WO2014090327A1 - Nouveaux enzymes pour une absorption gazeuse améliorée - Google Patents

Nouveaux enzymes pour une absorption gazeuse améliorée Download PDF

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WO2014090327A1
WO2014090327A1 PCT/EP2012/075529 EP2012075529W WO2014090327A1 WO 2014090327 A1 WO2014090327 A1 WO 2014090327A1 EP 2012075529 W EP2012075529 W EP 2012075529W WO 2014090327 A1 WO2014090327 A1 WO 2014090327A1
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seq
polypeptide
isolated
carbonic anhydrase
polynucleotide
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PCT/EP2012/075529
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English (en)
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Hans Kristian Kotlar
Svein Berg
Maria LIOLIOU
Alexander Wentzel
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Statoil Petoleum As
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Priority to PCT/EP2012/075529 priority Critical patent/WO2014090327A1/fr
Publication of WO2014090327A1 publication Critical patent/WO2014090327A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12F—RECOVERY OF BY-PRODUCTS OF FERMENTED SOLUTIONS; DENATURED ALCOHOL; PREPARATION THEREOF
    • C12F3/00—Recovery of by-products
    • C12F3/02—Recovery of by-products of carbon dioxide
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/14—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols by absorption
    • B01D53/1493—Selection of liquid materials for use as absorbents
    • 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.)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2252/00—Absorbents, i.e. solvents and liquid materials for gas absorption
    • B01D2252/60—Additives
    • B01D2252/602—Activators, promoting agents, catalytic agents or enzymes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/14—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols by absorption
    • B01D53/1456—Removing acid components
    • B01D53/1475—Removing carbon dioxide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/151—Reduction of greenhouse gas [GHG] emissions, e.g. CO2
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/59—Biological synthesis; Biological purification

Definitions

  • the present invention relates to new isolated polypeptides having carbonic anhydrase activity at elevated temperature and enhanced absorbing/desorbing abilities in regard to acidic components e.g. C0 2 from/to a gas mixture e.g. flue gas, natural gas or biogas.
  • a gas mixture e.g. flue gas, natural gas or biogas.
  • the present invention relates further to use of the isolated polypeptides.
  • Carbonic anhydrases (KEGG: EC 4.2.1.1) are widely spread in nature and catalyze the interconversion of carbon dioxide and water to bicarbonate and protons (or vice versa).
  • Carbonic anhydrase is one of the fastest enzymes known, with one molecule able to turnover a million molecules of bicarbonate per second. The enzyme will increase the kinetics in a reaction towards equilibrium but will not change equilibrium values.
  • Carbonic anhydrases are categorized in five distinct classes (alpha, beta, gamma, delta and epsilon) evolving from different origins, which may explain why between members of different families, no significant sequence similarity are found on the amino acid level.
  • One common feature among most known carbonic anhydrases is, however, a zinc ion in the catalytic site, by which the enzyme binds its substrate. The pKa is then lowered and allows for nucleophilic attack on the carbon dioxide group.
  • Co-factors other than zinc ions have been described for individual carbonic anhydrase enzymes. However, zinc ions are most preferred among the known carbonic anhydrase enzymes.
  • Carbon dioxide capture is an important step in both energy production and
  • the main problems with the existing technology are a low overall efficiency, a slow absorption rate of the gas (C0 2 ) and that most catalysts used up to date to increase absorption kinetics are toxic. Further, the most effective absorbents, such as amines, could harm the environment. Amines will decompose over time and generate a waste problem. Carbonates, for instance K 2 C0 3 and /or Na 2 C0 3 , are non toxic and could be used as absorbents instead of amines, but have much slower absorption rate than amines. NH 3 could, however, be used instead of amines.
  • the existing technology has also low energy efficiency, i.e. it consumes large amounts of energy, for heating, cooling and operating pumps, compressors, blowers etc. There is therefore a need for non-toxic effective absorbents/desorbents and/or catalysts.
  • One category of catalysts that has been suggested is enzymes. Enzymes are widely distributed in nature, and some are active in catalyzing C0 2 absorption/desorption during respiration. Enzymes may therefore prove to be both effective and non toxic catalysts. If enzymes are to be used as catalysts with the existing technology, flue gas from power plants has to be cooled down in a heat exchanger before it can be treated with enzymes.
  • thermostable enzymes will reduce the need to cool the flue gas and temperatures above 60°C will reduce the risk of microbial growth in the reactor(s).
  • WO 2010/014774 describes the extraction of C0 2 gas from a gas flow as catalyzed by the use of enzymes (carbonic anhydrase). Enzymes are also used in the desorption step.
  • the reactor may contain two or more different enzymes.
  • WO 2008/095057 and WO 2010/151787 refer to heat-stable carbonic anhydrases and their use in C0 2 extraction.
  • US 2009/0155889 describes a system and a method for absorbing C0 2 from a gas flow, where absorbent solution includes amines and the catalyst includes one or more enzymes.
  • US 2010/0086983 refers to a procedure to remove carbon dioxide from a gas flow using immobilized enzymes.
  • WO 2009/000025 shows a method to absorb C0 2 from a gas flow, whereby the absorption is catalyzed by the use of enzymes on a solid carrier.
  • US 6,143,556 refers to the use of enzymes to isolate specific gases from a gas flow. For this purpose, it is described using a bioreactor containing beads coated with enzymes. One or more different enzymes may be used and the carrier material may also include various types of material.
  • US 2008/0003662 refers to a method for separating carbon dioxide from a gas flow through the use of an enzyme (carbonic anhydrase).
  • WO 98/55210 discloses an apparatus and process for extraction of carbon dioxide from a gas flow.
  • the bioreactor contains an immobilized enzyme (carbonic anhydrase) that catalyses the process.
  • US 3,896,212 refers to the absorption of C0 2 in a gas flow by using different concentrations of catalyst, and that the amount is varied from the inlet to the outlet of absorbent, but does not show the use of enzymes.
  • the present invention relates to an isolated polypeptide having carbonic anhydrase activity as defined in any one of a) through e) or any combinations thereof: a) a polypeptide having an amino acid sequence corresponding to amino acid
  • SEQ ID NO:2 residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22 , SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or
  • polypeptide which is at least 60% identical to amino acid residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or a fragment of a) to b) having carbonic anhydrase activity, or a polypeptide encoded by nucleic acid sequence which hybridizes under medium stringency conditions with a polynucleotide sequence encoding a polypeptide of: i) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:
  • a further aspect of the invention relates to an isolated polypeptide or a mixture of polypeptides comprising at least one of the polypeptides according to the first aspect or embodiments thereof of the present invention.
  • Another aspect of the present invention relates to a composition
  • a composition comprising the isolated polypeptide or the mixture of polypeptides and an immobilizing agent.
  • Yet another aspect of the present invention relates to an isolated polynucleotide having the nucleotide sequence encoding the isolated polypeptide of the first aspect of the invention.
  • a further aspect relates to a nucleic acid construct comprising an operable linked control sequence directing the expression of the polynucleotide.
  • Yet a further aspect relates to a vector comprising the polynucleotide or the nucleic acid construct.
  • Another aspect relates to a host cell comprising the polynucleotide, the nucleic acid construct or the vector.
  • Yet another aspect relates to use of the isolated polypeptide or the composition for absorbing/desorbing an acidic component from/to an absorbing medium. Further aspects relates to use of the isolated polynucleotide, use of the nucleic acid construct, use of the vector and use of the host cell.
  • Figure 1 Illustrates gene synthesis and cloning of
  • Figure 2 Illustrates SDS-PAGE analysis of recombinant production of
  • pET16b negative control derived from a culture containing the empty expression plasmid pET16b; SI, crude extract; S2, heat- treated extract (20 min at 65 °C). Arrows indicate the expected target size of protein SCA04/SCA06b/SCA09/SCAl 1.
  • Figure 3 Illustrates carbonic anhydrase activity measurements using crude extracts from recombinant production of SCA04/SCA06b/SCA09/SCAl 1 in E. coli.
  • P buffer phosphate buffer A (reference);
  • pET16b negative control derived from a culture containing the empty expression plasmid pET16b.
  • Dilutions 1 : 10 and 1 :20 in buffer A prior to measurement are given behind the protein name where applicable.
  • Figure 4 Illustrates carbonic anhydrase activity measurements using crude extracts from recombinant production of SCA04/SCA06b/SCA09/SCAl 1 in E. coli after incubation at 23 °C (RT), 65 °C or 80 °C for 1 h or 5 h, as indicated.
  • Blue bars represent measurements diluted with ion free water containing 1 ⁇ ZnS0 4
  • red bars represent measurements diluted to 20 % (w/v) K 2 CO 3 , 1 ⁇ ZnS0 4 final concentration.
  • Figure 5A Illustrates specific activity of SCA04 as a function of substrate concentration. Data series 1 and 2 are shown in open squares and open diamonds respectively. All data were included in the calculations. Lines are calculated from the K m and V max found from non-linear fitting of the Michaelis-Menten equation.
  • Figure 5B Illustrates specific activity of SCA09 as a function of substrate concentration.
  • Data series 1 and 2 are shown in open squares and open diamonds, respectively. All data were included in the calculations. Lines are calculated from the K m and V max found from non-linear fitting of the Michaelis-Menten equation.
  • Figure 5C Illustrates specific activity of SCA11 as a function of substrate concentration.
  • Data series 1 and 2 are shown in open squares and open diamonds, respectively. All data were included in the calculations. Lines are calculated from the K m and V max found from non-linear fitting of the Michaelis-Menten equation.
  • Figure 6 Illustrates the relative C0 2 absorption rate as a function of C0 2 loading at different enzyme concentrations
  • Figure 7 Illustrates the impact of the absorbent concentration on the reaction kinetics.
  • Figure 8 Illustrates temperature stability of SCA04 at 80 °C
  • the acidic component might be, but is not limited to C0 2 .
  • other areas of use as improved oil recovery, biomass production, C0 2 storage or artificial lung support are suggested.
  • the isolated carbonic anhydrases according to the present invention have been isolated from microorganisms having high temperature oil and gas reservoirs as their natural habitat, also isolates from other habitats are however, possible.
  • the isolated carbonic anhydrases are able to perform their catalytic activity under elevated temperatures resulting in a reaction process with high energy efficiency.
  • Using the enzymes of the present invention in an absorber /desorber system may increase the efficiency at least 10 times with regard to the kinetic rate than existing technology, preferentially even higher. Making the technical process more efficient will have a significant impact on reducing the operational costs. Beneficial environmental advantages will also be achieved, in that the use of toxic absorbents/desorbents and catalysts may be avoided.
  • thermostable enzymes will reduce the need to cool the flue gas and temperatures above 60°C will reduce the risk of microbial growth in the reactor(s).
  • the catalyzed reaction depends on the C0 2 concentration in the gas.
  • C0 2 - absorption process the C0 2 concentration in the gas will decrease from the inlet to the outlet of the absorber. This will have an impact on how efficient the enzyme, or the mixture of enzymes, should be to catalyze the process.
  • a capture system where the concentration varies considerably in the gas phase from inlet to outlet, it will be beneficial to operate an absorber/desorber system where the enzyme or mixture of enzymes are optimized for each section.
  • the C0 2 concentration will be high, an enzyme or a mixture of enzymes with high K m value(s) is more efficient at high C0 2 content.
  • an enzyme or a mixture of enzymes with low K m value(s) should be the catalyst, as it is more efficient at low C0 2 content.
  • a further optimization of the reaction may be reached by using an immobilizing agent.
  • the enzyme for carbonic anhydrase based C0 2 capture should have a long life-time in the process, be very efficient, and have a suitable K m value for C0 2 and a suitable K m value for HCO 3 " which is different from the K m value for C0 2 .
  • the inventors identified the carbonic anhydrase enzymes with the above characterstics based on the protein sequence of the carbonic anhydrase from a putative micororganism selected from the group or any combination thereof: Methanocaldococcus
  • a synthetic gene was designed, and codon optimized for recombinant expression in E. coli.
  • the gene was further cloned by standard methods into a pUC vector and the desired coding sequence was confirmed by sequencing. Subsequently the gene was excised and cloned into an expression vector.
  • Heat shock competent cells of E. coli were transformed with the carbonic anhydrase gene, and an expression clone was picked and cultivated. The bacterial cells were disrupted and insoluble cell debris was pelleted by centrifugation, resulting in a crude extract containing the soluble protein.
  • the isolated carbonic anhydrases according to the present invention and their amino acid sequences are identified as follows: SCAOl (SEQ ID NO:26), SCA02 (SEQ ID NO:28), SCA03 (SEQ ID NO:30) SCA04 (SEQ ID NO:2), SCA05 (SEQ ID NO:6), SCA06b, (SEQ ID NO: 10) SCA07 (SEQ ID NO: 14), SCA09 (SEQ ID NO: 18), SCA10 (SEQ ID NO:32) and SCAl l (SEQ ID NO:22), and were identified in an oil reservoir metagenome derived DNA sequence database assembled from read sequences obtained by 454 pyrosequencing of the metagenomic DNA.
  • Said carbonic anhydrases were codon optimized for expression in E.coli and have the following sequence identity: SCA04 (SEQ ID NO:4), SCA05 (SEQ ID NO:8), SCA06b (SEQ ID NO: 12), SCA07 (SEQ ID NO: 16), SCA09 (SEQ ID NO:20) and SCAl l (SEQ ID NO:24)
  • the DNA sequences encoding the isolated carbonic anhydrases and the codon optimized anhydrases have the following identity: SCA04 (SEQ ID NOs: l and 3), SCA05 (SEQ ID NOs:5 and 7), SCA06b (SEQ ID NOs:9 and 11), SCA07 (SEQ ID NOs: 13 and 15), SCA09 (SEQ ID NOs: 17 and 19), SCAl l (SEQ ID NOs:21 and 23) respectively.
  • Further DNA sequences encoding the isolated carbonic anhydrases have the following identity: SCAOl (SEQ ID NO:25), SCA02 (SEQ ID NO:27), SCA03 (SEQ ID NO:29) and SCA10 (SEQ ID NO:31).
  • the present invention relates to an isolated polypeptide having carbonic anhydrase activity as defined in any one of a) through e) or any combinations thereof: a) a polypeptide having an amino acid sequence corresponding to amino
  • SEQ ID NO:2 residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or
  • polypeptide which is at least 60% identical to amino acid residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID N0: 16, SEQ ID N0: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32; or c) a fragment of a) or b) having carbonic anhydrase activity; or d) a polypeptide encoded by nucleic acid sequence which hybridizes under medium stringency conditions with a polynucleotide sequence encoding a polypeptide of: i) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO
  • degeneracy of the genetic code does not hybridize with the polynucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:3; but which codes for a polypeptide having an amino acid sequence according to a) or b).
  • polypeptide as defined in b) is preferably at least65%, identical to the amino acid residues selected from the group or any
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 70%, identical to the amino acid residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 75%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 80%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 85%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 90%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 95%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 96%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:
  • SEQ ID NO:6 SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 97%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 98%, identical to the amino acid residues selected from the group or any combinations thereof: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
  • SEQ ID NO: 18 SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • polypeptide as defined in b) is preferably at least 99%, identical to the amino acid residues selected from the group or any combinations thereof of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:32.
  • hybridization conditions in d) may be high stringency.
  • the present isolated polypeptides may further comprise putative metal ion binding sites comprising the following sites:
  • binding sites are believed to bind Zn 2+ but also other active binding sites involving e.g. Cd ions and Fe ions are possible.
  • the isolated polypeptide may further comprise a high K m value or a low K m value, for instance may the low K m value be chosen from the following range: from about 1 to about 25mM and the high K m value may be chosen from the following range: from about 25 to about 60mM.
  • the carbonic anhydrase activity may be maintained at a temperature above 65°C for at least one hour.
  • the activity may also be maintained for at least five hours in the temperature range of about 65°C to about 100°C, showing that the enzyme may perform its activity at high temperatures, i.e. temperatures of: above 40°C, preferably above 50°C, more preferably above 55°C, more preferably above 60°C, even more preferably above 65°C most preferably above 70°C, most preferably above 80°C, most preferably above 85°C, most preferably above 90°C and even most preferably above 100°C.
  • the carbonic anhydrase activity is maintained at a temperature of at least 80°C for at least two hours at a level corresponding to at least 90% of the initial activity.
  • carbonic anhydrase activity may be maintained at a temperature of above 65° C for at least 5 hours at a K 2 CO 3 concentration of 20% (w/v) even 80°C may be tolerated.
  • the K 2 CO 3 concentration of 20% (w/v) may have a stabilizing effect on the enzyme.
  • Another aspect of the invention relates to an isolated polypeptide or a mixture of isolated polypeptides comprising at least one of the isolated polypeptide identified above. Also its use in absorbing/desorbing an acidic component from/to an absorbing medium, wherein the acidic component may be C0 2, is an aspect of the present invention.
  • a composition comprising an immobilizing agent and the isolated polypeptide(s) as defined above.
  • the immobilizing agent may comprise a matrix, surface or substrates as for instance beads, fabrics, fibers, porous materials, CLEAs, structured or random packing, crystals such as monoliths or any combinations thereof.
  • the isolated polypeptides may be in an aqueous phase or immobilized on particles.
  • the enzyme(s) may (a) be but is not limited to dissolve in the absorbing liquid and flowing through the appropriate section of the absorber, (b) it may be immobilized on the respective section of the absorber, or (c) may be immobilized on particles floating inside the absorbing liquid.
  • composition may be used for absorbing/desorbing an acidic component from/to a gas mixture e.g. flue gas, natural gas or biogas, wherein the acidic component may be C0 2 .
  • a gas mixture e.g. flue gas, natural gas or biogas, wherein the acidic component may be C0 2 .
  • Yet another aspect of the present invention relates to an isolated polynucleotide having the nucleotide sequence encoding the polypeptide of the present invention.
  • nucleic acid construct may be operable linked to a control sequence directing the expression of the polynucleotide.
  • a vector comprising the polynucleotide or the nucleic acid construct.
  • a host cell comprising the said isolated polynucleotide is provided, the nucleic acid construct or the vector comprising the polynucleotide.
  • a further aspect of the present invention relates to the use of an isolated polypeptide according to the present invention or a composition as described above for
  • the acidic component may be C0 2
  • Yet another aspect of the present invention relates to the use of an isolated
  • nucleic acid construct having a nucleotide sequence encoding the polypeptide of the present invention.
  • a nucleic acid construct may be operable linked to a control sequence directing the expression of the polynucleotide.
  • nucleic acid construct relates to the use of a vector comprising the polynucleotide or the nucleic acid construct.
  • Another aspect of the present invention relates to the use of a host cell comprising the said isolated polynucleic acid, the nucleic acid construct or said vector.
  • isolated polypeptide refers to a 20-100% pure polypeptide determined by SDS-PAGE.
  • carbonic anhydrase activity as used herein is defined as an activity which catalyzes the conversion between carbon dioxide and bicarbonate.
  • thermostability is used herein to describe an enzyme that maintains activity over an elongated period of time at elevated temperatures.
  • the thermostability of the enzyme can be increased or enhanced in some way by immobilization, chemical modification (e.g. cross-linking) or use of stabilizing chemicals.
  • operably linked is defined as a configuration wherein a control sequence is placed in a position relative to the coding sequence of a
  • polynucleotide sequence and is thereby able to control the expression of the coding sequence.
  • sequence identity refers to a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. The two sequences to be compared must be aligned to the best possible fit with the insertion of gaps or alternatively, truncation at the ends of the protein sequences. Sequence identity has been calculated by the BLASTP program (Altschul et al, 1990).
  • gas mixture refers to the C0 2 containing gas stream.
  • a gas stream can be and is not limited to raw natural gas from oil or gas wells, syngas from the gasification of a carbon containing fuel, emission stream from combustion processes, flue gas from e.g. electric generation power plants, catalytic crackers, boilers etc, or biogas.
  • absorbent absorbing liquid
  • solvent solvent
  • absorbing liquid absorbing liquid
  • reacting liquid compound that has the ability to absorb C0 2 . It may comprise carbonates, and/or primary, and/or secondary and/or tertiary amines and/or blends thereof, and/or alkanolamines, and/or amino acid salts.
  • K m is defined in the present invention as the Michaelis-Menten constant.
  • Michaelis-Menten kinetics is a model of enzyme kinetics in the form of an equation describing the rate of enzymatic reactions by relating the reaction rate to the concentration of a substrate.
  • the Michaelis-Menten constant, K m is the substrate concentration at which the reaction rate is half of the maximum rate achieved by the system, at maximum (saturating) substrate concentrations.
  • V max is the maximum reaction rate
  • K m is the Michaelis-Menten constant.
  • absorbent absorbent
  • absorbing liquid absorbent
  • solvent solvent
  • catalyst is defined herein as any chemical entity that catalyses the hydration of carbon dioxide to bicarbonate. For the purposes of the present invention, it is closely related but not limited to the carbonic anhydrase family of enzymes.
  • immobilizing agent refers to an agent having the ability to stabilize enzyme(s) on e.g. on a matrix, surface or substrate. It can be at least partially composed of e.g. beads, fabrics, fibers, porous materials, structured or random packing, crystals or combinations thereof.
  • a synthetic gene including the coding sequence for a Factor Xa protease cleavable 8x His-tag was designed, and the gene was codon optimized for recombinant expression in E. coli.
  • a Pcil restriction site was introduced immediately upstream of the CA coding region.
  • the synthetic gene to which protective bases and appropriate restriction sites (5'-Xbal, 3'-BamRT) were added was synthesized by GenScript (Piscataway, NJ, USA) and cloned blunt-ended into EcoRV digested plasmid pUC57. Correct cloning was confirmed by restriction digest analysis, and the desired coding sequence was confirmed by sequencing (GenScript). Subsequently, the gene was excised from the pUC57 derivative and simultaneously cloned in two alternative ways into expression vector pET16b (Novagen).
  • the pUC57 resident carbonic anhydrase gene was excised from the pUC57 derivative using Ncol and BamHl and cloned into the similarly digested pET16b expression vector to give plasmid pSIN04-I/pSIN05-I/pSIN06b- I/pSIN07-I/pSIN09-I/pSINl l-I, respectively .
  • the carbonic anhydrase gene was excised from the pUC57 derivative using Pcil and BamHl and cloned into the compatible ends of an Ncol and BamHl digested pET16b vector fragment to give plasmid pSIN04-II/pSIN05- II/pSIN06b-II/pSIN07-II/pSIN09-II/pSINl l-II, respectively.
  • Heat-shock competent cells of E. coli BL21(DE3) were transformed with the carbonic anhydrase gene carrying pET16b derivative pSIN04-II/pSIN06b-II/pSIN09-II/pSINl 1- II, respectively.
  • One single clone was selected to inoculate 5 ml LB Amp 100 liquid medium.
  • the culture was incubated at 37 °C and 200 rpm overnight on a shaking incubator. 3 ml of this pre-culture was then used to inoculate 1000 ml LB medium supplemented with 100 ⁇ g/ml ampicillin in a baffled 3 1 shake flask.
  • the culture was incubated at 37 °C and 150 rpm while following culture growth by OD 6 oo measurement.
  • OD 6 oo of approximately 1.0
  • gene expression from the T7 promoter was induced by addition of 0.5 mM final concentration inducer isopropyl ⁇ -D-l- thiogalactopyranoside (IPTG) from a sterile stock solution.
  • IPTG isopropyl ⁇ -D-l- thiogalactopyranoside
  • 0.5 mM final concentration zinc sulphate was added from a sterile stock solution.
  • the gene expression induced culture was incubated for another 5 h at 16 °C and 150 rpm. Subsequently, cells were harvested by centrifugation (9000 xg, 4 °C, 15 min).
  • the wet weight of the cell paste was determined.
  • the pellet was re-suspended in a small volume of supernatant, transferred to 50 ml tubes and centrifuged again (6800 xg, 4 °C, 15 min). The supernatant was discarded, and the cell paste was stored at -80 °C prior to extraction.
  • the biomass was thawed on ice, and cells were re-suspended in 25 ml buffer A (50 mM potassium phosphate, pH 6.8, 1 ⁇ zinc sulphate) for each 5 g of wet weight. Cells were disrupted while held on ice by 5x1 min sonication using a Branson Sonifier (duty cycle 50 %, output control 3) with intermediate mixing.
  • a Branson Sonifier duty cycle 50 %, output control 3
  • the sonicated cell suspension was centrifuged (6800 xg, 4 °C, 15 min), and the supernatant was transferred to a fresh tube. An aliquot of this crude extract was incubated for 20 min at 65 °C on a water bath. The heated extract was centrifuged (6800 x g, 4 °C, 20 min), and the supernatant (heat treated crude extract) was transferred to a fresh tube. The resulting target enzyme enriched supernatant and the original crude extract were analysed for the presence of the target enzyme monomer using SDS-PAGE under reducing conditions. A similarly produced extract of E. coli carrying the empty expression vector pET16b was used as a negative control. The result of SDS-PAGE analysis is given in Figure 2.
  • Example 2 was added to the Tris buffer while mixing. After pH logging was started (WaveScan 2.0 controlling an Advantec USB-4711 A multifuntion module connected to the pH-meter's recorder output; channel range: +/-1.25mV), 9 ml C0 2 saturated ion- free water was added to the reaction vessel. C0 2 saturated water was prepared by bubbling C0 2 gas from dry ice in an isolation bottle through 0.5 L ion free water while stirring overnight. The decrease in pH was recorded at a resolution of 50 ms until a constant pH was obtained (25-80 s). The time needed for the pH to drop from pH 7.8 to pH 7.0 (dt) was determined and used as a measure for carbonic anhydrase enzymatic activity.
  • dt The time needed for the pH to drop from pH 7.8 to pH 7.0
  • Dilutions were incubated at room temperature ( ⁇ 23 °C), 65 °C or 80 °C for 1 h or 5 h. After incubation, samples were centrifuged in a microliter centrifuge (14,000 rpm, 4 °C, 5 min), and 285 ⁇ , of the cleared supernatant, corresponding to 100 ⁇ of undiluted crude extract, was used for activity measurement as described in Example 3.
  • SCA04/SCA06b/SCA09/SCAl 1 exhibited very different characteristics with respect to stability at high temperature and/or high salt concentration (Figure 4).
  • SCA04 was found to be very stable under all condition tested. Even after incubation for 5 h in 20 % (w/v) K 2 C0 3 , more than 50 % of the original activity was retained.
  • SCA11 was found to be relatively stable when incubated at 65 °C, though a clear decrease of activity over time was observed at this temperature. The combination of high temperature (65 °C) and 20 % (w/v) K 2 C0 3 was not tolerated, leading to a rapid loss of functionality. It had been shown before that SCA11 was in general quickly degraded at 80 °C (data not shown).
  • SCA06b was found to be stable at room temperature, but quantitatively degraded already after 1 h incubation at 65 °C. Interestingly, higher carbonic anhydrase activity was observed for SCA06b in the presence of 20 % (w/v) K 2 C0 3 . The high salt concentrations obviously had a stabilizing effect on the enzyme. This effect was also observed when SCA06b was incubated at 65 °C. SCA09 showed a relatively high stability when incubated at 65 °C or 80 °C, though some loss of activity was observed especially after 5 h of incubation. The additional presence of 20 % (w/v) K 2 C0 3 had no additional destabilizing effect at 65 °C, while after 5 h incubation at 80 °C, most of the activity was lost.
  • E. coli strains generated based on strain BL21(DE3) and carrying the respective CA encoding gene on a pET16b derived plasmid were pre-cultivated in 100 ml LB(g) medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 10 g/L glucose*H 2 0) containing 100 mg/1 ampicillin in baffled 500 ml shake flasks at 30 °C and 200 rpm on a shaking incubator. After approx.
  • Fermentations were performed at 30 °C and pH 6.8, automatically adjusted with 12.5 % NH 3 solution. Using an aeration rate of 0.35 to 1.5 wm, a minimum level of dissolved oxygen (DO) of 0.2 was maintained by automatic adjustment of the stirrer speed. After approx. 12 h of batch cultivation, exponential feeding was started using a 50 % glucose/MgS0 4 solution at an initial rate of 10 g/(L culture volume*h) [i.e. 7.5 g/(750 ml*h)] up to approx. 35 g/h. After that, constant feeding at 35 g/h was applied. Glucose levels were monitored manually when necessary and held limiting throughout the fed-batch phase.
  • CA gene expression was induced at an OD 6 oo of approx. 70 by addition of 0.75 ml 1 M IPTG and 1.5 ml 500 mM ZnS0 4 from sterile stock solutions.
  • Sample P0 was withdrawn immediately before induction, and subsequent samples [usually 5 ml in total for OD 6 oo measurement and biomass + supernatant (3x1 g)] were taken every 2-3 h. All samples were stored at -20 °C until analysis/extraction.
  • Approx. 6 h after induction of CA gene expression fermentation was stopped, and biomass was harvested by centrifugation in two centrifugation bags each. The supernatant was disposed, and the biomass was frozen and stored at -80 °C until further processing.
  • HCD fermentation derived biomass 48.3 g for SCA04, 77.6 g/L for SCA09, 61.6 g for SCA11
  • HCD fermentation derived biomass 48.3 g for SCA04, 77.6 g/L for SCA09, 61.6 g for SCA11
  • soluble CA enzyme result: crude extract
  • thermostable enzymes by heat treatment result: heat enriched crude extract
  • the quantification of enzymes SCA04, SCA09 and SCA11 in heat enriched crude extracts was performed by a combination of (i) the determination of total protein concentrations using the Bradford protein assay and bovine serum albumin (BSA) as a standard, and (ii) SDS-PAGE based band intensity quantification.
  • the Bradford assay was performed as follows: from a commercial stock solution of BSA (NEB, lO mg/ml) and a derived lOOx dilution (100 ⁇ g/ml), 800 ⁇ each of the dilutions of 0, 1, 5, 7.5 and 10 ⁇ g/ml BSA in ion free water were prepared and used as standards. Enriched crude extract samples were diluted 1 :10000, 1 :2000 and 1 : 1000 in 800 ⁇ final volume in order to fit the results to the linear OD595 detection range of the Spectramax microtiter plate reader. To each 800 ⁇ diluted samples and standards, 200 ⁇ Bradford color solution (Bio-Rad protein assay concentrate) was added and mixed thoroughly.
  • SDS-PAGE and quantification of the CA enzyme monomers of SCA04, SCA09 and SCA11 were performed as follows: six dilutions each of the respective enriched crude extracts were generated in a final volume of 20 ⁇ . This 20 ⁇ sample dilution and 10 ⁇ gel loading dye were mixed and boiled for 3 min. 25 ⁇ of the heated mixtures was then applied on 12 % Clare Page SDS-PA gels.
  • the protein standards used were the BioRad Dual color and Broad range standards. Lysozyme and BSA were used in dilution as further references. The gel images were analyzed using the ChemDoc software, and Image Reports were generated.
  • thermos bottle was filled with dry ice, and the developing gas was bubbled through a flask containing 500 ml ion free water while stirring. The system was left overnight to reach saturation, before the bottle was tightly closed and stored for at least one hour to overnight to equilibrate.
  • the C0 2 concentration in the substrate stock solution was determined by titration with 0.01 M NaOH in the presence of the pH indicator phenolphthalein and continued until the indicator turned pale pink (typically 33-36 ml for 10 ml C0 2 -saturated water).
  • Enzymatic activity was monitored by following the pH decrease after the addition of substrate solution and enzyme solution and subtracting the respective results from a control reaction where no enzyme was added. This decrease was linear between pH 8.3 and pH 7.3, and only values in this range were included in the calculation of the kinetic parameters.
  • the reaction mixture consisted of 12 ml buffer (20 mM Tris-S0 4 , 1 ⁇ ZnS0 4 , pH 8.3), 0.5-9 ml substrate stock solution (C0 2 -saturated water), 8.5-0 ml ion free water, and 0.1 ml enzyme solution or buffer (control). The total reaction volume in all cases was 21.1 ml. Buffer and ion free water were mixed, and the pH electrode was inserted in the reaction vessel.
  • the mixture was stirred at maximum stirrer speed, and the measurement/logging was started. After ⁇ 5 seconds, the substrate solution was added, and immediately afterwards, the enzyme was added. The decrease in pH was then monitored and logged at a resolution of 50 ms for about one minute.
  • the SCA04 enzyme was assayed using a 5-fold diluted enriched crude extract sample. For each substrate concentration, the activities were determined as the difference in slope values of the curve with enzyme added and the respective reference curve without enzyme added (buffer only). These values (in units of pH/10 per second) were then divided by the slope value -0.0143 pH/10 per mM, obtaining activities in the units of mM/s. By dividing these values by the concentration of functional enzyme in the system (correcting for dilutions and enzyme purity), activities in mmol/s per mg protein were calculated. Enzyme units (U) are often referred to as the amount of enzyme needed to produce 1 Mol product per minute (or second). Here, it is defined as the amount of enzyme needed to consume 1 Mol C0 2 per second, and specific activities (U/mg) were found by multiplying the mmol/s per mg protein- values with 1000.
  • the SCA09 enzyme was assayed using an undiluted enriched crude extract sample. For each substrate concentration, the activities were determined as the difference in slope values of the curve with enzyme added and the respective reference curve without enzyme added (buffer only). These values (in units of pH/10 per second) were then divided by the slope value -0.0158 pH/10 per mM, obtaining activities in the units of mM/s. By dividing these values by the concentration of functional enzyme in the system (correcting for dilutions and enzyme purity), activities in mmol/s per mg protein were calculated. Here, units are defined as the amount of enzyme needed to consume 1 Mol C0 2 per second, and specific activities (U/mg) were found by multiplying the mmol/s per mg protein- values with 1000.
  • the SCA11 enzyme was assayed using a 100-fold diluted enriched crude extract sample. For each substrate concentration, the activities were determined as the difference in slope values of the curve with enzyme added and the respective reference curve without enzyme added (buffer only). These values (in units of pH/10 per second) were then divided by the slope value -0.0143 pH/10 per mM, obtaining activities in the units of mM/s. By dividing these values by the concentration of functional enzyme in the system (correcting for dilutions and enzyme purity), activities in mmol/s per mg protein were calculated. Here, units are defined as the amount of enzyme needed to consume 1 Mol C0 2 per second, and specific activities (U/mg) were found by multiplying the mmol/s per mg protein- values with 1000.
  • Crude extract of E. coli containing recombinantly produced SCA04 was prepared as described in Example 2. Fractions of 500 ⁇ crude extract in 1.5 ml reaction vials were incubated on a heating block at 80 °C for 0, 15, 30, 60, 120, 180, 240 and 300 min, respectively. After incubation, samples were centrifuged in a microliter centrifuge (13,200 rpm, 22 °C, 10 min), and the cleared supernatant was used for carbonic anhydrase activity measurements at room temperature in triplicate as described in Example 3.
  • the mean result for the measurements of the sample incubated at 80 °C for 15 min was set as 100 % (0 min data excluded as an outlier) and the results for the other time-series samples were correlated to this.
  • the percentage of residual activity was plotted as a function of incubation time at 80 °C as presented in Figure 8. From the resulting curve, it can be derived that SCA04 tolerates incubation at 80 °C over extended periods of time with only minor loss of activity. After three hours of incubation, still more than 90 % of the initial carbonic anhydrase activity could be detected.

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Abstract

La présente invention concerne des polypeptides naturels isolés présentant une activité d'anhydrase carbonique à température élevée et des capacités d'absorption / de désorption améliorées des constituants d'acides tels que le CO2 provenant d'un mélange gazeux ou vers ledit mélange, par exemple le gaz de combustion, le gaz naturel ou le biogaz. La présente invention concerne également l'utilisation des polypeptides isolés.
PCT/EP2012/075529 2012-12-14 2012-12-14 Nouveaux enzymes pour une absorption gazeuse améliorée WO2014090327A1 (fr)

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