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Design of carbonic anhydrase with improved thermostability for CO2 capture via molecular simulations
Journal of CO₂ Utilization 38 (2020) 141–147
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Design of carbonic anhydrase with improved thermostability for CO2 capture via molecular simulations
T
Shenglan Wua,b, Jinrui Chena, Liang Mab, Kai Zhanga, Xiaoxiao Wanga,b, Yuping Weic, Jian Xua,b, Xia Xua,b,* a
Biochemical Engineering Research Center, Anhui University of Technology, Ma’anshan, Anhui, 243002, PR China School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma’anshan, Anhui, 243002, PR China c School of Life Science and Technology, Nanyang Normal University, Nanyang, Henan Province, 473061, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carbonic anhydrase Thermostability Molecular simulations Enzyme activity
Chemical absorption of CO2 is the most widely used method to capture CO2. However, high cost and slow CO2 absorption rate severely hinder its commercial utilization. Human carbonic anhydrase II (hCA II) having rapid hydratase reaction rate can potentially overcome these obstacles but being highly unstable against harsh process conditions. To enhance thermostability, a novel CA was developed based on molecular simulations. Substituting residue Leu204 with Lys has a comparable hydratase activity as the wild type (WT) hCA II, 1.98 × 106 s−1 for kcat and 2.36 × 108 M−1s−1 for kcat/Km. Importantly, the replacement of L204 K results in higher temperature resistance with 100 % retention at 45 °C and around 50 % at 55 °C while WT totally inactivates at 45 °C. Furthermore, L204 K is able to maintain its activity in the presence of anions and EDTA. The robustness and efficiency of this novel CA make it a competitive candidate for CO2 capture.
1. Introduction Carbon dioxide (CO2) mainly from the combustion of fossil fuels is primary greenhouse gas associated with global warming and climate change. Many attempts have been made to reduce the amount of CO2 emitted into atmosphere such as developing renewable energy sources, using low carbon fuels, CO2 capture and storage, and conversion of CO2 into fuels and chemicals [1]. In recent years, CO2 capture (CC) has become an extensively investigated area of research. There are a variety of approaches to capture CO2 including physical adsorption [2], membrane separation [3], hydrate formation [4], chemical adsorption, and monoethanolamine (MEA) absorption [5]. However, these CC options face certain technical and economic challenges [5]. Hence, alternatively, there is an enormous interest in using carbonic anhydrases (CAs) as biocatalysts for carbon sequestration [6–8] and production of valuable products [9–12]. Carbonic anhydrase, a zinc metalloenzyme, can catalyze CO2 hydration into bicarbonate (HCO3−) and also exhibits esterase activity [13–17]. γ-class CA from Methanosarcina thermophile (CAM) has the optimal activity for hydratase reaction at 55 °C with turnover rate of 105/s and inactivation at 75 °C [18]. CAs from Methanobacterium thermoautotrophicum (CAB) and Caminibacter mediatlanticus DSM 16658 are
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able to retain more than 40 % of their original hydratase activity at 85 °C but with low activity at 1.7 × 104 s−1 [19]. The enzymes from Bacillus subtilis and Citrobacter freundii are stable over a pH range of 7.0–11.0 and at temperature ≤ 60 °C but not given hydratase activity [20]. Human CA II with high turnover rate of 1.4 × 106 s−1 [21,22] has been identified as a potential biocatalyst for accelerating CO2 absorption from gas streams [23]. However, its application in this field has been limited due to poor stability and activity in harsh conditions such as high temperature, high concentration of organic amine and trace contaminants [24,25]. To overcome these problems, much effort has been made to develop an enzyme with high thermostability. To enhance thermostability of CA, disulfide bridges, ionic pair networks, proline substitutions and surface loop reduction have been proposed. 7 °C increase in hCA II temperature resistance was achieved by substituting three surface leucine residues (Leu100His, Leu224Ser and Leu240Pro) [26]. Further substitutions with Tyr7Phe and Asn67Gln improved proton transfer rate constants but not thermostability [27]. Replacing ALA23 and Leu203 with Cys also enhanced the melting temperature (Tm) of hCA II by 14 °C through a disulfide bridge at pH 7.8 but with a reduction in the rate constant for proton transfer [28]. Substituting Glu234 with Pro increased the rigidity of surface loops, only leading to 3 °C increase in Tm [28]. The melting temperature
Corresponding author at: Biochemical Engineering Research Center, Anhui University of Technology, No. 59 Hudong Road, Ma’anshan, Anhui, 243002, PR China. E-mail address: [email protected] (X. Xu).
https://doi.org/10.1016/j.jcou.2020.01.017 Received 20 December 2019; Received in revised form 13 January 2020; Accepted 15 January 2020 2212-9820/ © 2020 Elsevier Ltd. All rights reserved.
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cell debris were collected. The supernatant was loaded into Ni-nitrolotriacetate affinity chromatographic column (GE, USA). The column was first washed with 10 resin volumes of washing buffer (50 mM Tris, 150 mM NaCl, 50 mM imidazole at pH 7.5), and was then eluted with elution buffer (50 mM Tris, 150 mM NaCl, 50 mM imidazole, 250 mM imidazole at pH 7.5). The eluted fractions containing the target protein were concentrated using a centrifuge tube with a 3 KD cut-off membrane. Protein concentration was determined using a Bradford assay with bovine serum albumin (Sigma, USA) as a standard.
was slightly enhanced by improving compactness through deleting residues of 230–240 [28]. A directed evolution technology has been applied to dramatically enhance the properties of a β-class CA from Desulfovibrio vulgaris using high-throughput screening. The developed CA absorbed CO2 in the presence of 4.2 M alkaline amine at 87 °C, but with a lower activity than hCA II [29]. In recent years, four highly thermostable α-CAs, SspCA from Sulfurihydrogenibium yellowstonense YO3AOP1, SazCA from S ulphurihydrogenibium azorense, Thermovibrio ammonificans (TaCA) and Persephonella marina EX-H1 (PmCA), have been studied extensively. SazCA was demonstrated to have highest catalytic activity for the hydratase reaction with kcat of 4.4 × 106 s−1; kcat/Km of 3.5 × 108 M−1·s−1 at 20 °C and at pH 7.5 [30], more than twice greater than hCA II. The highly thermostable activity, which can retain CO2 hydration activity after 3 h incubation even 100 °C, may be caused by the substitution of Glu2 by His2 and Gln207 by His207 [31]. However, SazCA is quite sensitive to anions such as halide, hydrogen sulfate and sulfate anions, which can severely inhibit its CO2 hydration activity. The aim of this paper was to design a thermostable CA based on human CA II using molecular simulations. Structure stability of mutant hCA II was analyzed using dynamics molecular simulations. Kinetic characterization of mutant hCA II was determined. Esterase and hydratase activity were evaluated at different temperatures and different time intervals. Ability of chemical resistance to anions was also investigated.
2.3. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot analysis The tagged protein was determined using a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) [33,34]. Briefly, 20 μl sample with 5 μl loading buffer was boiled for 10 min and then added into electrophoresis wells. The expressed proteins were separated on the SDS-PAGE, transferred onto polyvinylidene fluoride membranes (Milipore, USA), and blocked with 5 % dried skimmed milk at room temperature. The membranes were incubated overnight with anti-His antibody (Santa Cruz, USA) at 4 °C, washed with TBST, and then incubated with horseradish peroxidase-conjugated IgG (Santa Cruz, USA) for 1 h at room temperature. After washing, bands were visualized using a chemiluminescence (ECL, Thermofisher, USA). 2.4. Activity assay
2. Materials and methods p-nitrophenol acetate (p-NPA) hydrolysis assay was employed to measures enzyme’s activity by monitoring the production of p-nitrophenol (p-NP) from p-NPA hydrolysis [35]. 50 μL of enzyme sample was added to a solution containing 50 μL of Na2HPO–NaH2PO4 buffer solution (0.1 M, pH 7.0) and 50 μL of p-NPA (3 mM) (Alading, China). The esterase activity was determined by measuring p-NP concentration in the hydrolysis solution at 25 °C for 6 min at 348 nm using a Microplate reader (Synergy, Biotek, USA). CO2 hydration activity assay was performed at room temperature as described by Kim et al. [22]. Briefly, to initiate the hydration reaction, CO2-saturated water was added to a mixture containing phosphate buffer saline (PBS, pH 8.35) and a 0.5 μg/ml enzyme with CO2 concentration ranging from 2 to 7 mM. An in-line pH measurement was adopted using a pH electrode connecting to a pH meter (pH 3 Leici, China). The hydratase reaction activity was determined by monitoring the time during the pH change, recorded by a computer. All the reactions for wild type hCA II and mutant hCA II were performed at least triplicate.
2.1. Molecular simulations Coordinate of hCA II was obtained from the RCSB protein data bank (PDB). A topology file of wild type hCA II was generated using the PRODRG2 server [32]. To investigate the stability of mutant hCA II, MD simulation was performed at 25 °C and 1 bar pressure for 10 ns using GROMACS 4.5.5 simulation package. During simulations, a Gromos96 G43a1 force field combined with simple point charge water model was adopted, and the default values for other settings were applied. Before the simulations, all of the bound water was removed. Root mean square deviation (RMSD) of the structures in the trajectory compared to the starting conformation was used to evaluate system stability. Rootmean-square-fluctuation (RMSF) is a measure of the flexibility of a residue, calculated by the square root of the variance of the fluctuation around the average position for the Cα atom of each residue. Simulation results were visualized using Visual Molecular Dynamics (VMD). All simulations were performed on an ASUS workstation (SAMSUNG, Korea).
2.5. Stability at different conditions
2.2. Recombinant protein expression and purification
The stability of mutant hCA II at different temperatures and in the presence of different chemical impurities was evaluated. To investigate the temperature effect on esterase activity and hydratase activity, the enzyme was kept at the temperature ranging from 37 to 95 °C. The thermostability of enzyme was monitored after 0.5, 1 and 2 h of incubation for at different temperatures. The WT hCA II was used as a control. To further look into the chemical stability of enzymes, the enzyme was incubated in the same buffer solution containing anions, SO42−, NO3−, SO32−, NO2- and EDTA-2Na. The activity of mutant hCA II samples stored in the same buffer solution without the presence of any impurities was used for comparison.
The complete gene sequences of wild type and mutant hCA II were designed, synthesized containing NdeI and HindIII restriction sites (GeneScript, USA), and then subcloned into a target vector pET-30a (+) containing a C-terminal and N-terminal hexahistidine (His) tag for E.coli expression. The constructed vector was transformed in BL21 (DE3) (Novagen, Merck, Shanghai, China) and incubated at ∼ 200 rpm for 60 min at 37 °C. After spreading on LB agar plate containing 50 μg/ml kanamycin (Sigma, USA), the plate was incubated upside down at 37 °C. The next day, single and well-isolated colonies were inoculated into 4 ml LB medium containing 50 g/ml kanamycin and were grown at 37 °C and 200 rpm until OD600 value was reached to 0.6-0.8. 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma, USA) was introduced and then the cells were cultured at 37 °C for 4 h. The cells were harvested using a centrifuge at 4000 rpm, re-suspended in lysis Buffer (50 mM Tris, 150 mM NaCl, 5 % glycerol pH 8.0) and disrupted using a sonicator (Xinzhi Biotech, Ningbo, China) for 10 min. The cell lysate was centrifuged at 15,000 rpm for 10 min, the supernatant and
3. Results 3.1. Analysis of hCA II The substitutions of critical residues can be effective to obtain active and stable hCA II variants for CO2 capture in the harsh conditions [9]. Hence, in this study, a mutant hCA II with thermostability by replacing 142
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increased from 298 K to 328 K. Then the root-mean-square deviation (RMSD) and root-mean-square-fluctuation (RMSF) of WT hCA II at 298 K and 328 K was determined. RSMD results revealed that WT hCAII was not stable at high temperature (Fig. 1c). RMSF, an indicator of flexibility of each residue in protein, showed that residues 18–28 (KDFPIAKGERQ), 62–65 (NGHA), 198–205 (LTTPPLLE), 232–236 (NGEGE), 240–246 (LMVDNW)and 252–254 (KNR) fluctuated more than the others (Fig. 1d), implicating that these liable portions may be responsible for the temperature sensitivity of WT hCA II. Furthermore, to evaluate which residues should be substituted, the position of these residues in WT hCA II was investigated. Only the residues in the regions of 62–65 and 197–205 were considered to be replaced, because compared with the residues of 18–28, 232–236, 240–248 and 252–254, the regions of 62–65 and 197–205 closed to the active site of hCA II (Fig. 1d).
3.2. Design hCA II with improved thermostability Here, the most fluctuated residue of A65 in the region of 62–65 and residues of L203 and L204 in the region of 198–205 were selected as the substitution candidates. In addition, L198, a hydrophobic amino acid like L203 and L204 was also chosen to be investigated. In order to shorten the distance between residue at the position 65 and active site, two amino acids of Lys and Leu both with long side chain were adopted to substitute A65. Leu at the positions of 198, 203 and 204 was considered to be replaced by Lys and Arg to increase protein potential which may lead to improvement in catalytic activity [36]. To evaluate the effects of residue substitution on hCA II, changes in the size of enzyme activity cavity, the distance between H64 and zinc, and the gyration radius were monitored using molecular dynamics simulations. As shown in Fig. 2a, the size of activity cavity in WT was dramatically increased when exposed to a high temperature from 298 K to 328 K. Compared with WT hCA II, the replacement of L204 with R/K and the substitution of A65 with K led to a reduction in the activity cavity size at 328 K. The distance between H64 and zinc after the substitution of L203R, L203 K, L198 K and L198R declined while for L204R, L204 K, A65 K and A65 l the values were similar to WT, suggesting that the replacement at the positions of 203 and 198 by R and K did not cause a negative effect on proton transfer network (Fig. 2b). Furthermore, the gyration radius indicating protein compactness was determined for the mutation and the WT hCA II. As shown in Fig. 2c, the substitution of L204 K and A65 K lowered the gyration radius. The reduction in the activity site size and gyration radius by L204 K was greater than L204R, and the distance between zinc and H64 after L204 K replacement was shorter than A65 K. Taken together, L204 K was selected for further experiments. Ball-and-stick diagrams of L204 K at 298 K and 328 K is shown in Fig. 2d.
3.3. Recombinant hCA II expression and purification The gene of designed hCA II was commercially synthesized. The pET30a–CAII gene was transformed into BL21 (DE3) competent cells. The confirmed clone examined by DNA sequencing was transformed into BL21 (DE3) for protein expression. E.coli without IPTG induced was used as a negative control. Analysis of CA expressed in E. coli using electrophoresis is shown in Fig. 3a. Due to the His-tag added to hCA II, the molecular weight of the CA should be 30.6 kD, confirmed by the band at around 30 kD in electrophoresis before and after purification (Fig. 3a and b). The CA extracted from cells was purified using Ni-NTA resin, as shown in Fig. 3c, a clear band at 30 kD was observed, indicating that the CA was successfully purified. The western blot with anti-His antibody was further applied to identify the purified protein. A positive result with anti-His was exhibited while no band for the negative control was observed (Fig. 3c and d).
Fig. 1. Human CA II analysis. (a) The typical feature of hCA II. (b) The active site of human CA II at 298 K and 328 K. (c) RSMD of human CA II at 298 K and 328 K. (d) RSMF of human CA II at 298 K and 328 K. (e) The location of residues with high flexibility at high temperature in the WT hCA II.
critical residues was developed based on molecular simulations. Before replacing the critical residues in hCA II, We first investigated the thermostability of WT hCA II using dynamic molecular simulations. hCA II has all the typical features of α-CA including the three histidine (H94, 96 and 119), the proton shuttle residue (H64) and the gatekeeping residues of E106 and T199 as shown in Fig. 1a. Temperature effects on the active site, a triangle as labeled in Fig. 1b, were monitored. The active site of hCA II became loose when the temperature 143
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Fig. 4. Kinetic parameter analysis for esterase reaction and hydratase reaction.
3.4. Enzyme activity analysis The purpose of the modification is to improve the thermostability of hCA II but not causing a reduction in enzyme activity. Hence, we first looked into the effect of the replacement of L204 by K on the activity of esterase reaction and hydratase reaction. Here the relative activity was used as an indicator for the comparison between the mutant and WT. The kinetic constants of esterase activity and hydratase activity for the mutant and WT hCA II were determined using p-NPA and CO2 as substrates, respectively (Fig. 4). Using p-NPA as substrate, the value for kcat/Km and Vmax of the mutant hCA II was around 1.7 fold and 16 times greater than the WT, respectively, indicating that the mutant has better esterase catalysis efficiency. For the hydratase reaction, the kinetic constants were derived from monitoring the pH changes during hydration. The turnover rate of WT was 1.44 × 106 s−1, consistent with previous research [9]. The substitution of L204 K led a slight improvement in the turnover rate, 1.98 × 106 s−1, around 37.5 % greater than the WT (Table 1).
Fig. 2. The designed CA analysis. (a) The distance between residue199-131, 199-67 and 131–67 in the WT and mutant CA. (b) The distance between His 64 and Zinc. (c) The turning radius of the WT and mutant. (d) Ball and stick diagram of L204 K active sites. The Zn is shown as a grey sphere. All simulations were repeated at least triplicate.
3.5. Thermostability of the designed hCA II The enzyme activity of the mutant hCA II was investigated at different temperatures. The WT hCA II was used as the comparison. L204 K led to an increase in the thermostability. About 90 % retention of esterase activity at 65 ℃ and 70 % at 95 ℃ after 2 h of incubation were observed (Fig. 5b) while the WT completely lost its activity when exposed to 55 ℃even for 0.5 h (Fig. 5a), consistent with the previous report [26]. Nearly 100 % hydratase activity at 45 ℃ and 50 % at 55 ℃ was retained while the WT completely lost its activity at 45 ℃ (Fig. 5c). 3.6. Responses to chemicals Here we studied the effect of some inorganic anions; NO3−, NO2−, SO42− and SO32− at the concentrations ranging from 0.167 mM to 3.33 mM on the enzyme activity at pH 7.4. The effect of anions on the esterase activity was different from the hydratase reaction activity. Like the WT, the presence of inorganic anions did not inhibit the esterase activity at the tested conditions (Fig. 6a). As an indicator for the capacity of capturing CO2, we further exploited the hydratase reaction activity in the presence of NO3−, NO2−, SO42− and SO32− and EDTA.
Fig. 3. Electrophoresis and Western Blot analysis of expressed designed CA. (a) Electrophoresis of expressed protein before purification. (b) Electrophoresis of expressed protein after purification. (c) Western Blot of expressed mutant before purification. (d) Western Blot of expressed mutant CA after purification. Columns 1–6 represent the precipitant of recombinant protein expressed with IPTG collected after cell disruption, the supernatant of recombinant protein expressed with IPTG collected from centrifuge after cell disruption, the precipitant without IPTG, the supernatant without IPTG induction, the precipitant with IPTG before cell disruption, the supernatant with IPTG induction before cell disruption.
Table 1 Comparison of the CO2 hydratase activities of mutant and WT hCAⅡ.
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Enzyme
kcat (s−1)
Km(mM)
kcat/Km(M−1s−1)
WT hCAⅡ mutant hCAⅡ
1.44 × 106 1.98 × 106
5.45 8.38
2.63 × 108 2.36 × 108
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capture. However, its activity is quite sensitive to temperature, especially for the hydration activity which inactivates at 45 °C [37]. This severely limits its applications in the field of CO2 capture from gas streams [23]. Hence, in this study, a mutant hCA II was designed based on molecular simulations to improve the thermostability of hCA II by the substitution of critical amino acids. The rational design of thermostable proteins is a challenge because the elements with regard to stability are various in different proteins. In previous reports, the thermostability of enzymes was enhanced by the strategies such as increasing surface loop rigidity [38] and surface compactness [39,40], decreasing surface hydrophobicity [25] and introducing conserved disulfide bridge [28]. Here, to improve the thermostability of hCA II without affecting its hydratase reaction activity, three parameters including the size of enzyme activity cavity, the distance between H64 and zinc and the gyration radius were used as criteria for assessing the stability and activity of hCA II before and after modification. The fluctuation of RMSD confirmed that the WT hCA II was not stable at high temperature (Fig. 1c), consistent with the previous report [37], in which WT hCA II exhibited inactivation when exposed to the temperature higher than 45 °C. The fluctuation of RSMF in the regions especially at the residues of 62–65 and 197–205 closed to the active site may lead to conformation changes of hCA II at 328 K, further causing changes in enzyme activity. Therefore, the residues in these regions were considered to be replaced. In comparison with the WT at 328 K, the substitution of L204 by K led to a decrease in the size of the activity cavity, suggesting that L204 K was more rigid than the WT, probably further resulting in the improvement in stability. The reduction in the distance between H64 and zinc after the modification implicated that the replacement of L204 by K might have positive effects on proton transfer network, which was further confirmed by the experiments (Table 1). L204 K led to a change in electrostatic potential around the active site, which may be a reason for an alternation of proton transfer rate and enzymatic activity [36]. Due toless gyration radius than L204R, L204 K exhibited more compactness, resulting in more stability of enzyme. Hence, L204 K was chosen for the further experiments. An enzyme with comparable activity to WT hCA II but with higher thermostability is beneficial for capturing CO2 from flue gas. In the previous research, the cysteine substitution resulted in 14 ℃ improvement in the melting temperature but causing a reduction in the activity of hCA II [28]. The replacement of three surface leucine residues led to similar activity but with a minor melting temperature increase by 7 ℃ [26]. Hence, in order to enhance the thermostability of hCA II, we proposed to replace L204 with K according to the simulations results. Experiment results exhibited that L204 K led to an increase in the thermostability with 50 % retention at 55 ℃ while the WT completely lost its activity when exposed to 45 ℃, consistent with the previous report [25]. L204 K had better catalysis efficiency in the hydratase reaction than WT hCA II and SspCA although less than SazCA [30]. This improvement in the catalysis efficiency may be caused by the changes in the distance between H64 and zinc, and water structure network, which further promoting subsequent proton transfer [25]. Importantly, the substitution not only enhanced the thermostablity of hCA II but also slightly improved the enzyme activity, associated with surface electrostatic potential [41]. To further improve the temperature resistance, enzyme immobilization can be further carried out [42,43]. A wide range of chemical species constitutes flue gas. The effect of some inorganic anions; NO3−, NO2−, SO42− and SO32− on the enzyme activity at pH 7.4 was evaluated. The hydratase reaction activity of the mutant was not affected by the presence of NO3−, NO2− and SO42− but inhibited by the presence of SO32-. However the presence of SO32- exhibited much less inhibition on L204 K than on SazCA [30]. The scarce inhibition by the anions on L204 K implicated that the anions are not directly bound to the active site or block the entrance of the cavity [44]. We further detected the resistance of mutant hCA II in the presence of EDTA. Although EDTA is not present in flue gas, the resistance to EDTA
Fig. 5. Thermostability of the WT and designed CA. (a) The WT thermostability of esterase activity after exposure to different temperatures for 0.5, 1 and 2 h. (b) The mutant thermostability of esterase activity after exposure to different temperatures for 0.5, 1 and 2 h. (c) The thermostability of hydratase reaction activity for the mutant and WT after exposure to 37, 45 and 55 °C for 2 h. Data represent mean values with error bar denoting the standard deviation of the means.
Fig. 6. Esterase and hydratase reaction activity of hCA II in the presence of various anions. (a) Esterase reaction activity of the WT and mutant in the presence of various anions at the concentrations ranging from 0.167 mM to 3.333 mM at room temperature. (b) Hydratase reaction activity of the mutant hCA II in the presence of various anions.
The hydratase reaction activity of the mutant was scarcely suppressed by the presence of NO3−, NO2− and SO42−, but slightly inhibited by SO32− and EDTA (Fig. 6b). 4. Discussion Due to the turnover rate much faster than the other types of CAs, hCA II has attracted more and more attention in the field of CO2 145
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is a critical property for hCA II [20]. The experimental results revealed that the replacement of L204 K displays a positive effect on withstanding the inhibition of EDTA, much better than CA from Bacillus subtilis [20]. In summary, a novel hCAII was developed based on molecular simulations. It has comparable hydratase activity as the WT hCA, 1.98 × 106 s−1 for kcat and 2.36 × 108 M−1s−1 for kcat/Km, but more importantly, it has higher thermostability with 100 % retention at 45 °C and around 50 % at 55 °C while WT totally inactivates at 45 °C. Furthermore, L204 K is able to maintain its activity in the presence of anions and EDTA. The robustness and efficiency of this novel CA make it a competitive candidate for CO2 capture.
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Alterio, et al., Crystal structure of the most catalytically effectivecarbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense, Bioorg. Med. Chem. Lett. 25 (2015) 2002–2006, https://doi.org/10.1016/j.bmcl.2015.02.068. [32] A.W. Schüttelkopf, D.M.F. Van Aalten, PRODRG: a tool for high-throughput crystallography of protein–ligand complexes, Acta Crystallogr. D 60 (2004) 1355–1363, https://doi.org/10.1107/S0907444904011679. [33] M. Boztas, P. Taslimi, M.A. Yavari, et al., Synthesis and biological evaluation of bromophenol derivatives with cyclopropyl moiety: ring opening of cyclopropane with monoester, Bioorg. Chem. 89 (2019) 103017, , https://doi.org/10.1016/j. bioorg.2019.103017. [34] U. Atmaca, R. Kaya, H.S. Karaman, et al., Synthesis of oxazolidinone from enantiomerically enriched allylic alcohols and determination of their molecular docking and biologic activities, Bioorg. 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CRediT authorship contribution statement Shenglan Wu: Validation, Investigation. Jinrui Chen: Investigation. Liang Ma: Investigation. Kai Zhang: Resources. Xiaoxiao Wang: Resources. Yuping Wei: Conceptualization. Jian Xu: Writing - review & editing. Xia Xu: Conceptualization, Supervision, Writing - original draft, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (91534107, 21576266 and 21978001), Wanjiang Scholar Program and Start Fund for Biochemical Engineering Research Center from Anhui University of Technology. References [1] D.Y.C. Leung, G. Caramanna, M.M. Maroto-Valer, An overview of current status of carbon dioxide capture and storage technologies, Renew. Sustain. Energy Rev. 39 (2014) 426–443, https://doi.org/10.1016/j.rser.2014.07.093. [2] A. Samanta, A. Zhao, G.K.H. Shimizu, et al., Post-combustion CO2 capture using solid sorbents: a review, Ind. Eng. Chem. Res. 51 (2011) 1438–1463, https://doi. org/10.1021/ie200686q. [3] A. Brunetti, F. Scura, G. Barbieri, E. Drioli, Membrane technologies for CO2 separation, J. Membr. Sci. 359 (2010) 115–125, https://doi.org/10.1016/j.memsci. 2009.11.040. [4] C.G. Xu, Z.Y. Chen, J. Cai, X.S. Li, Study on pilot-scale CO2 separation from flue gas by the hydrate method, Energy Fuel 28 (2013) 1242–1248, https://doi.org/10. 1021/ef401883v. [5] D. Singh, E. Croiset, P.L. Douglas, M.A. Douglas, Techno-economic study of CO2 capture from an existing coal-fired power plant: MEA scrubbing vs. O2/CO2 recycle combustion, Energy Convers. Manage. 44 (2003) 3073–3091, https://doi.org/10. 1016/S0196-8904(03)00040-2. [6] B. Liu, X. Luo, Z.W. Liang, et al., The development of kinetics model for CO2 absorption into tertiary amines containing carbonic anhydrase, AIChE J. 63 (2017) 4933–4943, https://doi.org/10.1002/aic.15833. [7] I.M. Power, A.L. Harrison, G.M. Dipple, Accelerating mineral carbonation using carbonic anhydrase, Environ. Sci. Technol. 50 (2016) 2610–2618, https://doi.org/ 10.1021/acs.est.5b04779. [8] S.H. Zhang, Y.Q. Lu, Surfactants facilitating carbonic anhydrase enzyme-mediated CO2 absorption into a carbonate solution, Environ. Sci. Technol. 51 (2017) 8537–8543, https://doi.org/10.1021/acs.est.7b00711. [9] A. Di Fiore, V. Alterio, S.M. Monti, et al., Thermostable carbonic anhydrases in biotechnological applications, Int. J. Mol. Sci. 16 (2015) 15456–15480, https://doi. org/10.3390/ijms160715456. [10] A. ElMekawy, H.M. Hegab, G. Mohanakrishna, et al., Technological advances in CO2 conversion electro-biorefinery: a step toward commercialization, Bioresour. Technol. Rep. 215 (2016) 357–370, https://doi.org/10.1016/j.biortech.2016.03. 023. [11] E.T. Hwang, H.M. Gang, J.Y. Chung, et al., Carbonic anhydrase assisted calcium carbonate crystalline composites as a biocatalyst, Green Chem. 14 (2012) 2216–2220, https://doi.org/10.1039/c2gc35444f. [12] A.B. Fulke, S.N. Mudliar, R. Yadav, et al., Bio-mitigation of CO2, calcite formation and simultaneous biodiesel precursors production using Chlorella sp, Bioresour. Technol. Rep. 101 (2010) 8473–8476, https://doi.org/10.1016/j.biortech.2010.06. 012.
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[38] K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, Y. Suzuki, Multiple proline substitutions cumulatively thermostabilize Bacillus Cereus atcc7064 Oligo-1,6Glucosidase : irrefragable proof supporting the proline rule, FEBS J. 226 (2008) 277–283, https://doi.org/10.1111/j.1432-1033.1994.tb20051.x. [39] C. Vieille, G.J. Zeikus, Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability, Microbiol. Mol. Biol. Rev. 65 (2001) 1–43, https:// doi.org/10.1128/MMBR.65.1.1-43.2001. [40] B. Villbrandt, G. Sagner, D. Schomburg, Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments, Protein Eng. Des. Sel. 10 (1997) 1281–1288, https://doi.org/10.1093/protein/10.11.1281. [41] S.S. Samantha, V.G. Alexey, V.G. Alexander, et al., Protein stability and surface electrostatics: a charged relationship, Biochemistry 45 (2006) 2761–2766, https://
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Contents lists available at ScienceDirect
Journal of CO2 Utilization journal homepage: www.elsevier.com/locate/jcou
Design of carbonic anhydrase with improved thermostability for CO2 capture via molecular simulations
T
Shenglan Wua,b, Jinrui Chena, Liang Mab, Kai Zhanga, Xiaoxiao Wanga,b, Yuping Weic, Jian Xua,b, Xia Xua,b,* a
Biochemical Engineering Research Center, Anhui University of Technology, Ma’anshan, Anhui, 243002, PR China School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma’anshan, Anhui, 243002, PR China c School of Life Science and Technology, Nanyang Normal University, Nanyang, Henan Province, 473061, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carbonic anhydrase Thermostability Molecular simulations Enzyme activity
Chemical absorption of CO2 is the most widely used method to capture CO2. However, high cost and slow CO2 absorption rate severely hinder its commercial utilization. Human carbonic anhydrase II (hCA II) having rapid hydratase reaction rate can potentially overcome these obstacles but being highly unstable against harsh process conditions. To enhance thermostability, a novel CA was developed based on molecular simulations. Substituting residue Leu204 with Lys has a comparable hydratase activity as the wild type (WT) hCA II, 1.98 × 106 s−1 for kcat and 2.36 × 108 M−1s−1 for kcat/Km. Importantly, the replacement of L204 K results in higher temperature resistance with 100 % retention at 45 °C and around 50 % at 55 °C while WT totally inactivates at 45 °C. Furthermore, L204 K is able to maintain its activity in the presence of anions and EDTA. The robustness and efficiency of this novel CA make it a competitive candidate for CO2 capture.
1. Introduction Carbon dioxide (CO2) mainly from the combustion of fossil fuels is primary greenhouse gas associated with global warming and climate change. Many attempts have been made to reduce the amount of CO2 emitted into atmosphere such as developing renewable energy sources, using low carbon fuels, CO2 capture and storage, and conversion of CO2 into fuels and chemicals [1]. In recent years, CO2 capture (CC) has become an extensively investigated area of research. There are a variety of approaches to capture CO2 including physical adsorption [2], membrane separation [3], hydrate formation [4], chemical adsorption, and monoethanolamine (MEA) absorption [5]. However, these CC options face certain technical and economic challenges [5]. Hence, alternatively, there is an enormous interest in using carbonic anhydrases (CAs) as biocatalysts for carbon sequestration [6–8] and production of valuable products [9–12]. Carbonic anhydrase, a zinc metalloenzyme, can catalyze CO2 hydration into bicarbonate (HCO3−) and also exhibits esterase activity [13–17]. γ-class CA from Methanosarcina thermophile (CAM) has the optimal activity for hydratase reaction at 55 °C with turnover rate of 105/s and inactivation at 75 °C [18]. CAs from Methanobacterium thermoautotrophicum (CAB) and Caminibacter mediatlanticus DSM 16658 are
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able to retain more than 40 % of their original hydratase activity at 85 °C but with low activity at 1.7 × 104 s−1 [19]. The enzymes from Bacillus subtilis and Citrobacter freundii are stable over a pH range of 7.0–11.0 and at temperature ≤ 60 °C but not given hydratase activity [20]. Human CA II with high turnover rate of 1.4 × 106 s−1 [21,22] has been identified as a potential biocatalyst for accelerating CO2 absorption from gas streams [23]. However, its application in this field has been limited due to poor stability and activity in harsh conditions such as high temperature, high concentration of organic amine and trace contaminants [24,25]. To overcome these problems, much effort has been made to develop an enzyme with high thermostability. To enhance thermostability of CA, disulfide bridges, ionic pair networks, proline substitutions and surface loop reduction have been proposed. 7 °C increase in hCA II temperature resistance was achieved by substituting three surface leucine residues (Leu100His, Leu224Ser and Leu240Pro) [26]. Further substitutions with Tyr7Phe and Asn67Gln improved proton transfer rate constants but not thermostability [27]. Replacing ALA23 and Leu203 with Cys also enhanced the melting temperature (Tm) of hCA II by 14 °C through a disulfide bridge at pH 7.8 but with a reduction in the rate constant for proton transfer [28]. Substituting Glu234 with Pro increased the rigidity of surface loops, only leading to 3 °C increase in Tm [28]. The melting temperature
Corresponding author at: Biochemical Engineering Research Center, Anhui University of Technology, No. 59 Hudong Road, Ma’anshan, Anhui, 243002, PR China. E-mail address: [email protected] (X. Xu).
https://doi.org/10.1016/j.jcou.2020.01.017 Received 20 December 2019; Received in revised form 13 January 2020; Accepted 15 January 2020 2212-9820/ © 2020 Elsevier Ltd. All rights reserved.
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cell debris were collected. The supernatant was loaded into Ni-nitrolotriacetate affinity chromatographic column (GE, USA). The column was first washed with 10 resin volumes of washing buffer (50 mM Tris, 150 mM NaCl, 50 mM imidazole at pH 7.5), and was then eluted with elution buffer (50 mM Tris, 150 mM NaCl, 50 mM imidazole, 250 mM imidazole at pH 7.5). The eluted fractions containing the target protein were concentrated using a centrifuge tube with a 3 KD cut-off membrane. Protein concentration was determined using a Bradford assay with bovine serum albumin (Sigma, USA) as a standard.
was slightly enhanced by improving compactness through deleting residues of 230–240 [28]. A directed evolution technology has been applied to dramatically enhance the properties of a β-class CA from Desulfovibrio vulgaris using high-throughput screening. The developed CA absorbed CO2 in the presence of 4.2 M alkaline amine at 87 °C, but with a lower activity than hCA II [29]. In recent years, four highly thermostable α-CAs, SspCA from Sulfurihydrogenibium yellowstonense YO3AOP1, SazCA from S ulphurihydrogenibium azorense, Thermovibrio ammonificans (TaCA) and Persephonella marina EX-H1 (PmCA), have been studied extensively. SazCA was demonstrated to have highest catalytic activity for the hydratase reaction with kcat of 4.4 × 106 s−1; kcat/Km of 3.5 × 108 M−1·s−1 at 20 °C and at pH 7.5 [30], more than twice greater than hCA II. The highly thermostable activity, which can retain CO2 hydration activity after 3 h incubation even 100 °C, may be caused by the substitution of Glu2 by His2 and Gln207 by His207 [31]. However, SazCA is quite sensitive to anions such as halide, hydrogen sulfate and sulfate anions, which can severely inhibit its CO2 hydration activity. The aim of this paper was to design a thermostable CA based on human CA II using molecular simulations. Structure stability of mutant hCA II was analyzed using dynamics molecular simulations. Kinetic characterization of mutant hCA II was determined. Esterase and hydratase activity were evaluated at different temperatures and different time intervals. Ability of chemical resistance to anions was also investigated.
2.3. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot analysis The tagged protein was determined using a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) [33,34]. Briefly, 20 μl sample with 5 μl loading buffer was boiled for 10 min and then added into electrophoresis wells. The expressed proteins were separated on the SDS-PAGE, transferred onto polyvinylidene fluoride membranes (Milipore, USA), and blocked with 5 % dried skimmed milk at room temperature. The membranes were incubated overnight with anti-His antibody (Santa Cruz, USA) at 4 °C, washed with TBST, and then incubated with horseradish peroxidase-conjugated IgG (Santa Cruz, USA) for 1 h at room temperature. After washing, bands were visualized using a chemiluminescence (ECL, Thermofisher, USA). 2.4. Activity assay
2. Materials and methods p-nitrophenol acetate (p-NPA) hydrolysis assay was employed to measures enzyme’s activity by monitoring the production of p-nitrophenol (p-NP) from p-NPA hydrolysis [35]. 50 μL of enzyme sample was added to a solution containing 50 μL of Na2HPO–NaH2PO4 buffer solution (0.1 M, pH 7.0) and 50 μL of p-NPA (3 mM) (Alading, China). The esterase activity was determined by measuring p-NP concentration in the hydrolysis solution at 25 °C for 6 min at 348 nm using a Microplate reader (Synergy, Biotek, USA). CO2 hydration activity assay was performed at room temperature as described by Kim et al. [22]. Briefly, to initiate the hydration reaction, CO2-saturated water was added to a mixture containing phosphate buffer saline (PBS, pH 8.35) and a 0.5 μg/ml enzyme with CO2 concentration ranging from 2 to 7 mM. An in-line pH measurement was adopted using a pH electrode connecting to a pH meter (pH 3 Leici, China). The hydratase reaction activity was determined by monitoring the time during the pH change, recorded by a computer. All the reactions for wild type hCA II and mutant hCA II were performed at least triplicate.
2.1. Molecular simulations Coordinate of hCA II was obtained from the RCSB protein data bank (PDB). A topology file of wild type hCA II was generated using the PRODRG2 server [32]. To investigate the stability of mutant hCA II, MD simulation was performed at 25 °C and 1 bar pressure for 10 ns using GROMACS 4.5.5 simulation package. During simulations, a Gromos96 G43a1 force field combined with simple point charge water model was adopted, and the default values for other settings were applied. Before the simulations, all of the bound water was removed. Root mean square deviation (RMSD) of the structures in the trajectory compared to the starting conformation was used to evaluate system stability. Rootmean-square-fluctuation (RMSF) is a measure of the flexibility of a residue, calculated by the square root of the variance of the fluctuation around the average position for the Cα atom of each residue. Simulation results were visualized using Visual Molecular Dynamics (VMD). All simulations were performed on an ASUS workstation (SAMSUNG, Korea).
2.5. Stability at different conditions
2.2. Recombinant protein expression and purification
The stability of mutant hCA II at different temperatures and in the presence of different chemical impurities was evaluated. To investigate the temperature effect on esterase activity and hydratase activity, the enzyme was kept at the temperature ranging from 37 to 95 °C. The thermostability of enzyme was monitored after 0.5, 1 and 2 h of incubation for at different temperatures. The WT hCA II was used as a control. To further look into the chemical stability of enzymes, the enzyme was incubated in the same buffer solution containing anions, SO42−, NO3−, SO32−, NO2- and EDTA-2Na. The activity of mutant hCA II samples stored in the same buffer solution without the presence of any impurities was used for comparison.
The complete gene sequences of wild type and mutant hCA II were designed, synthesized containing NdeI and HindIII restriction sites (GeneScript, USA), and then subcloned into a target vector pET-30a (+) containing a C-terminal and N-terminal hexahistidine (His) tag for E.coli expression. The constructed vector was transformed in BL21 (DE3) (Novagen, Merck, Shanghai, China) and incubated at ∼ 200 rpm for 60 min at 37 °C. After spreading on LB agar plate containing 50 μg/ml kanamycin (Sigma, USA), the plate was incubated upside down at 37 °C. The next day, single and well-isolated colonies were inoculated into 4 ml LB medium containing 50 g/ml kanamycin and were grown at 37 °C and 200 rpm until OD600 value was reached to 0.6-0.8. 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma, USA) was introduced and then the cells were cultured at 37 °C for 4 h. The cells were harvested using a centrifuge at 4000 rpm, re-suspended in lysis Buffer (50 mM Tris, 150 mM NaCl, 5 % glycerol pH 8.0) and disrupted using a sonicator (Xinzhi Biotech, Ningbo, China) for 10 min. The cell lysate was centrifuged at 15,000 rpm for 10 min, the supernatant and
3. Results 3.1. Analysis of hCA II The substitutions of critical residues can be effective to obtain active and stable hCA II variants for CO2 capture in the harsh conditions [9]. Hence, in this study, a mutant hCA II with thermostability by replacing 142
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increased from 298 K to 328 K. Then the root-mean-square deviation (RMSD) and root-mean-square-fluctuation (RMSF) of WT hCA II at 298 K and 328 K was determined. RSMD results revealed that WT hCAII was not stable at high temperature (Fig. 1c). RMSF, an indicator of flexibility of each residue in protein, showed that residues 18–28 (KDFPIAKGERQ), 62–65 (NGHA), 198–205 (LTTPPLLE), 232–236 (NGEGE), 240–246 (LMVDNW)and 252–254 (KNR) fluctuated more than the others (Fig. 1d), implicating that these liable portions may be responsible for the temperature sensitivity of WT hCA II. Furthermore, to evaluate which residues should be substituted, the position of these residues in WT hCA II was investigated. Only the residues in the regions of 62–65 and 197–205 were considered to be replaced, because compared with the residues of 18–28, 232–236, 240–248 and 252–254, the regions of 62–65 and 197–205 closed to the active site of hCA II (Fig. 1d).
3.2. Design hCA II with improved thermostability Here, the most fluctuated residue of A65 in the region of 62–65 and residues of L203 and L204 in the region of 198–205 were selected as the substitution candidates. In addition, L198, a hydrophobic amino acid like L203 and L204 was also chosen to be investigated. In order to shorten the distance between residue at the position 65 and active site, two amino acids of Lys and Leu both with long side chain were adopted to substitute A65. Leu at the positions of 198, 203 and 204 was considered to be replaced by Lys and Arg to increase protein potential which may lead to improvement in catalytic activity [36]. To evaluate the effects of residue substitution on hCA II, changes in the size of enzyme activity cavity, the distance between H64 and zinc, and the gyration radius were monitored using molecular dynamics simulations. As shown in Fig. 2a, the size of activity cavity in WT was dramatically increased when exposed to a high temperature from 298 K to 328 K. Compared with WT hCA II, the replacement of L204 with R/K and the substitution of A65 with K led to a reduction in the activity cavity size at 328 K. The distance between H64 and zinc after the substitution of L203R, L203 K, L198 K and L198R declined while for L204R, L204 K, A65 K and A65 l the values were similar to WT, suggesting that the replacement at the positions of 203 and 198 by R and K did not cause a negative effect on proton transfer network (Fig. 2b). Furthermore, the gyration radius indicating protein compactness was determined for the mutation and the WT hCA II. As shown in Fig. 2c, the substitution of L204 K and A65 K lowered the gyration radius. The reduction in the activity site size and gyration radius by L204 K was greater than L204R, and the distance between zinc and H64 after L204 K replacement was shorter than A65 K. Taken together, L204 K was selected for further experiments. Ball-and-stick diagrams of L204 K at 298 K and 328 K is shown in Fig. 2d.
3.3. Recombinant hCA II expression and purification The gene of designed hCA II was commercially synthesized. The pET30a–CAII gene was transformed into BL21 (DE3) competent cells. The confirmed clone examined by DNA sequencing was transformed into BL21 (DE3) for protein expression. E.coli without IPTG induced was used as a negative control. Analysis of CA expressed in E. coli using electrophoresis is shown in Fig. 3a. Due to the His-tag added to hCA II, the molecular weight of the CA should be 30.6 kD, confirmed by the band at around 30 kD in electrophoresis before and after purification (Fig. 3a and b). The CA extracted from cells was purified using Ni-NTA resin, as shown in Fig. 3c, a clear band at 30 kD was observed, indicating that the CA was successfully purified. The western blot with anti-His antibody was further applied to identify the purified protein. A positive result with anti-His was exhibited while no band for the negative control was observed (Fig. 3c and d).
Fig. 1. Human CA II analysis. (a) The typical feature of hCA II. (b) The active site of human CA II at 298 K and 328 K. (c) RSMD of human CA II at 298 K and 328 K. (d) RSMF of human CA II at 298 K and 328 K. (e) The location of residues with high flexibility at high temperature in the WT hCA II.
critical residues was developed based on molecular simulations. Before replacing the critical residues in hCA II, We first investigated the thermostability of WT hCA II using dynamic molecular simulations. hCA II has all the typical features of α-CA including the three histidine (H94, 96 and 119), the proton shuttle residue (H64) and the gatekeeping residues of E106 and T199 as shown in Fig. 1a. Temperature effects on the active site, a triangle as labeled in Fig. 1b, were monitored. The active site of hCA II became loose when the temperature 143
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Fig. 4. Kinetic parameter analysis for esterase reaction and hydratase reaction.
3.4. Enzyme activity analysis The purpose of the modification is to improve the thermostability of hCA II but not causing a reduction in enzyme activity. Hence, we first looked into the effect of the replacement of L204 by K on the activity of esterase reaction and hydratase reaction. Here the relative activity was used as an indicator for the comparison between the mutant and WT. The kinetic constants of esterase activity and hydratase activity for the mutant and WT hCA II were determined using p-NPA and CO2 as substrates, respectively (Fig. 4). Using p-NPA as substrate, the value for kcat/Km and Vmax of the mutant hCA II was around 1.7 fold and 16 times greater than the WT, respectively, indicating that the mutant has better esterase catalysis efficiency. For the hydratase reaction, the kinetic constants were derived from monitoring the pH changes during hydration. The turnover rate of WT was 1.44 × 106 s−1, consistent with previous research [9]. The substitution of L204 K led a slight improvement in the turnover rate, 1.98 × 106 s−1, around 37.5 % greater than the WT (Table 1).
Fig. 2. The designed CA analysis. (a) The distance between residue199-131, 199-67 and 131–67 in the WT and mutant CA. (b) The distance between His 64 and Zinc. (c) The turning radius of the WT and mutant. (d) Ball and stick diagram of L204 K active sites. The Zn is shown as a grey sphere. All simulations were repeated at least triplicate.
3.5. Thermostability of the designed hCA II The enzyme activity of the mutant hCA II was investigated at different temperatures. The WT hCA II was used as the comparison. L204 K led to an increase in the thermostability. About 90 % retention of esterase activity at 65 ℃ and 70 % at 95 ℃ after 2 h of incubation were observed (Fig. 5b) while the WT completely lost its activity when exposed to 55 ℃even for 0.5 h (Fig. 5a), consistent with the previous report [26]. Nearly 100 % hydratase activity at 45 ℃ and 50 % at 55 ℃ was retained while the WT completely lost its activity at 45 ℃ (Fig. 5c). 3.6. Responses to chemicals Here we studied the effect of some inorganic anions; NO3−, NO2−, SO42− and SO32− at the concentrations ranging from 0.167 mM to 3.33 mM on the enzyme activity at pH 7.4. The effect of anions on the esterase activity was different from the hydratase reaction activity. Like the WT, the presence of inorganic anions did not inhibit the esterase activity at the tested conditions (Fig. 6a). As an indicator for the capacity of capturing CO2, we further exploited the hydratase reaction activity in the presence of NO3−, NO2−, SO42− and SO32− and EDTA.
Fig. 3. Electrophoresis and Western Blot analysis of expressed designed CA. (a) Electrophoresis of expressed protein before purification. (b) Electrophoresis of expressed protein after purification. (c) Western Blot of expressed mutant before purification. (d) Western Blot of expressed mutant CA after purification. Columns 1–6 represent the precipitant of recombinant protein expressed with IPTG collected after cell disruption, the supernatant of recombinant protein expressed with IPTG collected from centrifuge after cell disruption, the precipitant without IPTG, the supernatant without IPTG induction, the precipitant with IPTG before cell disruption, the supernatant with IPTG induction before cell disruption.
Table 1 Comparison of the CO2 hydratase activities of mutant and WT hCAⅡ.
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Enzyme
kcat (s−1)
Km(mM)
kcat/Km(M−1s−1)
WT hCAⅡ mutant hCAⅡ
1.44 × 106 1.98 × 106
5.45 8.38
2.63 × 108 2.36 × 108
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capture. However, its activity is quite sensitive to temperature, especially for the hydration activity which inactivates at 45 °C [37]. This severely limits its applications in the field of CO2 capture from gas streams [23]. Hence, in this study, a mutant hCA II was designed based on molecular simulations to improve the thermostability of hCA II by the substitution of critical amino acids. The rational design of thermostable proteins is a challenge because the elements with regard to stability are various in different proteins. In previous reports, the thermostability of enzymes was enhanced by the strategies such as increasing surface loop rigidity [38] and surface compactness [39,40], decreasing surface hydrophobicity [25] and introducing conserved disulfide bridge [28]. Here, to improve the thermostability of hCA II without affecting its hydratase reaction activity, three parameters including the size of enzyme activity cavity, the distance between H64 and zinc and the gyration radius were used as criteria for assessing the stability and activity of hCA II before and after modification. The fluctuation of RMSD confirmed that the WT hCA II was not stable at high temperature (Fig. 1c), consistent with the previous report [37], in which WT hCA II exhibited inactivation when exposed to the temperature higher than 45 °C. The fluctuation of RSMF in the regions especially at the residues of 62–65 and 197–205 closed to the active site may lead to conformation changes of hCA II at 328 K, further causing changes in enzyme activity. Therefore, the residues in these regions were considered to be replaced. In comparison with the WT at 328 K, the substitution of L204 by K led to a decrease in the size of the activity cavity, suggesting that L204 K was more rigid than the WT, probably further resulting in the improvement in stability. The reduction in the distance between H64 and zinc after the modification implicated that the replacement of L204 by K might have positive effects on proton transfer network, which was further confirmed by the experiments (Table 1). L204 K led to a change in electrostatic potential around the active site, which may be a reason for an alternation of proton transfer rate and enzymatic activity [36]. Due toless gyration radius than L204R, L204 K exhibited more compactness, resulting in more stability of enzyme. Hence, L204 K was chosen for the further experiments. An enzyme with comparable activity to WT hCA II but with higher thermostability is beneficial for capturing CO2 from flue gas. In the previous research, the cysteine substitution resulted in 14 ℃ improvement in the melting temperature but causing a reduction in the activity of hCA II [28]. The replacement of three surface leucine residues led to similar activity but with a minor melting temperature increase by 7 ℃ [26]. Hence, in order to enhance the thermostability of hCA II, we proposed to replace L204 with K according to the simulations results. Experiment results exhibited that L204 K led to an increase in the thermostability with 50 % retention at 55 ℃ while the WT completely lost its activity when exposed to 45 ℃, consistent with the previous report [25]. L204 K had better catalysis efficiency in the hydratase reaction than WT hCA II and SspCA although less than SazCA [30]. This improvement in the catalysis efficiency may be caused by the changes in the distance between H64 and zinc, and water structure network, which further promoting subsequent proton transfer [25]. Importantly, the substitution not only enhanced the thermostablity of hCA II but also slightly improved the enzyme activity, associated with surface electrostatic potential [41]. To further improve the temperature resistance, enzyme immobilization can be further carried out [42,43]. A wide range of chemical species constitutes flue gas. The effect of some inorganic anions; NO3−, NO2−, SO42− and SO32− on the enzyme activity at pH 7.4 was evaluated. The hydratase reaction activity of the mutant was not affected by the presence of NO3−, NO2− and SO42− but inhibited by the presence of SO32-. However the presence of SO32- exhibited much less inhibition on L204 K than on SazCA [30]. The scarce inhibition by the anions on L204 K implicated that the anions are not directly bound to the active site or block the entrance of the cavity [44]. We further detected the resistance of mutant hCA II in the presence of EDTA. Although EDTA is not present in flue gas, the resistance to EDTA
Fig. 5. Thermostability of the WT and designed CA. (a) The WT thermostability of esterase activity after exposure to different temperatures for 0.5, 1 and 2 h. (b) The mutant thermostability of esterase activity after exposure to different temperatures for 0.5, 1 and 2 h. (c) The thermostability of hydratase reaction activity for the mutant and WT after exposure to 37, 45 and 55 °C for 2 h. Data represent mean values with error bar denoting the standard deviation of the means.
Fig. 6. Esterase and hydratase reaction activity of hCA II in the presence of various anions. (a) Esterase reaction activity of the WT and mutant in the presence of various anions at the concentrations ranging from 0.167 mM to 3.333 mM at room temperature. (b) Hydratase reaction activity of the mutant hCA II in the presence of various anions.
The hydratase reaction activity of the mutant was scarcely suppressed by the presence of NO3−, NO2− and SO42−, but slightly inhibited by SO32− and EDTA (Fig. 6b). 4. Discussion Due to the turnover rate much faster than the other types of CAs, hCA II has attracted more and more attention in the field of CO2 145
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is a critical property for hCA II [20]. The experimental results revealed that the replacement of L204 K displays a positive effect on withstanding the inhibition of EDTA, much better than CA from Bacillus subtilis [20]. In summary, a novel hCAII was developed based on molecular simulations. It has comparable hydratase activity as the WT hCA, 1.98 × 106 s−1 for kcat and 2.36 × 108 M−1s−1 for kcat/Km, but more importantly, it has higher thermostability with 100 % retention at 45 °C and around 50 % at 55 °C while WT totally inactivates at 45 °C. Furthermore, L204 K is able to maintain its activity in the presence of anions and EDTA. The robustness and efficiency of this novel CA make it a competitive candidate for CO2 capture.
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CRediT authorship contribution statement Shenglan Wu: Validation, Investigation. Jinrui Chen: Investigation. Liang Ma: Investigation. Kai Zhang: Resources. Xiaoxiao Wang: Resources. Yuping Wei: Conceptualization. Jian Xu: Writing - review & editing. Xia Xu: Conceptualization, Supervision, Writing - original draft, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (91534107, 21576266 and 21978001), Wanjiang Scholar Program and Start Fund for Biochemical Engineering Research Center from Anhui University of Technology. References [1] D.Y.C. Leung, G. Caramanna, M.M. Maroto-Valer, An overview of current status of carbon dioxide capture and storage technologies, Renew. Sustain. Energy Rev. 39 (2014) 426–443, https://doi.org/10.1016/j.rser.2014.07.093. [2] A. Samanta, A. Zhao, G.K.H. Shimizu, et al., Post-combustion CO2 capture using solid sorbents: a review, Ind. Eng. Chem. Res. 51 (2011) 1438–1463, https://doi. org/10.1021/ie200686q. [3] A. Brunetti, F. Scura, G. Barbieri, E. Drioli, Membrane technologies for CO2 separation, J. Membr. Sci. 359 (2010) 115–125, https://doi.org/10.1016/j.memsci. 2009.11.040. [4] C.G. Xu, Z.Y. Chen, J. Cai, X.S. Li, Study on pilot-scale CO2 separation from flue gas by the hydrate method, Energy Fuel 28 (2013) 1242–1248, https://doi.org/10. 1021/ef401883v. [5] D. Singh, E. Croiset, P.L. Douglas, M.A. Douglas, Techno-economic study of CO2 capture from an existing coal-fired power plant: MEA scrubbing vs. O2/CO2 recycle combustion, Energy Convers. Manage. 44 (2003) 3073–3091, https://doi.org/10. 1016/S0196-8904(03)00040-2. [6] B. Liu, X. Luo, Z.W. Liang, et al., The development of kinetics model for CO2 absorption into tertiary amines containing carbonic anhydrase, AIChE J. 63 (2017) 4933–4943, https://doi.org/10.1002/aic.15833. [7] I.M. Power, A.L. Harrison, G.M. Dipple, Accelerating mineral carbonation using carbonic anhydrase, Environ. Sci. Technol. 50 (2016) 2610–2618, https://doi.org/ 10.1021/acs.est.5b04779. [8] S.H. Zhang, Y.Q. Lu, Surfactants facilitating carbonic anhydrase enzyme-mediated CO2 absorption into a carbonate solution, Environ. Sci. Technol. 51 (2017) 8537–8543, https://doi.org/10.1021/acs.est.7b00711. [9] A. Di Fiore, V. Alterio, S.M. Monti, et al., Thermostable carbonic anhydrases in biotechnological applications, Int. J. Mol. Sci. 16 (2015) 15456–15480, https://doi. org/10.3390/ijms160715456. [10] A. ElMekawy, H.M. Hegab, G. Mohanakrishna, et al., Technological advances in CO2 conversion electro-biorefinery: a step toward commercialization, Bioresour. Technol. Rep. 215 (2016) 357–370, https://doi.org/10.1016/j.biortech.2016.03. 023. [11] E.T. Hwang, H.M. Gang, J.Y. Chung, et al., Carbonic anhydrase assisted calcium carbonate crystalline composites as a biocatalyst, Green Chem. 14 (2012) 2216–2220, https://doi.org/10.1039/c2gc35444f. [12] A.B. Fulke, S.N. Mudliar, R. Yadav, et al., Bio-mitigation of CO2, calcite formation and simultaneous biodiesel precursors production using Chlorella sp, Bioresour. Technol. Rep. 101 (2010) 8473–8476, https://doi.org/10.1016/j.biortech.2010.06. 012.
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