the genetic code does not hybridize with the polynucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31;

but which codes for a polypeptide having an amino acid sequence according to a) or b).

2.

The isolated polypeptide according to claim 1 , wherein the polypeptide has a high K_m or a low K_m value.

3.

The isolated polypeptide according to claim 2, wherein said low K_m is chosen from the range: from about 1 to about 25mM and said high K_m value is chosen from the range: form about 25 to about 60mM.

4.

The isolated polypeptide of any one of claims 1-3, wherein the carbonic anhydrase activity is maintained at a temperature of above 65°C for at least one hour.

5.

The isolated polypeptide of any one of claims 1-3, wherein the carbonic anhydrase activity is maintained at a temperature of at least 80°C for at least two hours at a level corresponding to at least 90% of the initial activity.

6.

An isolated polypeptide or a mixture of isolated polypeptides comprising at least one of the polypeptides according to any one of claims 1-5.

7.

The isolated polypeptide or the mixture of isolated polypeptides according to claim 6, for the use of absorbing/desorbing an acidic component from/to a gas mixture.

8.

The isolated polypeptide or the mixture of isolated polypeptides according to claim 7, wherein the acidic component is C0₂.

9.

A composition comprising the isolated polypeptide or the mixture of polypeptides according to any one of claims 1-8 and an immobilizing agent.

10.

The composition according to claim 9, wherein said immobilizing agent comprises a matrix, a surface or a substrate.

11.

The composition according to claim 10, wherein said matrix, surface or substrate comprises beads, fabrics, fibers, porous materials, CLEAs, structured or random packing, crystals such as monoliths or any combinations thereof.

12.

The composition according to any one of claims 8-11, for the use of

absorbing/desorbing an acidic component from/to an absorbing medium.

13.

The composition according to claim 12, wherein the acidic component is C0₂.

14.

An isolated polynucleotide having a nucleotide sequence which encodes a polypeptide according to any one of claims 1-8.

15.

A nucleic acid construct comprising an operable linked control sequence directing the expression of a polynucleotide according to claim 14.

16.

A vector comprising the polynucleotide of claim 14 or the nucleic acid construct of claim 15.

17.

A host cell comprising the polynucleotide of claim 14, nucleic acid construct of claim 15 or the vector of claim 16.

18.

Use of an isolated polypeptide according to any one of claims 1-8 or a composition according to any one of claims 9-13 for absorbing/desorbing an acidic component from/to an absorbing medium.

19.

The use according to claim 18, wherein the acidic component is carbon dioxide.

5 20.

Use of an isolated polynucleotide according to claim 14.

21.

Use of a nucleic acid construct according to claim 15.

10

22.

Use of a vector according to claim 16.

23.

i s Use of a host cell according to claim 17.