WO2014090328A1 - Absorption/desorption of acidic components such as e.g. co2 by use of at least one catalyst - Google Patents

Abstract

Method for removal of CO₂ from a gas mixture (201), said method comprising at least one absorption step and optionally at least one desorption step wherein an absorbent (202) is used to absorb the CO₂ and at least one carbonic anhydrase enzyme is used to catalyze the absorption, wherein at least one carbonic anhydrase enzyme with a high K_m value (catalyst 2) is used as catalyst(s) in an absorption section (section 2) where the CO₂ concentration is high and at least one carbonic anhydrase enzyme with a low Km value (catalyst 1) is used as catalyst(s) in an absorption section (section 1) where the CO₂ concentration is lower than the high CO₂ concentration.

Description

Absorption/desorption of acidic components such as e.g. C0₂ by use of at least one catalyst

INTRODUCTION

The present invention generally relates to the absorption/desorption of acidic components, e.g. carbon dioxide (C0₂), from a gas mixture, e.g. flue gas, natural gas or biogas by use of at least one catalyst for improved absorption or absorption and desorption of said acidic components.

BACKGROUND OF THE INVENTION

Gas absorption is used to separate specific gas components from a gas mixture, such as the removal of C0₂ from flue gas, natural gas or biogas. An absorbent can absorb the gas components either by physical or by chemical solubility. A typical example of chemical absorption is the use of amine solutions to absorb C0₂. Since C0₂ is an acidic gas, the absorbent should have high pH as the absorbed C0₂ is then converted to bicarbonate (HC0₃ ^~) and carbonate (C0₃ ^2~) and this increases the capacity. Other examples of chemical absorbents are solutions of K₂C0₃, Na₂C0₃, NH₃, (NH₄)₂(C0₃) and aminoacid salts.

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

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

One problem with gas absorption is the slow reaction rate in the absorber. To increase the reaction rate, various catalysts/promotors can be added, such as piperazine.

An additional problem is the environmental aspect. Amines, which are most widely used because they absorb C0₂ faster than for example K₂C0₃ and NH₃, will release some amine components to the environment. Some amines are classified as "red" chemicals (chemicals which pose an environmental hazard), and may also

decompose/react into other types of products that are even more toxic. Many of the catalysts/promoters used are also classified as toxic.

The main problems with the existing technology are low overall efficiency, a slow absorption rate of gas, such as e.g. C0₂ and that most catalysts used up to date to increase absorption kinetics are toxic. Further, the most effective absorbents, such as amines, could harm the environment. Amines will decompose over time and generate a waste problem. Carbonates, for instance K₂CO₃ and /or Na₂C03, are non-toxic and could be used as absorbents instead of amines, but have much slower absorption rate than amines. N¾ could also be used instead of amines.

Also, the existing technology has 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 and/or catalysts. One category of catalysts that has been suggested is enzymes. Enzymes are widely distributed in nature and are active in catalyzing C0₂ absorption/desorption during respiration. Enzymes may therefore prove to be both effective and non-toxic catalysts for gas absorption/desorption processes, e.g. absorption/desorption of C0₂. One possible group of enzymes comes from the carbonic anhydrase family of enzymes.

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 similarities are found on the amino acid level. One common feature among the 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.

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

It is an object of the present invention to provide a method for enhanced

absorption/desorption of acidic components, such as e.g. carbon dioxide (C0₂) from a gas mixture, flue gas, natural gas or biogas by use of at least one catalyst for improved absorption or absorption and desorption of said components which may at least partly overcome the above mentioned problems. C0₂ capture processes and the use of enzymes as catalysts in said processes have been described in:

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

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

US 2009/0155889 describes a system and a method for absorbing CO₂ from a gas flow, where the 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 CO₂ 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 CO₂ in a gas flow by using different concentrations of catalyst, and that the amount is varied from the inlet to the outlet of absorbent, but does not show the use of enzymes.

SUMMARY OF THE INVENTION

The above problems are solved by the present invention. The objects of the invention are to increase the reaction rate, i.e. the absorption/desorption rate of CO₂, to obtain a process with high energy efficiency, and to avoid the use of toxic absorbents and toxic catalysts.

Said objects of the invention are achieved by the method according to the appended claims.

The present invention concerns a method for removal of C0₂ from a gas mixture, said method comprising at least one absorption step and optionally at least one desorption step wherein an absorbent is used to absorb the C0₂ and at least one carbonic anhydrase enzyme is used to catalyze the absorption, wherein at least one carbonic anhydrase enzyme with a high K_m value is used as catalyst(s) in an absorption section where the C0₂ concentration is high and at least one carbonic anhydrase enzyme with a low K_m value is used as catalyst(s) in an absorption section where the C0₂ concentration is lower than the high C0₂ concentration.

The low K_m is selected from the following range: from about 1 to about 25 mM and the high K_m value is selected from the following range: from about 25 to about 60 mM.

The enzymes are preferably selected from at least one of the following categories: extremophilic, thermophilic, hyperthermophilic, psychrophilic.

In a preferred embodiment, the at least one carbonic anhydrase enzyme is selected from the following isolated polypeptides having carbonic anhydrase activity:

Methanocaldococcus fervens AG86, Psychromonas ingrahamii 37, Desulfovibrio sp ND132, Sulfurospirillum deleyianum DSM 6946, Methanococcus aeolicus Nankai-3 and Pelobacter carbinolicus DSM 2380.

The at least one catalyst may be (a) dissolved in the absorbent and flowing through the appropriate absorption section and/or (b) immobilized on the respective absorption section and/or (c) immobilized on particles floating inside the absorbent.

In an embodiment of the invention, the at least one catalyst may be immobilized on a matrix, surface or substrate such as beads, fabrics, fibers, porous materials, CLEAs, structured or random packing, or crystals such as monoliths or combination thereof.

The absorbent used may comprise carbonates, amines, amino acid salts or blends thereof. The H in said at least one absorption step(s) is preferably selected from one of the following ranges: 7.0-11.0 and the pH in the at least one desorption step(s) is preferably < 7.0.

The temperature in said at least one absorption step(s) is preferably selected from one of the following ranges: 5 to 90 °C, 20 to 90 °C, 70 to 90°C, and the temperature in said at least one desorption step(s) is preferably selected from one of the following ranges: 80 to 140°C, 100 to 110°C.

In an embodiment of the invention, the at least one enzyme is used as a catalyst in said at least one desorption step and said at least one enzyme is the same or different as the at least one enzyme used as catalyst in said at least one absorption step.

DESCRIPTION OF THE FIGURES

Figure 1 shows, in a schematic view, an embodiment of the process according to the present invention;

Figure 2 shows, in a schematic view, an embodiment of the process according to the present invention;

Figure 3 shows, in a schematic view, an embodiment of the process according to the present invention.

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

Figure 6 A 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.

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.

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.

illustrates the relative C0₂ absorption rate as a function of C0₂ loading at different enzyme concentrations;

illustrates the impact of the absorbent concentration on the reaction kinetics.

DETAILED DESCRIPTION OF THE INVENTION

It is suggested to use at least one catalyst such as at least one carbonic anhydrase (CA) enzyme to increase absorption and/or desorption in an absorption and/or a desorption unit to make the absorption and/or desorption system work more effectively. The catalyst(s) might be, but is not limited to, enzymes. Different types of metalloenzymes that can mimic the catalytic activity of the active site of the natural enzyme can also be used. With the process according to the present invention, the C0₂ capture will be improved.

The catalyzed reaction depends on the C0₂ concentration in the gas mixture, such as e.g. flue gas, natural gas, biogas. In a C0₂-absorption process the C0₂ concentration in the gas will decrease from the inlet to the outlet of the absorber. This will have an impact on how efficient the catalyst such as an enzyme or mixture of enzymes, should be to catalyze the process. In a capture system where the concentration varies considerably in the gas phase from inlet to outlet, it will be beneficial to operate the absorber with at least one section where the catalyst(s) are optimized for each section.

The inventors surprisingly detected that using an enzyme or a mixture of enzymes with different K_m values at different sections in the absorber/desorber system, made the absorption/desorption process more efficient. At the inflow of an absorber system, the C0₂ concentration will be high; an enzyme or a mixture of enzymes with high K_m value(s) is more efficient at high C0₂ content. At the outlet part, with lower C0₂ concentration, an enzyme or a mixture of enzymes with low K_m value(s) should be the catalyst, as it is more efficient at low CO₂ content.

One aspect of the present invention relates to a method for removal of CO₂ from a gas mixture, e.g. flue gas, natural gas or biogas, said method comprising at least one absorption step and optionally at least one desorption step wherein an absorbent is used to absorb the CO₂ and at least one carbonic anhydrase enzyme is used to catalyze the absorption, wherein at least one carbonic anhydrase enzyme with a high K_m value is used as catalyst(s) in an absorption section where the CO₂ concentration is high and at least one carbonic anhydrase enzyme with a low K_m value is used as catalyst(s) in an absorption section where the CO₂ concentration is lower than the high CO₂

concentration.

The enzyme for carbonic anhydrase based CO₂ capture should have a long life-time in the process, be very efficient, and have a suitable K_m value for CO₂ and a suitable K_m value for HCO₃ ^" which is different from the K_m value for CO₂.

A high K_m value denotes an enzyme with low affinity to CO₂; thus an enzyme with high K_m should be used at high CO₂ concentrations in the gas stream, i.e. close to the inlet of the gas in the absorber (bottom). An enzyme with lower K_m can be used close to the outlet of the absorber column (top), where the concentration of CO₂ in the gas phase is lower.

The low K_m value is chosen from the following range: from about 1 to about 25 mM and the high K_m value is chosen from following range: from about 25 to about 60 mM.

The enzymes that are used as catalysts in the present process are chosen from at least one of the following categories: extremophilic, thermophilic, hyperthermophilic, psychrophilic. In an embodiment of the invention, the enzyme catalyst would typically be a hyperthermophilic carbonic anhydrase. Potentially new types of metallocene catalysts, mimicking the active site of the enzyme, having a Zn or Cd atom, could also be used. The enzymes can be derived from high temperature oil and/ or natural gas reservoirs, from environmental samples like hydrothermal vents, or can be combinations of chimeric enzymes, i.e. engineered from two or more different thermophilic organisms. Further, the thermophilic enzyme(s) can be used in combination with psychrophilic enzyme(s) for low temperature absorption/desorption processes.

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

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

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

The isolated carbonic anhydrases according to the present invention are identified as follows: SCAOl, SCA02, SCA03, SCA04, SCA05, SCA06b, SCA07, SCA09, SCA10 and SCAl 1 and were identified in an oil reservoir metagenome derived DNA sequence database assembled from read sequences obtained by 454 pyrosequencing of the metagenomic DNA. Sequences were found similar to sequences from: Methanocaldococcus FS406-22, Thermococcus AM4, Sulfurovum NB C37- \, Methanocaldococcus fervens AG86, Psychromonas ingrahamii 37, Desulfovibrio sp ND132, Sulfur o spirillum deleyianum DSM 6946, Methanococcus aeolicus Nankai-3 and Pelobacter carbinolicus DSM 2380 respectively. Said carbonic anhydrases were also codon optimized for expression in E. coli.

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, but also other sources 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. The absorption /desorption system according to the present invention may be at least 10 times more efficient with regard to kinetic rate than existing technology, preferentially even higher. Making the technical process more efficient will have a significant impact on reducing the operational cost. Environmental benefits will also be achieved in that the use of toxic absorbents/desorbents and catalysts may be avoided.

In one or more embodiments the carbonic anhydrase activity may be maintained at a temperature above 65°C for at least one hour. The activity may also be maintained for at least 5 hours in the temperature range of about 65°C to about 80°C, showing that the enzyme may perform its activity at high temperatures i.e. temperatures selected from a group consisting 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.

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

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

In existing technology, gas from power plants has to be cooled down in a heat exchanger before it can be treated with enzymes. The higher the operating temperature of the enzymatic process, the smaller the heat exchanger can be. The use of

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

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

The enzymes to be used as catalysts can be dissolved in an aqueous phase or immobilized on small particles. More specifically, the catalysts could (a) be dissolved in the absorbing liquid and flowing through the appropriate absorption section, (b) they could be immobilized on the respective absorption section, or (c) could be immobilized on particles floating inside the absorbing liquid.

The enzymes used as catalysts could be immobilized on particles to be recycled in two separate sections. Such particles could be, but are not limited to, microbeads in the form of uniform polymer particles such as for instance Ugelstad particles in the size range 1 - 50 μιη, possibly also magnetic particles for easier separation. Specially stabilized enzymes increase the kinetics. The stabilization could also be done by other nano- devices, such as monoliths, micro-crystals, cross-linked enzyme aggregates (CLEAs) etc. In particular, specialized particles or support that will increase stabilization and lifetime of the enzyme is suggested.

The immobilization step increases the longevity of the enzyme and might also stabilize its function at higher temperatures. The immobilization can be on a matrix, surface or substrate such as beads, fabrics, fibers, porous materials, CLEAs, structured or random packing, crystals such as monoliths or combination thereof. The stabilization of these particular types of enzymes is new, as well as the combination of enzymes with different K_m values.

Carbonic anhydrases are a family of enzymes that catalyzes both the hydration of C0₂ and the dehydration of HCO₃ ^". Higher pH promotes hydration while lower pH promotes dehydration. More specifically, pH values > 7 promotes hydration, whereas pH values < 7 promotes dehydration.

The pH in the at least one absorption step(s) may be selected from one of the following ranges: 7.0-11.0, or 7.5-9.0 and the pH in the at least one desorption step(s) may be < 7.0.

The temperature in the at least one absorption step(s) may be selected from one of the following ranges: 5 to 90 °C, 20 to 90 °C, 70 to 90°C, and the temperature in the at least one desorption step(s) may be selected from one of the following ranges: 80 to 140 °C, 100 to 110 °C.

The process according to the invention may be run at a temperature range between from about 30 °C to about 150 °C. The temperature range for the absorption step may be from about 60 °C to about 90 °C and from about 100 °C to about 110 °C for the desorption step. If needed, an enzyme that can be the same as or different from an enzyme used in the at least one absorption step can be introduced in the at least one desorption step of the process to speed up the rate.

The absorption /desorption system according to the present invention may be at least 10 times more efficient with regard to kinetic rate than the existing technology, preferentially even higher. Due to either the reduction in absorber and/or desorber size compared to existing plants or no use of amine-components as absorbents, or the combination of the two, the overall system will be much more environmentally friendly and efficient than existing plants.

Potential recycling of the enzyme catalyst, possibly with a small make up - addition will make economics of the enzyme catalyst beneficial.

With the combination of efficient catalysts it is possible to design more compact plants. The design of a C0₂ capture process is closely linked to the kinetics of C0₂ absorption into the absorbent. The size of an absorber is a limiting factor in the design of a capture plant. The absorber size affects the plant foot-print, required solvent mass flow, cooling and heating requirements, pumping capacity, pipe sizing and lengths, pressure drops etc. More important, limitation in absorber size also influences the range of available absorbents for capturing C0₂. Thus, the C0₂ absorption kinetics has significant impact on capital expenditure (CAPEX), operating expenditure (OPEX) and energy efficiency. Efficient catalysts make it possible to use alternative absorbents, such as K₂C0₃ or Na₂C0₃ solutions, which are cheap to acquire, non-proprietary and non-toxic and will never decompose to harmful by-products. Furthermore, there might be a more efficient use of the buffer system to generate C0₃ ^2" or to strip away the HC0₃ ^". Due to the fast reaction kinetics, volumes and size can be reduced.

Due to the smaller size of the plants, one can reduce the amount of required chemicals, resulting in a more energy and cost efficient process. It enables the possibility to use new and/or more efficient solvents.

New heat stable carbonic anhydrases having the ability to increase the reaction rate, i.e. the absorption/desorption of an acidic component are identified and isolated. The acidic component might be, but are not limited to C0₂, also other areas of use are suggested; improved oil recovery, biomass production, C0₂ storage or artificial lung support.

It is suggested to use at least one carbonic anhydrase enzyme to increase absorption and/or desorption in an absorption and/or a desorption unit to make the absorption and/or desorption system work more effectively. Utilizing the carbonic anhydrase enzyme(s) according to the present invention, the C0₂ capture will be improved. The invention is valid for both flue gas capture systems (atmospheric pressure) and C0₂ capture from natural gas at elevated or high pressure. In an embodiment of the invention, the at least one enzyme is used as a catalyst in said at least one desorption step and said at least one enzyme is the same or different as the at least one enzyme used as catalyst in said at least one absorption step.

DEFINITIONS

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

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

_ Vmax [S]

° K_m+ [S] wherein

Vo is the initial reaction rate,

V_max is the maximum reaction rate,

[S] is the substrate concentration,

K_m is the Michaelis-Menten constant.

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

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

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

The term "extremophilic enzyme" is used herein to describe enzymes that exist and are stable under or even might require physically and/or geochemically extreme conditions that are detrimental to most life on earth.

The term "hyperthermophilic enzyme" is used herein to describe enzymes that exist and are stable at temperatures between e.g. 60-122°C, with optimal temperatures above e.g. 80°C.

The term "thermophilic enzyme" is used herein to describe enzymes that exist and are stable at temperatures between e.g. 45-122°C, with optimal temperatures between e.g. 60-80°C.

The term "psychrophile" (also known as a "cryophile") is defined herein as an organism that is capable of reproduction and growth at low temperatures, typically in the range e.g. -10 to 20°C.

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

The term "make-up addition" denotes herein a stream that adds a regulated amount of enzyme or enzyme mixture to compensate for enzyme that denaturates or loses its activity during the absorption process.

The term "stabilization" refers herein to immobilization of the enzyme(s) on a matrix, surface or substrate. It can be at least partially composed of beads, fabrics, fibers, porous materials, structured or random packing, crystals or combinations thereof.

The term "immobilizing agent" as used herein refers to an agent having the ability to stabilize an enzyme on e.g. 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. The term "carbonic anhydrase activity" as used herein is defined as an activity which catalyzes the conversion between carbon dioxide and bicarbonate.

The present invention will be more understood by referring to the following examples. The examples provided intend to show the range of applicability of the invention, and do not limit its scope.

EXAMPLES

Example 1

Figure 1 - Streams:

101 : C0₂ containing feed gas

102: absorbent stream, optionally regenerated from the desorber

103: catalyst 1 stream

104: lean solvent to absorber 1 (102+103)

105: rich solvent leaving absorber 1

106: removal of catalyst 1 (optional)

107: rich solvent from absorber 1, with optionally removed catalyst 1

108: catalyst 2 stream

109: lean solvent to absorber 2 (107+108)

110: gas out from absorber 1, going in to absorber 2

111 : CO₂ depleted gas stream

112: rich solvent leaving absorber 2

113: removal of catalyst 2 (optional)

114: rich solvent from absorber 2, with optionally removed catalyst 2

115: catalyst 3 stream (optional)

116: solvent to desorber/regenerator (114+115)

117: regenerated solvent, optionally containing catalyst 3

118: removal of catalyst 3 (optional)

120: CO₂ rich gas stream leaving desorber

CO₂ containing gas 101 is fed to a first absorber, catalyst 1 stream 103 with suitable K_n value enters the first absorber with the solvent stream 102, gas out from the first absorber 110 is fed to a second absorber, catalyst 1 is optionally removed 106 from the rich solvent 105 leaving the first absorber, the rich solvent with optionally removed catalyst 1 stream 107 and catalyst 2 stream 108 with suitable K_m value different from the K_m value of catalyst 1 is fed to the second absorber as lean solvent 109, C0₂ depleted gas 111 exits the second absorber, catalyst 2 is optionally removed 113 from the rich solvent stream 112 leaving the second absorber, the solvent from the second absorber with optionally removed catalyst 2 stream 114 and catalyst 3 stream 115 enters a desorber/regenerator as the solvent stream 116, C0₂ rich gas stream 120 leaves the desorber, the regenerated solvent stream comprising catalyst 117 leaves the desorber, and catalyst 3 is optionally removed through stream 118 from the regenerated solvent stream 117, and the solvent stream 102 is optionally returned to the first absorber.

Catalyst 1 , catalyst 2 and catalyst 3 can be the same or different types of carbonic anhydrase or blends thereof. They can be dissolved in an aqueous phase or immobilized on small particles.

Example 2

Figure 2 - Streams:

201 : C0₂ containing feed gas

202: absorbent stream, optionally regenerated from the desorber

203 : C0₂ depleted gas stream

204: rich solvent leaving absorber

205 : heated solvent to desorber

206: C0₂ rich gas stream leaving desorber

C0₂ containing gas 201 is fed to an absorber with at least one section, catalyst 1 with a suitable K_m value either enters the absorber with the absorbent stream 202, or is already immobilized in the absorption column, catalyst 2 with a different K_m value than catalyst 1 either enters a second section in the absorber or is already immobilized in the absorption column, the two catalysts being active at different sections of the absorber related to the concentration of C0₂ in the gas, C0₂ depleted gas stream 203 exits the absorber, rich solvent 204 leaves the absorber, a heat exchanger may optionally adjust the solvent temperature before solvent 205 enters the desorber with catalyst 3, C0₂ rich gas stream 206 leaves the desorber, and the solvent 202 is optionally returned to the absorber.

Catalyst 1, catalyst 2, and catalyst 3 can be the same or different types of carbonic anhydrase or blends thereof. They can be dissolved in an aqueous phase or immobilized on small particles. Example 3

Figure 3 - Streams:

301 : C0₂ containing feed gas

302: absorbent stream, optionally regenerated from the desorber

303: catalysts 1 & 2

304: lean solvent to absorber (302 + 303)

305: CO₂ depleted gas stream

306: rich solvent leaving absorber

307: optional removal of catalysts 1 & 2

308: solvent, optionally heated, to desorber

309: CO₂ rich gas stream

CO₂ containing gas 301 is fed to an absorber with at least one section, solvent stream 302 is optionally regenerated from the desorber. The solvent stream 302 and the streams of catalysts 1 & 2 with different K_m values 303 enter the absorber as lean solvent stream 304 to absorber, CO₂ depleted gas stream 305 exits the absorber, rich solvent stream 306 leaves the absorber, catalysts 1 & 2 are optionally removed 307, a heat exchanger may optionally adjust the solvent temperature before the solvent stream 308 enters the desorber, CO₂ rich gas stream 309 leaves the desorber, and the solvent stream 302 is optionally returned to the absorber.

Catalyst 1 and catalyst 2 can be the same or different types of carbonic anhydrase or blends thereof. They can be dissolved in an aqueous phase or immobilized on small particles.

Example 4

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

The following procedure was used to determine carbonic anhydrase activity, representing a modified version of the carbonic anhydrase activity assay as described by Wilbur, 1948, J. Biol. Chem. 176, 147-154: On a magnet stirrer at room temperature, a 50 ml plastic beaker (reaction vessel) containing a small stirrer magnet was filled with 12 ml 20 mM Tris-S0₄ buffer pH 8,3, 1 μΜ ZnS0₄. A calibrated pH electrode connected to a PHM210 pH meter (Radiometer Analytical) was installed in the buffer solution to allow for both continuous pH measurement as well as thorough mixing of the vessel's content at 750 rpm. 100 μΐ culture crude extract prepared in buffer A (50 mM potassium phosphate, pH 6.8, 1 μΜ zinc sulfate) was added to the Tris buffer while mixing. After pH logging was started (WaveScan 2.0 controlling an Advantec USB-4711A multifuntion module connected to the pH-meter's recorder output; channel range: +/-1.25mV), 9 ml C0₂ saturated ion- free water was added to the reaction vessel. C0₂ saturated water was prepared by bubbling C0₂ gas from dry ice in an isolation bottle through 0.5 L ion free water while stirring overnight. The decrease in pH was recorded at a resolution of 50 ms until a constant pH was obtained (25-80 s). The time needed for the pH to drop from pH 7.8 to pH 7.0 (dt) was determined and used as a measure for carbonic anhydrase enzymatic activity. Three independent measurements were regularly performed in order to define the standard deviation between individual measurements, regularly leading to a variation of less than 5 %. 100 μΐ pure buffer A or 100 μΐ of a reference crude extract (prepared from a similarly treated culture containing the empty expression vector pET16b) was used as a negative control. A spontaneous pH change from pH 7.8 to pH 7.0 in the absence of enzymatic activity was regularly obtained within 18-35 s. The result is given in Figure 4A. Activity units were calculated

USing the formula U=(dt_pH7.8-pH7-0, buffer A-dt_pH7.8-pH7-0, e_Xtract)/dt_pH7.8-pH7-0, extract ^X factor of dilution with buffer A and correlated to 1 ml sample volume (Figure 4B).

All four proteins SCA04/SCA06b/SCA09/SCAl 1 could be shown to exhibit carbonic anhydrase activity (Figure 4B). Measuring at room temperature (23 °C), SCAl 1 showed the highest activity, followed by SCA04. SCA06b and SCA09 activity could be detected, though at very low levels. No activity was observed in the negative control extract (Figure 4B).

Example 5

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

In order to evaluate the stability of recombinantly produced

SCA04/SCA06b/SCA09/SCAl 1, respectively, at 65 °C and/or 80 °C and/or at high salt concentration [20 % (w/v) K₂C0₃], 175 μΐ, of crude extract were diluted with 325 μΐ, 30.8 % (w/v) K₂C0₃, 1 μΜ ZnS0₄, pH8.3 to give a final concentration of 20 % (w/v) K₂C0₃ in the diluted extract. In parallel, 175 μΐ, crude extract were diluted with 325 μΐ, ion free water containing 1 μΜ ZnS0₄. Dilutions were incubated at room temperature (-23 °C), 65 °C or 80 °C for 1 h or 5 h. After incubation, samples were centrifuged in a microliter centrifuge (14,000 rpm, 4 °C, 5 min), and 285 μΐ, of the cleared supernatant, corresponding to 100 μΐ of undiluted crude extract, was used for activity measurement as described in Example 4. For the ion free water/ 1 μΜ ZnS0₄ diluted samples, the time for the pH to drop from pH 7.8 to pH 7.0 (dt_pH7.8-_PH7.o) was determined, while for the respective high salt samples, due to a higher initial pH, the drop time from pH 8.2 to pH 7.4 (dtpH8.2 pH7.4) was determined, both corresponding to an almost linear decrease of pH over time. Dilutions (1 : 10, 1 : 100 or 1 :200) were applied where necessary in order to obtain dt values between 5 and 20 seconds. Activity units were calculated using the formula U=(dtpH8.2-pH7.4, ref-dt_pH8.2-pH7.4, e_Xtract)/dt_pH8.2-pH7.4, extract ^X factor of dilution With

20 % (w/v) K₂C0₃, 1 μΜ ZnS0₄, pH8.3 for the samples containing 20 % (w/v) K₂C0₃ and U=(dtpH7.8-pH7.0, ref-dtpH7.8-pH7.0, e_Xtract)/dt_pH7.8-pH7.0, extract ^X factor of dilution With ion free water containing 1 μΜ ZnS0₄ for the samples diluted with ion free water containing 1 μΜ ZnS0₄. The reference measurements for the two different conditions (ref) were obtained from measuring similarly diluted crude extract from a culture containing the empty expression vector pET16b incubated for 1 h at 65 °C. The calculated activity units were correlated to 1 ml extract volume. The results are presented in Figure 5. The four enzymes SCA04/SCA06b/SCA09/SCAl 1 exhibited very different characteristics with respect to stability at high temperature and/or high salt concentration (Figure 5). SCA04 was found to be very stable under all condition tested. Even after incubation for 5 h in 20 % (w/v) K₂C0₃, more than 50 % of the original activity was retained. SCA11 was found to be relatively stable when incubated at 65 °C, though a clear decrease of activity over time was observed at this temperature. The combination of high temperature (65 °C) and 20 % (w/v) K₂C0₃ was not tolerated, leading to a rapid loss of functionality. It had been shown before that SCA11 was in general quickly degraded at 80 °C (data not shown). SCA06b was found to be stable at room temperature, but quantitatively degraded already after 1 h incubation at 65 °C. Interestingly, higher carbonic anhydrase activity was observed for SCA06b in the presence of 20 % (w/v) K₂C0₃. The high salt concentrations obviously had a stabilizing effect on the enzyme. This effect was also observed when SCA06b was incubated at 65 °C. SCA09 showed a relatively high stability when incubated at 65 °C or 80 °C, though some loss of activity was observed especially after 5 h of incubation. The additional presence of 20 % (w/v) K₂C0₃ had no additional destabilizing effect at 65 °C, while after 5 h incubation at 80 °C, most of the activity was lost.

Example 6

Determination of K_m values of SCA04, SCA09 and SCA11

(i) Recombinant production in E. coli and preparation of heat enriched crude extracts containing SCA04, SCA09 and SC All For the recombinant production of the enzymes SCA04, SCA09 and SCA11 in E. coli, high cell density (HCD) fed-batch fermentation experiments in bench-top scale fermentors were performed. E. coli strains generated based on strain BL21(DE3) and carrying the respective CA encoding gene on a pET16b derived plasmid were pre- cultivated in 100 ml LB(g) medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 10 g/L glucose*H₂0) containing 100 mg/1 ampicillin in baffled 500 ml shake flasks at 30 °C and 200 rpm on a shaking incubator. After approx. 10 h of incubation, 125 μΐ of the grown cultures on this medium were used to inoculate 100 ml Hi.l inoculation medium (8.6 g/L Na₂HP0₄x2H₂0, 3 g/L KH₂P0₄, 1 g/L NH₄C1, 0.5 g/L NaCl, 0.06 g/L Fe(III) citrate hydrate, 0.003 g/L H₃B0₄, 0.015 g/L MnCl₂x4H₂0, 0.0084 g/L

EDTAx2H₂0, 0.0015 g/L CuCl₂x2H₂0, 0.0025 g/L Na₂Mo₄0₄x2H₂0, 0.0025 g/L CoCl₂x6H₂0, 0.008 g/L Zn(CH₃COO)₂x2H₂0, 10 g/L glucose, 0.6 g/L MgS0₄x7H₂0) containing 100 mg/1 ampicillin in baffled 500 ml shake flasks which was then incubated at 30 °C and 200 rpm to an OD600 of approx. 6 (after approx. 18 h). 750 ml Hf.l medium (16.6 g/L KH₂P0₄, 4 g/L (NH₄)₂HP0₄, 2.1 g/L citric acid, 0.075 g/L Fe(III) citrate hydrate, 0.0038 g/L H₃B0₄, 0.0188 g/L MnCl₂x4H₂0, 0.0105 g/L EDTAx2H₂0, 0.0019 g/L CuCl₂x2H₂0, 0.0031 g/L Na₂Mo0₄x2H₂0, 0.0031 g/L CoCl₂x6H₂0, 0.01 g/L Zn(CH₃COO)₂x2H₂0, 25 g/L glucose, 1.5 g/L MgS0₄x7H₂0) containing 100 mg/1 ampicillin were then inoculated to a calculated final OD₆oo of 0.05.

Fermentations were performed at 30 °C and pH 6.8, automatically adjusted with 12.5 % NH₃ solution. Using an aeration rate of 0.35 to 1.5 wm, a minimum level of dissolved oxygen (DO) of 0.2 was maintained by automatic adjustment of the stirrer speed. After approx. 12 h of batch cultivation, exponential feeding was started using a 50 % glucose/MgS0₄ solution at an initial rate of 10 g/(L culture volumexh) [i.e.

7.5 g/(750 mlxh)] up to approx. 35 g/h. After that, constant feeding at 35 g/h was applied. Glucose levels were monitored manually when necessary and held limiting throughout the fed-batch phase. CA gene expression was induced at an OD₆oo of approx. 70 by addition of 0.75 ml 1 M IPTG and 1.5 ml 500 mM ZnS0₄ from sterile stock solutions. Sample P0 was withdrawn immediately before induction, and subsequent samples [usually 5 ml in total for OD₆oo measurement and biomass + supernatant (3^χ 1 g)] were taken every 2-3 h. All samples were stored at -20 °C until

analysis/extraction. Approx. 6 h after induction of CA gene expression, fermentation was stopped, and biomass was harvested by centrifugation in two centrifugation bags each. The supernatant was disposed, and the biomass was frozen and stored at -80 °C until further processing. Based on the time series samples withdrawn after induction of gene expression, recombinant production of active enzyme was confirmed by SDS- PAGE and using the carbonic anhydrase pH assay. HCD fermentation derived biomass (48.3 g for SCA04, 77.6 g/L for SCA09, 61.6 g for SCA11) was processed using the following procedure for the extraction of soluble CA enzyme (result: crude extract) and the subsequent enrichment of the thermostable enzymes by heat treatment (result: heat enriched crude extract): each 10 g wet weight of biomass was re-suspended in 20 ml buffer A (50 mM potassium phosphate, 1 μΜ ZnSC"4, pH 6,8) by pipetting and/or whirl-mixing (e.g. in a 50 ml tube). While placed on ice, the biomass suspension was sonicated (Branson Sonifier, flat tip, duty cycle 50 %, output control 4) for 10 x 1 min with thorough mixing after each minute of sonication. The treated sample was centrifuged (20 min, 20000 xg, 4 °C), and the supernatant was transferred to a fresh reaction tube. The supernatant (crude extract) was stored at -80 °C (alternatively short term storage at 4 °C). To produce a crude extract enriched for the thermostable CA enzyme, an aliquot of the respective crude extract was heated for 25 min at 65 °C in a water bath while inverting the tube several times during incubation to ensure thorough heating. After incubation, the sample was centrifuged (15 min, 20000 xg, 4 °C), and the supernatant (=heat enriched crude extract) was stored in aliquots a 1-1.5 ml at -80 °C.

(ii) Determination of enzyme concentrations and purities ofSCA04, SCA09 and SC All in heat enriched crude extracts

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

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

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

concentrations, the concentrations of the CA enzymes in the enriched crude extract were calculated to be 6.84 mg/ml (SCA04), 12.16 mg/ml (SCA09) and 15.84 mg/ml

(SCA11).

(Hi) Production of C0₂-saturated water

To produce C0₂-saturated water as substrate stock solution for subsequent CA activity measurements for K_m determination, a thermos bottle was filled with dry ice, and the developing gas was bubbled through a flask containing 500 ml ion free water while stirring. The system was left overnight to reach saturation, before the bottle was tightly closed and stored for at least one hour to overnight to equilibrate. The C0₂

concentration in the substrate stock solution was determined by titration with 0.01 M NaOH in the presence of the pH indicator phenolphthalein and continued until the indicator turned pale pink (typically 33-36 ml for 10 ml C0₂-saturated water).

(iv) Measuring enzymatic activity and data conversion

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

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

(v) K_m determination ofSCA04

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

In Figure 6A, the specific activities of SCA04 are plotted as a function of the substrate concentration. Two individual series were included, plotted as open squares and open diamonds, respectively, while the sum of the two is presented as closed diamonds. Values for the Michaelis-Menten kinetic parameters K_m and V_max were determined by using the Microsoft Excel solver tool to minimize the sum of squared deviations between measured and values calculated from the Michaelis-Menten equation including all measurements.

(vi) K_m determination ofSCA09

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

In Figure 6B, the specific activities of SCA09 are plotted as a function of substrate concentration. Two individual series were included, plotted in open squares and open diamonds, respectively, while the sum of the two is seen in closed diamonds. Values for the Michaelis-Menten kinetic parameters K_m and V_max were determined by using the Microsoft Excel solver tool to minimize the sum of squared deviations between measured and values calculated from the Michaelis-Menten equation including all measurements.

(vii) K_m determination ofSCAll

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

In Figure 6C, the specific activities of SCA11 are plotted as a function of substrate concentration. Two individual series were included, plotted in open squares and open diamonds, respectively, while the sum of the two is seen in closed diamonds. Values for the Michaelis-Menten kinetic parameters K_M and V_max were determined by using the Microsoft Excel solver tool to minimize the sum of squared deviations between measured and values calculated from the Michaelis-Menten equation including all measurements. (νίίί) Summary

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

Table 1. Kinetics parameters for SCA04, SCA09 and SCA11

SCA04 SCA09 SCA11

9.1 205977 6.1 15752 48.8 8295138

Example 7

Effect of the amount of enzyme added

To demonstrate the impact of free carbonic anhydrase on the reaction kinetics, tests were conducted in a stirred cell at enzyme concentrations of 1 , 2 and 4 wt% in a 20 wt% K₂CO₃ solution. The initial loading of the solution was adjusted to 0.1 and 0.2 mole C0₂/mole K₂CO₃. The enzyme used is a variant of carbonic anhydrase from human erythrocyte cells type II. A known amount of absorption solution was placed in the cell, and let equilibrate with pure C0₂ under constant atmospheric pressure. The volume of C0₂ absorbed is logged as a function of time and is used to calculate the rate of absorption. The results were compared to tests conducted without the presence of enzyme (points presented as 0 wt%) and are expressed as relative C0₂ absorption rate, i.e. as the ratio of absorption rate with enzyme to absorption rate in the absence of enzyme. The results indicate that the enzyme enhances the absorption rate for all tested K₂CO₃ solutions. As the initial loading of the solution increases, there is a decrease in the relative rate of absorption. The enzymes succeed to increase the kinetics by a noticeable and important factor, but it is expected that as the loading increases further, the enhancement will be reduced mainly due to the fact that as C0₂ is absorbed in the solution, accumulation of HCO₃ ^" species reduces the rate of absorption. Figure 7 illustrates the relative C0₂ absorption rate as a function of C0₂ loading at different enzyme concentrations. Example 8

Effect of the solvent concentration

To demonstrate the impact of the solvent concentration on the reaction kinetics, tests were conducted in a stirred cell with a fixed amount of enzyme (4 wt%) at different K₂CO₃ concentrations. The results are shown in Figure 8, which illustrates the relative CO₂ absorption rate as a function of the K₂CO₃ concentration (wt%) at an amount of 4 wt% enzyme.

Having described preferred embodiments of the invention it will be apparent to those skilled in the art that other embodiments incorporating the concepts may be used. These and other examples of the invention illustrated above are intended by way of example only and the actual scope of the invention is to be determined from the following claims.

Claims

C l a i m s 1.

Method for removal of C0₂ from a gas mixture, said method comprising at least one absorption step and optionally at least one desorption step wherein an absorbent is used to absorb the C0₂ and at least one carbonic anhydrase enzyme is used to catalyze the absorption,

c h a r a c t e r i s e d i n that

at least one carbonic anhydrase enzyme with a high K_m value is used as catalyst(s) in an absorption section where the C0₂ concentration is high and at least one carbonic anhydrase enzyme with a low K_m value is used as catalyst(s) in an absorption section where the C0₂ concentration is lower than the high C0₂ concentration.

2.

Method according to claim 1 ,

characterised in that

said low K_m is selected from the following range: from about 1 to about 25 mM and said high K_m value is selected from the following range: from about 25 to about 60 mM.

3.

Method according to claim 1 ,

characterised in that

said enzymes are selected from at least one of the following categories: extremophilic, thermophilic, hyperthermophilic, psychrophilic.

4.

Method according to claim 1 ,

characterised in that

said at least one carbonic anhydrase enzyme(s) is(are) selected from the following isolated polypeptides having carbonic anhydrase activity:

Methanocaldococcus fervens AG86, Psychromonas ingrahamii 37, Desulfovibrio sp ND132, Sulfurospirillum deleyianum DSM 6946, Methanococcus aeolicus Nankai-3 and Pelobacter carbinolicus DSM 2380.

5.

Method according to claim 1 ,

characterised in that

said at least one catalyst is being (a) dissolved in said absorbent and flowing through the appropriate absorption section and/or (b) immobilized on the respective absorption section and/or (c) immobilized on particles floating inside said absorbent.

6.

Method according to claim 5,

characterised in that

said at least one catalyst is being immobilized on a matrix, surface or substrate such as beads, fabrics, fibers, porous materials, CLEAs, structured or random packing, or crystals such as monoliths or combination thereof.

7.

Method according to claim 1 ,

characterised in that

said absorbent used comprises carbonates, amines, aminoacid salts or blends thereof.

8.

Method according to claim 1 ,

characterised in that

the pH in said at least one absorption step(s) is selected from the following range: 7.0- 11.0, and that the pH in said at least one desorption step(s) is < 7.0.

9.

Method according to claim 1 ,

characterised in that

the temperature in said at least one absorption step(s) is selected from one of the following ranges: 5 to 90 °C, 20 to 90 °C, 70 to 90°C, and the temperature in said at least one desorption step(s) is selected from one of the following ranges: 80 to 140°C, 100 to 110°C.

10.

Method according to claim 1 ,

characterised in that

at least one enzyme is used as a catalyst in said at least one desorption step and that said at least one enzyme is the same or different as the at least one enzyme used as catalyst in said at least one absorption step.