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WO2011066304A2 - Polypeptides génétiquement modifiés de l'anhydrase carbonique de classe bêta et utilisations de ceux-ci - Google Patents

Polypeptides génétiquement modifiés de l'anhydrase carbonique de classe bêta et utilisations de ceux-ci Download PDF

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WO2011066304A2
WO2011066304A2 PCT/US2010/057843 US2010057843W WO2011066304A2 WO 2011066304 A2 WO2011066304 A2 WO 2011066304A2 US 2010057843 W US2010057843 W US 2010057843W WO 2011066304 A2 WO2011066304 A2 WO 2011066304A2
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residue
position corresponding
carbonic anhydrase
polar
polypeptide
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WO2011066304A3 (fr
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Louis Clark
Sabrina Zimmerman
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Codexis Inc
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Codexis Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide

Definitions

  • the present disclosure relates to ⁇ -class carbonic anhydrase polypeptides and uses thereof.
  • the present disclosure further relates to nucleic acids encoding carbonic anhydrase polypeptides, expression systems for the production of carbonic anhydrase polypeptides, as well as to methods and bioreactors for the capture and sequestration of carbon dioxide using the recombinant carbonic anhydrase polypeptides of the present disclosure.
  • CA carbonic anhydrase
  • CA In the forward or “hydration” reaction, CA combines carbon dioxide and water to provide bicarbonate and a proton, or depending on the pH, to provide carbonate (CO 3 "2 ) and two protons. In the reverse, or “dehydration” reaction, CA combines bicarbonate and a proton to provide carbon dioxide and water.
  • Carbonic anhydrases are metalloenzymes that typically have Zn +2 in the active site. However carbonic anhydrases having e.g. Co 2 or Cd 2 in the active site have been reported. At least three classes of carbonic anhydrases have been identified in nature.
  • the a-class carbonic anhydrases are found in vertebrates, bacteria, algae, and the cytoplasm of green plants. Vertebrate a-carbonic anhydrases are among the fastest enzymes known, exhibiting a turnover number (k cat ) (the number of molecules of substrate converted by an enzyme to product per catalytic site per unit of time) of 10 6 sec "1 .
  • the ⁇ -class carbonic anhydrases are found in bacteria, algae, and chloroplasts, while ⁇ -class carbonic anhydrases are found in Archaea and some bacteria. Although carbonic anhydrases of each of these classes have similar active sites, they do not exhibit significant overall amino acid sequence homology and they are structurally distinguishable from one another. Hence, these three classes of carbonic anhydrase provide an example of convergent evolution.
  • carbonic anhydrase could be used as a biological catalyst to accelerate the capture of carbon dioxide produced by produced by combustion of fossil fuels.
  • carbonic anhydrases found in nature are not ideally suited for use in such applications. Accordingly, there is a need in the art for carbonic anhydrases that can effectively hydrate carbon dioxide at elevated temperatures and at alkaline pH for extended periods of time in the presence of relatively high concentrations of carbonate.
  • carbonic anhydrases should also be stable to variations in pH, e.g., stable not only at a relatively alkaline pH suitable for hydration and sequestration of carbon dioxide but also at a relatively acidic pH suitable for subsequent release and/or recapture of the hydrated and/or sequestered carbon dioxide.
  • the present disclosure provides heat-stable recombinant carbonic anhydrase polypeptides that are capable of catalyzing the hydration of carbon dioxide at elevated temperatures.
  • the present disclosure also provides recombinant carbonic anhydrases that are capable of catalyzing the hydration of carbon dioxide in the presence of relatively high concentrations of carbonate or amine solvents such as the solvent AMP.
  • the present disclosure provides heat-stable recombinant carbonic anhydrases that are capable of catalyzing the hydration of carbon dioxide at elevated temperatures in the presence of relatively high concentrations of carbonate.
  • the present disclosure also provides polynucleotides encoding the recombinant carbonic anhydrase polypeptides of the disclosure, methods and hosts cells for the expression of those polypeptides, as well as methods and bioreactors for using the presently-disclosed polypeptides.
  • the recombinant carbonic anhydrase polypeptides described herein have an amino acid sequence that has one or more amino acid differences as compared to a wild-type reference carbonic anhydrase (e.g., polypeptides of SEQ ID NO: 2 or 4) that result in an improved property of the enzyme.
  • a wild-type reference carbonic anhydrase e.g., polypeptides of SEQ ID NO: 2 or 4
  • the engineered carbonic anhydrase polypeptides have an improved property as compared to the naturally- occurring wild-type carbonic anhydrase enzymes obtained from Methanobacterium thermoautotrophicus str. Delta H (" thermoautotrophicus"; SEQ ID NO: 2).
  • Improvements in an enzyme property include increases in thermostability, solvent stability, base stability, enzyme activity at elevated pH, increased level of expression, and enzyme stability and/or activity during pH variations, as well as reduced product inhibition (e.g., product inhibition by carbonate or bicarbonate). Improvements in an enzyme property also include increased stability, solubility, and/or activity in the presence of additional reagents useful for absorption or sequestration of carbon dioxide, including, for example, calcium ions, aqueous carbonate solutions, amines such as ammonia, monoethanolamine (“MEA”), methyldiethanolamine, piperazine, piperazine mono- and diethanolamine and 2-aminomethylpropanolamine ("AMP").
  • MEA monoethanolamine
  • AMP 2-aminomethylpropanolamine
  • a recombinant carbonic anhydrase polypeptide of the present disclosure is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2, and has, at the position corresponding to the indicated position of SEQ ID NO: 2, at least one of the following features (e.g., amino acid substitutions): residue at position corresponding to X2 is a cysteine (C); residue at position corresponding to X8 is a non-polar residue; residue at position corresponding to XI 0 is a non-polar residue; residue at position corresponding to X13 is a cysteine (C); residue at position corresponding to X17 is
  • residue at position corresponding to X126 is an aromatic residue; residue at position corresponding to X147 is an aromatic residue; residue at position corresponding to X153 is an acidic residue; residue at position corresponding to XI 55 is a constrained residue; or residue at position corresponding to XI 59 is a polar residue.
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is C; residue at position corresponding to X8 is L; residue at position corresponding to X10 is G; residue at position corresponding to X13 is C; residue at position corresponding to XI 7 is V; residue at position corresponding to X22 is G or V; residue at position corresponding to X23 is Y; residue at position corresponding to X24 is E; residue at position corresponding to X25 is
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is a cysteine (C); residue at position corresponding to X10 is a non-polar residue; residue at position corresponding to X13 is a cysteine (C); residue at position corresponding to X17 is a non-polar residue; residue at position corresponding to X22 is a non-polar residue; residue at position corresponding to X23 is an aromatic residue; residue at position corresponding to X24
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is C; residue at position corresponding to X10 is G; residue at position corresponding to X13 is C; residue at position corresponding to X17 is V; residue at position corresponding to X22 is G or V; residue at position corresponding to X23 is Y; residue at position corresponding to X24 is E; residue at position corresponding to X25 is P; residue at position corresponding to X28 is P
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is a cysteine (C); residue at position corresponding to X8 is a non-polar residue; residue at position corresponding to XI 0 is a non-polar residue; residue at position corresponding to X13 is a cysteine (C); residue at position corresponding to XI 7 is a non-polar residue; residue at position corresponding to X22 is a non-polar residue; residue at position at position corresponding to X2
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is C; residue at position corresponding to X8 is L; residue at position corresponding to X10 is G; residue at position corresponding to X13 is C; residue at position corresponding to XI 7 is V; residue at position corresponding to X22 is G or V; residue at position corresponding to X25 is P; residue at position corresponding to X28 is P; residue at position corresponding to X40 is T
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one of the following features or sets of features: (a) residue at position corresponding to X2 is a cysteine; (b) residue at position corresponding to X8 is a non-polar residue; residue at position corresponding to X85 is a basic residue; and residue at position corresponding to X126 is an aromatic residue; (c) residue at position corresponding to X10 is a non-polar residue; and residue at position corresponding to X75 is a non-polar residue; (a) residue at position corresponding to
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one of the following features or sets of features: (a) residue at position corresponding to X2 is C; (b) residue at position corresponding to X8 is L; residue at position corresponding to X85 is K; and residue at position corresponding to X126 is F; (c) residue at position corresponding to X10 is G; and residue at position corresponding to X75 is L; (d) residue at position corresponding to X8 is L; and residue at position corresponding to X75
  • a recombinant carbonic anhydrase polypeptide of the present disclosure comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO:2, and has at least one of the following amino acid substitutions: R2C, I8L, D10G, R13C, D17V, D22V, D22G, L23Y, S24E, D25P, H28P, L32S, S40T, G53K, D69E, A75L, A76V, N85K, V106A, N121D, S123K, V126F, S 147H, S 153D, G155P, and
  • a recombinant carbonic anhydrase comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence corresponding to SEQ ID NO: 2, wherein the recombinant carbonic anhydrase polypeptide amino acid sequence includes any one set of the specified amino acid substitution combinations presented in Table 2.
  • these recombinant carbonic anhydrase polypeptides can have mutations at other amino acid residues.
  • the recombinant carbonic anhydrase polypeptides of the present disclosure are capable of hydrating carbon dioxide to bicarbonate with improved activity relative to a reference ⁇ -class carbonic anhydrase polypeptide of SEQ ID NO: 2.
  • improved activity is determined following heat challenge in basic solvents. For example, in some embodiments after heating the recombinant carbonic anhydrase polypeptide and the reference polypeptide in a solution at a temperature of 75°C for about 30 minutes, wherein the solution is selected from a solution comprising 150 mM K 2 CO 3 at pH 10.9, and a solution comprising 1 M AMP at pH 9.7.
  • the recombinant carbonic anhydrase polypeptides exhibit an activity (e.g., rate of catalysis) that is greater than that of the polypeptide of SEQ ID NO: 2.
  • exemplary polypeptides with such properties include, but are not limited to, polypeptides which comprise an amino acid sequence corresponding to SEQ ID NO: 6, 8, 10, 16, 18, 22, 24, 26, 28, 30, 36, 40, 42, and 44.
  • the recombinant carbonic anhydrase polypeptides of the disclosure exhibit activity at least 1.05-times, 1.1 -times, 1.2-times, 1.3-times, 1.4-times increased relative to the activity polypeptide of SEQ ID NO: 2.
  • the recombinant carbonic anhydrase polypeptides of the disclosure exhibit at least 1.1 -fold increased activity relative to the activity of a reference polypeptide of SEQ ID NO: 2.
  • Exemplary polypeptides with such properties include, but are not limited to, polypeptides which comprise an amino acid sequence corresponding to SEQ ID NO: 6, 8, 10, 18, 22, 24, 26, 28, 30, 40, 42, and 44.
  • the recombinant carbonic anhydrase polypeptides of the disclosure exhibit activity that is at least 1.2-fold increased activity relative to the activity of a reference polypeptide of SEQ ID NO: 2.
  • Exemplary polypeptides with such properties include, but are not limited to, polypeptides which comprise an amino acid sequence corresponding to SEQ ID NO: 6, 8, 18, 22, 24, 26, 28, 40, 42, and 44.
  • the recombinant carbonic anhydrase polypeptides of the disclosure exhibit activity that is at least 1.3-fold increased activity relative to the activity of a reference polypeptide of SEQ ID NO: 2.
  • Exemplary polypeptides with such properties include, but are not limited to, polypeptides which comprise an amino acid sequence corresponding to SEQ ID NO: 6, 8, 24, 26, 40, 42, and 44.
  • the recombinant carbonic anhydrase polypeptides of the disclosure exhibit activity that is at least 1.4-fold increased activity relative to the activity of a reference polypeptide of SEQ ID NO: 2.
  • Exemplary polypeptides with such properties include, but are not limited to, polypeptides which comprise an amino acid sequence corresponding to SEQ ID NO: 6, 24, and 42.
  • the present disclosure provides polynucleotides encoding the engineered carbonic anhydrases described herein or polynucleotides that hybridize to such polynucleotides under highly stringent conditions.
  • the polynucleotide can include promoters and other regulatory elements useful for expression of the encoded engineered carbonic anhydrases, and can utilize codons optimized for specific desired expression systems.
  • the polynucleotides encode a recombinant carbonic anhydrase polypeptide having at least the following amino acid sequence as compared to the amino acid sequence of SEQ ID NO: 2, and further comprising at least one acid substitution selected from the group of amino acid substitutions and additions provided in Table 2.
  • Exemplary polynucleotides include, but are not limited to, a polynucleotide sequence of any of SEQ ID NOs: 5, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45.
  • the present disclosure provides a polynucleotide encoding a recombinant carbonic anhydrase polypeptide capable of improved expression in a host cell relative to a reference polynucleotide encoding a ⁇ -class carbonic anhydrase polypeptide of SEQ ID NO: 4.
  • the polynucleotide capable of improved expression in a host cell encodes a polypeptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 4 and five amino terminal residues corresponding to XI through X5 of SEQ ID NO: 2.
  • the polynucleotide capable of improved expression encodes a recombinant carbonic anhydrase of at least 176 amino acids, and in further embodiments can comprise one or more of the amino acid residue differences relative to SEQ ID NO: 2 described herein.
  • these substitutions at one or more positions are selected from X2, X8, X10, X13, X17, X22, X23, X24, X25, X28, X32, X40, X53, X69, X75, X76, X85, X106, X121, X123, X126, X147, X153, X155, and X159, and can include any one of R2C, I8L, D10G, R13C, D17V, D22V, D22G, L23Y, S24E, D25P, H28P, L32S, S40T, G53K, D69E, A75L, A76V, N85K, V106A, N121D, S123K, V126F, S147H, S 153D, G155P, and L159S.
  • the present disclosure provides host cells comprising the polynucleotides and/or expression vectors described herein.
  • the host cells may be M. thermoautotrophicus or they may be a different organism, such as E. coli, Saccharomyces cerevisiae, filamentous fungal organism (e.g., Aspergillus spp.), or a Bacillus spp. (e.g., B. amyloliquefaciens, B. licheniformis, B. megaterium, B. stearothermophilus, and B. subtilis).
  • the host cells can be used for the expression and isolation of the engineered carbonic anhydrase enzymes described herein, or, alternatively, they can be used directly for carrying out the reactions of Scheme 1.
  • the disclosure provides a method of producing a recombinant carbonic anhydrase polypeptide of the present disclosure, wherein said method comprises the steps of: (a) transforming a host cell with an expression vector polynucleotide encoding the recombinant carbonic anhydrase polypeptide; (b) culturing said transformed host cell under conditions whereby said recombinant carbonic anhydrase polypeptide is produced by said host cell; and (c) recovering said recombinant carbonic anhydrase polypeptide from said host cells.
  • the method of producing the recombinant carbonic anhydrase may be carried out wherein said expression vector comprises a secretion signal, and said cell is cultured under conditions whereby the recombinant carbonic anhydrase polypeptide is secreted from the cell.
  • the expression vector comprises a polynucleotide encoding a secretion signal.
  • a single recombinant carbonic anhydrase enzyme may be used or, alternatively, mixtures of two or more recombinant carbonic anhydrase enzymes may be used.
  • recombinant carbonic anhydrase polypeptides of the present disclosure exhibit improved the improved property of increased thermal stability and/or increased stability to solvent or base.
  • the recombinant carbonic anhydrase polypeptides of the present disclosure exhibit increased activity in the presence of basic solvents, e.g., the recombinant carbonic anhydrase polypeptides of the present disclosure retain substantially more enzymatic activity when assayed in the presence of C0 3 ⁇ 2 at a concentration within a range of from about 0.1 M C0 3 ⁇ 2 to about 5 M C0 3 ⁇ 2 , from about 0.2 M C0 3 ⁇ 2 to about 4 M C0 3 "2 , or from about 0.3 M C0 3 "2 to about 3 M C0 3 "2 .
  • the recombinant carbonic anhydrase polypeptides of the present disclosure exhibit increased
  • thermotolerance ⁇ e.g., thermostability
  • the recombinant carbonic anhydrase polypeptides of the present disclosure retain substantially more enzymatic activity after exposure to a temperature within the range of from about 60°C to about 90°C, or within a range of from 70°C to about 80°C.
  • the recombinant carbonic anhydrase polypeptides of the present disclosure are used in methods for the absorption and/or desorption of carbon dioxide produced, for example, by the combustion of fossil fuels.
  • a recombinant carbonic anhydrase polypeptides of the present disclosure is used to catalyze the hydration of carbon dioxide to provide a solution comprising bicarbonate and/or carbonate ions, e.g. depending on the pH of that solution.
  • the bicarbonate and/or carbonate containing solution can be recovered (e.g., isolated) and contacted with a recombinant carbonic anhydrase polypeptide of the present disclosure to release the carbon dioxide.
  • the recombinant carbonic anhydrase polypeptides of the present disclosure is immobilized on a solid surface and one or both of the hydration and dehydration reactions are carried out in a bioreactor.
  • the hydration reaction is performed at a relatively alkaline pH while the dehydration is carried out at a relatively acidic pH.
  • the present disclosure provides a method for removing carbon dioxide from a gas stream comprising the step of contacting the gas stream with a solution comprising a recombinant carbonic anhydrase polypeptide having an improved property of the disclosure under suitable conditions, whereby carbon dioxide from the gas stream is dissolved in the solution and converted to hydrated carbon dioxide.
  • the method is carried out wherein the solution is aqueous, or an aqueous co-solvent system.
  • the solution used is aqueous-solvent system comprising an organic solvent comprising an amine compound selected from monoethanolamine (MEA), methyldiethanolamine (MDEA), and
  • the methods can be carried out wherein the recombinant carbonic anhydrase polypeptide is immobilized on a surface, for example a surface on a particle in the solution.
  • the method further comprises the step of isolating the solution comprising the hydrated carbon dioxide and contacting the isolated solution with hydrogen ions and a recombinant carbonic anhydrase polypeptide, thereby converting the hydrated carbon dioxide to carbon dioxide gas and water.
  • FIGURE 5 The stability of purified (1 mg/mL) CabOOl ("Cab long") polypeptide of SEQ ID NO: 2 in varying AMP concentrations ranging from 25 mM to 3 M at room temperature (no heat) and after heat challenge of 75°C or 80°C for 30 minutes.
  • the present disclosure is directed to recombinant carbonic anhydrases having improved properties, particularly as compared to those of their parent, the carbonic anhydrase of SEQ ID NO: 2.
  • the present disclosure is also directed to the use of such carbonic anhydrases in methods for the capture and sequestration of carbon dioxide generated by combustion of fossil fuel.
  • the present disclosure is further directed to the use of such carbonic anhydrases in bioreactors useful for not only for sequestration (hydration) of carbon dioxide generated by fossil fuel burning power plants but also for the subsequent recovery (dehydration) of that previously-sequestered carbon dioxide.
  • Carbonic anhydrase and “CA” are used interchangeably herein to refer to a polypeptide having an enzymatic capability of carrying out the reactions depicted in Scheme 1.
  • Carbonic anhydrase as used herein include naturally occurring (wild type) carbonic anhydrases as well as non- naturally occurring engineered polypeptides generated by human manipulation.
  • Protein polypeptide
  • peptide a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post- translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation,
  • D- and L-amino acids include D- and L-amino acids, and mixtures of D- and L-amino acids.
  • Coding sequence refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.
  • Naturally occurring or wild-type refers to the form found in nature.
  • a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.
  • Recombinant or “engineered” or “non-naturally occurring” when used with reference to, e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.
  • Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.
  • Percentage of sequence identity refers to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Determination of optimal alignment and percent sequence identity is performed using the BLAST and BLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.
  • the BLAST analyses involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915).
  • Reference sequence refers to a defined sequence to which another sequence is compared.
  • a reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence.
  • a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide.
  • two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides over a comparison window to identify and compare local regions of sequence similarity.
  • reference sequence is not intended to be limited to wild-type sequences, and can include engineered or altered sequences.
  • a “reference sequence” can be a previously engineered or altered amino acid sequence.
  • Comparison window refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.
  • “Corresponding to”, “reference to” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
  • the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence.
  • a given amino acid sequence such as that of an engineered carbonic anhydrase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.
  • “Increased enzymatic activity” or “increased activity” refers to an improved property of an engineered enzyme, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of carbonic anhydrase) as compared to a reference enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of K m , V max or k cat , changes of which can lead to increased enzymatic activity.
  • the carbonic anhydrase activity can be measured by any one of standard assays used for measuring carbonic anhydrase activity, such as change in C0 2 , H + , or carbonate concentration. Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when enzymes in cell lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.
  • Conversion refers to the enzymatic transformation of a substrate to the corresponding product.
  • Percent conversion refers to the percent of the substrate that is converted to the product within a period of time under specified conditions.
  • the "enzymatic activity” or “activity” of a carbonic anhydrase polypeptide can be expressed as “percent conversion” of the substrate to the product.
  • thermo stable refers to a polypeptide that is resistant to inactivation when exposed to a set of temperature conditions (e.g., 40-80°C) for a period of time (e.g., 0.5-24 hrs) compared to the untreated enzyme, thus retaining a certain level of residual activity (e.g., more than 60% to 80% for example) after exposure to elevated temperatures.
  • a set of temperature conditions e.g. 40-80°C
  • a period of time e.g., 0.5-24 hrs
  • Hydrophilic Amino Acid or Residue refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et ah, 1984, J. Mol. Biol. 179: 125-142.
  • Genetically encoded hydrophilic amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gin (Q), Asp (D), Lys (K) and Arg (R).
  • Acidic Amino Acid or Residue refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Glu (E) and Asp (D).
  • Base Amino Acid or Residue refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK value of greater than about 6 when the amino acid is included in a peptide or polypeptide.
  • Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion.
  • Genetically encoded basic amino acids include Arg (R) and Lys (K).
  • Poly Amino Acid or Residue refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • Genetically encoded polar amino acids include Asn (N), Gin (Q), Ser (S) and Thr (T).
  • Hydrophobic Amino Acid or Residue refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of greater than zero according to the normalized consensus
  • Genetically encoded hydrophobic amino acids include Pro (P), He (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A) and Tyr (Y).
  • Aromatic Amino Acid or Residue refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W). Although owing to the its heteroaromatic ring side chain His (H) is classified as an aromatic residue, it may also be classified as a basic residue owing to pKa of its heteroaromatic nitrogen atom.
  • Non-polar Amino Acid or Residue refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and which has bonds in which the pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar).
  • Genetically encoded non-polar amino acids include Gly (G), Leu (L), Val (V), He (I), Met (M) and Ala (A).
  • Aliphatic Amino Acid or Residue refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala (A), Val (V), Leu (L) and He (I).
  • the amino acid Cys (C) is unique in that it can form disulfide bridges with other Cys (C) amino acids or other sulfanyl- or sulfhydryl-containing amino acids.
  • the ability of Cys (and other amino acids with -SH containing side chains) to exist in a polypeptide in either the reduced free -SH or oxidized disulfide-bridged form affects whether it contributes net hydrophobic or hydrophilic character to the polypeptide. While Cys exhibits a hydrophobicity of 0.29 according to the normalized consensus scale of Eisenberg (Eisenberg et al., 1984, supra), it is to be understood that for purposes of the present disclosure, Cys is classified into its own unique group.
  • the amino acid Pro (P) is conformationally constrained nature. Although it has hydrophobic properties, as used herein, Pro (P) or other similar residues is classified as a "conformationally constrained”.
  • Hydroxyl-containing Amino Acid or Residue refers to an amino acid or residue containing a hydroxyl (-OH) moiety. Genetically-encoded hydroxyl-containing amino acids include Ser (S) and Thr (T). While L-Tyr (Y) contains a hydroxyl moiety, it is classified herein as an aromatic amino acid or residue.
  • amino acid difference or “residue difference” refers to a change in the residue at a specified position of a polypeptide sequence when compared to a reference sequence.
  • a residue difference at position X3, where the reference sequence has a glutamine refers to a change of the residue at position X3 to any residue other than glutamine.
  • an enzyme can include one or more residue differences relative to a reference sequence, where multiple residue differences typically are indicated by a list of the specified positions where changes are made relative to the reference sequence.
  • Constant amino acid substitutions refer to the interchangeability of residues having similar side chains, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids.
  • an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain is substituted with another amino acid with a basis side chain, e.g., lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid
  • Non-conservative substitution refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain.
  • an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.
  • “Deletion” refers to modification of the polypeptide by removal of one or more amino acids from the reference polypeptide.
  • Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the polypeptide while retaining enzymatic activity and/or retaining the improved properties of an engineered carbonic anhydrase enzyme.
  • Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.
  • Insertions refers to modification of the polypeptide by addition of one or more amino acids to the reference polypeptide.
  • the improved engineered carbonic anhydrase enzymes comprise insertions of one or more amino acids to the naturally occurring carbonic anhydrase polypeptide as well as insertions of one or more amino acids to other improved carbonic anhydrase polypeptides. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.
  • Fragment refers to a polypeptide that has an amino-terminal and/or carboxy- terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can typically have about 80%, 90%, 95%, 98%, and 99% of the full-length carbonic anhydrase polypeptide, for example the polypeptide of SEQ ID NO:2.
  • isolated polypeptide refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides.
  • the term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis).
  • the improved recombinant carbonic anhydrase enzymes may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the improved recombinant carbonic anhydrase enzyme can be an isolated polypeptide.
  • substantially pure polypeptide refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight.
  • a substantially pure carbonic anhydrase composition will comprise about 60 % or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition.
  • Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
  • the isolated improved recombinant carbonic anhydrase polypeptide is a substantially pure polypeptide composition.
  • Heterologous polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.
  • Codon optimized refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest.
  • the polynucleotides encoding the recombinant carbonic anhydrase enzymes may be codon optimized for optimal production from the host organism selected for expression.
  • Control sequence is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present disclosure.
  • Each control sequence may be native or foreign to the polynucleotide of interest.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • operably linked is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest.
  • Fusion construct refers to a nucleic acid comprising the coding sequence for a first polypeptide and the coding sequence (with or without introns) for a second polypeptide in which the coding sequences are adjacent and in the same reading frame such that, when the fusion construct is transcribed and translated in a host cell, a polypeptide is produced in which the C-terminus of the first polypeptide is joined to the N-terminus of the second polypeptide.
  • a “fusion polypeptide” refers to the polypeptide product of the fusion construct.
  • the recombinant (or engineered) carbonic anhydrase (“CA”) enzymes of the present disclosure are those having an improved property when compared with a naturally-occurring, wild-type carbonic anhydrase enzyme obtained from Methanobacterium thermoautotrophicus str. Delta H (SEQ ID NO: 2).
  • Enzyme properties for which improvement is desirable include, but are not limited to, enzymatic activity, thermal stability, pH activity profile, refractoriness to inhibitors, e.g. product inhibition by bicarbonate and/or carbonate, refractoriness to inhibition by other reaction components, such as monoethanolamine, methyldiethanolamine, and 2-aminomethylpropanolamine, and solvent stability.
  • the improvements can relate to a single enzyme property, such as enzymatic activity, or a combination of different enzyme properties, such as enzymatic activity and
  • thermostability
  • the engineered carbonic anhydrase with improved enzyme property is described with reference to Methanobacterium thermoautotrophicus str. Delta H (SEQ ID NO:2).
  • the amino acid residue position is determined in these carbonic anhydrases beginning from the initiating methionine (M) residue (i.e., M represents residue position 1), although it will be understood by the skilled artisan that this initiating methionine residue may be removed by biological processing machinery, such as in a host cell or in vitro translation system, to generate a mature protein lacking the initiating methionine residue.
  • amino acid residue position at which a particular amino acid or amino acid change is present in an amino acid sequence is sometimes describe herein in terms "Xn", or “residue n", where n refers to the residue position.
  • a substitution mutation which is a replacement of an amino acid residue in a residue corresponding to a residue of a reference sequence, for example the naturally occurring carbonic anhydrase of SEQ ID NO: 2, with a different amino acid residue is denoted as follows “X (number) Z,” where X is the amino acid found in the wild type enzyme of M. thermoautotrophicus (SEQ ID NO: 2) at position “number” and Z is the amino acid found at position "number” of the "mutant” enzyme, i.e.
  • D7S refers to an instance in which the "wild type" amino acid aspartic acid at position 7 of SEQ ID NO: 2 has been replaced with the amino acid serine.
  • mutations are sometimes described as a mutation of a residue "to a" type of amino acid.
  • SEQ ID NO: 2 residue 8 (isoleucine (I)) can be mutated "to a” polar residue.
  • residue 22 can be mutated from aspartic acid "to an" asparagine.
  • the recombinant carbonic anhydrase polypeptides herein can have a number of modifications to the reference sequence (M. thermoautotrophicus of SEQ ID NO: 2) to result in an improved carbonic anhydrase enzyme property.
  • the number of modifications to the amino acid sequence can comprise one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 9 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the reference enzyme sequence.
  • the number of modifications to the naturally occurring polypeptide or an engineered polypeptide that produces an improved carbonic anhydrase property may comprise from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-1 1, 1-12, 1- 14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40 modifications of the reference sequence.
  • the modifications can comprise insertions, deletions, substitutions, or combinations thereof.
  • the modifications comprise amino acid substitutions to the reference sequence i.e. the sequence of M. thermoautotrophicus carbonic anhydrase of SEQ ID NO: 2.
  • Substitutions that can produce an improved carbonic anhydrase property may be at one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, 9 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 15% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the reference enzyme sequence.
  • the number of substitutions to the naturally occurring polypeptide or an engineered polypeptide that produces an improved carbonic anhydrase property can comprise from about 1-2, 1 - 3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-1 1, 1- 12, 1-14, 1-15, 1-16, 1- 18, 1-20, 1-22, 1-24, 1-25, 1-30, 1- 35 or about 1-40 amino acid substitutions of the reference sequence.
  • the improved property of the recombinant carbonic anhydrase polypeptide is an increase in its ability to a substrate to product. In some embodiments, the improved property of the recombinant carbonic anhydrase polypeptide is with respect to an increase in rate of conversion of the substrate carbon dioxide to the product bicarbonate under certain conditions (e.g., increased temperature, increased base concentration, or increased amount of a solvent). This improvement in enzymatic activity can be manifested by the ability to use less of the improved polypeptide as compared to the wild-type or other reference sequence(s) to reduce or convert the same amount of product.
  • the improved property of the recombinant carbonic anhydrase polypeptide is with respect to its stability or thermostability. In some embodiments, the recombinant carbonic anhydrase polypeptide has more than one improved property, such as a combination of enzyme activity and thermostability.
  • the recombinant carbonic anhydrase polypeptides are equivalent to or improved as compared to wild-type of SEQ ID NO: 2 with respect to their activity, i.e., rate or ability of convert substrate to product under certain conditions.
  • Exemplary polypeptides that are capable of converting the substrate (CO 2 ) to the product (HCO 3 1 ) with an activity (e.g., rate of conversion) that is equivalent to or improved over wild-type include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to any one of SEQ ID NOs: 6, 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 36, 38, 40, 42, 44, and 46.
  • Exemplary polypeptides that are capable of converting the substrate to the product at a rate that is at least about 1.1 -fold improved as compared to the wild-type include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to SEQ ID NO: 6, 8, 10, 18, 22, 24, 26, 28, 30, 40, 42, and 44.
  • Exemplary polypeptides that are capable of converting the substrate to the product at a rate that is at least about 1.2-fold improved as compared to the wild-type include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to SEQ ID NO: 6, 8, 18, 22, 24, 26, 28, 40, 42, and 44.
  • Exemplary polypeptides that are capable of converting the substrate to the product at a rate that is at least about 1.3-fold improved as compared to the wild-type include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to SEQ ID NO: 6, 8, 24, 26, 40, 42, and 44.
  • Exemplary polypeptides that are capable of converting the substrate to the product at a rate that is at least about 1.4-fold improved as compared to the wild-type include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to SEQ ID NO: 6, 24, and 42.
  • Table 2 below provides a list of the SEQ ID NOs for and specific amino acid residue difference for specific exemplifications of recombinant (or engineered) carbonic anhydrases having improved properties relative to the reference polypeptide of SEQ ID NO: 2.
  • Improved enzyme properties e.g., improved activity, thermal stability, base stability, solvent stability, exhibited by these engineered polypeptides are described in the Examples below (see e.g., Example 7, Table 4, Fig. 4).
  • each row lists two SEQ ID NOs, where the odd number refers to the nucleotide sequence that encodes the amino acid sequence provided by the even number. All sequences below are derived from the wild-type Methanobacterium thermoautotrophicus str. Delta H carbonic anhydrase sequences (SEQ ID NO: 1 and SEQ ID NO: 2).
  • the improved recombinant carbonic anhydrase polypeptides herein comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical as compared to SEQ ID NO:2 and comprises at least one amino acid substitution listed in Table 2.
  • Such improved carbonic anhydrase polypeptides disclosed herein may further comprise additional modifications, including substitutions, deletions, insertions, or combinations thereof.
  • substitutions can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.
  • these recombinant carbonic anhydrase polypeptides can have optionally from about 1 - 2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-1 1, 1-12, 1-14, 1-15, 1- 16, 1- 18, 1-20, 1-22, 1-24, 1-25, 1- 30, 1-35 or about 1-40 amino acid residue differences at other amino acid residues.
  • the number of modifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.
  • the present disclosure also contemplates engineered carbonic anhydrase polypeptides comprising mutations based on locations or regions in the structure of the parent polypeptide.
  • a variant of a parent polypeptide can include an amino acid substitution at a particular residue at a location in the structure of the parent polypeptide as identified in Table 3.
  • Exemplary substitutions at each of the relevant locations include those identified in Table 2.
  • a recombinant carbonic anhydrase polypeptide of the present disclosure is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2, and has, at the position corresponding to the indicated position of SEQ ID NO: 2, at least one of the following features (e.g., amino acid substitutions): residue at position corresponding to X2 is a cysteine (C); residue at position corresponding to X8 is a non-polar residue; residue at position corresponding to X10 is a non-polar residue; residue at position corresponding to X13 is a cysteine (C); residue at position corresponding to X17 is a non
  • residue at position corresponding to X126 is an aromatic residue; residue at position corresponding to X147 is an aromatic residue; residue at position corresponding to X153 is an acidic residue; residue at position corresponding to XI 55 is a constrained residue; or residue at position corresponding to XI 59 is a polar residue.
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is C; residue at position corresponding to X8 is L; residue at position corresponding to X10 is G; residue at position corresponding to X13 is C; residue at position corresponding to XI 7 is V; residue at position corresponding to X22 is G or V; residue at position corresponding to X23 is Y; residue at position corresponding to X24 is E; residue at position corresponding to X25 is
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is a cysteine (C); residue at position corresponding to X10 is a non-polar residue; residue at position corresponding to X13 is a cysteine (C); residue at position corresponding to X17 is a non-polar residue; residue at position corresponding to X22 is a non-polar residue; residue at position corresponding to X23 is an aromatic residue; residue at position corresponding to X24
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is C; residue at position corresponding to X10 is G; residue at position corresponding to X13 is C; residue at position corresponding to X17 is V; residue at position corresponding to X22 is G or V; residue at position corresponding to X23 is Y; residue at position corresponding to X24 is E; residue at position corresponding to X25 is P; residue at position corresponding to X28 is P
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one or more of the following features: residue at position corresponding to X2 is C; residue at position corresponding to X8 is L; residue at position corresponding to X10 is G; residue at position corresponding to X13 is C; residue at position corresponding to XI 7 is V; residue at position corresponding to X22 is G or V; residue at position corresponding to X25 is P; residue at position corresponding to X28 is P; residue at position corresponding to X40 is T
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one of the following features or sets of features: (a) residue at position corresponding to X2 is a cysteine; (b) residue at position corresponding to X8 is a non-polar residue; residue at position corresponding to X85 is a basic residue; and residue at position corresponding to X126 is an aromatic residue; (c) residue at position corresponding to XI 0 is a non-polar residue; and residue at position corresponding to X75 is a non-polar residue
  • the recombinant carbonic anhydrase polypeptide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2 and comprises an amino acid sequence that includes one of the following features or sets of features: (a) residue at position corresponding to X2 is C; (b) residue at position corresponding to X8 is L; residue at position corresponding to X85 is K; and residue at position corresponding to X126 is F; (c) residue at position corresponding to X10 is G; and residue at position corresponding to X75 is L; (d) residue at position corresponding to X8 is L; and residue at position corresponding to X75
  • a recombinant carbonic anhydrase polypeptide of the present disclosure comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO:2, and has at least one of the following amino acid substitutions: R2C, I8L, D10G, R13C, D17V, D22V, D22G, L23Y, S24E, D25P, H28P, L32S, S40T, G53K, D69E, A75L, A76V, N85K, V106A, N121D, S123K, V126F, S 147H, S 153D, G155P, and
  • the present disclosure provides a recombinant carbonic anhydrase polypeptide capable of hydrating carbon dioxide to bicarbonate with improved activity relative to a reference ⁇ -class carbonic anhydrase polypeptide of SEQ ID NO:2, wherein the recombinant carbonic anhydrase polypeptide comprises an amino sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any one of the engineered carbonic anhydrase polypeptides of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46.
  • the amino acid differences are at positions other than those positions identified as having amino acid residue differences for the specific recombinant carbonic anhydrase polypeptide sequence as shown in Table 2 (e.g., positions X13, X76, and X121 for SEQ ID NO: 42).
  • any of the recombinant carbonic anhydrase polypeptides disclosed herein may further comprise additional modifications, including substitutions, deletions, insertions, or combinations thereof.
  • the substitutions can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.
  • these carbonic anhydrase polypeptides can have optionally from about 1-2, 1-3, 1-4, 1- 5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-1 1, 1-12, 1-14, 1-15, 1- 16, 1- 18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40 amino acid differences relative to the reference polypeptide of SEQ ID NO: 2 at positions other than X2, X8, XI 0, XI 3, XI 7, X22, X23, X24, X25, X28, X32, X40, X53, X69, X75, X76, X85, X106, X121, X123, X126, X147, X153, X155, and/or X159.
  • the number of additional amino acid differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.
  • the present disclosure provides a recombinant carbonic anhydrase polypeptide comprising a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a portion of the reference sequence of SEQ ID NO:2, the portion comprising a contiguous sequence of 25, 50, 75, 100, or more than 100 contiguous amino acids of SEQ ID NO:2.
  • a recombinant carbonic anhydrase polypeptide of the present disclosure comprises a sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO:2 or any one of the engineered carbonic anhydrase polypeptides of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46, and comprises additional amino acids at the amino-terminus and/or the carboxyl terminus.
  • the improved engineered carbonic anhydrase enzymes can comprise deletions of the naturally occurring carbonic anhydrase polypeptides as well as deletions of other improved carbonic anhydrase polypeptides.
  • each of the improved engineered carbonic anhydrase enzymes described herein can comprise deletions of the polypeptides described herein.
  • the deletions can comprise one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the recombinant carbonic anhydrase polypeptides, as long as the functional activity of the recombinant carbonic anhydrase activity is maintained.
  • the deletions can comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-1 1, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40 amino acid residues.
  • the recombinant carbonic anhydrase polypeptides of the disclosure can be in the form of fusion polypeptides in which the carbonic anhydrase polypeptides are fused to other polypeptides, such as antibody tags (e.g., myc epitope) or purifications sequences (e.g., His tags).
  • the recombinant carbonic anhydrase polypeptides can be used with or without fusions to other polypeptides.
  • polypeptides described herein are not restricted to the genetically encoded amino acids.
  • polypeptides described herein may be comprised, either in whole or in part, of naturally-occurring and/or synthetic non-encoded amino acids.
  • non-encoded amino acids of which the polypeptides described herein may be comprised include, but are not limited to: the D-enantiomers of the genetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr); a-aminoisobutyric acid (Aib); ⁇ -aminohexanoic acid (Aha); ⁇ -aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3-chlorophenylalanine (Mcf
  • penicillamine Pen
  • Teic l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
  • Thi ⁇ -2-thienylalanine
  • Mso methionine sulfoxide
  • nArg N(w)-nitroarginine
  • hLys homolysine
  • phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer); phosphothreonine (pThr);
  • homoaspartic acid hAsp
  • homoglutanic acid hGlu
  • l-aminocyclopent-(2 or 3)-ene-4 carboxylic acid pipecolic acid (PA), azetidine-3-carboxylic acid (ACA); l-aminocyclopentane-3-carboxylic acid
  • allylglycine aOly
  • propargylglycine pgGly
  • homoalanine hAla
  • norvaline nVal
  • homoleucine hLeu
  • homovaline hVal
  • homoisolencine hlle
  • homoarginine hArg
  • N acetyl lysine AcLys
  • 2,4 diaminobutyric acid Dbu
  • 2,3-diaminobutyric acid Dab
  • N-methylvaline MeVal
  • homocysteine hCys
  • homoserine hSer
  • Hyp hydroxyproline
  • homoproline hPro
  • amino acids or residues bearing side chain protecting groups may also comprise the polypeptides described herein.
  • protected amino acids include (protecting groups listed in parentheses), but are not limited to: Arg(tos), Cys(methylbenzyl), Cys
  • Non-encoding amino acids that are conformationally constrained of which the polypeptides described herein may be composed include, but are not limited to, N-methyl amino acids (L- configuration); l-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3- carboxylic acid; homoproline (hPro); and 1 -aminocyclopentane-3 -carboxylic acid.
  • the present disclosure provides polynucleotides encoding the engineered carbonic anhydrase enzymes.
  • the polynucleotides may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide.
  • Expression constructs containing a heterologous polynucleotide encoding the engineered carbonic anhydrase can be introduced into appropriate host cells to express the corresponding carbonic anhydrase polypeptide.
  • the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences presented in Table 2.
  • the polynucleotide comprises a nucleotide sequence encoding a recombinant carbonic anhydrase polypeptide with an amino acid sequence that has at least about 80% or more sequence identity, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more sequence identity to any of the engineered carbonic anhydrase polypeptides described herein, i.e., a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46.
  • the codons are preferably selected to fit the host cell in which the protein is being produced.
  • preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells.
  • the polynucleotide of SEQ ID NO: 1 could be codon optimized for expression in E. coli, but otherwise encode the naturally occurring carbonic anhydrase of Methanobacterium thermoautotrophicus sir. Delta H.
  • codon optimized polynucleotides encoding the recombinant carbonic anhydrase enzymes may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full length coding region.
  • the polynucleotides encoding the engineered carbonic anhydrase are selected from SEQ ID NO: 5, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45. In some embodiments, the polynucleotides encoding the engineered carbonic anhydrases are capable of hybridizing under highly stringent conditions to a polynucleotide comprising SEQ ID NO: 35, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45.
  • polynucleotides encode some of the polypeptides represented by the amino acid sequences listed in Table 2.
  • the polynucleotides comprise polynucleotides that encode the polypeptides described herein but have about 80% or more sequence identity, about 85% or more sequence identity, about 90% or more sequence identity, about 95% or more sequence identity, about 98% or more sequence identity, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered carbonic anhydrase.
  • the reference polynucleotide is selected from polynucleotide sequences represented by SEQ ID NO: 5, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45.
  • the present disclosure provides a polynucleotide encoding a recombinant carbonic anhydrase polypeptide capable of improved expression in a host cell relative to a reference polynucleotide encoding a 171 amino acid ⁇ -class carbonic anhydrase polypeptide of SEQ ID NO: 4 (described in Example 2 as "Cab002").
  • the polynucleotide capable of improved expression in a host cell encodes a polypeptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 4 and five amino terminal residues corresponding to XI through X5 of SEQ ID NO: 2.
  • the polynucleotide capable of improved expression in a host cell is capable of producing at least 1.5-fold to 2.0-fold more protein than expressed by the reference polynucleotide encoding the 171 amino acid ⁇ -class carbonic anhydrase polypeptide of SEQ ID NO: 4.
  • the polynucleotide capable of improved expression in host cell encodes a recombinant carbonic anhydrase of at least 176 amino acids, and in further embodiments can comprise one or more of the amino acid residue differences relative to SEQ ID NO: 2 described herein.
  • these substitutions at one or more positions are selected from X2, X8, X10, X13, X17, X22, X23, X24, X25, X28, X32, X40, X53, X69, X75, X76, X85, X106, X121, X123, X126, X147, X153, X155, and X159, and can include any one of R2C, I8L, D10G, R13C, D17V, D22V, D22G, L23Y, S24E, D25P, H28P, L32S, S40T, G53K, D69E, A75L, A76V, N85K, V106A, N121D, S123K, V126F, S 147H, S 153D, G155P, and L159S.
  • An isolated polynucleotide encoding an improved recombinant carbonic anhydrase polypeptide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector.
  • the techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. Guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2006.
  • suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa- Kamaroff et al., 1978, Proc.
  • suitable promoters for directing the transcription of the nucleic acid constructs of the present disclosure include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for
  • useful promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO- 1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokinase
  • ADH2/GAP Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • Saccharomyces cerevisiae 3-phosphoglycerate kinase Other useful promoters for yeast host cells are described by Romanos et al, 1992, Yeast 8:423
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
  • exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
  • Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3 -phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
  • the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell.
  • the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used.
  • Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
  • Suitable leaders for yeast host cells are obtained from the genes for
  • ADH2/GAP dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
  • Exemplary polyadenylation sequences for filamentous fungal host cells can be from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, wad Aspergillus niger alpha-glucosidase.
  • Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol Cell Bio 15:5983-5990.
  • the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of an engineered carbonic anhydrase polypeptide and directs the encoded polypeptide into the cell's secretory pathway (e.g., a secretion signal).
  • the 5'-end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide.
  • the 5'-end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence.
  • the foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.
  • the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide.
  • any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.
  • an engineered carbonic anhydrase polypeptide of the invention can be operably linked to a signal sequence derived from a bacterial species such as a signal peptide sequence derived from a Bacillus (e.g., B.
  • Exemplary signal peptide coding regions useful for secretion bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NC1B 1 1837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (e.g., the signal peptides nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.
  • Exemplary signal peptide coding regions useful for secretion from filamentous fungal host cells can be the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
  • Exemplary signal peptides useful for secretion from yeast host cells can be from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et ah, 1992, supra.
  • the control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (WO 95/33836).
  • the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
  • regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • suitable regulatory sequences include the lac, tac, and trp operator systems.
  • suitable regulatory systems include, as examples, the ADH2 system or GAL1 system.
  • suitable regulatory sequences include the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.
  • regulatory sequences are those which allow for gene amplification.
  • these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals.
  • the nucleic acid sequence encoding the recombinant carbonic anhydrase polypeptide of the present disclosure would be operably linked with the regulatory sequence.
  • the present disclosure is also directed to a recombinant expression vector comprising a polynucleotide encoding an engineered carbonic anhydrase polypeptide or a variant thereof, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced.
  • the various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites.
  • the nucleic acid sequence of the present disclosure may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide sequence.
  • 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 vectors may be linear or closed circular plasmids.
  • the expression 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.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • the expression vector of the present invention preferably contains one or more selectable markers, which permit easy selection of transformed cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
  • Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • amdS acetamidase
  • argB ornithine carbamoyltransferase
  • bar phosphinothricin acetyltransferase
  • hph hygromycin phosphotransferase
  • niaD nitrate reductase
  • Embodiments for use in an Aspergillus cell include the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
  • the expression vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.
  • the expression vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell.
  • the additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s).
  • the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with 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.
  • the integrational elements may be non-encoding or encoding nucleic acid sequences.
  • the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • bacterial origins of replication are P15A ori or the origins of replication of plasmids pBR322, pUC19, pACYC177 (which plasmid has the P15A ori), or pACYC184 permitting replication in E. coli, and pUBl 10, pE194, pTA1060, or ⁇ permitting replication in Bacillus.
  • origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS 1 , ARS4, the combination of ARS 1 and CEN3, and the combination of ARS4 and CEN6.
  • the origin of replication may be one having a mutation which makes it's functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proc Natl Acad Sci. USA 75: 1433).
  • More than one copy of a nucleic acid sequence of the present invention may be inserted into the host cell to increase production of the gene product.
  • An increase in the copy number of the nucleic acid sequence 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 nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • Suitable commercial expression vectors include p3xFLAGTMTM expression vectors from Sigma- Aldrich Chemicals, St. Louis MO., which includes a CMV promoter and hGH polyadenylation site for expression in mammalian host cells and a pBR322 origin of replication and ampicillin resistance markers for amplification in E. coli.
  • Other suitable expression vectors are Bacillus megaterium shuttle vector pMM1525 (Boca Scientific Inc.
  • Boca Raton, FL pBluescriptll SK(-) and pBK-CMV, which are commercially available from Stratagene, LaJolla CA, and plasmids which are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (Lathe et al, 1987, Gene 57: 193-201).
  • the present disclosure provides a host cell comprising a polynucleotide encoding a recombinant carbonic anhydrase polypeptide of the present disclosure, the polynucleotide being operatively linked to one or more control sequences for expression of the recombinant carbonic anhydrase enzyme in the host cell.
  • Host cells for use in expressing the recombinant carbonic anhydrase polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E.
  • yeast cells e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201 178)
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells
  • plant cells e.g., CHO, COS, BHK, 293, and Bowes melanoma cells
  • the host cell is a bacterial host cell of the Bacillus species, e.g., B. thuringiensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B.coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis , B. clausii, B. stearothermophilus, B. halodurans and B. amyloliquefaciens .
  • Bacillus species e.g., B. thuringiensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B.coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis , B. clausii, B. stearothermophil
  • Polynucleotides for expression of the recombinant carbonic anhydrase may be introduced into cells by various methods known in the art. Techniques include among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells will be apparent to the skilled artisan.
  • An exemplary host cell is Escherichia coli W31 10.
  • the expression vector was created by operatively linking a polynucleotide encoding an improved recombinant carbonic anhydrase into the plasmid pCKl 10900 ⁇ see, US application publication 20040137585) operatively linked to the lac promoter under control of the lacl repressor.
  • the expression vector also contained the PI 5a origin of replication and the chloramphenicol resistance gene.
  • Cells containing the subject polynucleotide in Escherichia coli W31 10 were isolated by subjecting the cells to chloramphenicol selection.
  • Another exemplary host cell is Escherichia coli BL21.
  • the naturally-occurring carbonic anhydrase enzyme that catalyzes the hydration reaction is obtained (or derived) from
  • thermoautotrophicus sir. Delta H Methanobacterium thermoautotrophicus sir. Delta H.
  • polynucleotide sequence is codon optimized to enhance expression of the recombinant carbonic anhydrase in a specified host cell.
  • the parental polynucleotide sequence encoding the wild-type carbonic anhydrase polypeptide of M. thermoautotrophicus (SEQ ID NO: 1), can be assembled from oligonucleotides based upon that sequence or from oligonucleotides comprising a codon-optimized coding sequence for expression in a specified host cell, e.g., an E. coli host cell.
  • the polynucleotide can be cloned into an expression vector, placing the expression of the recombinant carbonic anhydrase gene under the control of the lac promoter and lacl repressor gene. Clones expressing the active carbonic anhydrase in E. coli can be identified and the genes sequenced to confirm their identity.
  • the engineered carbonic anhydrase can be obtained by subjecting the polynucleoticde encoding the naturally occurring carbonic anhydrase to mutagenesis and/or directed evolution methods, as discussed above.
  • An exemplary directed evolution technique is mutagenesis and/or DNA shuffling as described in Stemmer, 1994, Proc Natl Acad Sci USA 91 : 10747- 10751 ; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. 6,537,746.
  • directed evolution procedures include, among others, staggered extension process (StEP), in vitro recombination (Zhao et al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al, 1994, PCR Methods Appl. 3:S 136-S140), and cassette mutagenesis (Black et al, 1996, Proc Natl Acad Sci USA 93:3525-3529).
  • Methodologies for screening and identifying polypeptides for desired activities are useful in the preparation of new compounds such as modified enzymes and/or new pharmaceuticals.
  • Directed evolution can be used to discover or enhance activity of polypeptides of commercial interest. For example, if the activity of a known catalyst is insufficient for a commercial process, directed evolution and/or other protein engineering technologies may be used to make appropriate
  • Improvements to the catalyst to improve activity on the substrate of interest can be developed to enhance an active enzyme and/or to optimize a microbe/enzyme for scaled-up production.
  • Current methodologies are often limited by time and cost factors. In some instances, it may take months or years, at great expense, to find a new polypeptide with the desired activity, if one is ever found. Furthermore, the number of polypeptide variants that must be screened is often cumbersome. Thus, there is a long felt need for compositions and methods used to identify novel polypeptide variants having a desired activity.
  • the clones obtained following mutagenesis treatment are screened for engineered carbonic anhydrase having a desired improved enzyme property.
  • Measuring enzyme activity from the expression libraries can be performed using the standard biochemistry technique of monitoring changes in pH, either directly or indirectly, as indicated in the Examples.
  • activity of the carbonic anhydrases of the disclosure may be measured using either the forward or reverse reactions depicted in Scheme 1.
  • the improved enzyme property desired is thermal stability
  • enzyme activity may be measured after subjecting the enzyme preparations to a defined temperature for a defined period of time and measuring the amount of enzyme activity remaining after heat treatments.
  • Clones containing a polynucleotide encoding a recombinant carbonic anhydrase are then isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.
  • the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence.
  • polynucleotides and oligonucleotides of the invention can be prepared by chemical synthesis using, e.g., the classical phosphoramidite method described by Beaucage et al, 1981, Tet Lett 22: 1859-69, or the method described by Matthes et al, 1984, EMBO J.
  • oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • essentially any nucleic acid can be obtained from any of a variety of commercial sources, such as The Midland Certified Reagent Company, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGen Inc.
  • the disclosure provides a method of producing a recombinant carbonic anhydrase polypeptide of the present disclosure, wherein said method comprises the steps of: (a) transforming a host cell with an expression vector polynucleotide encoding the recombinant carbonic anhydrase polypeptide; (b) culturing said transformed host cell under conditions whereby said recombinant carbonic anhydrase polypeptide is produced by said host cell; and (c) recovering said recombinant carbonic anhydrase polypeptide from said host cells.
  • the method of producing the recombinant carbonic anhydrase may be carried out wherein said expression vector comprises a secretion signal, and said cell is cultured under conditions whereby the recombinant carbonic anhydrase polypeptide is secreted from the cell.
  • the expression vector comprises a polynucleotide encoding a secretion signal.
  • Engineered carbonic anhydrase enzymes expressed in a host cell can be recovered from the cells and or the culture medium using any one or more of the well known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra- centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available under the trade name CelLytic BTM from Sigma- Aldrich of St. Louis MO.
  • Chromatographic techniques for isolation of the recombinant carbonic anhydrase polypeptide include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art.
  • affinity techniques may be used to isolate the improved carbonic anhydrase enzymes.
  • any antibody which specifically binds the recombinant carbonic anhydrase polypeptide may be used.
  • various host animals including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a polypeptide of the disclosure.
  • the polypeptide may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette Guerin) and Corynebacterium parvum.
  • BCG Bacilli Calmette Guerin
  • Corynebacterium parvum bacilli Calmette Guerin
  • a carbonic anhydrase of the present disclosure can be used to hydrate carbon dioxide in the form of bicarbonate and a proton, which in turn, will be converted to carbonate and/or a mixture of bicarbonate and carbonate at an elevated pH (e.g., about pH 9.0 to about pH 1 1.0).
  • a carbonic anhydrase of the disclosure can be used to dehydrate sequestered carbon dioxide by reaction at a relatively acidic pH.
  • the present disclosure provides methods for removing (e.g., extracting and sequestering) carbon dioxide from a gas stream comprising the step of contacting the gas stream with a solution comprising a recombinant carbonic anhydrase polypeptide of a recombinant carbonic anhydrase of the disclosure having an improved property (e.g., increased activity and/or thermostability) under suitable conditions, whereby carbon dioxide is removed from the gas stream by dissolving into the solution where it is converted to hydrated carbon dioxide by the recombinant carbonic anhydrase.
  • an improved property e.g., increased activity and/or thermostability
  • the method can comprise the further step of isolating the solution comprising the hydrated carbon dioxide and contacting the isolated solution with hydrogen ions and a recombinant carbonic anhydrase polypeptide, thereby converting the hydrated carbon dioxide to carbon dioxide gas and water.
  • the solution can be removed from contact with the gas stream (e.g., isolated after some desired level of hydrated carbon dioxide is reached) and further treated with a recombinant carbonic anhydrase to convert the bicarbonate in solution into carbon dioxide gas, which is then released from the solution and captured e.g., into a pressurized chamber.
  • the methods of carrying out the carbonic anhydrase-catalyzed hydration reactions described herein may be carried out under a range of suitable conditions.
  • suitable conditions can be determined by routine experimentation that includes, but is not limited to, contacting the solution containing the carbonic anhydrase polypeptide with C0 2 at an experimental condition (e.g., amine concentration, temperature, C0 2 loading) and then detecting the relevant activity (e.g., rate of C0 2 absorption), for example, using the methods described in the Examples provided herein.
  • carbonic anhydrase polypeptides disclosed herein are biocatalysts having an improved property (e.g., thermal stability, solvent stability, and/or base stability) that allows them to accelerate the absorption of carbon dioxide gas into a solution and/or accelerate subsequent desorption from the solution under a range of conditions.
  • the gas streams from which C0 2 removal is desirable are at elevated temperatures, and upon contacting a solution, as in the method disclosed herein, heat is also transferred and the solution temperature also is elevated.
  • the suitable conditions for carrying out the method comprise an elevated solution temperature.
  • elevated temperature further underscores the importance of using thermostable carbonic anhydrase polypeptides such as those disclosed herein.
  • the method of carbonic anhydrase catalyzed hydration (absorption) is typically carried out at a temperature in the range of from about 25°C to about 85°C or higher.
  • the reaction is carried out at a temperature in the range of from about 40°C to about 80°C. In still other embodiments, it is carried out at a temperature in the range of from about 50°C to about 75°C.
  • the carbonic anhydrase catalyzed dehydration (stripping) is typically carried out at a temperature in the range of from about 25°C to about 85°C or higher, optionally at reduced pressure.
  • the carbonic anhydrases and associated methods for removing (e.g., extracting and sequestering) carbon dioxide from a gas stream disclosed herein can be used in existing systems that use a solution for absorbing carbon dioxide from e.g., flue gas.
  • Equipment, processes, and methods for carbon dioxide capture and sequestration using solutions into which carbon dioxide is absorbed (i.e., captured by diffusing from gas stream into the liquid solution) and/or from which carbon dioxide is desorbed (i.e., extracted by diffusing from liquid solution into gas phase) are described in e.g., U.S. Pat. Nos.
  • the methods of removing carbon dioxide from a gas stream can be carried out wherein the solution is aqueous, or an aqueous co-solvent system.
  • the solution used is aqueous-solvent system comprising an organic solvent selected from monoethanolamine (“MEA”), methyldiethanolamine (“MDEA”), and
  • the co-solvent system comprises a ratio of water to organic solvent from about 90: 10 (v/v) to about 10:90 (v/v), in some embodiments, from about 80:20 to about 20:80 (v/v), in some embodiments, from about 70:30 (v/v) to about 30:70 (v/v), and in some embodiments, from about 60:40 (v/v) to about 40:60 (v/v).
  • the methods of removing carbon dioxide from a gas stream can be carried out wherein the recombinant carbonic anhydrase polypeptide is immobilized on a surface, for example wherein the enzyme is linked to the surface of a solid-phase particle in the solution.
  • Methods for linking (covalently or non-covalently) enzymes to solid-phase particles e.g., porous or non-porous beads, or solid supports
  • Methods for treating a gas stream using immobilized enzymes are described in e.g., U.S. patent no. 6,143,556, U.S. patent publication no.
  • the methods for removing carbon dioxide from a gas stream can be carried out wherein the recombinant carbonic anhydrase polypeptide is immobilized on a surface, for example wherein the enzyme is linked to the surface of a solid-phase particle (e.g., beads) in the solution.
  • the methods using immobilized polypeptides can be carried out wherein the method further comprises a step of isolating or separating the immobilized carbonic anhydrase from the solution.
  • the solution After separating the immobilized carbonic anhydrase from the solution, the solution can be treated to conditions that may inactivate the enzyme, e.g., desorption of CO 2 at high temperatures. Further, the separately retained immobilized enzyme can be added to another solution and reused.
  • the method can be carried out wherein the carbonic anhydrase comprises the improved property at least 1.2-fold, at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 25-fold increased activity of hydrating carbon dioxide or dehydrating bicarbonate under suitable conditions.
  • the suitable conditions used in the method can comprise a concentration of the carbonic anhydrase polypeptide of from about 0.1 g/L to about 10 g/L, about 0.25 g/L to about 7.5 g/L, about 0.5 g/L to about 5 g/L, less than 10 g/L, less than about 5 g/L, or less than about 2.5 g/L.
  • the recombinant carbonic anhydrase polypeptides having improved activity, thermal stability, solvent stability and/or base stability, relative the reference polypeptide of SEQ ID NO: 2 described herein, including those exemplified in Table 2, can be used in the methods of removing carbon dioxide from a gas stream.
  • the methods can be carried out using a recombinant carbonic anhydrase polypeptide that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence of SEQ ID NO: 2, and has, at the position corresponding to the indicated position of SEQ ID NO: 2, at least one of the following features (e.g., amino acid substitutions): residue at position corresponding to X2 is a cysteine (C); residue at position corresponding to X8 is a non-polar residue; residue at position corresponding to X10 is a non-polar residue; residue at position corresponding to X13 is a cysteine (C); residue at position corresponding to X17 is
  • the methods can use an improved recombinant carbonic anhydrase polypeptide of the present disclosure that comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the Methanobacterium thermoautotrophicus str.
  • Delta H carbonic anhydrase of SEQ ID NO:2 and that further comprises, as compared to the amino acid sequence of the Methanobacterium thermoautotrophicus str.
  • Delta H carbonic anhydrase of SEQ ID NO:2 at least one amino acid substitution selected from the group consisting of: R2C, I8L, D10G, R13C, D17V, D22V, D22G, L23Y, S24E, D25P, H28P, L32S, S40T, G53K, D69E, A75L, A76V, N85K, V106A, N121D, S123K, V126F, S 147H, S 153D, G155P, and L159S.
  • the recombinant carbonic anhydrase polypeptide used to carry out the method of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46.
  • the foregoing recombinant carbonic anhydrase polypeptides useful in the method of removing carbon dioxide may further comprise additional modifications, including substitutions, deletions, insertions, or combinations thereof.
  • the substitutions can be non-conservative
  • these carbonic anhydrase polypeptides can have optionally from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1- 10, 1-1 1, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40 mutations at other amino acid residues.
  • the number of modifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.
  • the recombinant carbonic anhydrase polypeptides useful for carrying a method of removing carbon dioxide exhibit an activity that is equivalent to or greater than that of the polypeptide of SEQ ID NO: 2.
  • said improved activity is determined after a heat challenge of heating the recombinant carbonic anhydrase polypeptide and the reference polypeptide in a solution at a temperature of 75°C for about 30 minutes, wherein the solution is selected from a solution comprising 150 mM K 2 CO 3 at pH 10.9, and a solution comprising 1 M AMP at pH 9.7.
  • Exemplary polypeptides with such properties include, but are not limited to, polypeptides which comprise an amino acid sequence corresponding to SEQ ID NO: 6, 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 36, 38, 40, 42, 44, and 46.
  • the recombinant carbonic anhydrase polypeptides useful in the methods of removing carbon dioxide of the disclosure exhibit activity at least 1.05-times, 1.1 -times, 1.2-times, 1.3-times, 1.4-times increased relative to the activity polypeptide of SEQ ID NO: 2.
  • Exemplary polypeptides exhibiting at least about 1.1 -fold improved activity as compared to the wild- type of SEQ ID NO: 2 include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to SEQ ID NO: 6, 8, 10, 18, 22, 24, 26, 28, 30, 40, 42, and 44.
  • Exemplary polypeptides exhibiting at least about 1.2-fold improved activity as compared to the wild-type of SEQ ID NO: 2 include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to SEQ ID NO: 6, 8, 18, 22, 24, 26, 28, 40, 42, and 44.
  • Exemplary polypeptides exhibiting at least about 1.3-fold improved activity as compared to the wild-type of SEQ ID NO: 2 include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to SEQ ID NO: 6, 8, 24, 26, 40, 42, and 44.
  • Exemplary polypeptides exhibiting at least about 1.4-fold improved activity as compared to the wild-type of SEQ ID NO: 2 include but are not limited to, polypeptides that comprise the amino acid sequences corresponding to SEQ ID NO: 6, 24, and 42.
  • the methods of the present disclosure use a recombinant carbonic anhydrase comprising the amino acid sequence of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46.
  • the carbonic anhydrase-catalyzed hydration reactions described herein are carried out in a solvent or solution.
  • suitable solvents include water (e.g., aqueous solution), and mixtures of water and an organic reagent or solvent (e.g., monoethanolamine (MEA), methyldiethanolamine (MDEA), and 2-aminomethylpropanolamine (AMP), and the like) or aqueous carbonate mixtures.
  • aqueous solvents including water and aqueous co- solvent systems, are used.
  • Exemplary aqueous co-solvent systems have water and one or more organic solvent.
  • an organic solvent component of an aqueous co-solvent system is selected such that it does not completely inactivate the recombinant carbonic anhydrase enzyme.
  • Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified engineered carbonic anhydrase enzyme in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.
  • the organic solvent component of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases.
  • the ratio of water to organic solvent in the co-solvent system is in the range of from about 90: 10 (v/v) to about 10:90 (v/v), and typically from about 80:20 (v/v) and 20:80 (v/v).
  • the co-solvent system may be pre-formed prior to addition to the reaction mixture, or it may be formed in situ in the reaction vessel.
  • the aqueous solvent may be pH-buffered or unbuffered.
  • hydration of carbon dioxide can be carried out at a pH of about pH 9 or above or at a pH of about pH 10 or above, usually in the range of from about 8 to about 1 1.
  • release (dehydration) of captured carbon dioxide e.g., as bicarbonate
  • the dehydration is carried out at a pH of about 8 or below, often in the range of from about pH 6 to about pH 8.
  • the pH of the reaction mixture may change.
  • the pH of the reaction mixture may be maintained at a desired pH or within a desired pH range by the addition of an acid or a base during the course of the reaction.
  • the pH may be controlled by using an aqueous solvent that comprises a buffer.
  • Suitable buffers to maintain desired pH ranges are known in the art and include, for example, carbonate, HEPES, triethanolamine buffer, and the like. Combinations of buffering and acid or base addition may also be used.
  • the present disclosure has illustrated the use of the method in solutions comprising a high concentration of an organic solvent comprising an amine compound (e.g., AMP) and/or carbonate ion.
  • an organic solvent comprising an amine compound (e.g., AMP) and/or carbonate ion.
  • solutions comprising other compounds are known to facilitate the absorption of CO 2 from a gas stream, and it is contemplated that the present methods could be used with such solutions.
  • solutions comprising a variety of different organic solvents comprising amine compounds are known.
  • solvents comprising amine compounds that facilitate C0 2 absorption from a gas stream into a solution are described in e.g., PCT Publ. No.
  • the methods of removing carbon dioxide from a gas stream can be carried out wherein the solution comprises an organic solvent that comprises an amine compound, preferably an amine compound that exhibits improved thermodynamic and kinetic properties for the absorption of CO 2 .
  • the suitable conditions comprise a solution of a co-solvent system comprising an amine compound
  • the amine compound can be selected from: 2-amino-2-2-(2- aminoethylamino)ethanol (AEE), 2-amino-2-hydroxymethyl- l,3-propanediol (AHPD), methyl- 1- propanol (AMP), diethanolamine (DEA), diisopropanolamine (DIP A), N-hydroxyethylpiperazine (HEP), N-methyldiethanolamine (MDEA), monoethanolamine (MEA), N-methylpiperazine (MP), piperazine, piperidine, 2-(2-tert-butylaminoethoxy)ethanol (TBEE), triethanolamine (TEA), triisopropanolamine (TIA), tris, 2-(2-aminoethoxy)ethanol, 2-(2-tert-butylaminopropoxy)ethanol, 2- (2-tert-amylaminoethoxy)ethanol,
  • AEE 2-amino-
  • the amine compound is selected from AMP, MEA, MDEA, and TIA, and in one preferred embodiment the solution comprises the amine compound MDEA.
  • the suitable conditions can comprise an amine compound concentration of from about 1 M to about 10 M, from about 2 M to about 8 M, from about 2.5 M to about 6.5 M, from about 3 M to about 5 M, at least about 2 M, at least about 3 M, at least about 4.2 M, or at least about 5 M.
  • Another known process for capturing CO 2 from a gas stream uses a solution containing a high concentration of ammonia. Due to the high volatility of ammonia vapor the process is typically run at relatively low temperatures, and is sometimes referred to as a "chilled ammonia" process. Methods and conditions of the chilled ammonia process for CO 2 capture from a flue gas stream are described in e.g., U.S. Pat. No. 7,641,717 B2, and U.S. Pat. Publ. No. 2009/0155889A1, each which is hereby incorporated by reference herein.
  • a solution containing ammonia is used to facilitate carbon dioxide absorption from the gas streams.
  • ammonia solutions can be used under suitable conditions comprising an ammonia concentration of about 1 M to about 8 M, from about 2 M to about 7 M, from about 3 M to about 6 M, at least about 1 M, at least about 2 M, at least about 3 M, at least about 4 M, or at least about 5 M, or at least about 5.6 M.
  • the solution comprising ammonia can be used at chilled temperatures (e.g., for absorption) and/or elevated temperatures (e.g., for desorption of carbon dioxide).
  • the method using a solution comprising ammonia can be carried out wherein the suitable conditions comprise a solution temperature of from about 0°C to about 20°C, from about 0°C to about 10°C, from about 5°C to about 15°C, from about 8°C to about 12°C, less than about 15°C, or less than about 10°C.
  • the method of removing CO 2 from a gas stream can be carried out wherein the suitable conditions comprise a solution comprising carbonate ion at a concentration of about 0.1 M CO 3 2" to about 5 M CO 3 2" , from about 0.2 M C0 3 2" to about 4 M C0 3 2" , or from about 0.3 M C0 3 2" to about 3 M C0 3 2" , at least about 0.2 M Na 2 C0 3 , at least about 0.4 M Na 2 C0 3 , or at least about 1 M Na 2 C0 3 .
  • the suitable conditions comprise a solution comprising carbonate ion at a concentration of about 0.1 M CO 3 2" to about 5 M CO 3 2" , from about 0.2 M C0 3 2" to about 4 M C0 3 2" , or from about 0.3 M C0 3 2" to about 3 M C0 3 2" , at least about 0.2 M Na 2 C0 3 , at least about 0.4 M Na 2 C0 3 , or at least about 1 M Na 2 C
  • the engineered carbonic anhydrase enzyme may be added to the reaction mixture in the form of the purified enzymes, whole cells transformed with a gene encoding the enzyme, and/or cell extracts and/or lysates of such cells.
  • the method is carried out wherein the carbonic anhydrase polypeptide is added to the solution in the form of a powder (e.g., shake-flask powder, or DSP powder).
  • the powder may contain the polypeptide in partially purified or a highly purified form.
  • whole cells transformed with a gene encoding the engineered carbonic anhydrase enzyme or cell extracts and/or lysates thereof may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., a crude paste) forms.
  • the cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment or the like, followed by a desalting procedure prior to lyophilization (e.g., ultrafiltration, dialysis, and the like).
  • any of the cell preparations may be stabilized by crosslinking using known crosslinking agents, such as, for example, glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, and the like) or by the crosslinking of protein crystals or precipitated protein aggregate particles.
  • known crosslinking agents such as, for example, glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, and the like) or by the crosslinking of protein crystals or precipitated protein aggregate particles.
  • the ability of the carbonic anhydrase polypeptide to accelerate C0 2 absorption into or desorption from a solution can be affected by the mole ratio of CO 2 to other compounds already present in the solution, which is also referred to as the CO 2 loading of the solution and can be denoted by the mole ratio of CO 2 to the moles of the relevant CO 2 absorption mediating compound in the solution (e.g., AMP solvent), which is denoted by the term "a.”
  • the carbonic anhydrase polypeptides of the present disclosure can be used under a range of loading conditions which can be varied depending on the particular CO 2 absorption mediating compound used in the solution.
  • Example 1 Construction of Two Genes Encoding Wild Type ⁇ -Class Carbonic Anhydrase Enzymes (CabOO 1 and Cab002) of Methanothermobacter thermoautotrophicus strain Delta H and Expression Vectors
  • M. thermoautotrophicus (M. thermoautotrophicus) (see e.g., Smith et ah, J. Bacterid. 179(22) 7135-55 (1997)) indicated two possible starting methionine codons that would result in two versions of the genes, one 15 base pairs longer at the 5' end. The resulting two versions of the mature protein would differ in length by 5 residues.
  • Two DNA constructs coding for the carbonic anhydrase from M. thermoautotrophicus were synthesized based upon the genome sequence disclosed as GenBank Accession No. NC 000916.
  • a single microbial colony of E. coli containing a plasmid carrying the carbonic anhydrase gene of interest (e.g., CabOOl, Cab002) was inoculated into 50 ml Luria Bertani broth containing 30 ⁇ g/mL chloramphenicol and 1% glucose. Cells were grown overnight (at least 16 h) in an incubator at 30°C with shaking at 250 rpm.
  • the culture was diluted into 250 mL 2YT (16 g/L bacto-tryptone, lOg/L yeast extract, 5 g/L sodium chloride30 ⁇ g/mL chloramphenicol) in 1 L flask to an optical density at 600 nm (OD600) of 0.1 and allowed to grow at 30°C.
  • Expression of the carbonic anhydrase gene was induced with 1 mM IPTG, and ZnS0 4 added to a final concentration of 0.5 mM when the OD 6 oo of the culture was 0.6 to 0.8 and then the broth was incubated overnight (at least 16 h). Cells were harvested by centrifugation (5000 rpm, 15 min, 4°C) and the supernatant discarded.
  • the cell pellet was resuspended with 3 mL of lysis buffer per gram of cell wet weight and allowed to incubate at room temperature.
  • the lysis buffer consisted of 25 mM HEPES, 0.5 mg/mL lysozyme and 0.25 mg/mL polymyxin B sulfate (PMBS), pH 8.2.
  • the resuspended cells were then passed (two passes) through a Constant Systems Cell Disruptor System (Constant Systems, UK), at a pressure of 33.6 kpsi. Soluble and insoluble cell contents were separated by centrifugation at 12,000 rpm for 20 minutes at 4°C. The clarified lysate was then lyophilized and stored at -20 °C.
  • the activity assay was carried out in Whatman 96-well plates with 300 volume wells (GE Healthcare, Inc. Piscataway, NJ).
  • the assay reaction mix was prepared by adding 20 of the polypeptide containing solution (e.g., clarified lysate or previously heat-challenged clarified lysate) to a plate well containing 180 of the assay solution: 150 mM K 2 CO 3 , pH 10.9, 400 ⁇ phenolphthalein.
  • the assay reaction mix then was allowed to equilibrate in a 20% CO 2 atmosphere for 25 minutes at room temperature (e.g., 25°C).
  • the assay plates also include negative reaction contained "negative lysates" from E coli BL21 cells transformed with pCKl 10900 vector alone. The negative lysates typically exhibited some carbonic anhydrase activity due to the presence of some residual E. coli "background” carbonic anhydrase activity.
  • CabOOl SEQ ID NO: 1
  • Example 5 Activity and Stability (thermal and base) Characteristics of Purified ⁇ - class Carbonic Anhydrase, CabOO 1
  • SEQ ID NO: 2 was carried out by heat treatment as described in e.g., Smith, et al., Journal of
  • CabOOl was also challenged at 75°C and 85°C for 30 minutes at increasing carbonate (K 2 CO 3 ) concentrations ranging from 150 mM to 1000 mM and assayed for activity. As shown in Fig. 4, CabOOl maintained activity for 30 minutes at 85°C at up to 800 mM carbonate concentration. At concentrations higher than 800 mM carbonate CabOOl rapidly inactivated.
  • K 2 CO 3 carbonate
  • AirPoreTM microporous tape (Qiagen, Inc., Valencia, California). These overnight cultures were diluted 40-fold into fresh 2YT (24g/L yeast extract, 12 g/L bacto-tryptone containing 30 ⁇ g/ml chloramphenicol) in deep 96 well plates and after 2.5 hours of growth at 250 rpm shaker 30°C (OD600 should equal 0.7-0.8), 1/10 volume 10 mM IPTG (isopropyl thiogalactoside) and 5mM ZnS0 4 were added (ImM final IPTG and 0.5mM final ZnS0 4 ). The cultures were allowed to grow another 5 hours at 30°C.
  • 2YT 24g/L yeast extract, 12 g/L bacto-tryptone containing 30 ⁇ g/ml chloramphenicol
  • Lysis buffer contained 25 mM HEPES buffer (pH 8.3), 0.5 mg/ml PMBS (polymixin B sulfate), 0.2 mg/ml lysozyme, 1 mM DTT (dithiothreitol).
  • the plate was centrifuged at 4000 rpm, 4°C, for 25 minutes.
  • the clarified lysate was purified by heat treatment as described in Example 5.
  • Heat challenge assays were used to screen the engineered polypeptides prepared as in Example 6 for improved thermal and solvent stability relative to wild-type CabOO 1. Two conditions were screened: (a) 150 mM K 2 C0 3 , pH 10.9; and (b) 1 M AMP, pH 9.7.
  • Clarified lysate containing the engineered polypeptide was incubated for 30 minutes at 75°C in solution containing either: (a) 150 mM K 2 C0 3 , pH 10.9; or (b) 1M AMP, pH 9.7. Then 20 ⁇ , of the heat challenged sample was assayed using the C0 2 dehydration activity assay as described in Example 3. The most improved engineered CabOOl polypeptides as determined by screening are shown in Table 4 below.
  • engineered CabOOl polypeptides can be prepared that include various combinations of the above-identified beneficial residue differences R2C, I8L, D10G, R13C, D17V, D22V, D25P, H28P, S40T, D69E, A75L, N85K, N121D, S147H, G155P, and L159S.
  • Screening of such combinatorial libraries of engineered polypeptides can be carried out under same or increasing challenge conditions (e.g., thermal stability, base stability, solvent stability) to identify additional engineered polypeptides with improved properties, wherein the polypeptides comprise these beneficial residue differences and/or additional amino acid differences.
  • challenge conditions e.g., thermal stability, base stability, solvent stability
  • Libraries of engineered polypeptides can also be created using an improved "backbone” based on any one of the above-identified sequences having improved thermal and/or solvent stability shown in Table 4.

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Abstract

La présente invention concerne des enzymes d'anhydrase carbonique de classe ß génétiquement modifiées possédant des propriétés améliorées par comparaison à une anhydrase carbonique de classe ß de type sauvage d'origine naturelle et les utilisations de celles-ci pour la séquestration du dioxyde de carbone ainsi que pour la libération de dioxyde de carbone à partir d'une composition comprenant du bicarbonate. L'invention concerne également des polynucléotides codant pour les enzymes anhydrase carbonique génétiquement modifiées et des cellules hôtes capables d'exprimer les enzymes anhydrase carbonique génétiquement modifiées.
PCT/US2010/057843 2009-11-25 2010-11-23 Polypeptides génétiquement modifiés de l'anhydrase carbonique de classe bêta et utilisations de ceux-ci Ceased WO2011066304A2 (fr)

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US8354262B2 (en) 2010-06-30 2013-01-15 Codexis, Inc. Chemically modified carbonic anhydrases useful in carbon capture systems
WO2013028512A1 (fr) * 2011-08-19 2013-02-28 Novozymes, Inc. Microorganismes recombinants pour production d'acides dicarboxyliques en c4
US8420364B2 (en) 2010-06-30 2013-04-16 Codexis, Inc. Highly stable beta-class carbonic anhydrases useful in carbon capture systems
US8497103B2 (en) 2010-06-21 2013-07-30 Novozymes, Inc. Methods for C4-dicarboxylic acid production in filamentous fungi
US8518676B2 (en) 2010-06-21 2013-08-27 Novozymes, Inc. Polypeptides having C4 dicarboxylic acid transporter activity and polynucleotides encoding same
US8617859B2 (en) 2010-06-04 2013-12-31 Novozymes, Inc. C4 dicarboxylic acid production in filamentous fungi
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US8741611B2 (en) 2009-09-01 2014-06-03 Novozymes, Inc. Methods for improving malic acid production in filamentous fungi
WO2014090327A1 (fr) * 2012-12-14 2014-06-19 Statoil Petoleum As Nouveaux enzymes pour une absorption gazeuse améliorée
EP3344764A4 (fr) * 2015-09-03 2019-04-10 CO2 Solutions Inc. Variants d'anhydrase carbonique dethermovibrio ammonificans
WO2020010427A1 (fr) * 2018-07-12 2020-01-16 Garnica Leonardo Procédé respiratoire inverse pour l'absorption de dioxyde de carbone

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WO2010081007A2 (fr) * 2009-01-09 2010-07-15 Codexis, Inc. Polypeptides d'anhydrase carbonique et leurs utilisations

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US8741611B2 (en) 2009-09-01 2014-06-03 Novozymes, Inc. Methods for improving malic acid production in filamentous fungi
US8617859B2 (en) 2010-06-04 2013-12-31 Novozymes, Inc. C4 dicarboxylic acid production in filamentous fungi
US8497103B2 (en) 2010-06-21 2013-07-30 Novozymes, Inc. Methods for C4-dicarboxylic acid production in filamentous fungi
US8518676B2 (en) 2010-06-21 2013-08-27 Novozymes, Inc. Polypeptides having C4 dicarboxylic acid transporter activity and polynucleotides encoding same
US9303275B2 (en) 2010-06-21 2016-04-05 Novozymes, Inc. Polypeptides having C4 dicarboxylic acid transporter activity and polynucleotides encoding same
US8354262B2 (en) 2010-06-30 2013-01-15 Codexis, Inc. Chemically modified carbonic anhydrases useful in carbon capture systems
US8569031B2 (en) 2010-06-30 2013-10-29 Codexis, Inc. Chemically modified carbonic anhydrases useful in carbon capture systems
US8420364B2 (en) 2010-06-30 2013-04-16 Codexis, Inc. Highly stable beta-class carbonic anhydrases useful in carbon capture systems
US8354261B2 (en) 2010-06-30 2013-01-15 Codexis, Inc. Highly stable β-class carbonic anhydrases useful in carbon capture systems
US8512989B2 (en) 2010-06-30 2013-08-20 Codexis, Inc. Highly stable beta-class carbonic anhydrases useful in carbon capture systems
US8722387B2 (en) 2011-02-28 2014-05-13 Novozymes, Inc. Microorganisms for C4-dicarboxylic acid production
US9447440B2 (en) 2011-02-28 2016-09-20 Novozymes, Inc. Microorganisms for C4-dicarboxylic acid production
US8663956B2 (en) 2011-08-19 2014-03-04 Novozymes, Inc. Recombinant microorganisms for production C4-dicarboxylic acids
WO2013028512A1 (fr) * 2011-08-19 2013-02-28 Novozymes, Inc. Microorganismes recombinants pour production d'acides dicarboxyliques en c4
WO2014090327A1 (fr) * 2012-12-14 2014-06-19 Statoil Petoleum As Nouveaux enzymes pour une absorption gazeuse améliorée
EP3344764A4 (fr) * 2015-09-03 2019-04-10 CO2 Solutions Inc. Variants d'anhydrase carbonique dethermovibrio ammonificans
WO2020010427A1 (fr) * 2018-07-12 2020-01-16 Garnica Leonardo Procédé respiratoire inverse pour l'absorption de dioxyde de carbone

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