Heat-stable carbonic anhydrases and their use

Abstract

The present invention relates to use of heat-stable carbonic anhydrase in CO 2 extraction, e.g., from flue gas, natural gas or biogas. Furthermore, the invention relates to isolated polypeptides having carbonic anhydrase activity at elevated temperatures and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides.

1 . A method of using a heat-stable alpha-class or gamma-class carbonic anhydrase of bacterial or archaeal or fungal origin, but excluding gamma-class carbonic anhydrase from  Methanosarcina thermophila  strain TM-1 (DSM 1825), comprising:
 contacting a carbon dioxide-containing medium with the heat stable carbonic anhydrase, wherein carbon dioxide is extracted from the carbon dioxide-containing medium.   
     
     
         2 . A method in accordance with  claim 1  where the carbonic anhydrase is an alpha-class carbonic anhydrase. 
     
     
         3 . A method in accordance with  claim 2 , wherein the alpha-carbonic anhydrase is at least 60% identical to the carbonic anhydrase from  Bacillus clausii  KSM-K16 (NCBI acc. No. Q5WD44 or SEQ ID NO: 14) or to the carbonic anhydrase from  Bacillus halodurans  (NCBI acc. No. Q9KFW1 or SEQ ID NO: 16). 
     
     
         4 . A method in accordance with  claim 2 , where the alpha-class carbonic anhydrase is an isolated polypeptide selected from the group consisting of:
 a) a polypeptide having an amino acid sequence which has at least 94% identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or at least 91% identity with the amino acid sequence of SEQ ID NO: 6, or at least 96% identity with the amino acid sequence of SEQ ID NO: 8, or at least 89% identity with the amino acid sequence of SEQ ID NO: 10, or at least 97% identity with the amino acid sequence of SEQ ID NO: 12;   b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with:
 i) a polynucleotide sequence encoding a mature polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, 
 ii) a polynucleotide sequence selected from the group consisting of regions of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature enzyme, 
 iii) the cDNA sequence contained in a polynucleotide sequence selected from the group consisting of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature enzyme, 
 iv) a subsequence of (i), (ii) or (iii) of at least 100 contiguous nucleotides, or 
 v) a complementary strand of (i), (ii), (iii) or (iv); and 
   c) a fragment of (a) or (b) having carbonic anhydrase activity.   
     
     
         5 . A method in accordance with  claim 2 , wherein the alpha-class carbonic anhydrase is an isolated polypeptide having an amino acid sequence which has at least 97% identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or a functional fragment thereof. 
     
     
         6 . A method of using heat-stable beta-class carbonic anhydrase that is at least 60% identical to the beta-class carbonic anhydrase from  Bacillus clausii  KSM-K16 identified as NCBI acc. No. Q5WE01, or from  Bacillus halodurans  (NCBI acc. No. Q9K7S3), comprising:
 contacting a carbon dioxide-containing medium with the heat-stable beta-class carbonic anhydrase, wherein carbon dioxide is extracted from the carbon dioxide-containing medium.   
     
     
         7 . A method in accordance with  claim 1 , where the heat-stable carbonic anhydrase maintain activity at temperatures above 45° C. for at least 15 minutes. 
     
     
         8 . A method in accordance with  claim 1 , where the heat-stable carbonic anhydrase is used in a bioreactor. 
     
     
         9 . A method in accordance with  claim 8 , wherein the bioreactor comprises a contained liquid membrane (CLM). 
     
     
         10 . A method in accordance with  claim 8 , wherein the membrane liquid is a bicarbonate buffer with a pH of at least 9.0. 
     
     
         11 . A method in accordance with  claim 1 , wherein the carbon dioxide-containing medium is a gas. 
     
     
         12 . A method in accordance with  claim 1 , wherein the carbon dioxide-containing medium is a multiphase mixture. 
     
     
         13 . A method in accordance with  claim 11 , where the carbonic dioxide-containing gas is emitted from combustion or fermentation. 
     
     
         14 . A method in accordance with  claim 13 , where the gas is a flue gas. 
     
     
         15 . A method in accordance with  claim 11 , where the carbonic dioxide-containing gas is a raw natural gas or a syngas. 
     
     
         16 . A method in accordance with  claim 11 , where the carbonic dioxide-containing gas is a biogas. 
     
     
         17 . A method in accordance with  claim 1 , where the carbon dioxide-containing medium is a liquid. 
     
     
         18 . A method in accordance with  claim 1 , wherein the extraction of carbon dioxide is performed at temperatures between 45° C. and 60° C. 
     
     
         19 . An isolated polypeptide having carbonic anhydrase activity at elevated temperatures, selected from the group consisting of:
 a) a polypeptide having an amino acid sequence which has at least 94% identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or at least 91% identity with the amino acid sequence of SEQ ID NO: 6, or at least 96% identity with the amino acid sequence of SEQ ID NO: 8, or at least 89% identity with the amino acid sequence of SEQ ID NO: 10, or at least 97% identity with the amino acid sequence of SEQ ID NO: 12;   b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with:
 i) a polynucleotide sequence encoding a mature polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, 
 ii) a polynucleotide sequence selected from the group consisting of regions of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature enzyme, or a sequence differing there from by virtue of the degeneracy of the genetic code, 
 iii) the cDNA sequence contained in a polynucleotide sequence selected from the group consisting of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature enzyme, or a sequence differing there from by virtue of the degeneracy of the genetic code, 
 iv) a subsequence of (i), (ii) or (iii) of at least 100 contiguous nucleotides, or 
 v) a complementary strand of (i), (ii), (iii) or (iv); and 
   c) a fragment of (a) or (b) having carbonic anhydrase activity.   
     
     
         20 . The polypeptide according to  claim 19  having an amino acid sequence which has at least 97% identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or a functional fragment thereof. 
     
     
         21 . The polypeptide according to  claim 19 , wherein the polynucleotide encoding the polypeptide is selected from the group of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature enzyme, or a sequence differing there from by virtue of the degeneracy of the genetic code. 
     
     
         22 . The polypeptide according to  claim 19 , where the carbonic anhydrase activity is maintained at temperatures above 45° C. for at least 15 minutes. 
     
     
         23 . The polypeptide according to  claim 22 , where the carbonic anhydrase activity is maintained at temperatures above 45° C. for at least 30 days. 
     
     
         24 . The polypeptide according to  claim 19  where a tag which aids the purification or immobilization of the polypeptide is added to the polypeptide. 
     
     
         25 . The polypeptide according to  claim 24 , where the tag is a polyhistidine tag. 
     
     
         26 . A composition comprising the polypeptide according to  claim 19 . 
     
     
         27 . The composition according to  claim 26 , wherein the carbonic anhydrase is immobilized on a matrix. 
     
     
         28 . The composition according to  claim 27 , where that matrix is selected from the group beads, fabrics, fibers, hollow fibers, membranes, particulates, porous surfaces, rods, and tubes. 
     
     
         29 . The composition according to  claim 26 , characterized by being applicable in the capture of carbon dioxide. 
     
     
         30 . An isolated polynucleotide having a nucleotide sequence which encodes for the polypeptide defined in  claim 19 . 
     
     
         31 . An isolated polynucleotide obtained by:
 a) hybridizing a population of DNA under medium stringency conditions with
 i) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, 
 ii) a cDNA sequence contained in a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, or 
 iii) a complementary strand of (i) or (ii); and 
   b) isolating the hybridizing polynucleotide, which encodes a polypeptide having carbonic anhydrase activity at elevated temperatures.   
     
     
         32 . A recombinant expression vector comprising the nucleic acid construct of  claim 31 . 
     
     
         33 . A recombinant host cell comprising the recombinant expression vector of  claim 32 . 
     
     
         34 . A method for producing the polypeptide of  claim 19  comprising:
 a) cultivating a strain, which in its wild-type form is capable of producing the polypeptide, to produce the polypeptide; and 
 b) recovering the polypeptide. 
 
     
     
         35 . A method for producing a polypeptide of  claim 19  comprising:
 a) cultivating a recombinant host cell as defined in  claim 34  under conditions conducive for production of the polypeptide; and 
 b) recovering the polypeptide.