Human carbonic anhydrase II with increased physical stability

Abstract

An isolated polypeptide having carbonic anhydrase activity, the sequence of which corresponds to modified human carbonic anhydrase II is described. The isolated polypeptide comprises the mutations A23C, S99C, L202C, C205S and V241C and the polypeptide has increased physical stability compared to wild type carbonic anhydrase II. Further, the polypeptide comprises disulfide bridges between C23 and C202 and/or between C99 and C241.

Claims

1. An isolated polypeptide having carbonic anhydrase activity, the sequence of which corresponds to modified human carbonic anhydrase II, wherein the polypeptide comprises the mutations A23C, S99C, L202C, C205S and V241C relative to wild type human carbonic anhydrase II having the amino acid sequence of SEQ ID NO:9, has increased physical stability compared to wild type carbonic anhydrase II and further comprises disulfide bridges between C23 and C202 and/or between C99 and C241; and wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 8.

2. The isolated polypeptide having carbonic anhydrase activity according to claim 1, having a thermodynamic stability increased by 23.5 kJ/mol compared to wild type carbonic anhydrase II.

3. The isolated polypeptide having carbonic anhydrase activity according to claim 1, having a melting point increased by 18.5 C. compared to wild type carbonic anhydrase II.

4. The isolated polypeptide having carbonic anhydrase activity according to claim 1, having an activation energy of unfolding increased by 25 kJ/mol compared to wild type carbonic anhydrase II.

5. The isolated polypeptide having carbonic anhydrase activity according to claim 1, having a rate of unfolding in water at 21 C. that is about 22.000 times slower compared to wild type human carbonic anhydrase II.

6. The isolated polypeptide having carbonic anhydrase activity according to claim 1, having a half-life of 86 days at 60 C., 8 days at 65 C. and 1.6 days at 70 C.

7. The isolated polypeptide having carbonic anhydrase activity according to claim 1, wherein the isolated polypeptide maintains its increased physical stability compared to wild type carbonic anhydrase II in aqueous solutions of ethanol amines, comprising methyldiethanolamine (MDEA), monoethanolamine (MEA), diethanolamine (DEA), and aminoethoxyethanol.

8. A method of using an isolated polypeptide having carbonic anhydrase activity according to claim 1 for extraction of carbon dioxide from a carbon dioxide containing medium.

9. The method according to claim 8, wherein the isolated polypeptide having carbonic anhydrase activity is used in a bioreactor.

10. A method of preparing an isolated polypeptide of SEQ ID NO: 8, according to claim 1 comprising acceleration of the formation of disulfide bridges by incubation of the polypeptide at elevated temperatures of 25-60 C. in the presence of an oxidizing agent at a pH of 7-10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph illustrating the definition of difference in free energy, between the unfolded state (U) and the native folded state (F) of a protein (G.sub.FU1). The graph further illustrates how the thermodynamic stability can be increased by stabilizing the folded state (G.sub.FU2).

(2) FIG. 2 illustrates the relationship between thermodynamic and thermal stability and that knowledge about the thermodynamic stability at a single temperature does not give any information about the melting temperature (T.sub.m) of a protein. The G.sub.FU (T) function of a hypothetical thermolabile protein () with its melting temperature (T.sub.m) and the possible increase in T.sub.m by up shifting (), right shifting () and flattening () of the G.sub.FU (T) function (adapted from ref. 8).

(3) FIG. 3 is a graph illustrating the definition of activation energy of unfolding (E.sub.A, unfolding) of a protein determined by the difference in free energy between the folded state (F) and the transition state (ts.sup.#) on the unfolding pathway.

(4) FIG. 4 is a graph illustrating how the thermodynamic stability (G.sub.FU) for a protein is increased by restricting the freedom of the unfolded state by incorporation of a disulfide bridge (U.sub.SS), thus placing the unfolded state on a higher energy level.

(5) FIG. 5 is a graph illustrating the resulting increase in both thermo-dynamic stability (G.sub.FU, SS) and activation energy for unfolding (E.sub.A,SS), for a protein with a disulfide bridge inserted at positions that affect both the freedom of the unfolded state as well as the folding pathway and thereby the transition state (). Comparison is made with an unmodified reference wild type (wt) protein ().

(6) FIG. 6 is a graph illustrating the enzyme variants resistance to unfolding in a denaturing agent as fraction of unfolded protein as a function of Gu-HCl concentration for SEQ ID NO: 2 (), 4 (.square-solid.), 6 () and 8 incubated over night () and SEQ ID NO: 8 incubated 2-5 days (.circle-solid.).

(7) FIG. 7 A-C illustrates the life times at 60 C. (FIG. 7A), 65 C. (FIG. 7B) and 70 C. (FIG. 7C) for SEQ ID NO:2 (), 4 (.square-solid.), 6 () and 8 (.circle-solid.). Note that SEQ ID NO 2 is only measured at 60 C. as it is instantly inactivated already at this temperature (FIG. 7A) and that SEQ ID NO: 4 is only measured at 60 and 65 C. (FIGS. 7A and 7B). The only variant having an appreciable life time at all temperatures is the polypeptide of SEQ ID NO: 8.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

(8) One aspect of the present invention is to provide an enzyme that has a high enough physical stability to make bioreactors, that are designed and capable of extracting CO.sub.2 from a CO.sub.2-containing medium, practical and economically feasible.

(9) The present disclosure provides an engineered, highly efficient, human carbonic anhydrase II variant that has an increased physical stability as determined by thermodynamic, thermal and kinetic stability as well as prolonged life time.

(10) The stabilized human carbonic anhydrase II according to the present invention has a thermodynamic stability increased by 23.5 kJ/mol.

(11) The present invention further provides an engineered human carbonic anhydrase II that is heat-stable and is able to catalyze the hydration of CO.sub.2 at normal and elevated temperatures over long periods of time. The heat stability of the present invention provides a carbonic anhydrase that has a melting point of 77.5 C. and maintains 100% CO.sub.2 hydration activity for at least 15 min at 70 C. and more than 20% residual CO.sub.2 hydration activity at 95 C. for at least 15 min.

(12) The present invention also provides an engineered kinetically stabilized human carbonic anhydrase II that has an activation energy for unfolding (E.sub.A, unfolding) increased by 25 kJ/mol and a rate of unfolding (k.sub.U) at ambient temperature that is about 22 000 times slower than the wild type enzyme.

(13) The present disclosure further provides an engineered human carbonic anhydrase II that maintains its relative stabilization properties in relation to the wild-type enzyme also in solutions other than buffered aqueous solutions e.g. ethanolamine solutions.

(14) The present invention also provides a method to economically and effectively produce the engineered human carbonic anhydrase II according to the present invention.

(15) The present invention further provides polynucleotides encoding the wild-type and the engineered human carbonic anhydrase II according to the invention.

(16) The present invention relates to a genetically engineered variant of the enzyme human carbonic anhydrase II having the amino acid sequence according to SEQ ID NO: 8, having substantially increased physical stability, as defined by increased thermal, thermodynamic and kinetic stability, and as compared to those of its parent enzymes having the amino acid sequence of SEQ ID NO: 2, 4 and 6. The nucleotide sequences corresponding to SEQ ID NO: 2, 4, 6 and 8 are shown in SEQ ID NO: 1, 3, 5 and 7, respectively. The increased physical stability provides the enzyme properties that allows the enzyme to be used, with an increased life-time, at elevated temperatures (i.e. higher than 37 C.) and in media other than buffered aqueous solutions (e.g. in methyldiethanolamine solutions).

(17) Furthermore, the combination of SEQ ID NO: 2, 4 and 6 leads to the properties of SEQ ID NO: 8 that allows it to be produced in an economically viable way. One aspect of the invention is the use of stable carbonic anhydrases as catalysts in bioreactors for capture and sequestration of CO.sub.2 from CO.sub.2-containing gases, liquids or multiphase mixtures. The present invention is of particular importance when a prolonged life-time is desired and/or when the temperature of the CO.sub.2-containing medium is above the melting point of naturally occurring or commercially available carbonic anhydrases. The present invention is additionally useful both for sequestration (hydration) of CO.sub.2 and subsequent recovery of bicarbonate (dehydration) of the previously sequestered CO.sub.2.

(18) Definitions

(19) Carbonic anhydrase and the abbreviation CA is used interchangeably to refer to a polypeptide having enzymatic E.0 4.2.1.1 activity and that is capable of catalyzing the inter-conversion of carbon dioxide and water to bicarbonate and a proton.

(20) Human carbonic anhydrase II and HCA II is used interchangeably to denote the iso-form 2 variant of human carbonic anhydrase II. Wild type human carbonic anhydrase II has the amino acid sequence defined in SEQ ID NO: 9 (GenBank accession number NM 000067.2).

(21) Wild-type or naturally occurring refers to the form of polypeptide or polynucleotide sequence that can be found in nature and has not been intentionally modified by human manipulation.

(22) Pseudo-wild-type human carbonic anhydrase II (HCA II.sub.pwt) refers to a variant of human carbonic anhydrase II with characteristics indistinguishable from the wild type human carbonic anhydrase II with the naturally occurring cysteine in position 205 exchanged by genetic manipulation to instead code for the amino acid serine (C205S). Conventional denotation of human carbonic anhydrase iso-form sequences sometimes refers to positions relative to the positions in human carbonic anhydrase I and numbering can thus differ between different publications. However, unless otherwise stated all positions defined in this text refers to the sequences and positions as defined in SEQ ID NO: 1-8.

(23) Modified polypeptides according to the invention involves polypeptides having more mutations, truncated variants of the polypeptides, and polypeptides having one or more amino acids added at the N- or C-terminal part of the polypeptide.

EXAMPLES

Example 1

Selection of Mutation Positions

(24) The positions chosen for mutation and introduction of cysteines were based on the findings of two earlier variants of HCA II.sub.pwt. Although not a valid measure of physical stability.sup.[15], for one variant (SEQ ID NO: 4) the midpoint of denaturation in increasing concentrations of a chemical denaturant (guanidine hydrochloride) was increased.sup.[16]. In another variant (SEQ ID NO: 6) the thermodynamic stability was increased at ambient temperature (23 C.).sup.[17]. In these two individually engineered disulfide bridge variants of HCA II.sub.pwt, cysteine in position 99 makes a disulfide bridge with cysteine in position 241 in one variant (SEQ ID NO: 4) and in the other variant (SEQ ID NO: 6) cysteine in position 23 makes a disulfide bridge with cysteine in position 202. However, all other important parameters concerning stability for these variants were unknown. Since the following information cannot simply be deduced from knowing the midpoint concentration of unfolding for one component (SEQ ID NO: 2) or the thermodynamic stability at ambient temperatures of the other component (SEQ ID NO: 6), the thermodynamic stability, the melting point, the stability in 30% ethanol amine solution, the kinetic stability, the unfolding rates and the lifetime at elevated temperatures of both the individual variants (SEQ ID NO: 4 and 6) were determined according to the following examples. From the collective information gained for the individual variants (SEQ ID NO: 4 and 6) in example 6-10 in this document, it is understood that both variants individually possess properties that are beneficial for carbonic anhydrases to be used in an industrial process designed to capture CO.sub.2. Thus, a combination of the two variants could tentatively lead to an enzyme variant with several of the necessary properties enhanced. However, as can be understood from the background art, this cannot be acclaimed without the necessary design of a combined variant and the characterization thereof.

(25) Furthermore, a combination of the two disulfide bridges could very well also lead to that the protein can no longer fold or the cysteines make disulfide bonds with the wrong partner and thereby fold to a non-native state. For one of the variants (SEQ ID NO: 6) it was also earlier found that an out of the ordinary chemical method was needed to form the disulfide bridge under an acceptable time scale, which would hamper the large scale production of the enzyme.sup.[16]. For the efficient large-scale production of the enzyme the earlier proposed methods would be hard to implement to an economically feasible industrial production process of the enzyme. Thus, based on the experimental findings of the two single-disulfide variants in this document, a novel double-disulfide variant (SEQ ID NO: 8) was designed (example 2), produced (example 3-5) and characterized with regards to important properties such as activity, physical stability and lifetime (example 6-11).

Example 2

Site-directed Mutagenesis of HCA II

(26) All variants were produced by the same methods. As a template for further modifications, a nucleotide (SEQ ID NO: 1) coding for a well known variant of HCA II with the only cysteine in the polypeptide sequence at position 205 (SEQ ID NO: 2) replaced with a serine, was used.sup.[18]. The use of this variant prevents faulty disulfide bridges from being formed between any introduced new cysteine and the otherwise single naturally occurring cysteine in position 205. This variant of HCA II has further properties that are indistinguishable from the wild type HCA II and is therefore identified as a pseudo-wild-type human carbonic anhydrase II (HCA II.sub.pwt). The nucleotide sequence coding for HCA II.sub.pwt was cloned into the plasmid pACA, a vector for T7-RNA polymerase-directed expression. The production of T7 RNA polymerase is in turn under control by a lac promoter, thus production of the cloned HCA II protein can be activated by addition of lactose or analogs such as IPTG. The plasmid was maintained in a laboratory expression strain of E. coli (BL21/DE3). Plasmids were prepared by using the Qiagen plasmid preparation kit according to the manufacturer's instructions. Mutagenesis oligo-nucleotides were designed and ordered to specification from DNA technology AS (Denmark). The HCA II.sub.pwt nucleotide sequence, contained in the purified plasmids, was thereafter subject to site-directed mutagenesis using the aforementioned DNA oligomers and the QuickChange site-directed mutagenesis kit from Stratagene. After purification of the treated plasmids, aliquots of the plasmids was sent for sequencing (GATC Gmbh, Germany) for verification of correct desired sequence and mutations. After verification the plasmids was used to transform a new set of BL21/DE3 cells which were grown to a cell density of approx. OD 1 at A.sub.660 in 20 ml 2LB medium. The cells were transferred in aliquots of 500 L to Eppendorf tubes and mixed with 500 L 50% glycerol and frozen in liquid nitrogen. The E. coli stocks were thereafter stored at 70 C.

Example 3

Protein Production

(27) All variants were produced by the same methods. 215 mL of over-night cultures of 50 mL of transformed BL21/DE3, containing plasmids carrying the mutated HCA II.sub.pwt, and grown in LB medium at 37 C., was transferred and used to inoculate 21.5 L of LB medium in shake bottles. The cells were allowed to grow at 37 C. to a cell density of approx. OD 0.8 at A.sub.660 and were then supplemented with IPTG and ZnSO.sub.4 to a final concentration of 1 mM, respectively and the cells were left to produce the protein over night. The cells of the culture broths were sedimented by centrifugation at 3.000g and the supernatant was discarded. The cells were resuspended in 40 mL of 10 mM tris-H.sub.2SO.sub.4, pH 9.0. The cell suspension was thereafter subjected to ultrasonication to break the cell walls and release the cell content. The cell suspension was thereafter centrifuged at 10.000g for 30 min and the supernatant containing the produced mutated HCA II.sub.pwt was collected. The pH of the supernatant was adjusted to an approx. pH of 9 with tris base. The supernatant was mixed with approx. 10 mL of an affinity gel for HCA II (BioRad CM agarose with a sulfonamide coupled to the matrix) and allowed to stand for 30 min before being applied to a chromatography column. The gel was washed with several bed volumes of 10 mM tris-H.sub.2SO.sub.4, pH 9.0 under monitoring of the A.sub.280. When no more change in A.sub.280 could be detected the protein was eluted with 10 mM tris-H.sub.2SO.sub.4, pH 7.0 and 0.5 M azide. The eluate was collected and transferred to dialysis tubes with a molecular weight cut-off of 10 kDa (Millipore) and then dialyzed against 510 L of dialysis buffer (10 mM tris-H.sub.2SO.sub.4, pH 7.5) with at least 8 h between each change of buffer. The dialyzed protein solutions were then collected and concentrated in centrifugation tubes with a molecular cut-off of 10 kDa.

(28) Concentration of the protein sample was determined by A.sub.280 measurement using an extinction coefficient of .sub.280=55 400 M.sup.1 cm.sup.1. The protein sample was further analyzed for purity by overloading of protein sample (10 pg per well) onto a SDS-PAGE. After the SDS-PAGE run the proteins in the gel were stained with commassie brilliant blue. For each produced protein sample it was found that no other protein band could be visually detected. Thus, since the proteins were considered to be pure the mutated variants of HCA II could be subject to further analysis.

Example 4

Detection of Free Cysteines

(29) All variants containing cysteines were analyzed by the same methods. Free cysteines, i.e. non-productive cysteines that had not formed a cystine residue with its expected partner and thus had not formed a stabilizing disulfide bridge, was detected by 7-chloro-4-nitrobenzofurazan (NBD-CI). Protein, tris-H.sub.2SO.sub.4 pH 7.5 and guanidine hydrochloride (Gu-HCl) were mixed to a final concentration of 17.1 M, 0.1 M and 5 M, respectively. Free cysteines were detected with a time scan of 30 min at 420 nm using a spectrophotometer (Hitachi U-2001) after addition of a tenfold excess of NBD-CI (171 M). As a reference, a sample of HCA II.sub.pwt (that has no cysteine amino acid residue) was run. If there are free cysteines, the NBD-CI will react with the thiol group and form a cysteine-NBD moiety that absorbs light in the visual wavelength (turns yellow). With an extinction coefficient of .sub.420=13 000 M.sup.1 cm.sup.1 for the cysteine-NBD moiety, one free cysteine per protein will give an absorbance A.sub.420 of 0.22 at the used concentration of protein after the reaction, and four free cysteines will thus give an absorbance A.sub.420 of 0.88. The only disulfide variant that did not show increase of absorbance at 420 nm after the reaction was the single disulfide bridge variant SEQ ID NO: 4 which thus had no free cysteines and a single disulfide bond fully formed. The other single-disulfide variant (SEQ ID NO: 6) was, as earlier found, not able to spontaneously form its disulfide bridge.sup.[16, 17]. More importantly, it was subsequently found that the novel double-disulfide variant (SEQ ID NO: 8) also had about 50% of free cysteins (2 out of 4 cysteines not forming a disulfide bridge). Most likely, this indicates that one disulfide bridge had formed spontaneously, whereas the other disulfide bridge was not formed during production of the enzyme of SEQ ID NO: 8. Thus, a method to form the missing disulfide bridge needed to be developed.

Example 5

Formation of Disulfide Bridges of SEQ ID NO: 6 and 8

(30) Due to low resistance of the reduced form towards unfolding in guanidine hydrochloride (C.sub.m, FU of 0.7 M Gu-HCl).sup.[17], the disulfide bridge of SEQ ID NO: 6 was formed by a chemical method as has previously been described in the literature.sup.[16], resulting in a protein with both cysteines reacted in a correct disulfide bridge and with a retained native and active conformation. However, the double-disulfide bridge variant of SEQ ID NO: 8 had only one out of two disulfide bridges formed. Most likely, it was the disulfide bridge of SEQ ID NO: 4 that had formed and the disulfide bridge of SEQ ID NO: 6 that had not formed, analogously to the behavior of the individual disulfide bridge variants. Nevertheless, regardless of which of the two disulfide bridges that had formed, each will individually lead to a higher thermal stability of the protein (see example 9). Thus, instead of using the earlier described chemical method to increase the structural flexibility to facilitate for the cysteines to find each other, the formation of the second disulfide bridge in SEQ ID NO: 8 could be accomplished by allowing the reaction to take place at elevated temperatures.

(31) Therefore, an alternative scheme to the chemical method used to form the disulfide bridge of SEQ ID NO: 6 was developed for the double-disulfide bridge variant of SEQ ID NO: 8. Since the melting point of the least stabilized variant (SEQ ID NO: 4) with a formed disulfide bridge is increased by 7.5 C. and is unaffected by incubation at temperatures <55 C. (see example 9), the double disulfide variant of SEQ ID NO: 8 could effectively be incubated at 50 C. to induce formation of the second disulfide bridge donated from SEQ ID NO: 6. For the purpose of verifying this approach an experimental assay was designed. Two stock solutions containing 85.5 M of protein (SEQ ID NO: 8 with only one disulfide bridge formed) in 50 mM tris-H.sub.2SO.sub.4 pH 8.5 supplemented with a 100 fold concentration of oxidized dithiotreitol (DTT) was prepared. One solution was incubated at room temperature whereas the other was incubated in a heated cabinet at 50 C. At certain time points aliquots of the stock solutions were withdrawn and measured for free cysteines as described in example 4. It was found that by incubating the sample at 50 C. this method yielded 100% disulfide bridge formation of SEQ ID NO: 8 within 24 hours. At this time the sample incubated at room temperature had only formed approx. 20% of the disulfide bridges. The samples were further analyzed by SDS-PAGE which revealed that no dimers had been formed during the thermal process, indicating that correct disulfide bridges had been formed. In terms of applicability of the enzyme of SEQ ID NO: 8 this is a very important result as it makes the large-scale production of the variant feasible. Partly because, as compared to the earlier described chemical method, less amount of costly chemicals is needed since no addition of Gu-HCl is necessary in the process.

(32) Furthermore, the completed enzyme product does not need down stream processing to be cleaned from the denaturing agent Gu-HCl. Yet more, the reaction rate with SEQ ID NO: 8 and the described thermal method is faster (24 h for 100% disulfide bridge formation) than the chemical method as it takes place at elevated temperatures. This can be compared to the rate of disulfide bridge formation in SEQ ID NO: 6 using the earlier described chemical method (100 h for 100% disulfide bridge formation).

Example 6

Stability against Unfolding by Denaturing Agents in Aqueous Solution and in 30% Methyldiethanolamine

(33) Aliquotes of 0.85 M solutions of each of the described HCA II variants of SEQ ID NO: 2, 4, 6 and 8 were incubated in room temperature over night (approx 18 hours) in increasing amount of the denaturant Gu-HCl (0-6 M) in buffered solutions (0.1 M tris-H.sub.2SO.sub.4 pH 7.5). For the methyldiethanolamine (MDEA) measurements the solution also contained MDEA at a final concentration of 30%. Fluorescence spectra were recorded for each variant at all Gu-HCl concentrations chosen in a spectrofluorometer (Jobin-Yvon Fluoromax 4). Excitation wavelength was 295 nm and three accumulative emission spectra were recorded for each sample between 310-400 nm. From the spectra the wavelength shift was determined. The data was normalized and the fractional change as a function of Gu-HCl concentration was determined. From this it was determined at what Gu-HCl concentration the midpoint of unfolding (C.sub.m, FU) occurred for each variant in both media (table 1). For samples incubated in buffered aqueous solution the values for HCA II.sub.pwt (SEQ ID NO: 2) and the two single disulfide bridge variants (SEQ ID NO: 4 and 6) reached earlier found values (C.sub.m, FU of 1.0, 1.40 and 1.85 M Gu-HCl, respectively). Unexpectedly, the subsequently produced novel SEQ ID NO: 8 variant reached an apparent very high C.sub.m, FU of 2.6 M Gu-HCl, which is higher than the sum of each individual stabilizing disulfide bridge (1.0+0.4+0.85=2.25 M GuHCl). This could mean one of two things. Either there were some synergistic effect in the stability making the double disulfide bridge enzyme of SEQ ID NO: 8 in fact more stable against denaturation by Gu-HCl than the sum of stabilization of the two contributing single disulfide bridge variants.

(34) Alternatively, the kinetic stability of SEQ ID NO: 8 was increased so that the rate of unfolding was slower and thus the protein sample of SEQ ID NO: 8 did not reach equilibrium in 18 h. Therefore, the very same samples were incubated for an additional 24 h (total of 42 h) before data was collected again. For SEQ ID NO: 8 it was found that the curve had shifted to lower concentrations and stopped at a C.sub.m, FU of 2.25 M Gu-HCl. Thus, although the SEQ ID NO: 8 enzyme was not as resistant to denaturants as the initial result indicated, this is still a considerable increase in stability as determined by C.sub.m, FU and compared to SEQ ID NO: 2, 4 and 6. Furthermore, this also proves that the combination of disulfide bridge variants of SEQ ID NO: 4 and 6 is achievable since the protein can still fold and the stability reaches the sum of each individual stabilizing disulfide bridge (2.25 M GuHCl) and thus no stabilizing effects regarding resistance to denaturing agents are lost from combining the two.

(35) The slow equilibration that was found for SEQ ID NO: 8 is a behavior that to our knowledge has not earlier been demonstrated for any earlier variants of HCA II. The behavior implies that the double disulfide variant of SEQ ID NO: 8 has a high kinetic barrier to unfolding. This would then lead to that the unfolding rate of SEQ ID NO: 8 is slower than for each of the individual disulfide bridge variants of SEQ ID NO: 4 and 6, and thus that the equilibrium between the folded and unfolded state takes longer time to reach than for each individual disulfide bridge variant (SEQ ID NO: 4 and 6).

(36) TABLE-US-00001 TABLE 1 Midpoint concentration of unfolding in increasing concentration of Gu-HCl SEQ SEQ SEQ SEQ ID NO 8 SEQ ID NO 8 ID ID ID (INCUBATION (INCUBATION NO 2 NO 4 NO 6 3-5 DAYS) OVER NIGHT) C.sub.M,FU_(H2O) 1.0 1.4 1.85 2.25 2.6 (M Gu-HCl) C.sub.M,FU_(30% MDEA) 0.3 0.75 1.2 1.4 (M Gu-HCl)

(37) The stability against denaturing agents in 30% MDEA follows the same trend, i.e. SEQ ID NO: 2 has the lowest C.sub.m, FU followed by SEQ ID NO: 4, 6 and 8 respectively (see table 1). Thus, this result confirms that although MDEA generally destabilizes the proteins, the relative increase in stability against denaturation in Gu-HCl from introduced disulfide bridges is almost unaffected by the properties of the surrounding media, as earlier described. Therefore, also in 30% MDEA, the protein according to SEQ ID NO: 8 has a considerably higher stability than the HCA II.sub.pwt variant (SEQ ID NO: 2) has. Thus, the protein variants of SEQ ID NO: 6 and 8 are more stable even in 30% MDEA than SEQ ID NO: 2 is even in buffered aqueous solution.

Example 7

Thermodynamic Stability in Aqueous Solution

(38) The equilibrium constant data (K) in the transition region, for each enzyme variant, obtained in example 6 was used to calculate the thermodynamic stability of the respective enzyme variant in purely buffered aqueous solution according to the relationship G=RT ln(K) by the linear extrapolation method.sup.[19].

(39) TABLE-US-00002 TABLE 2 Thermodynamic stability in buffered aqueous solution at ambient temperature (21 C.). SEQ SEQ SEQ Thermodynamic ID ID ID SEQ ID NO 8 stability NO 2 NO 4 NO 6 (inc. 3-5 days) G.sub.(H2O) 30.5 39 46 54 (kJ/mol) G.sub.(H2O) 8.5 12.5 23.5 (kJ/mol) relative to SEQ ID NO 2

(40) Clearly, the increased resistance of SEQ ID NO: 8 to denaturation by Gu-HCl as judged by C.sub.m, FU values is an effect of a significantly increased thermodynamic stability. Furthermore, the increase in thermodynamic stability of SEQ ID NO: 8 is slightly larger than the sum of increased stability of SEQ ID NO: 4 and 6 (8.5+12.5<23.5), indicating a small synergistic effect.

Example 8

Activity Assays of Carbonic Anhydrases

(41) Activity assays were used in order to measure the change in enzyme activity in response to changes in conditions (denaturing agents and temperature) to reveal melting temperatures (T.sub.m), unfolding rates, kinetic stability and life time at elevated temperatures. Activity assays are also important to establish the absolute activity of the protein variants of SEQ ID NO: 4, 6 and 8, in relation to the pseudo-wild-type enzyme, since what is desired is an as high as possible catalytic activity and efficiency also in the engineered variants.

(42) Several variants of colorometric CO.sub.2-hydration activity assays have earlier been described in literature.sup.[20, 21, 22] which are all based on the enzymatic reaction which leads to the production of bicarbonate and protons from carbon dioxide and water. Thus, enzymatic activity of carbonic anhydrase will give a faster decrease in pH than the spontaneous reaction and can be monitored if the reaction takes place in a buffer containing the pH indicator bromothymol blue (BTB).

(43) An aqueous stock solution saturated with CO.sub.2 was prepared by bubbling ice cooled deionized water with CO.sub.2 through a gas diffuser for at least 1 hour prior to use. To monitor the CO.sub.2-hydration activity per mg of enzyme, 2 ml of 25 mM veronal-H.sub.2SO.sub.4, pH 8.2, containing 20 mg/L of BTB was mixed with 1 ml deionized water and 30 L of protein (8.5 M) in a small beaker placed in an ice-bath on top of a magnetic stirrer. All solutions were kept on ice prior to use. The reaction was started by the addition of 2 ml of the CO.sub.2 saturated solution to the stirred buffer solution. Simultaneously with the addition of CO.sub.2 saturated solution a stop watch was started and the time to reach pH 6.5 was determined by comparison of color to a reference sample containing 2 ml 0.2 M Na-phosphate buffer pH 6.5, 2 ml 25 mM veronal H.sub.2SO.sub.4, pH 8.2, containing 20 mg/L of BTB and 1 ml deionized water. The time to reach pH 6.5 was measured for the catalyzed reactions (t.sub.c) and for the un-catalyzed blank reactions (t.sub.b) and the following equation was used to determine activity units (A.U.) per mg enzyme:

(44) $A.U./\mathrm{mg}=\frac{\left(\frac{t_b}{t_c}\right)-1}{\mathrm{mg}\mathrm{enzyme}}$

(45) In all CO.sub.2-hydration experiments the amount of enzyme in the activity assay was 7.5 g. The CO.sub.2-hydration activity of the three disulfide variants (SEQ ID NO: 4, 6 and 8) at the conditions of measurement (0 C.) was found to be 105, 82 and 75 percent respectively of the activity of the HCA II.sub.pwt variant. Thus, all disulfide bridge variants remain highly active with regards to CO.sub.2 hydration.

Example 9

Thermal Stability Assay

(46) For each enzyme variant (SEQ ID NO: 2, 4, 6 and the subsequently produced SEQ ID NO: 8) stock solutions of 8.5 M enzyme in 10 mM tris-H.sub.2SO.sub.4 pH 7.0 were prepared. Aliquotes of 70 L enzyme solutions was placed in thin-walled PCR tubes. In order to prevent increase in enzyme concentration in the enzyme solutions after incubation, due to evaporation and condensation of liquid in the PCR tube, a PCR thermocycler with a heated top was used (GeneAmp PCR-system 9600, Applied Biosystems). For each enzyme variant and target temperature a sample was placed in the thermocycler which was programmed for constant ramping to the set temperature (55 to 95 C.) and incubated for 15 min or 2 hours. After incubation the samples were allowed to cool to room temperature for 10 min before measurements of the residual enzymatic activity according to example 8. All experiments were performed in duplicates. The resulting residual activity after thermal treatment for 15 minutes and 2 hours is presented in table 3 and 4, respectively. Clearly, all engineered variants have higher thermostability than the pseudo-wildtype enzyme (SEQ ID NO: 2). Furthermore, the variant with the highest thermostability is the constructed double disulfide bridge variant (SEQ ID NO: 8). To calculate the approximate T.sub.m (i.e. the temperature at which 50% residual activity remain) of each variant, data from the 15 min incubation was fitted to a sigmoidal function (Table-Curve, Jandel Scientific) and is presented in Table 5.

(47) However, it is important to note that the T.sub.m values obtained are only apparent melting points. Nevertheless, the increase in T.sub.m (T.sub.m) of modified variants (SEQ ID NO: 4, 6 and 8), as compared to HCA II.sub.pwt (SEQ ID NO: 2), represents accurate values. The reason for this is that thermal denaturation of the enzymes is not an equilibrium process but is an example of irreversible inactivation where the enzymes aggregate after unfolding at temperatures close to or above their respective melting points. Thus, what is actually monitored in the activity assay is how large population of enzyme molecules that have yet not unfolded and aggregated at the respective temperature. Consequently, in this case of thermal stability, the kinetic stability is as important as the thermodynamic stability in deciding the behavior at elevated temperatures. Thus, in comparison to the other variants, SEQ ID NO: 8 has two striking characteristics. Firstly, it has an exceptionally high melting point of approximately 77.5 C. which is an increase of 18.5 C. compared to the pseudo-wild-type variant, and even higher than the approx. 70 C. of the -carbonic anhydrase from Methanosarcina thermophila. Secondly, the enzyme of SEQ ID NO: 8 has an apparent residual activity of above 20% at temperatures far beyond its melting point of 77.5 C. This is almost certainly a result of a remarkably high kinetic stability, resulting in that not even incubation for 15 min at 95 C. is enough to completely inactivate all enzyme molecules. Clearly, this is a valuable feature if the enzyme is to be used in e.g. a temperature phased process were the temperature is continuously altered between low and high temperatures since the high kinetic stability of SEQ ID NO: 8 allows the enzyme to survive short bursts of temperatures far beyond its melting temperature.

(48) TABLE-US-00003 TABLE 3 Percent remaining CO.sub.2-hydration activity after 15 min incubation ENZYME VARI- TEMPERATURE ( C.) ANT 55 60 65 70 75 80 85 90 95 SEQ ID 100 22 2 0 0 ND ND ND ND NO 2 SEQ ID 100 100 77 4 0 0 ND ND ND NO 4 SEQ ID 100 100 100 80 2 1 ND ND ND NO 6 SEQ ID 100 100 100 100 76 26 24 23 22 NO 8

(49) TABLE-US-00004 TABLE 4 Percent remaining CO.sub.2-hydration activity after 2 h incubation ENZYME VARI- TEMPERATURE ( C.) ANT 55 60 65 70 75 80 85 90 95 SEQ ID 100 2 1 0 0 ND ND ND ND NO 2 SEQ ID 100 95 50 1 0 0 ND ND ND NO 4 SEQ ID 100 100 100 39 0 0 ND ND ND NO 6 SEQ ID 100 100 100 92 38 7 7 ND ND NO 8

(50) TABLE-US-00005 TABLE 5 Melting points and increase in melting point of enzyme variants Enzyme variant T.sub.m T.sub.m SEQ ID NO: 2 59 SEQ ID NO: 4 66.5 7.5 SEQ ID NO: 6 71.5 12.5 SEQ ID NO: 8 77.5 18.5

Example 10

Kinetic Stability of Engineered HCA II Variants

(51) In order to determine the unfolding rates (k.sub.U) and activation energy for unfolding (E.sub.A, unfolding) of the respective enzyme variant, chemical denaturation was employed. For the unfolding assay each enzyme variant was subjected to increasing concentrations of Gu-HCl, starting from a concentration of 0.2-0.3 M above their respective midpoint concentration of unfolding (C.sub.m, FU, see table 1, example 6) in steps of 0.1 M. For example HCA II.sub.pwt, with a C.sub.m, FU of 1.0 M Gu-HCl, was denatured in Gu-HCl concentrations of 1.2, 1.3, 1.4, and 1.5 M Gu-HCl. Stock solutions of Gu-HCl, to reach the final assay concentration, were prepared and protein stock solutions of 0.5 mg/ml were prepared for each enzyme variant. 2.5 l of enzyme was mixed with 47.5 l of Gu-HCl to reach the targeted Gu-HCl concentration and a protein concentration of 0.025 mg/ml (8.5 M). Each protein variant and each Gu-HCl concentration samples were prepared at room temperature (21 C.), from stock solutions, to monitor residual activity after 10 and 30 sec, and 1, 2, 5, 30, 45, and 60 min. Activity measurements were done as described in example 8 (with 30 l of sample), with the difference that the buffered BTB solution was supplemented with 0.5 mM of the metal chelator EDTA to prevent refolding of enzymes in the assay. Refolding could otherwise occur as both protein and the denaturing agent (Gu-HCl) is diluted in the activity assay. EDTA binds Zn.sup.2+ which is released from unfolded enzymes and present in solution, and which is necessary for the activity HCA II. Thus, the addition of EDTA to the assay freezes the state of the sample so that residual CO.sub.2-hydration activity can be measured. When the residual activity is plotted as a function of time for each Gu-HCl concentration the unfolding rate (k.sub.U) at that very Gu-HCl concentration can be calculated by fitting the data to a single exponential term according to y=ae.sup.kx. To calculate the unfolding rate constant in aqueous solution the natural logarithm of the measured rate constants for the respective enzyme variant is plotted against Gu-HCl concentration and the linearized data is extrapolated to 0 M Gu-HCl (giving the ln k.sub.U at 0 M Gu-HCl). The free energy of activation (G.sup.#), that is, in this case, the free energy of activation for unfolding (E.sub.A, unfolding), can be calculated using the Arrhenius equation, G.sup.#=RT[ln(k.sub.B/h)ln(k.sub.U/T)], where R is the gas constant, 8.314 J.Math.mol.sup.1.Math.K.sup.1, k.sub.B/h is the constant 2.08358.Math.10.sup.10, T is the absolute temperature in Kelvin and k.sub.U is the rate constant for unfolding. The results from the measurements of kinetic stability are presented in table 6.

(52) TABLE-US-00006 TABLE 6 Unfolding kinetics data of enzyme variants at 21 C. SEQ ID SEQ ID SEQ ID SEQ ID NO 2 NO 4 NO 6 NO 8 Lnk.sub.U,H2O 9.30 14.1 12.2 19.3 k.sub.U,H2O (min.sup.1) 9.1 * 10.sup.5 7.5 * 10.sup.7 5.1 * 10.sup.6 4.2 * 10.sup.9 TIMES SLOWER 122 18 22000 UNFOLDING AS COMPARED TO SEQ ID NO 2 E.sub.A,unfolding 96 kJ/mol 108 kJ/mol 103 kJ/mol 121 kJ/mol E.sub.A,unfolding 12 kJ/mol 7 kJ/mol 25 kJ/mol (INCREASE AS COMPARED TO SEQ ID NO 2)

(53) For all stabilized disulfide bridge variants (SEQ ID NO: 4, 6 and 8) there is an obvious decrease in unfolding rate (k.sub.U (H2O)) which culminates in the very slow unfolding of the constructed SEQ ID NO: 8 that unfolds 22.000 times slower than the HCA II.sub.pwt variant (SEQ ID NO: 2) in aqueous media at 21 C.

(54) What is important is that the enzyme of SEQ ID NO: 8 behaves as a completely new variant of HCA II. SEQ ID NO: 4 was found to confer a high increase in kinetic stability (E.sub.A, unfolding of 12 kJ/mol) and a lower increase in thermodynamic stability (G.sub.FU of 8.5 kJ/mol), and behaves thus as an enzyme with an engineered disulfide bridge with an altered folding pathway and thereby a transition state at a higher energy level. Contrary to SEQ ID NO: 4, the enzyme of SEQ ID NO: 6 was found to have a lower increase in kinetic stability (E.sub.A, unfolding of 7 kJ/mol) but possesses a high thermodynamic stabilization (G.sub.FU of 12.5 kJ/mol). Thus, this variant has an unfolded state placed on an even higher level of free energy. On the other hand it has a folding pathway that is only slightly altered and therefore a transition state with only a slightly higher energy level than for the enzyme of SEQ ID NO: 2. However, for the enzyme of SEQ ID NO: 8 there is both a very high kinetic stabilization (E.sub.A, unfolding of 25 kJ/mol) and a very high increase in thermodynamic stability (G.sub.FU of 23.5 kJ/mol). For the thermodynamic stability the increase is close, although not identical, to the sum of increased stability of SEQ ID NO: 4 and 6. However, the very large increase in kinetic stability is an unpredictable effect that stems from the successful introduction of two disulfide bridges in the enzyme, at positions that forces the protein to fold via an unexplored pathway, which differs from the enzymes of SEQ ID NO: 2, 4 and 6, while at the same time the folding ability and enzymatic activity is retained.

Example 11

Life-time at Elevated Temperatures Assayed by Esterase Activity Measurements

(55) The increased thermal, thermodynamic and kinetic stability of the double disulfide bridge variant of SEQ ID NO: 8 should render it a high life time at elevated temperatures. For practical reasons this was monitored by the esterase activity of the enzyme. Stock solutions of 2.5 mg/ml of each enzyme variant were prepared in 10 mM tris-H.sub.2SO.sub.4 pH 7.0 in tubes with screw cap and sealing to prevent evaporation. These were placed in a heated cabinet at the desired temperature (60, 65 or 70 C.) and aliquotes were withdrawn at different time points for measurement of residual esterase activity. Esterase activity was assayed by adding 6 L of protein sample to 1.44 mL of reaction buffer (50 mM tris-H.sub.2SO.sub.4, pH 8.5 with an ionic strength of 0.1 M adjusted with Na.sub.2SO.sub.4) in a cuvette. The sample was supplemented with reagent, 60 L of 30 mM para-nitrophenyl acetate (pNPA) in ice cold acetone, and briefly mixed before esterase activity was measured at 348 nm in a spectrophotometer. The increase in absorbance of the catalyzed reaction was monitored for 60 seconds and the increase in absorbance of a blank reaction (no enzyme added) was then subtracted. The apparent second-order rate constant (k) was calculated according to earlier described methodology.sup.[23].

(56) As a time zero reference the esterase activity of each enzyme stock solution was determined before heat treatment. The esterase activity of the three disulfide variants (SEQ ID NO: 4, 6 and 8) at the conditions of measurement (approx. 21 C.) was found to be 99, 86 and 78 percent respectively, compared to the activity of the HCA II.sub.pwt variant. Thus, all disulfide bridge variants remain highly active also with regards to the esterase activity and to the approximate same degree as CO.sub.2 hydration activity (example 8). The residual activity of each variant at each temperature was plotted against time and fitted to a single exponential term (y=a.Math.e.sup.kx) to obtain the rate constant for unfolding for each enzyme variant at the three temperatures. Since the inactivation is a first-order rate process, the half-life (t.sub.1/2) of each enzyme variant at each temperature can be calculated by t.sub.1/2=ln 2/k. The results of the life time experiments are presented in table 7-9 and FIG. 7.

(57) TABLE-US-00007 TABLE 7 Percent remaining esterase activity after 15 min incubation TEMPERATURE ( C.) ENZYME VARIANT 60 65 70 SEQ ID NO 2 1 0 0 SEQ ID NO 4 102 61 1 SEQ ID NO 6 104 83 82 SEQ ID NO 8 99 96 91

(58) TABLE-US-00008 TABLE 8 Percent remaining esterase activity after 2 h incubation TEMPERATURE ( C.) ENZYME VARIANT 60 65 70 SEQ ID NO 2 0 ND ND SEQ ID NO 4 103 50 ND SEQ ID NO 6 104 72 39 SEQ ID NO 8 106 89 80

(59) TABLE-US-00009 TABLE 9 Inactivation rate constants (k.sub.U), inactivation half time (t.sub.1/2) and t.sub.1/10 of enzyme variants at 60-70 C. TEMPERATURE ENZYME 60 C. 65 C. 70 C. VARIANT k.sub.U (h.sup.1) t.sub.1/2 (h) t.sub.1/10 (h) k.sub.U(h.sup.1) t.sub.1/2 (h) t.sub.1/10 (h) k.sub.U(h.sup.1) t.sub.1/2 (h) t.sub.1/10 (h) SEQ ID NO 2 SEQ ID NO 4 0.0205 35 112 0.346 2 6.6 SEQ ID NO 6 0.00255 272 903 0.0475 15 48 0.434 2 5.3 SEQ ID NO 8 0.000337 2057 6832 0.00381 182 604 0.0180 38 127 (86 days) (285 days) (8 days) (25 days) (1.6 days) (5.3 days)
The increased physical stability, engineered into the double disulfide bridge variant of SEQ ID NO: 8, results in a much slower inactivation rate and thus a much longer life time at increased temperatures than the other variants. At 60 C. the half time of activity inactivation is 86 days for the double disulfide bridge variant of SEQ ID NO: 8 which can be compared to HCA II.sub.pwt (SEQ ID NO: 2) which is instantly unfolded and inactivated at the same temperature. Furthermore, the time for the activity to fall down to 1/10 for SEQ ID NO: 8 is approximately 285 days at 60 C. That is, if two different reactors used the same amount of the engineered HCA II of SEQ ID NO: 8 and an enzyme with 1/10 of activity (as for example Cam), respectively, the reactor with SEQ ID NO: 8 would need 285 days to even fall to the low starting value of the Cam reactor. Thus, it is not only the physical stability per se that is important for an enzyme's practicability, but also the activity and efficiency of the protein. Even at 70 C., where all other variants are quickly unfolded and inactivated, the enzyme of SEQ ID NO: 8 has an appreciable slow inactivation and increased life time with a half life of 1.6 days. The ability to withstand temperatures in the range of 60-70 C. with a long life time is an extremely important feature of SEQ ID NO: 8. At for example modern incinerator plants the flue gas cleaning consists of so many steps that the flue gas is cooled down to approx. 60-70 C. before it reaches the smokestack.sup.[24]. Thus, any enzyme that is to be used in a CO.sub.2-capturing bioreactor at an incinerator plant should preferably also be stable and active in the temperature range of 60-70 C., which is thus fulfilled by the enzyme of SEQ ID NO: 8. Furthermore, the combination of disulfide bridges of SEQ ID NO: 8 has been engineered into an enzyme (HCA II.sub.pwt) that belongs to the structurally conserved superfamily of -carbonic anhydrases. To find structurally related proteins, a database search of the three dimensional structure of HCA II (PDB ID 2cba) was executed against the Conserved Domain Database (CDD).sup.[25], which includes alignments of conserved protein domains to known 3-dimensional protein structures in the Molecular Modeling Database (MMDB). The search resulted in 4977 protein sequences with related conserved domains and 438 related solved structures from the -carbonic anhydrase superfamily. Thus, the combination of the structural motifs of the disulfide bridges between position C23-C202 and C99-C241 in the SEQ ID NO:8 variant of HCA II.sub.pwt can be identified and most likely be grafted also into other members of the -carbonic anhydrase superfamily by homology modeling as earlier described. Indeed, the significantly increased stability of SEQ ID NO: 6 was originally accomplished by homology modeling between HCA II and the distantly related homologous -carbonic anhydrase from Neisseria gonorrhoeae (NGCA, 38.5% sequence identity) which has a naturally occurring disulfide bridge. By homology modeling it was found that the positions for the disulfide bridge in NGCA (sequence positions of C28 and C181) had their three dimensionally structurally equivalent positions in HCA II at the positions A23 and L202. Thus, by homology modeling against a distantly related homologous enzyme a geometrically correct disulfide bridge could be grafted from NGCA into the correct positions in HCA II.sup.[17].

REFERENCES

(60) 1) Zimmerman, S. A. and Ferry, J. G. (2008). The - and -classes of carbonic anhydrase. Current pharmaceutical design 14, 716-721. 2) Alber, B. E., Ferry, J. G. (1994). A carbonic anhydrase from the archaeon Methanosarcina thermophila. Proceedings of the National Academy of Science 91, 6909-6913. 3) IPCC, 2007: Climate Change 2007: Synthesis Report. Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, Pachauri, R. K and Reisinger, A. (eds.)]. IPCC, Geneva, Switzerland. 4) Zimmerman, S. A., Tomb, J.-F., Ferry, J. G. (2010). Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the class of carbonic anhydrases. Journal of Bacteriology, 192, 1353-1360. 5) Silverman, D. N. and Lindskog, S. (1988). The catalytic mechanism of carbonic anhydrase: Implications of a rate-limiting protolysis of water. Accounts of chemical research, 21, 30-36. 6) Ferry, J. G. (2010). The class of carbonic anhydrase. Biochimica et Biophysica Acta, 1804, 374-381. 7) MacAuley, S. R., Zimmerman, S. A., Apolinario, E. E., Eyilia, C., Hou, Y.-M., Ferry, J. G. and Sowers, K. R. (2009). The archetype -class carbonic anhydrase (Cam) contains iron when synthesized in vivo. Biochemistry, 48, 817-819. 8) Sterner, R. and Liebl, W. (2001). Thermophilic adaptation of proteins. Critical Reviews in Biochemistry and Molecular Biology 36, 39-106. 9) Eijsink, V. G. H., Bjork, A., Gseidnes, S., Sirevg, R., Synstad, B, van den Burg, B. and Vriend, G. (2004). Rational engineering of enzyme stability. Journal of Biotechnology 113, 105-120. 10) Wang, T. W., Zhu, H., Ma, X. Y., Ma, Y. S., Wei, D. Z. (2006). Structure-based stabilization of an enzyme: The case of penicillin acylase from Alcaligenes faecalis. Protein and Peptide Letters 13, 177-183. 11) Qi, X., Guo, Q., Wei, Y., Xu, H. Huang, R. (2012). Enhancement of pH stability and activity of glycerol dehydratase from Klebsiella pneumoniae by rational design. Biotechnology Letters 34, 339-346. 12) Brndn, C. and Tooze, J. In Introduction to Protein Structure. 2.sup.nd Ed. ISBN 0-8153-2305-0. Garland publishing, NY. 13) Lin, C., Chao, Y., Xiangshan, Z., Yuanxing, Z. (2009). Rational introduction of disulfide bond to enhance optimal temperature of Lipomyces starkeyi alpha-dextranase expressed in Pichia pastoris. Journal of Microbiology and Biotechnology 19, 1506-1513 14) Pellequer, J.-L., Chen, S.-W., W. (2006). Multi-template approach to modeling engineered disulfide bonds. ProteinsStructure Function and Bioinformatics 65, 192-202. 15) Shortle, D. (1996). The denatured state (the other half of the folding equation) and its role in protein stability FASEB J. 10, 27-34. 16) Karlsson, M., Mrtensson, L.-G., Karlsson, C and Carlsson, U. (2005). Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations. Biochemistry 44, 3487-3493. 17) Mrtensson, L.-G., Karlsson, M. and Carlsson, U. (2002). Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond. Biochemistry 41, 15867-15875. 18) Freskgrd, P.-O., Carlsson, U., Mrtensson, L.-G., Jonsson, B.-H. (1991). Folding around the C-terminus of human carbonic anhydrase IIKinetic characterization by use of a chemically reactive SH-group introduced by protein engineering. FEBS letters 289, 117-122. 19) Pace, C. N. and Shaw, K. L. (2000). Linear extrapolation method of analyzing solvent denaturation curves. Proteins 41, 1-7. 20) Wilbur, K. M. and Anderson, N. G. (1948). Electrometric and colorometric determination of carbonic anhydrase. Journal of biological chemistry 176, 147-154. 21) Rickli, E. E., Ghazanfar, S. A. S., Gibbons, B. H. and Edsall, J. T. (1964), Carbonic anhydrase from human erythrocytes. Preparation and properties of two enzymes. Journal of biological chemistry, 239, 1066-1078. 22) Khalifah R. G. (1971). The carbon dioxide hydration activity of carbonic anhydrase. Journal of biological chemistry 246, 2561-2573. 23) Armstrong J. M., Wyers, D. W., Verpoorte, J. A. and Edsall, J. T. (1966). Purification and properties of human erythrocyte carbonic anhydrases. Journal of biological chemistry 241, 5137-5149. 24) Cimini, S., Prisciandaro, M. and Barba, D. (2005). Simulation of a waste incineration process with flue-gas cleaning and heat recovery sections using Aspen plus. Waste Management 25, 171-175. 25) National Center for Biotechnology Information. URL: http://www.ncbi.nlm.nih.gov/