WO2014012181A1

WO2014012181A1 - Method for preparing surface modified carbonic anhydrase with enhanced activity and/or stability

Info

Publication number: WO2014012181A1
Application number: PCT/CA2013/050552
Authority: WO
Inventors: Julie Gingras; Eric Madore; Mathieu Simard; Normand Voyer; Glenn R. Kelly
Original assignee: Co2 Solutions Inc.
Priority date: 2012-07-16
Filing date: 2013-07-16
Publication date: 2014-01-23

Abstract

A method for obtaining surface modified carbonic anhydrases having improved activity and/or stability is provided. The method comprises modifying surface functional groups of a carbonic anhydrase having an initial surface charge by reaction with a reagent while varying a predetermined reaction parameter, whereby the reaction allows obtaining a set of modified carbonic anhydrases with respective surface charges different than the initial surface charge; testing the stability and/or activity of the modified carbonic anhydrases; and identifying modified carbonic anhydrases having improved stability and/or activity. The method may also 10 comprise surface modifying a plurality of different carbonic anhydrases in the presence of at least one reagent; testing the stability and/or activity of resulting modified carbonic anhydrases; and identifying the modified carbonic anhydrases having improved stability and/or activity. A surface modified carbonic anhydrase having improved stability and/or activity is also provided. The modified carbonic anhydrase is useful biocatalyst for CO 2 hydration or desorption.

Description

METHOD FOR PREPARING SURFACE MODIFIED CARBONIC ANHYDRASE

WITH ENHANCED ACTIVITY AND/OR STABILITY

FIELD OF THE INVENTION

The present invention generally relates to the field of enzyme stabilization and/or activation and more particularly to a method for preparing and identifying carbonic anhydrases having improved activity and/or stability. The invention also relates to surface modified carbonic anhydrases having improved activity and/or stability and uses thereof.

BACKGROUND OF THE INVENTION

Enzymes are useful biocatalysts that are used increasingly in industrial processes. They possess several advantages over traditional chemical catalysts, e.g. heavy metal catalysts, such as operating in less toxic solvents and being environmentally benign. In addition, enzymes can catalyze very efficiently numerous chemical processes that cannot be achieved by conventional chemical processes. They differ from most other catalysts in that they are highly specific for their substrates.

The enzyme carbonic anhydrase ("CA") (EC 4.2.1 .1 ), catalyzes the following reversible reaction:

C0₂ + H₂0 HCO3- + H⁺ Hence, CA is able to catalyze the "hydration" of carbon dioxide to provide bicarbonate and a proton, or depending on the pH, to provide carbonate (CO3^"2) and two protons. In the reverse, or "dehydration" reaction, CA combines bicarbonate and a proton to provide carbon dioxide and water.

The major drawback limiting the use of carbonic anhydrase enzymes as biocatalysts is their sensitivity to denaturation, degradation and/or deactivation under the conditions usually utilized in industrial processes. For example, the enzymes activity is affected by temperature, pressure, chemical environment (e.g. pH), and the substrates concentration.

Enzyme immobilization is one method used to enhance enzyme activity and/or stability. For example, the enzyme is adsorbed on a matrix, entrapped/encapsulated in insoluble beads or microspheres, or covalently bonded to a matrix through a chemical reaction (cross-linkage). However, these methods may show some drawbacks. For example, the active site of the immobilized enzyme may be blocked by the matrix when the enzyme is adsorbed thereon, thus reducing the activity of the enzyme. When the enzyme is entrapped in insoluble beads or microspheres, it may happen that the arrival of the substrate and the exit of products are hindered.

The covalent modification of functional groups at the surface of an enzyme is another known process that leads to enzyme with altered efficiency and stability to denaturation and degradation. However, it is not possible to predict the outcomes of chemical surface modifications which depend on various parameters including for example the nature of the reagent, the level of modification and/or the resulting total surface charge. Thus, current methods used to enhance enzyme stability are not predictable and do not stabilize enzymes enough towards miscellaneous conditions to prevent or retard denaturation significantly.

Thus, there is a need for a method to obtain carbonic anhydrases with enhanced stability and/or activity.

There is a need for surface modified carbonic anhydrases with enhanced stability and/or activity.

There is also a need for surface modified carbonic anhydrases with enhanced stability and/or activity that can be used for the absorption of carbon dioxide and/or desorption of carbon dioxide.

SUMMARY OF THE INVENTION

The present invention responds to the above-identified need by providing a method for preparing and identifying carbonic anhydrases having improved activity and/or stability, involving chemical modifications of functional groups on the surface the carbonic anhydrases.

In one aspect, there is provided a method for preparing and identifying surface modified carbonic anhydrases having an improved activity and/or stability comprising: a) providing a carbonic anhydrase having an initial surface charge; b) modifying surface functional groups of the carbonic anhydrase, comprising subjecting the carbonic anhydrase to at least one reaction with a reagent and varying at least one predetermined reaction parameter, whereby the reaction allows obtaining a set of modified carbonic anhydrases with respective surface charges different than the initial surface charge; c) testing the stability and/or activity of the set of surface modified carbonic anhydrases; and

d) identifying surface modified carbonic anhydrases of the set having an improved stability and/or activity.

In another aspect, there is provided a method for preparing and identifying surface modified carbonic anhydrases having an improved activity and/or stability comprising: a) providing a plurality of different carbonic anhydrases each having an initial surface charge; b) modifying surface functional groups of each of the carbonic anhydrase, comprising separately subjecting each carbonic anhydrase to at least one reaction with a reagent thereby obtaining a set of modified carbonic anhydrases with respective surface charges different than the initial surface; c) testing the stability and/or activity of each surface modified carbonic anhydrases of the set; and

d) identifying surface modified carbonic anhydrases of the set having an improved stability and/or activity. In an optional aspect, when a plurality of different carbonic anhydrases are surface modified, the modification step may further comprise varying at least one predetermined reaction parameter.

In another optional aspect of the above method, the at least one predetermined reaction parameter comprises a reagent concentration, a carbonic anhydrase concentration, a pH at which the reaction is performed, a solvent in which the reaction is performed, a reaction temperature, a reaction time, or any combination thereof. For example, the at least one predetermined reaction parameter comprises a reagent concentration.

In another optional aspect, the modifying step of the above method comprises: i. covalent modification of the functional groups by reaction with a monofunctional reagent, or ii. covalent stapling of the functional groups by reaction with a bifunctional reagent, or iii. covalent stitching of the functional groups by reaction with a multifunctional reagent, the multifunctional reagent having at least three reactive groups capable to react with at least three surface functional groups of the carbonic anhydrase.

In another optional aspect, the surface functional groups that are modified comprise neutral or protonated amino groups; neutral or deprotonated carboxylic acid groups; neutral or protonated guanidino groups; or any combination thereof. In another optional aspect, the surface functional groups that are modified comprise neutral or protonated amino groups and the covalent modification, covalent stapling or covalent stitching comprises an acylation reaction, an imination reaction, or a thioureation reaction.

In another optional aspect, the surface functional groups comprise lysine amino functional groups, amino terminal groups of the peptide chains of the carbonic anhydrase, or primary amino groups resulting from a previous modification. In another optional aspect, the modifying reaction comprises an acylation that is performed using: i. an aliphatic, aromatic or heteroaromatic acid anhydride, acyl halide, or acid activated ester as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diacyl halide or diacid activated ester as the at least one bifunctional reagent; or iii. a polyacyl halide having at least three acyl halide groups or a polyacid activated ester having at least three acid activated ester groups as the at least one multifunctional reagent, wherein the acyl halide groups of the polyacyl halide or the acid activated ester groups of the polyacid activated ester are linked to each other by an aliphatic, aromatic or heteroaromatic group.

In another optional aspect, the modifying reaction comprises an imination that is performed using: i. an aliphatic, aromatic or heteroaromatic monoaldehyde as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic dialdehyde as the at least one bifunctional reagent; or iii. a polyaldehyde having at least three aldehyde groups as the at least one multifunctional reagent, wherein the aldehyde groups of the polyaldehyde are linked to each other by an aliphatic, aromatic or heteroaromatic group.

In another optional aspect, the modifying reaction comprises a thioureation that is performed using: i. an aliphatic, aromatic or heteroaromatic isothiocyanate reagent as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diisothiocyanate reagent as the at least one bifunctional reagent; or iii. a polyisothiocyanate reagent having at least three isothiocyanate groups as the at least one multifunctional reagent, wherein the isothiocyanate groups of the polyisothiocyanate reagent are linked to each other by an aliphatic, aromatic or heteroaromatic group.

In another optional aspect, the aliphatic group of the acylation, imination or thioureation reagent comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S.

In another optional aspect, the aromatic and heteroaromatic groups of the acylation, imination or thioureation reagent comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from O, N and S. In another optional aspect, the acylation is performed with activated esters comprising fluorophenol esters, nitrophenol esters, hydroxybenzotriazole esters, N- hydroxy succinimide esters, or isourea esters.

In another optional aspect, the acylation is performed with diacid activated ester comprising [bis(succinimidyl) penta(ethyleneglycol)] BS(PEG)5 or [bis(succinimidyl) nona(ethyleneglycol)] BS(PEG)9. In another optional aspect, the diacid activated ester concentration is varied to obtain a [diacid activated ester : amino] molar ratio ranging from 0.2 to 20.

In another optional aspect, the acylation is performed with an acid anhydride comprising acetic anhydride, phtalic anyhydride or succinic anhydride.

In another optional aspect, the acid anhydride concentration is varied to obtain a [acid anhydride : amino] molar ratio ranging from 2 to 200.

In another optional aspect, the imination is performed using a monoaldehyde being acetaldehyde, propionaldehyde, butyraldehyde or benzaldehyde, or using a dialdehyde being glutaraldehyde, or using a polyaldehyde being dextran polyaldehyde or a polyethylene glycol polyaldehyde.

In another optional aspect, the modifying reaction involves stapling surface amino groups using glutaraldehyde as the bifunctional reagent and the concentration of glutaraldehyde is varied to obtain a [glutaraldehyde : amino] molar ratio ranging from 0.5 to 200. In another optional aspect, the modifying reaction involves stitching surface amino groups using dextran polyaldehyde as the multifunctional reagent and the concentration of dextran polyaldehyde is varied to obtain a [dextran polyaldehyde : carbonic anhydrase] (w/w) ratio ranging from 0.3 to 13.8.

In another optional aspect, the surface functional groups that are modified comprise neutral or deprotonated carboxylic acid groups and the covalent modification, covalent stapling or covalent stitching comprises an esterification reaction or an amidation reaction.

In another optional aspect, the carboxylic acid groups that are modified comprise carboxylic acid groups of glutamic or aspartic acids, of the C-terminal group of the peptide chains of the carbonic anhydrase, or carboxylic acid groups resulting from a previous modification.

In another optional aspect, the modifying reaction comprises an esterification that is performed using: i. an aliphatic, aromatic or heteroaromatic alcohol as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diol as the at least one bifunctional reagent; or iii. a polyalcohol having at least three alcohol groups as the at least one multifunctional reagent, wherein the alcohol groups of the polyalcohol are linked to each other by an aliphatic, aromatic or heteroaromatic group. In another optional aspect, the modifying reaction comprises an amidation that is performed using:

i. an aliphatic, aromatic or heteroaromatic amine as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diamine as the at least one bifunctional reagent; or iii. a polyamine having at least three amino groups as the at least one multifunctional reagent, wherein the amino groups of the polyamine are linked to each other by an aliphatic, aromatic or heteroaromatic group.

In another optional aspect, the aliphatic group of the esterification or amidation reagent comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S.

In another optional aspect, the aromatic and heteroaromatic groups of the esterification or amidation reagent comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from 0, N and S.

In another optional aspect, the carboxylic acid groups are activated prior to modification, stapling or stitching, by reaction with an activating reagent comprising 0-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or a carbodiimide reagent alone or with an additive. In another optional aspect, the carbodiimide reagent is chosen from the group consisting of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1 - ethyl-3-(3'-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).

In another optional aspect, the additive used in combination with the carbodiimide reagent is chosen from the group consisting of p-nitrophenol (PNP), pentafluorophenol (PFP), hydroxybenzotriazole (HOBT) and sulfo-N- hydroxysuccinimide (sulfo-NHS). In another optional aspect, the modifying reaction involves stapling carboxylic acid groups of glutamic or aspartic acids using ethylene diamine as the bifunctional reagent. The ethylene diamine concentration may be varied to obtain a [ethylene diamine : carboxylic acid group] molar ratio ranging from 2 to 200.

In another optional aspect, the surface functional groups that are modified comprise neutral or protonated guanidino groups and the covalent modification, covalent stapling or covalent stitching comprises a reaction between the guanidino groups and an alpha-diketo compound. In another optional aspect, the guanidino groups are present on arginine or are guanidino groups resulting from a previous modification.

In another optional aspect, the modifying reaction involves reacting neutral or protonated guanidino groups on the surface of the carbonic anhydrase with: i. an aliphatic, aromatic or heteroaromatic alpha-diketo compound as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic alpha-diketo compound having two alpha-diketo groups as the at least one bifunctional reagent; or iii. an alpha-diketo compound having at least three alpha-diketo groups as the at least one multifunctional reagent, wherein the alpha-diketo groups are linked to each other by an aliphatic, aromatic or heteroaromatic group.

In another optional aspect, the aliphatic group of the alpha-diketo compounds comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S.

In another optional aspect, the aromatic and heteroaromatic groups of the alpha- diketo compounds comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from 0, N and S.

In another optional aspect, the step of modifying involves stitching aldehyde groups resulting from a reaction between carbonic anhydrase surface amino groups and an aldehyde compound, and wherein polyethylenimine is used as the multifunctional reagent. The aldehyde compound may be glutaraldehyde.

In another optional aspect, the carbonic anhydrase is a free carbonic anhydrase, an immobilized carbonic anhydrase or an aggregated carbonic anhydrase.

In another optional aspect, the carbonic anhydrase is a monomeric or multimeric carbonic anhydrase. In another optional aspect, the carbonic anhydrase is a wild-type carbonic anhydrase, a mutant carbonic anhydrase or a variant carbonic anhydrase.

According to another aspect, there is provided a surface modified carbonic anhydrase having an improved stability and/or activity which is prepared and identified by the above defined method.

According to another aspect, there is provided a use of a surface modified carbonic anhydrase having an improved stability and/or activity as defined above in an industrial process comprising CO2 capture by CO2 hydration.

According to another aspect, there is provided a use of a surface modified carbonic anhydrase having an improved stability and/or activity as defined above, in an industrial process comprising CO2 desorption by bicarbonate dehydration.

According to another aspect, there is provided a process for treating a C0₂- containing gas comprising contacting the C0₂-containing gas with an absorption solution comprising water in the presence of a carbonic anhydrase as defined above, whereby the carbonic anhydrase catalyzes the hydration reaction of dissolved C0₂ into bicarbonate ions and hydrogen ions within the aqueous solution to produce a bicarbonate loaded solution.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram representing examples of enzyme's modifications according to the method of the present invention. Figure 2 is a diagram representing the percentage of modified amino groups as measured by TNBS on the surface of a carbonic anhydrase for different reagent: NH₂ [R:NH₂] molar ratios, wherein the reagent is an anhydride monofunctional reagent.

Figure 3 is a diagram representing the effect of chemically modifying surface amino groups with different anhydride monofunctional reagents on carbonic anhydrase activity for different reagent: NH₂ [R:NH₂] molar ratios. Figure 4 is a diagram representing the effect of stapling surface amino groups with a bifunctional dialdehyde reagent on carbonic anhydrase activity for different reagent: NH₂ [R:NH₂] molar ratios.

Figure 5 is a diagram representing the effect of stapling surface amino groups with BS(PEG)₅ or BS(PEG)₉ bifunctional reagent on carbonic anhydrase activity after challenge for different reagent: NH₂ [R:NH₂] molar ratios.

Figure 6 is a diagram representing the effect of stapling surface amino groups with BS(PEG)₉ bifunctional reagent on carbonic anhydrase activity after challenge of 3 days at 70 Ό in 0.3 M sodium carbonate for a reage nt:NH₂ [R:NH₂] molar ratio of 5: 1 , where a larger quantity of enzyme is used (100 mg).

Figure 7 is a diagram representing the effect of stitching surface amino groups with a dextran polyaldehyde reagent on carbonic anhydrase activity and stability at ratios reagent: carbonic anhydrase [R:CA] ranging from 0.3 to 13.8 (w/w). Figure 8 is a diagram representing the effect of chemical modification on immobilized carbonic anhydrase activity and stability where a monofunctional, a bifunctional or a multifunctional reagent is used for the modification.

DETAILED DESCRIPTION OF THE INVENTION

The method for obtaining carbonic anhydrases having improved stability and/or activity will be described. The method broadly comprises chemically modifying exposed functional groups at the surface of the carbonic anhydrase and identifying the carbonic anhydrases which have an improved stability and/or activity.

"Carbonic anhydrase" or "CA" as used herein refers to a carbonic anhydrase or an analogue thereof and include naturally occurring, modified, recombinant and/or synthetic CA, including chemically modified enzymes, enzyme aggregates, cross- linked enzymes, enzyme particles, enzyme-polymer complexes, polypeptide fragments, enzyme-like chemicals such as small molecules mimicking the active site of carbonic anhydrase enzymes and any other functional analogue of the enzyme carbonic anhydrase. The expressions "improved activity", "enhanced activity" or "increased activity" as used herein mean that the activity of the chemically modified CA, i.e. its capability of hydrating CO2 into bicarbonate and/or desorbing CO2 by bicarbonate dehydration, is increased compared to the activity of the unmodified CA.

"Improved stability", "enhanced stability" or "increased stability" as used herein mean that the stability of the chemically modified CA in challenging conditions is increased compared to the stability of the unmodified CA in the same challenging conditions. The stability can be determined as the activity of the CA (i.e. its capability of hydrating CO2 into bicarbonate and/or desorbing CO2 by bicarbonate dehydration) after the CA has been challenged. Challenging conditions are exposure conditions known to affect the stability of the CA. For example, the stability of the CA is determined after exposure to certain compounds/reagents/ions (e.g. amine compound, ammonia and/or carbonate ions), high temperatures, or any other conditions that usually lead to denaturation or inactivation of CA. According to one embodiment of the method, a carbonic anhydrase having an initial surface charge is chemically modified by reacting the functional groups at the surface thereof with at least one reagent while varying at least one predetermined reaction parameter. The reaction allows obtaining a set of modified carbonic anhydrases with respective surface charges different than the initial surface charge. As will be detailed hereinafter, the functional groups at the surface of the carbonic anhydrase can be modified by more than one reagent. Moreover, different reaction parameters may be varied during the reaction for a given reagent. The method thus allows producing several surface modified carbonic anhydrases. According to another embodiment, the method involves providing a plurality of carbonic anhydrases each having an initial surface charge, and chemically modifying functional groups at the surface of each one of the carbonic anhydrases by reaction with at least one reagent. The reaction allows obtaining a set of modified carbonic anhydrases with respective surface charges different than their initial surface charge. The carbonic anhydrases which are reacted in this embodiment are different from each other. For example, a plurality of different carbonic anhydrases CA-1 , CA-2, CA-3... and CA-i may be reacted independently (i.e. each carbonic anhydrase being placed in a separate reaction vessel) with the same first reagent R1 to provide corresponding surface modified carbonic anhydrases CA-1/R1 , CA-2/R1 , CA-3/R1 ... and CA-i/RI , respectively. In an optional embodiment, the modifying step may also be carried out by further varying at least one predetermined reaction parameter, thus allowing the preparation of further modified carbonic anhydrases (CA-1/R1 )_a, (CA-1/R1 )_b, ... (CA-1/R1 )_n, (CA- 2/R1 )_a, (CA-2/R1 )_b, ... (CA-2/R1 )_n, (CA-3/R1 )_a, (CA-3/R1 )_b, ... (CA-3/R1 )_n... (CA- i/RI )_a, (CA-i/RI )_b, ... and (CA-i/RI )_n. After modification with the first reagent R1 , each of the resulting modified carbonic anhydrase could be further modified with at least one other reagent, with or without varying a predetermined reaction parameter, thus leading to a further set of modified carbonic anhydrases. Each modification, with or without varying the predetermined reaction parameter, will modify the surface charge of each of the carbonic anhydrase.

In one embodiment, the chemical modification involves covalent modification of the surface functional groups of the CA by reaction with a monofunctional reagent, covalent stapling of pairs of functional groups by reaction with a bifunctional reagent, or covalent stitching of surface functional groups by reaction with a multifunctional reagent having at least three reactive groups capable to react with the surface functional groups of the CA.

For a given reagent, the reaction may be performed by varying at least one reaction parameter. The reaction parameter which may be varied include for example the reagent concentration, the enzyme concentration, the pH at which the reaction is performed, the nature of the solvent in which the reaction is performed, the reaction temperature or the reaction time, influencing the reaction conditions and outcomes. In varying one reaction parameter, it is possible to obtain a set of modified CA having different surface charges. For example, positive charges on the surface of the CA may be changed to neutral or to negative charges. Alternatively, negative charges on the surface of the CA may be changed to neutral or to positive charges. Neutral charges may also be changed to positive or negative charges. The modification may also change the hydrophobicity or hydrophilicity of the surface of the CA. By varying one given parameter of the reaction, it is possible to gradually change from 0 to 100% of the accessible reactive functional groups on the surface of the CA. For the purpose of clarity, one will refer to the reagent concentration as the given parameter of the reaction which may be varied to obtain the modified enzymes with various surface charges in the following description. However, one could choose to vary other parameter such as for example the enzyme concentration, the pH at which the reaction is performed, the nature of the solvent, the reaction temperature or reaction time, or any other parameter influencing the reaction.

For the purpose of clarity, one will also refer to the modification of a given CA in the following description. However, in one embodiment of the method, the modification can also be performed starting from a plurality of different CA, each CA being modified separately. When a plurality of different CA is used, the modification may be performed by reacting the surface functional groups of each CA, with or without varying a reaction parameter. In a preferred embodiment, each of the plurality of CA is reacted while varying a reaction parameter. The surface of the given CA may thus be modified in a systematic manner using a first reagent at different concentrations to provide a first set of modified carbonic anhydrases. This first set of modified carbonic anhydrases would include a first modified CA obtained when the modification is performed at a first concentration of the first reagent, a second modified CA obtained when the modification is performed at a second concentration of the first reagent, a third modified CA obtained when the modification is performed at a third concentration of the first reagent, and so on. In the first set of modified carbonic anhydrases which is so obtained, each modified CA has a proper surface charge different than the initial surface charge of the CA.

Further modifications of remaining functional groups on the surface of the CA (either unreacted functional groups or other functional groups) or of new functional groups introduced on the surface of the CA following the reaction with the first reagent, can be performed by applying the method on at least one of the modified carbonic anhydrases obtained in the first set, using a second reagent at different concentrations (or by varying any other parameter of the reaction). In this way, a second set of modified carbonic anhydrases having been modified by two different reagents is obtained. In an alternative, the second set of modified carbonic anhydrases can be obtained using the same reagent as the first reagent but varying another reaction parameter than the reagent concentration. Each modified CA of the second set will have a proper surface charge different than the surface charge of the at least one selected modified enzyme of the first set. The reaction with the second reagent at different concentrations or the second reaction with the first reagent but varying another reaction parameter than the concentration can be done on two or more of the modified carbonic anhydrases of the first set, thus leading to a library of modified carbonic anhydrases.

As mentioned above, the first reagent may be a monofunctional reagent modifying the surface charge of the enzyme, or a bifunctional or multifunctional reagent modifying the surface charge of the enzyme and providing rigidity to the enzyme. The second reagent could also be any of a monofunctional, bifunctional or multifunctional reagent. In one embodiment, the first reagent is a monofunctional reagent modifying the surface charge of the enzyme and the second reagent is a bifunctional or multifunctional reagent further providing rigidity to the enzyme.

The monofunctional reagent is thus capable to modify the surface of the CA by reacting with at least one functional group present on the surface thereof. More particularly, the monofunctional reagent is capable to modify at least one functional group present on the surface of the CA by formation of a covalent bond. The bifunctional reagent comprises two reactive groups wherein each of the two reactive groups is capable to bond covalently to a functional group on the surface of the CA. Thus, the bifunctional reagent is capable to covalently staple a pair of distant functional groups on the surface of the CA. The functional groups on the surface of the CA which are stapled may be the same or different. The reactive groups of the bifunctional reagent may also be the same or different.

The multifunctional reagent comprises three or more reactive groups wherein each reactive group is capable to bond covalently to a functional group on the surface of the CA. Thus, the multifunctional reagent is capable to covalently stitch together distant functional groups on the surface of the CA. The functional groups on the surface of the CA which are stitched may be the same or different. The reactive groups of the multifunctional reagent may also be the same or different.

Figure 1 is a scheme showing examples of modifications which can be performed on surface groups of a given CA enzyme, according to various embodiments of the method. In this figure five different possible ways of modifying the surface of one CA enzyme are shown. However, one should not limit the present method to these examples as many other modifications could be performed on the CA. Moreover, the modifications exemplified in Figure 1 could be carried out starting from a plurality of different CA.

According to a first embodiment shown in Figure 1 , the enzyme is first reacted with Monofunctional reagent 1 to provide a Surface Modified Enzyme with reagent 1 (SME w/ reagent 1 ). Then, the SME w/ reagent 1 is reacted in a second step with Monofunctional reagent 2 to provide a Surface Modified Enzyme with both reagents 1 and 2 (SME w/ reagents 1 and 2). The surface groups which are modified with Monofunctional reagent 1 can be identical or different than the surface groups which are modified with Monofunctional reagent 2. For example, Monofunctional reagent 1 could react with amino groups and Monofunctional reagent 2 with carboxylic acid groups. But, both reagents could also react with the same functional groups (amino groups or carboxylic acid groups). For example, Monofunctional reagent 2 could react with unreacted groups remaining on the surface of the enzyme after reaction with Monofunctional reagent 1 .

According to a second embodiment shown in Figure 1 , the enzyme is first reacted with Bifunctional reagent 1 to provide a Surface Stapled Enzyme with reagent 1 (SSE w/ reagent 1 ). Then, the SSE w/ reagent 1 is reacted in a second step with another Bifunctional reagent 2 to provide a Surface Stapled Enzyme with both reagents 1 and 2 (SSE w/ reagents 1 and 2). The pairs of surface groups which are modified with Bifunctional reagent 1 can be identical or different than the pairs of surface groups which are modified with Bifunctional reagent 2.

According to a third embodiment shown in Figure 1 , the enzyme is first reacted with Monofunctional reagent 1 to provide a Surface Modified Enzyme with reagent 1 (SME w/ reagent 1 ). Then, the SME w/ reagent 1 may be reacted in a second step with either Bifunctional reagent 2 or Multifunctional reagent 2. When the second modification is performed in the presence of Bifunctional reagent 2, one obtains a Surface Modified Enzyme with reagent 1 and Stapled with reagent 2 (SME w/ reagent 1 and Stapled w/ reagent 2). When the second modification is performed in the presence of Multifunctional reagent 2, one obtains a Surface Modified Enzyme with reagent 1 and Stitched with reagent 2 (SME w/ reagent 1 and Stitched w/ reagent 2). In this embodiment, the Monofunctional reagent 1 will react for example with amino groups or carboxylic acid groups on the surface of the enzyme, thereby modifiying the surface charge of the enzyme. Then, reaction with either Bifunctional reagent 2 or Multifunctional reagent 2 will further provide rigidity to the enzyme.

According to a fourth embodiment shown in Figure 1 , the enzyme is reacted with Multifunctional reagent 1 to provide a Surface Stitched Enzyme with reagent 1 (SStE w/ reagent 1 ). With such a modification, the surface charge of the enzyme can be modified while stitching further provides rigidity to the enzyme.

The modification of the CA can be performed on a portion of the surface functional groups. Alternatively, substantially all of the surface functional groups could be modified. The percentage of modified surface functional groups on the surface of the CA will depend on the reaction parameter which is varied when performing the modification reaction. Hence, the percentage of modified surface functional groups may depend on the concentration of the mono-, bi-, multifunctional reagent(s), the concentration of the CA, the pH at which the reaction is performed, the reaction temperature, the reaction time or the type of solvent used in the reaction. In one embodiment, the percentage of modified surface functional groups depends on the concentration ratio of reagent to surface functional groups [reagent : surface functional group]. In another embodiment, the percentage of modified surface functional groups depends on the concentration ratio of reagent to CA [reagent : CA].

As mentioned above, varying reaction parameters while performing the surface modification modifies the surface charge of the CA. The nature of the reagent and the nature of the functional groups which are modified also affect the surface charge of the CA, its hydrophobicity and/or hydrophilicity. Thus, positive charges on the surface of the CA can be changed to negative and/or neutral charges through the modification. Alternatively, negative charges on the surface of the CA can be changed to positive and/or neutral charges through the modification. Neutral charges can also be changed to either positive or negative charges. Hydrophilicity on the surface of the CA may be increased if the surface of the modified CA is electrically charged and/or the mono-, bi- or multi- functional reagent comprises hydrophilic groups. Hydrophobicity can be increased using mono- bi- or multi- functional reagent comprising hydrophobic groups (e.g. aromatic reagents and/or reagents having long aliphatic chains). In one embodiment, the surface functional groups of the CA which are modified are neutral or protonated amino groups (i.e. positively charged amino groups), neutral or deprotonated carboxylic acid groups (i.e. negatively charged carboxylic acid groups), and/or neutral or protonated guanidino groups.

Several types of reactions can be performed for modifying the surface of the CA. In one embodiment, the covalent modification, stapling or stitching involves an acylation reaction, an imination reaction, a thioureation reaction, an esterification reaction, an amidation reaction, a reaction between a guanidino group and an alpha-diketo compound or any combination of these reactions. The reaction conditions for performing these modifications are known to the skilled person in the art.

In one embodiment, the functional groups on the surface of the CA which are modified comprise neutral or protonated amino groups and the covalent modification, stapling or stitching involves an acylation, an imination or a thioureation reaction. The amino functional groups which are modified may be present on lysine or may be amino terminal groups of the CA enzyme's peptide chains. The amino functional groups involved in the acylation, imination or thioureation reaction may also be primary amino groups resulting from a previous modification made on the surface of the CA.

The monofunctional reagents which may be used for carrying out the acylation reaction include aliphatic, aromatic or heteroaromatic acid anhydride, acyl halide, or acid activated ester. The bifunctional reagent for the acylation reaction may be aliphatic, aromatic or heteroaromatic diacyl halides or diacid activated esters. The multifunctional reagent could be a polyacyl halide or polyacid activated ester where the acyl halide or acid activated ester groups are linked to each other by aliphatic, aromatic or heteroaromatic groups.

In one embodiment, the aliphatic groups are linear, branched or cyclic groups having from 1 to 18 carbon atoms and optionally including heteroatoms selected from 0, N and S. In another embodiment, the aromatic and heteroaromatic groups comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from O, N and S. In another embodiment, the activated esters used as the acylation reagents are chosen from fluorophenol esters, nitrophenol esters, hydroxybenzotriazole esters, N-hydroxy succinimide esters, and isourea esters.

In another embodiment, the reaction involves stapling amino groups on the surface of the CA by an acylation reaction using [bis(succinimidyl) penta(ethyleneglycol)] BS(PEG)5 or [bis(succinimidyl) nona(ethyleneglycol)] BS(PEG)9 as bifunctional reagents. The reaction with BS(PEG)5 or BS(PEG)9 may be carried out by varying the concentration of BS(PEG)5 or BS(PEG)9 to reach a [diacid activated ester : amino functional group] molar ratio ranging from 0.2 to 20.

In another embodiment, the acylation reaction is performed using an acid anhydride selected from acetic anhydride, phtalic anyhydride and succinic anhydride. When the concentration of the acid anhydride is the parameter which is varied during the reaction, it can be varied to obtain a [acid anhydride : amino functional group] molar ratio ranging from 2 to 200.

When the reaction is an imination reaction, the monofunctional reagent can be an aliphatic, aromatic or heteroaromatic aldehyde, the bifunctional reagent can be an aliphatic, aromatic or heteroaromatic dialdehyde, and the multifunctional reagent can be a polyaldehyde having at least three aldehyde functions, where the aldehyde groups are connected to each other by an aliphatic, aromatic or heteroaromatic group. In one embodiment, the aliphatic group may be a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from O, N and S and the aromatic and heteroaromatic groups may comprise one, two or three fused or linked cycles. The heteroatoms of the heteroaromatic groups may be O, N or S. In another embodiment, the monofunctional aldehyde is acetaldehyde, propionaldehyde, butyraldehyde or benzaldehyde. In another embodiment, the dialdehyde is glutaraldehyde. Examples of polyaldehyde include dextran polyaldehyde or a polyethylene glycol polyaldehyde.

In another embodiment, the reaction involves stapling surface amino groups using glutaraldehyde as the bifunctional reagent and the concentration of glutaraldehyde is varied to obtain a [glutaraldehyde : amino functional group] molar ratio ranging from 0.5 to 200.

In a further embodiment, the reaction involves stitching surface amino groups using dextran polyaldehyde as the multifunctional reagent and the concentration of dextran polyaldehyde is varied to obtain a [dextran polyaldehyde : carbonic anhydrase] (w/w) ratio ranging from 0.3 to 13.8.

When the reaction is a thioureation reaction, the monofunctional reagent can be an aliphatic, aromatic or heteroaromatic isothiocyanate and the bifunctional reagents used for stapling functional amino groups can be an aliphatic, aromatic or heteroaromatic diisothiocyanate reagents. Examples of multifunctional reagents which can be used for stitching amino groups on the surface of the CA by a thioureation reaction include polyisothiocyanate reagents having at least three isothiocyanate groups, where the isothiocyanate groups are connected to each other by an aliphatic, aromatic or heteroaromatic group. The aliphatic group may be a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from O, N and S. Moreover, the aromatic and heteroaromatic groups may comprise one, two or three fused or linked cycles. The heteroatoms of the heteroaromatic groups may be 0, N or S.

According to another embodiment of the method, the functional groups which are modified on the surface of the CA are neutral or deprotonated carboxylic acid groups and the covalent modification, covalent stapling and covalent stitching involve an esterification or amidation reaction.

In another embodiment, the carboxylic acid groups which are modified are carboxylic acid groups of glutamic or aspartic acids or of the C-terminal group of the CA peptide chains. The carboxylic acid groups which are esterified or amidated may also be carboxylic acid groups resulting from a previous modification made on the surface of the CA.

In some embodiment, the carboxylic acid groups on the surface of the CA may be activated prior to be modified by esterification or amidation. Such an activation may be done by reaction with an activating reagent such as 0-benzotriazole-N,N,N',N'- tetramethyluronium hexafluorophosphate (HBTU) or a carbodiimide reagent used alone or with an additive. In one embodiment, the carbodiimide reagent is chosen from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1 -ethyl-3- (3'-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). In another embodiment, the additive used in combination with the carbodiimide reagent is p- nitrophenol (PNP), pentafluorophenol (PFP), hydroxybenzotriazole (HOBT) or N- hydroxysuccinimide (NHS).

When the modification involves an esterification of carboxylic groups on the surface of the CA, the monofunctional reagent may be an aliphatic, aromatic or heteroaromatic monoalcohol, the bifunctional reagent may be an aliphatic, aromatic or heteroaromatic diol and the multifunctional reagent may be a polyol having at least three alcohol functions where the alcohol functions are linked to each other by an aliphatic, aromatic or heteroaromatic group.

And when the modification involves an amidation reaction, it may be performed using an aliphatic, aromatic or heteroaromatic monoamine as monofunctional reagent, an aliphatic, aromatic or heteroaromatic diamine as bifunctional reagent, or a polyamine having at least three amino groups as multifunctional reagent, where the amino groups are linked to each other by an aliphatic, aromatic or heteroaromatic group. The aliphatic groups of the esterification and amidation reagents may be linear, branched or cyclic groups having from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S. The aromatic and heteroaromatic groups of such reagents may have one, two or three fused or linked cycles, and their heteroatoms may be selected from 0, N and S.

In one embodiment, the modification involves stapling carboxylic acid groups of glutamic or aspartic acids on the surface of the CA using ethylene diamine as bifunctional reagent or polyethylenimine as multifunctional reagent. In another embodiment, carboxylic acid groups of glutamic or aspartic acids on the surface of the CA are modified using the ethylene diamine in a concentration which is varied to obtain a [ethylene diamine : carboxylic acid group] molar ratio ranging from 2 to 200.

In a further embodiment of the method, the covalent modification on the surface of the CA involves reacting surface guanidinium groups with an aliphatic, aromatic or heteroaromatic alpha-diketo compound as monofunctional reagent. When the modification involves stapling pairs of the enzyme surface functional groups, one may use an aliphatic, aromatic or heteroaromatic alpha-diketo compound having two alpha-diketo groups as bifunctional reagent. For stitching the CA surface functional groups, one may use an aliphatic, aromatic or heteroaromatic alpha- diketo compound having at least three alpha-diketo groups as multifunctional reagent. The aliphatic group may be a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S and the aromatic and heteroaromatic groups may comprise one, two or three fused or linked cycles. The heteroatoms of the heteroaromatic groups may be 0, N or S. In one embodiment, the guanidinium groups which are modified on the surface of the CA are present on arginine. The guanidinium groups may also result from a previous modification made on the surface of the CA.

According to another embodiment, the step of modifying the surface of the CA may involve stitching aldehyde groups resulting from a reaction between CA surface amino groups and an aldehyde compound, such as glutaraldehyde, and using polyethylenimine as the multifunctional reagent.

The surface modified carbonic anhydrases resulting from the modification step of the method may have an increased activity and/or stability. Hence, the method further includes a step of testing the carbonic anhydrases for their activity and/or stability after modification. For example, the modified enzyme can be tested for its activity towards CO2 hydration into bicarbonate and/or towards CO2 desorption by bicarbonate dehydration. The stability of the carbonic anhydrase can be measured as its activity in challenging conditions or after having subjected the modified carbonic anhydrase to challenging conditions. For example, the challenging conditions include high temperatures, exposure of the carbonic anhydrase to chemicals or salts, or any other conditions that usually lead to denaturation or inactivation.

Once the activity and/or stability of the modified carbonic anhydrases has been determined, the next step of the process involves identifying the modified carbonic anhydrases for which the activity and /or the stability is increased compared to the activity and/or stability of the unmodified carbonic anhydrase, in the same conditions. This last step allows determining which surface modified carbonic anhydrases have improved activity and/or stability and obtaining surface modified carbonic anhydrases which could be good biocatalysts candidates.

As mentioned above carbonic anhydrase or an analogue thereof can be used in the present method to provide modified carbonic anhydrases or analogues thereof having improved activity and/or stability. The carbonic anhydrase which is modified may be a free carbonic anhydrase, an immobilized carbonic anhydrase or an aggregated carbonic anhydrase. The carbonic anhydrase is said to be free when it is provided directly as part of a formulation or solution. Alternatively, the carbonic anhydrase may be fixed to a solid non-porous packing material, on or in a porous packing material, on or in a porous material coated on a particle or a packing material, on or in particles or may be provided as aggregates. It should be noted that the carbonic anhydrase used in a free state may be in a pure form or may be in a mixture including impurities or additives such as other proteins, salts and other molecules coming from the carbonic anhydrase production process.

Furthermore, the surface modified carbonic anhydrases obtained from a free or an aggregated carbonic anhydrase may be immobilized on to a solid non-porous packing material, on or in a porous packing material, on or in particles. Immobilization of the carbonic anhydrase, before or after modification, can be done by entrapping the carbonic anhydrase inside or fixing it to a porous coating material that is provided around a support that is porous or non-porous. The carbonic anhydrase may be immobilized directly onto the surface of a support (porous or non-porous) or may be present as cross linked carbonic anhydrase aggregates (CLEAs) or cross linked carbonic anhydrase crystals (CLECs). CLEA comprises precipitated carbonic anhydrase molecules forming aggregates that are then cross- linked using chemical agents. The CLEA may or may not have a 'support' or 'core' made of another material which may or may not be magnetic. CLEC comprise carbonic anhydrase crystals and cross linking agent and may also be associated with a 'support' or 'core' made of another material. When a support is used, it may be made of polymer, alumina, ceramic, metal(s), silica, solgel, chitosan, cellulose, alginate, polyacrylamide, carbon-based materials, nanoporous and mesoporous silicates, magnetic particles, titanium oxide, zirconium oxide, and/or other materials known in the art to be suitable for immobilization or carbonic anhydrase support.

The carbonic anhydrase which is modified according to the present method may be a monomeric or multimeric carbonic anhydrase. A multimeric carbonic anhydrase may be any of a dimer, trimer, tetramer, hexamer, octamer or any other multimeric carbonic anhydrase. The multimer can be a homomultimer or a heteromultimer. It can be a wild-type carbonic anhydrase, a mutant carbonic anhydrase or a variant carbonic anhydrase.

The carbonic anhydrases having an improved activity and/or stability obtained by the above method may be useful as biocatalysts in industrial processes involving CO2 hydration or dehydration. They are particularly useful in processes involving CO₂ capture by CO₂ hydration, or processes comprising CO₂ desorption by bicarbonate dehydration.

For example, the surface modified carbonic anhydrases are well-suited for removing C0₂from a CO₂ containing effluent, such as a gaseous or liquid effluent.

The modified carbonic anhydrases may thus be used in a process for treating a C0₂-containing gas. The process comprises contacting a C0₂-containing gas with an absorption solution comprising water in the presence of the surface modified carbonic anhydrase enzyme obtained by the above method. In the process, the surface modified carbonic anhydrase catalyzes the hydration reaction of dissolved C0₂ into bicarbonate ions and hydrogen ions within the aqueous solution to produce a bicarbonate loaded solution. In the presence of an alkaline pH, bicarbonate ions may be converted into carbonate ions and be further precipitated in the presence of bivalent cations such as magnesium or calcium to form the corresponding magnesium or calcium carbonate. In another embodiment, the bicarbonate ions in solution may be converted into precipitates by contact with monovalent cations, such as for example sodium, potassium, ammonium or cesium to form the corresponding sodium, potassium, ammonium or cesium bicarbonate.

In one embodiment, the absorption solution further comprises one or more absorption compounds to aid in the CO₂ absorption and to combine with the catalytic effects of the carbonic anhydrase. United States Patent No. 7,740,689 describes a formulation and method for absorbing C0₂ from a gas using a solution containing an absorption compound and carbonic anhydrase. In addition, international PCT patent application Nos. PCT/CA2010/001212, PCT/CA2010/001213 and PCT/CA2010/001214 describe using carbonic anhydrase in combination with absorption compounds to enhance CO2 capture. The above patent and applications are incorporated herein by reference along with the following references: United States Patent No. 6.908.507, United States Patent No. 7.176.017, United States Patent No. 6.524.843, United States Patent No. 6.475.382, United States Patent No. 6.946.288, United States Patent No. 7.596.952, United States Patent No. 7.514.056, United States Patent No. 7.521 .217, United States Patent Application No. 61/272.792 and United States Patent Application No. 61/344.869, which are all currently held by the Applicant. Various reactors and processes described in the preceding references may be used in connection with the process for treating a C0₂-containg gas using a surface modified carbonic anhydrase according to the present invention.

While the above method is described in connection with the obtaining of modified carbonic anhydrases having improved activity and/or stability, it could also be applied to any other enzymes. For example, enzymes such as hydrolases, oxidoreductases, ligases, isomerases, lyases and transferases could be modified using the above described chemical modifications and then tested for their activity and/or stability to identify enzymes having improved activity and/or stability.

EXAMPLES AND METHODS

Example 1 : Preparation of surface modified carbonic anhydrase using a monofunctional reagent

Modification of lysine groups with an anhydride compound The goal is to identify the effect of charge modifications (from positive to neutral or from positive to negative) on enzyme (carbonic anhydrase) activity. Different ratios of anhydride to NH₂ were used to obtain enzyme with different level of modification.

To a 1 -10 mg/ml enzyme solution in 0.5 M carbonate buffer at pH 8.5, the modifying compound is gradually added over a period of 30 minutes while stirring.

The pH of the solution is monitored and kept at 8.5 by addition of 2 M NaOH if needed. At the end of the reaction, the surface modified enzyme is dialyzed into a 0.1 M carbonate buffer at pH 8.5 (8 centrifugations with 10 kDa filter, 5 minutes at 5000 RPM). The concentration of the modifying compound is varied to obtain a [compound: NH₂] molar ratio (or [R:NH₂] ratio, with R for reagent) ranging from 0 to 200, wherein NH₂ is the total amount of NH₂ groups present on the enzyme (a protein contains several NH₂ groups that can be modified). The level of modification is determined using the TNBS test (trinitrobenzenesulfonic acid test).

The following anhydrides were tested: acetic, succinic and phtalic anhydride. Succinic and phtalic anhydrides transform positively charged amino groups on the surface of the enzyme into negatively charged groups. Furthermore, phtalic anhydride introduces an hydrophobic aromatic group at the surface of the enzyme. Acetic anhydride on the other hand transforms positively charged NH₂ groups on the surface of the enzyme into neutral groups. Figure 2 shows the extent of modified amino groups as measured by TNBS. With a ratio of 2, 0-20% of the NH₂ groups present on the carbonic anhydrase are modified. With a ratio of 200, about 90% of the NH₂ groups are modified.

Figure 3 shows the effect of chemical surface modification on the carbonic anhydrase activity. All three anhydrides have a positive effect on enzyme activity as non-challenged modified enzyme display a FIONE over 1 (a non-modified enzyme has a FIONE value of 1 ). Figure 3 also shows the effect of chemical surface modification on the carbonic anhydrase stability which is measured as its activity after challenge at 85 Ό for 10 min in 2M MDEA (N-methyldiethanolamine). The stability is affected by these modifications.

Example 2: Preparation of surface stapled carbonic anhydrase using a bifunctional reagent

Modification of lysine groups with a dialdehyde compound

To a 1 -10 mg/ml enzyme solution in 0.2 M carbonate buffer at pH 8.5, the dialdehyde is added and the reaction is performed over 60 minutes while stirring. At the end of the reaction, the surface modified enzyme is dialyzed into a 0.1 M carbonate buffer pH 8.5 (8 centrifugations with 10 kDa filter, 5 minutes at 5000 RPM). The concentration of the modifying compound is varied to obtain [compound: NH₂] molar ratio ranging from 0 to 200. The level of modification is determined using the TNBS test. Optionally, the resulting enzyme can be reduced using NaBH₄ or NaBH₃CN.

In the present example, glutaraldehyde was used as the dialdehyde. The goal was therefore to attach together pairs of NH₂ groups at the surface of the carbonic anhydrase, using various [glutaraldehyde: NH₂] ratios. Figure 4 shows the effect of surface modification of the carbonic anhydrase with glutaraldehyde on the activity and stability of the enzyme. Activity is measured before and after the enzyme has been challenged. Challenge is 10 minutes at 85 °C in 2 M MDEA. This modification has a positive effect on the enzyme activity, regardless of the [glutaraldehyde: NH₂] molar ratio as indicated by a FIONE over 2. At low ratios (ranging from about 0.5 to 8), surface modification using glutaraldehyde has a positive effect on enzyme stability.

Example 3: Preparation of surface stapled carbonic anhydrase using a bifunctional reagent

Modification of lysine groups with BS(PEG)s [Bis(succinimidyl) pentafethylene glycol)] and BS(PEG)9 [Bis(succinimidyl) nonafethylene glycol)] compounds

To a 0.5-10 mg/ml enzyme solution in 0.1 M carbonate buffer at pH 8.5, the BS(PEG)n is added and the reaction is performed over 30 minutes while stirring. At the end of the reaction, the surface modified enzyme is dialyzed into a 0.1 M carbonate buffer pH 8.5 (volume exchanged 5 times using a 30 kDa MWCO membrane). The concentration of the modifying compound is varied to obtain [compound: NH₂] molar ratio ranging from 0 to 20.

The goal was to attach together pairs of NH₂ groups at the surface of the carbonic anhydrase, using various [BS(PEG)n:NH₂] ratios.

Figure 5 shows the effect of surface modification of the carbonic anhydrase with BS(PEG)5 and BS(PEG)g on the stability of the enzyme which is measured as its activity after the enzyme has been challenged. Challenge is 24 hours at 60 °C in 0.3 M sodium carbonate pH 10. This modification has a positive effect on the enzyme stability when the ratios [BS(PEG)n:NH₂] is > 1 .

The previous experiment was performed with around 1 mg of enzyme. We repeated it at larger scale, with over 100 mg enzymes, using BS(PEG)₉ at a [BS(PEG)₉:NH₂] ratio of 5. Figure 6 shows the effect of modification with BS(PEG)₉ of carbonic anhydrase, at larger scale, on its the activity and stability. This modification increases both its activity and stability.

Example 4: Preparation of surface stitched carbonic anhydrase using a multifunctional reagent

Modification of lysine groups with a dextran polyaldehyde compound

Dextran polyaldehyde is produced by partial oxidation of dextran using sodium metaperiodate. In brief, dextran is dissolved in 100 mM phosphate buffer pH 7 and sodium metaperiodate is added in amount necessary to oxidize the dextran at 40%. The product is purified by precipitation in ethanol. To a 1 -10 mg enzyme in 50 mM HEPES pH 8.2, the dextran polyaldehyde (compound) is added and the reaction is performed over 60 minutes while stirring. At the end of the reaction, the surface modified enzyme is reduced using NaCNBH₃. The concentration of the modifying compound is varied to obtain [compound:enzyme] (w/w) ratio ranging from 0.3 to 13.8.

The goal was to attach together several NH₂ groups at the surface of the CA enzyme, using various [dextran polyaldehyde:enzyme] ratios.

Figure 7 shows the effect of surface modification of the carbonic anhydrase with dextran polyaldehyde on the activity and stability of the enzyme. Activity is measured before and after the enzyme has been challenged. Stability corresponds to the activity of the enzyme after challenge. Challenge is 1 to 14 days at 60°C in 2 M MDEA (N-methyldiethanolamine). This modification has a neutral effect on the enzyme activity. At ratios > 1 , this modification has a positive effect on enzyme stability. Example 5: Surface modification of an immobilized carbonic anhydrase using different reagents

The goal is to identify the effect of different kind of modifications on the immobilized enzyme activity and stability. Anhydride acetic at different ratio aa:enzyme, PEI (polyethylenimine) at different ratio PEI:enzyme and dextran polyaldehyde at different oxidation level, were used to obtain enzyme with different level of modification.

The carbonic anhydrase was immobilized on alumina particles (50 nanometers) using the procedure described in US patent publication No. 201 1 -0097781 . In brief, the alumina particles were functionalized with 3-aminopropyltriethoxysilane. Those were then treated sequentially with glutaraldehyde, polyethylenimine, glutaraldehyde again, enzyme and sodium borohydride.

Modification with acetic anhydride:

To the immobilized enzyme in 0.5 M carbonate buffer at pH 8.5, the modifying compound is gradually added over a period of 30 minutes while stirring. The pH of the solution is monitored and kept at 8.5 by addition of 2 M NaOH if needed. At the end of the reaction, the surface modified immobilized enzyme is cleaned with water by centrifugation (3 centrifugations, 5 minutes at 5000 RPM). The concentration of the modifying compound is varied to obtain a [compound:enzyme] (w/w) ratio (or [R:enzyme] ratio, with R for reagent) ranging from 0 to 2.

Modification with PEI:

To the immobilized enzyme in 0.5 M carbonate buffer at pH 8.5, glutaraldehyde and PEI were sequentially added as described in US 201 1 -0097781. At the end of the reaction, the surface modified enzyme is reduced using NaBH₄ or NaCNBH₃ and cleaned with water by centrifugation (3 centrifugations, 5 minutes at 5000 RPM). The concentration of glutaraldehyde and PEI were varied to obtain different modification level.

Modification with dextran polyaldehyde:

Dextran polyaldehyde is produced by partial oxidation of dextran using sodium metaperiodate. In brief, dextran is dissolved in 100 mM phosphate buffer pH 7 and sodium metaperiodate is added in amount necessary to oxidize the dextran at 10%, 40% or 80%. The product is purified by precipitation in ethanol. To the immobilized enzyme in 50 mM HEPES pH 8.2, the dextran polyaldehyde is added and the reaction is performed over 60 minutes while stirring. At the end of the reaction, the surface modified enzyme is reduced using NaCNBH₃ and cleaned with water by centrifugation (3 centrifugations, 5 minutes at 5000 RPM). The concentration of the modifying compound is kept constant [3.4g oxidized dextran/g enzyme] but the oxidation level of dextran is varied from 10 to 80%.

Figure 8 shows the effect of chemical surface modification on the activity and stability of the immobilized carbonic anhydrase. The PEI modification has a positive effect on enzyme activity. The positive effect on activity is proportional to the amount of PEI used. Dextran polyaldehyde has a neutral or negative effect on the activity. The anhydride acetic modification has a positive effect on enzyme activity. The positive effect on activity is proportional to the amount of anhydride used. None of these modifications had a positive effect on stability.

Example 6: Preparation of surface modified carbonic anhydrase using a monofunctional reagent

Modification of glutamic or aspartic acid with EDC/NHS/Ethylene diamine The goal is to identify the effect of charge modifications (from negative to positive) on enzyme activity. Different ratios of ethylene diamine to NH₂ were used to obtain enzyme with different level of modification.

To a 1 -10 mg/ml enzyme solution in 0.2 M MES buffer pH 5, 0.02 M EDC, 0.006 M sulfo-NHS and different concentration of ethylene diamine are added. The reaction is performed at 25°C for 120 minutes while stirring.

At the end of the reaction, the surface modified enzyme is dialyzed into a 0.1 M carbonate buffer at pH 8.5 (8 centrifugations with 10 kDa filter, 5 minutes at 5000 RPM). The concentration of the modifying compound is varied to obtain a [compound:COOH] ratio (or [R:COOH] ratio, with R for reagent) ranging from 0 to 200, wherein COOH is the total amount of COOH groups present on the enzyme (a protein contains several COOH groups that can be modified). The level of modification is determined using the TNBS test (trinitrobenzenesulfonic acid test).

No significant effect was observed.

Example 7: Method of Modification of enzyme surface arginine guanidine group with a diketo-compound

Procedure 1: Preparation of Dihydroxycyclohexane-Arg (DHCH-Arg)-modified enzymes

Modification of arginine residues was carried out as described in Patthy, L. & Smith, E. L, J. Biol. Chem., 1975, 250, 557-564 and Patthy, L. & Smith, E. L, J. Biol. Chem. 1975, 250, 565-569. Briefly, cyclohexadione (CHD; 33- or 100-fold molar excess per mol of arginine) was added to a 0.1 mM solution of an enzyme in 40 ul of 0.2 M sodium borate (pH 9) (50 and 150 mM CHD, respectively). Reactions were carried out for 120 min at 37 . Re actions were terminated by cooling to 0 Ό and adding 5 μΙ of 50% acetic acid. Arg-modified enzymes were recovered by usual procedures analyzed immediately by MS to determine the level of Arg modification. This procedure can be adapted to any other diketo-compound. Depending on the enzyme and the diketo reagent, longer reaction time may be required to achieve the desired level of Arg-modification.

Procedure 2: Modification of Arg of enzyme using a diketo reagent

This procedure is adapted from the procedure of Gauthier, M. A. & Klok, H-A., Biomacromolecules 201 1 , 12, 482-493. A diketo reagent (150 μηποΙ) in 1 .5 mL H₂0 solution at 20 Ό is transferred to 2 mL of a borat e buffer solution and the pH adjusted to 9. An enzyme (0.1 1 μηιοΙ) was then added and the solution stirred for 2-24 h at room temperature. The reaction mixture was then purified using an appropriate procedure and the Arg-modified enzyme was analyzed by HPLC and MS. To obtain different level of modification of Arg residues, a larger excess of the diketo reagent (i.e., 5-50-fold excess relative to arginine residues at the surface of the enzyme) was used. In addition, the modification can also be performed at pH 7.4 in 100 mM sodium phosphate buffer rather than in borate buffer.

Procedure 3: Modification of Arg of enzyme using a diketo reagent

This procedure is adapted from the procedure of Saraiva, M. A.; Borges, C. M. & Florencio, M. H., J. Mass Spectrom. 2006, 41 755-770. 1 mL of buffer solution (200 mM) at pH 7.5 was added to 1 mL of the aqueous solution of the enzyme (1 mM). To this mixture was also added, separately, 1 mL of the aqueous solution of a dicarbonyl(diketo) reagent (for example glyoxal, methylglyoxal, phenylglyoxal and diacetyl (2-100 mM), and the resulting mixture was agitated at 50 Ό for 24 h. Aliquots were taken at fixed time periods and analyzed. At the end of the reaction, sample were treated and analyzed as above.

Claims

1 . A method for preparing and identifying surface modified carbonic anhydrases having an improved activity and/or stability comprising: a) providing a carbonic anhydrase having an initial surface charge; b) modifying surface functional groups of the carbonic anhydrase, comprising subjecting the carbonic anhydrase to at least one reaction with a reagent and varying at least one predetermined reaction parameter, whereby the reaction allows obtaining a set of modified carbonic anhydrases with respective surface charges different than the initial surface charge; c) testing the stability and/or activity of the set of surface modified carbonic anhydrases; and

2. The method claim 1 , wherein the at least one predetermined reaction parameter comprises a reagent concentration, a carbonic anhydrase concentration, a pH at which the reaction is performed, a solvent in which the reaction is performed, a reaction temperature, a reaction time, or any combination thereof.

3. The method claim 2, wherein the at least one predetermined reaction parameter comprises a reagent concentration.

4. The method of any one of claims 1 or 3, wherein the step of modifying comprises: i. covalent modification of the functional groups by reaction with a monofunctional reagent, or ii. covalent stapling of the functional groups by reaction with a bifunctional reagent, or iii. covalent stitching of the functional groups by reaction with a multifunctional reagent, the multifunctional reagent having at least three reactive groups capable to react with at least three surface functional groups of the carbonic anhydrase.

5. The method of any one of claims 1 to 4, wherein the surface functional groups that are modified comprise neutral or protonated amino groups; neutral or deprotonated carboxylic acid groups; neutral or protonated guanidino groups; or any combination thereof.

6. The method of claim 5, wherein the surface functional groups that are modified comprise neutral or protonated amino groups and the covalent modification, covalent stapling or covalent stitching comprises an acylation reaction, an imination reaction, or a thioureation reaction.

7. The method of claim 6, wherein the surface functional groups comprise lysine amino functional groups, amino terminal groups of the peptide chains of the carbonic anhydrase, or primary amino groups resulting from a previous modification.

8. The method of claim 6 or 7, wherein the reaction comprises an acylation that is performed using: i. an aliphatic, aromatic or heteroaromatic acid anhydride, acyl halide, or acid activated ester as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diacyl halide or diacid activated ester as the at least one bifunctional reagent; or iii. a polyacyl halide having at least three acyl halide groups or a polyacid activated ester having at least three acid activated ester groups as the at least one multifunctional reagent, wherein the acyl halide groups of the polyacyl halide or the acid activated ester groups of the polyacid activated ester are linked to each other by an aliphatic, aromatic or heteroaromatic group.

9. The method of claim 6 or 7, wherein the reaction comprises an imination that is performed using: i. an aliphatic, aromatic or heteroaromatic monoaldehyde as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic dialdehyde as the at least one bifunctional reagent; or iii. a polyaldehyde having at least three aldehyde groups as the at least one multifunctional reagent, wherein the aldehyde groups of the polyaldehyde are linked to each other by an aliphatic, aromatic or heteroaromatic group.

10. The method of claim 6 or 7, wherein the reaction comprises a thioureation that is performed using: i. an aliphatic, aromatic or heteroaromatic isothiocyanate reagent as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diisothiocyanate reagent as the at least one bifunctional reagent; or iii. a polyisothiocyanate reagent having at least three isothiocyanate groups as the at least one multifunctional reagent, wherein the isothiocyanate groups of the polyisothiocyanate reagent are linked to each other by an aliphatic, aromatic or heteroaromatic group.

1 1 . The method of any one of claims 8 to 10, wherein the aliphatic group comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S.

12. The method of any one of claims 8 to 10, wherein the aromatic and heteroaromatic groups comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from O, N and S.

13. The method of claim 8, wherein the activated esters comprise fluorophenol esters, nitrophenol esters, hydroxybenzotriazole esters, N-hydroxy succinimide esters, or isourea esters.

14. The method of claim 8, wherein the diacid activated ester is [bis(succinimidyl) penta(ethyleneglycol)] BS(PEG)5 or [bis(succinimidyl) nona(ethyleneglycol)] BS(PEG)9.

15. The method of claim 14, wherein the diacid activated ester concentration is varied to obtain a [diacid activated ester : amino] molar ratio ranging from 0.2 to 20.

16. The method of claim 8, wherein the acid anhydride is acetic anhydride, phtalic anyhydride or succinic anhydride.

17. The method of claim 16, wherein the acid anhydride concentration is varied to obtain a [acid anhydride : amino] molar ratio ranging from 2 to 200.

18. The method of claim 9, wherein the monoaldehyde is acetaldehyde, propionaldehyde, butyraldehyde or benzaldehyde, the dialdehyde is glutaraldehyde and the polyaldehyde is dextran polyaldehyde or a polyethylene glycol polyaldehyde.

19. The method of claim 18, wherein the reaction involves stapling surface amino groups using glutaraldehyde as the bifunctional reagent and the concentration of glutaraldehyde is varied to obtain a [glutaraldehyde : amino] molar ratio ranging from 0.5 to 200.

20. The method of claim 18, wherein the reaction involves stitching surface amino groups using dextran polyaldehyde as the multifunctional reagent and the concentration of dextran polyaldehyde is varied to obtain a [dextran polyaldehyde : carbonic anhydrase] (w/w) ratio ranging from 0.3 to 13.8.

21 . The method of any one of claims 1 to 4, wherein the surface functional groups that are modified comprise neutral or deprotonated carboxylic acid groups and the covalent modification, covalent stapling or covalent stitching comprises an esterification reaction or an amidation reaction.

22. The method of claim 21 , wherein the carboxylic acid groups comprise carboxylic acid groups of glutamic or aspartic acids, of the C-terminal group of the peptide chains of the carbonic anhydrase, or carboxylic acid groups resulting from a previous modification.

23. The method of claim 21 or 22, wherein the reaction comprises an esterification that is performed using: i. an aliphatic, aromatic or heteroaromatic alcohol as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diol as the at least one bifunctional reagent; or iii. a polyalcohol having at least three alcohol groups as the at least one multifunctional reagent, wherein the alcohol groups of the polyalcohol are linked to each other by an aliphatic, aromatic or heteroaromatic group.

24. The method of claim 21 or 22, wherein the reaction comprises an amidation that is performed using:

25. The method of claim 23 or 24, wherein the aliphatic group comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S.

26. The method of claim 23 or 24, wherein the aromatic and heteroaromatic groups comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from 0, N and S.

27. The method of any one of claims 21 to 26, wherein the carboxylic acid groups are activated prior to modification, stapling or stitching, by reaction with an activating reagent comprising 0-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or a carbodiimide reagent alone or with an additive.

28. The method of claim 27, wherein the carbodiimide reagent is chosen from the group consisting of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1 -ethyl-3-(3'-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).

29. The method of claim 27 or 28, wherein the additive used in combination with the carbodiimide reagent is chosen from the group consisting of p-nitrophenol

(PNP), pentafluorophenol (PFP), hydroxybenzotriazole (HOBT) and sulfo-N- hydroxysuccinimide (sulfo-NHS).

30. The method of claim 24, wherein the reaction involves stapling carboxylic acid groups of glutamic or aspartic acids using ethylene diamine as the bifunctional reagent.

31 . The method of claim 30, wherein the ethylene diamine concentration is varied to obtain a [ethylene diamine : carboxylic acid group] molar ratio ranging from 2 to 200.

32. The method of any one of claims 1 to 4, wherein the surface functional groups that are modified comprise neutral or protonated guanidino groups and the covalent modification, covalent stapling or covalent stitching comprises a reaction between the guanidino groups and an alpha-diketo compound.

33. The method of claim 32, wherein the guanidino groups are present on arginine or are guanidino groups resulting from a previous modification.

34. The method of claim 32 or 33, wherein the reaction involves reacting neutral or protonated guanidino groups on the surface of the carbonic anhydrase with: i. an aliphatic, aromatic or heteroaromatic alpha-diketo compound as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic alpha-diketo compound having two alpha-diketo groups as the at least one bifunctional reagent; or iii. an alpha-diketo compound having at least three alpha-diketo groups as the at least one multifunctional reagent, wherein the alpha-diketo groups are linked to each other by an aliphatic, aromatic or heteroaromatic group.

35. The method of claim 34, wherein the aliphatic group comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from O, N and S.

36. The method of claim 34, wherein the aromatic and heteroaromatic groups comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from 0, N and S.

37. The method of claim 4, wherein the step of modifying involves stitching aldehyde groups resulting from a reaction between carbonic anhydrase surface amino groups and an aldehyde compound, and wherein polyethylenimine is used as the multifunctional reagent.

38. The method of claim 37, wherein the aldehyde compound is glutaraldehyde.

39. The method of any one of claims 1 to 38, wherein the carbonic anhydrase is a free carbonic anhydrase, an immobilized carbonic anhydrase or an aggregated carbonic anhydrase.

40. The method of any one of claims 1 to 39, wherein the carbonic anhydrase is a monomeric or multimeric carbonic anhydrase.

41 . The method of any one of claims 1 to 40, wherein the carbonic anhydrase is a wild-type carbonic anhydrase, a mutant carbonic anhydrase or a variant carbonic anhydrase.

42. A method for preparing and identifying surface modified carbonic anhydrases having an improved activity and/or stability comprising: a) providing a plurality of different carbonic anhydrases each having an initial surface charge; b) modifying surface functional groups of each of the carbonic anhydrase, comprising separately subjecting each carbonic anhydrase to at least one reaction with a reagent thereby obtaining a set of modified carbonic anhydrases with respective surface charges different than the initial surface; c) testing the stability and/or activity of each surface modified carbonic anhydrases of the set; and

43. The method of claim 42, further comprising varying at least one predetermined reaction parameter during the modifying step.

44. The method of claim 43, wherein the predetermined reaction parameter comprises a reagent concentration, a carbonic anhydrase concentration, a pH at which the reaction is performed, a solvent in which the reaction is performed, a reaction temperature, a reaction time, or any combination thereof.

45. The method of claim 44, wherein the at least one predetermined reaction parameter comprises a reagent concentration.

46. The method of any one of claims 42 or 45, wherein the step of modifying comprises: i. covalent modification of the functional groups by reaction with a monofunctional reagent, or ii. covalent stapling of the functional groups by reaction with a bifunctional reagent, or iii. covalent stitching of the functional groups by reaction with a multifunctional reagent, the multifunctional reagent having at least three reactive groups capable to react with at least three surface functional groups of the carbonic anhydrase.

47. The method of any one of claims 42 to 46, wherein the surface functional groups that are modified comprise neutral or protonated amino groups; neutral or deprotonated carboxylic acid groups; neutral or protonated guanidino groups; or any combination thereof.

48. The method of claim 47, wherein the surface functional groups that are modified comprise neutral or protonated amino groups and the covalent modification, covalent stapling or covalent stitching comprises an acylation reaction, an imination reaction, or a thioureation reaction.

49. The method of claim 48, wherein the surface functional groups comprise lysine amino functional groups, amino terminal groups of the peptide chains of the carbonic anhydrase, or primary amino groups resulting from a previous modification.

50. The method of claim 48 or 49, wherein the reaction comprises an acylation that is performed using: i. an aliphatic, aromatic or heteroaromatic acid anhydride, acyl halide, or acid activated ester as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diacyl halide or diacid activated ester as the at least one bifunctional reagent; or iii. a polyacyl halide having at least three acyl halide groups or a polyacid activated ester having at least three acid activated ester groups as the at least one multifunctional reagent, wherein the acyl halide groups of the polyacyl halide or the acid activated ester groups of the polyacid activated ester are linked to each other by an aliphatic, aromatic or heteroaromatic group.

51 . The method of claim 48 or 49, wherein the reaction comprises an imination that is performed using: i. an aliphatic, aromatic or heteroaromatic monoaldehyde as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic dialdehyde as the at least one bifunctional reagent; or iii. a polyaldehyde having at least three aldehyde groups as the at least one multifunctional reagent, wherein the aldehyde groups of the polyaldehyde are linked to each other by an aliphatic, aromatic or heteroaromatic group.

52. The method of claim 48 or 49, wherein the reaction comprises a thioureation that is performed using: i. an aliphatic, aromatic or heteroaromatic isothiocyanate reagent as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diisothiocyanate reagent as the at least one bifunctional reagent; or iii. a polyisothiocyanate reagent having at least three isothiocyanate groups as the at least one multifunctional reagent, wherein the isothiocyanate groups of the polyisothiocyanate reagent are linked to each other by an aliphatic, aromatic or heteroaromatic group.

53. The method of any one of claims 50 to 52, wherein the aliphatic group comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S.

54. The method of any one of claims 50 to 52, wherein the aromatic and heteroaromatic groups comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from O, N and S.

55. The method of claim 50, wherein the activated esters comprise fluorophenol esters, nitrophenol esters, hydroxybenzotriazole esters, N-hydroxy succinimide esters, or isourea esters.

56. The method of claim 50, wherein the diacid activated ester is [bis(succinimidyl) penta(ethyleneglycol)] BS(PEG)5 or [bis(succinimidyl) nona(ethyleneglycol)] BS(PEG)9.

57. The method of claim 56, wherein the diacid activated ester concentration is varied to obtain a [diacid activated ester : amino] molar ratio ranging from 0.2 to 20.

58. The method of claim 50, wherein the acid anhydride is acetic anhydride, phtalic anyhydride or succinic anhydride.

59. The method of claim 58, wherein the acid anhydride concentration is varied to obtain a [acid anhydride : amino] molar ratio ranging from 2 to 200.

60. The method of claim 51 , wherein the monoaldehyde is acetaldehyde, propionaldehyde, butyraldehyde or benzaldehyde, the dialdehyde is glutaraldehyde and the polyaldehyde is dextran polyaldehyde or a polyethylene glycol polyaldehyde.

61 . The method of claim 60, wherein the reaction involves stapling surface amino groups using glutaraldehyde as the bifunctional reagent and the concentration of glutaraldehyde is varied to obtain a [glutaraldehyde : amino] molar ratio ranging from 0.5 to 200.

62. The method of claim 60, wherein the reaction involves stitching surface amino groups using dextran polyaldehyde as the multifunctional reagent and the concentration of dextran polyaldehyde is varied to obtain a [dextran polyaldehyde : carbonic anhydrase] (w/w) ratio ranging from 0.3 to 13.8.

63. The method of any one of claims 42 to 47, wherein the surface functional groups that are modified comprise neutral or deprotonated carboxylic acid groups and the covalent modification, covalent stapling or covalent stitching comprises an esterification reaction or an amidation reaction.

64. The method of claim 63, wherein the carboxylic acid groups comprise carboxylic acid groups of glutamic or aspartic acids, of the C-terminal group of the peptide chains of the carbonic anhydrase, or carboxylic acid groups resulting from a previous modification.

65. The method of claim 63 or 64, wherein the reaction comprises an esterification that is performed using: i. an aliphatic, aromatic or heteroaromatic alcohol as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic diol as the at least one bifunctional reagent; or iii. a polyalcohol having at least three hydroxyl groups as the at least one multifunctional reagent, wherein the hydroxyl groups of the polyalcohol are linked to each other by an aliphatic, aromatic or heteroaromatic group.

66. The method of claim 63 or 64, wherein the reaction comprises an amidation that is performed using:

67. The method of claim 65 or 66, wherein the aliphatic group comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S.

68. The method of claim 65 or 66, wherein the aromatic and heteroaromatic groups comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from 0, N and S.

69. The method of any one of claims 63 to 68, wherein the carboxylic acid groups are activated prior to modification, stapling or stitching, by reaction with an activating reagent comprising 0-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or a carbodiimide reagent alone or with an additive.

70. The method of claim 69, wherein the carbodiimide reagent is chosen from the group consisting of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1 -ethyl-3-(3'-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).

71 . The method of claim 69 or 70, wherein the additive used in combination with the carbodiimide reagent is chosen from the group consisting of p-nitrophenol (PNP), pentafluorophenol (PFP), hydroxybenzotriazole (HOBT) and sulfo-N- hydroxysuccinimide (sulfo-NHS).

72. The method of claim 66, wherein the reaction involves stapling carboxylic acid groups of glutamic or aspartic acids using ethylene diamine as the bifunctional reagent.

73. The method of claim 72, wherein the ethylene diamine concentration is varied to obtain a [ethylene diamine : carboxylic acid group] molar ratio ranging from 2 to 200.

74. The method of any one of claims 42 to 47, wherein the surface functional groups that are modified comprise neutral or protonated guanidino groups and the covalent modification, covalent stapling or covalent stitching comprises a reaction between the guanidino groups and an alpha-diketo compound.

75. The method of claim 74, wherein the guanidino groups are present on arginine or are guanidino groups resulting from a previous modification.

76. The method of claim 74 or 75, wherein the reaction involves reacting neutral or protonated guanidino groups on the surface of the carbonic anhydrase with: i. an aliphatic, aromatic or heteroaromatic alpha-diketo compound as the at least one monofunctional reagent; ii. an aliphatic, aromatic or heteroaromatic alpha-diketo compound having two alpha-diketo groups as the at least one bifunctional reagent; or iii. an alpha-diketo compound having at least three alpha-diketo groups as the at least one multifunctional reagent, wherein the alpha-diketo groups are linked to each other by an aliphatic, aromatic or heteroaromatic group.

77. The method of claim 76, wherein the aliphatic group comprises a linear, branched or cyclic group comprising from 1 to 18 carbon atoms and optionally heteroatoms selected from 0, N and S.

78. The method of claim 76, wherein the aromatic and heteroaromatic groups comprise one, two or three fused or linked cycles, and the heteroatoms of the heteroaromatic groups are selected from O, N and S.

79. The method of claim 46, wherein the step of modifying involves stitching aldehyde groups resulting from a reaction between carbonic anhydrase surface amino groups and an aldehyde compound, and wherein polyethylenimine is used as the multifunctional reagent.

80. The method of claim 79, wherein the aldehyde compound is glutaraldehyde.

81 . The method of any one of claims 42 to 80, wherein each of the carbonic anhydrases of the plurality of carbonic anhydrases is independently a free carbonic anhydrase, an immobilized carbonic anhydrase or an aggregated carbonic anhydrase.

82. The method of any one of claims 42 to 81 , wherein each of the carbonic anhydrases of the plurality of carbonic anhydrases is independently a monomeric or multimeric carbonic anhydrase.

83. The method of any one of claims 42 to 82, wherein each of the carbonic anhydrases of the plurality of carbonic anhydrases is independently a wild-type carbonic anhydrase, a mutant carbonic anhydrase or a variant carbonic anhydrase.

84. A surface modified carbonic anhydrase having an improved stability and/or activity which is prepared and identified by the method of any one of claims 1 to 83.

85. Use of a surface modified carbonic anhydrase having an improved stability and/or activity as defined in claim 84 or prepared and identified according to the method of any one of claims 1 to 83, in an industrial process comprising CO2 capture by CO2 hydration.

86. Use of a surface modified carbonic anhydrase having an improved stability and/or activity as defined in claim 84 or prepared and identified according to the method of any one of claims 1 to 83, in an industrial process comprising C0₂ desorption by bicarbonate dehydration.

87. A process for treating a C0₂-containing gas comprising contacting the C0₂- containing gas with an absorption solution comprising water in the presence of a carbonic anhydrase as defined in claim 84 or prepared and identified according to the method of any one of claims 1 to 83, whereby the carbonic anhydrase catalyzes the hydration reaction of dissolved C0₂ into bicarbonate ions and hydrogen ions within the aqueous solution to produce a bicarbonate loaded solution.