Engineering of Microbial Carbonic Anhydrase for Enhanced Carbon Sequestration


Carbonic anhydrase (CA) is a zinc-containing enzyme that presents in both eukaryotes and prokaryotes and rapidly catalyses the hydration reaction of C02 to HCOJ and proton (H+), and vice versa (Di Fiore et ah, 2015). As per the latest measurement of C02 by NOAA in 2017, the concentration of C02 in the atmosphere was about 400ppm, and it is rising by 2ppm annually (NOAA, 2017) due to rapid industrial development, uncontrolled CO, emissions from various industries, burning of fossil fuels, and uncontrolled human growth. CO, has a significant impact on climate change and global warming. The increased CO, concentration in the atmosphere since the post-industrial era can correlate with increasing global surface temperatures (Giri and Pant, 2018). Over the last century (1906-2005), the average global temperature data revealed that the average temperature increases by 0.7°C + 0.2°C over that period (Jansen et al., 2007). In the last decade, the sequestration of CO, by microbial CA have been attracted much attention as an alternative way to carbon management technology due to high reaction rate of CA (104—106s_l) and high tolerance rate towards high CO, concentration (Bose and Satyanarayana, 2017). However, low' thermal stability, poor activity, and reusability of CA have some industrial limitations over its optimum utilization. Protein engineering has been widely applied to alter the existing protein structure or enzyme in order to improve its catalytic properties for various environmental, industrial, and pharmaceutical applications. Exploiting CA activity by CA engineering is an attractive technique to speed up its diverse application in biomimetic route for CO, sequestration from the combustion fuel gases w'ith its improved environmental compatibility, potential economic viability, activity, and stability (Jo et al., 2016; Giri and Pant, 2019a). Recent advancement of experimental and computational CA engineering can lead to the developments of the large-scale industrial biocatalyst. Various proteins have been successfully engineered by several methods such as site-directed mutagenesis, directed evolution, protein immobilization, biological and chemical modifications, active site-directed substitutions, ProSAR: amino acid contributions, and rational design of substrate specificity (Kazlauskas and Bornscheuer, 2009; Kumar et al., 2018; Verma et al., 2019). Directed evolution is an effective technique for the major changes in protein active site with efficient biocatalytic performances (Alvizo et al., 2014; Boone et al., 2013; Zhang et al., 2016; Zuo et al., 2018). Other methods like ProSAR: amino acid contributions are fast and provide an easier w'ay to alter enzymes with optimal activity (Lippow' and Tidor, 2007; Yao et al., 2015; Chen et al., 2015; Zhang et al., 2017). This chapter focuses on mainly microbial CA engineering techniques and briefly addresses the environmental applications of the immobilized CAs. Methods involving the modification and expression of the CA gene, immobilization, amino acid modification, engineering in de novo disulphide bond in free microbial CA, and CA immobilization are significantly promising techniques (Kumar et al., 2015; Zhang et al., 2016; Wu et al., 2016; Chen et al., 2016; Kumar et al., 2018a, 2018b) and are cost-effective processes for the overall CO, mitigation (Kambar and Ozdemir, 2010). Several studies of engineered recombinant microbial CA (Del Prete et al., 2012; Joseph et al., 2010), directed evolution (Alvizo et al., 2014; Jo et al., 2016), and immobilization (Sharma et al., 2011; have expressed and modified CA, which is an attempt to overcome its activity, stability, and recovery issue. CA engineering involves the following three steps: (i) engineering strategies or selecting protein changes such as directed evolution, rational design; (ii) making these changes (mutagenesis); and (iii) screening or selection (Kazlauskas and Bornscheuer, 2009). Choosing different strategies can result in various benefits or drawbacks in each of these steps (Figure 5.1).


Microbial CA belongs to three genetically different classes a, (3, and у that contain a tetrahedrally active catalytic Zn (II) ion in the active site. Three imidazole groups of histidines (His-94, 96, and 119) and a hydroxyl molecule in a- and y-CA are attached with the metal ion in the active site. In the (3-CA, one histidine and two

Proposed CA engineering methods based on the amount of available information of target CA

FIGURE 5.1 Proposed CA engineering methods based on the amount of available information of target CA.

cysteine residues are attached with the metal ion present in the active site (Capasso and Supuran, 2015). y-CA not only contains Fe(II) ion but can also actively bind with Zn(II) orCo(II) ions (Supuran and Capasso, 2017). Leu-198, Val-( 121, 143,207), and Trp-209, a cluster of hydrophobic amino acids, are responsible for carbon dioxide binding and participate in the proton shuttling process (Ferry, 2010). A cluster of hydrophilic amino acids of CA (Asn-62, His-64, Tyr-7, and Asn-67) are responsible for the orientation of C02, during CO, catalysis (Domsic and McKenna, 2010). CAs from Sulfurihydrogenibium yellowstonense, Thermovibrio ammonificans, Neisseria gonorrhoeae, and Sulfurihydrogenibium azorense are the only known three-dimensional structures that belong to a-CA (Di Fiore et al., 2013; Supuran and Capasso, 2017), and the X-ray crystal structure of CAs is determined from many bacterial species such as Mycobacterium tuberculosis, Citrobacter freundii, Pseudomonas spp., Haemophilus influenzae, Vibrio cholerae, Salmonella enterica, and Escherichia coli (Ferraroni et al., 2015; Giri et al., 2018; Giri and Pant, 2019a). Only one CAM (Carbonic Anhydrase Methanosarcina) from methane-producing archaea Methanosarcina thermophila has been crystallized until now (Kisker et al., 1996).

Catalytic Activity of CA

Despite their differences in structure, CAs have a common mechanism of action. The catalytic reaction of CA is a two-step ping-pong reaction that catalyses CO,, which is expressed as follows (Supuran and Capasso, 2017):

In the catalytic mechanism of CA, the histidine residue accepts a proton (H+) which is formed from the water molecules. Nucleophilic attack by zinc-bounded hydroxyl group on the molecule of carbon dioxide takes place, which results in enzyme-bicarbonate complex formation (Silverman and Lindskog, 1988). In a-CA, His-64 receives a proton from the active site of water molecules by its interference with the molecule of zinc-bounded water molecule (Tripp et ah, 2001). Lys-91 and Tyr-131 near the active site cavity of CA isozyme can also influence the kcal and can be targeted to incorporate these imidazole analogues. Kimber et al. (2000) analysed this observation and divided (3-class into two subclasses, the ‘Cab type’ and ‘plant type’, found from Methanothermobacter thermoautotrophicum and Pisum sativum CA, respectively.


The development of recombinant technology by the expression of high-yielding CA gene into the targeted microorganism is a promising biotechnological method to enhance carbon sequestration potential of microbial CA. The methods of CA engineering have been effective towards CO, sequestration.

Genetic Engineering

Recently, genetic engineering of CA has gained considerable research interest because of climate change and energy crisis. The natural capability of CO, fixation by CA can be genetically engineered to achieve a desired result with improved activity and/or stability and generate high-value bioproducts (Ducat et ah, 2011; Chen et ah, 2012). Jo et ah (2013) expressed CA from N. gonorrhoeae (ngCA) in the periplasm of E. coli and successfully demonstrated the whole-cell catalyst as an effective catalyst for CO, sequestration (Figure 5.2). In another study, engineered yeast (Saccharomyces cerevisiae) is also used for the enhancement of CO, hydration reaction (Barbero et ah, 2013). Recombinant CAs from different microorganisms such as Hahellachejuensis, N. gonorrhoeae, and Thalassiosira weissflogii are expressed in E. coli, which after expression results in high pH and temperature stability, doubled esterase activity, and accelerated rate of calcite crystal formation (Ki et ah, 2013). Fan et ah (2011) studied ice nucleation protein (INP) from Pseudomonas syringae as surface-anchoring support for the expression ofa-CA from Helicobacter pylori on the outer membrane of E. coli, which served as resourceful biocatalyst for CO, sequestration with significantly increased CO, removal rate. Del Prete et ah (2012) purified a-CA from V. cholerae (VchCA) and cloned, which is four times more active than the other microbial CAs. a-Type CA protein (CAA1) of Mesorhizobium loti coded by msi040 gene is expressed in nitrogen-fixing and free-living bacteria for CO, fixation in associated root nodules (Kalloniati et ah, 2009).

Design concept for the C0 mitigation by genetic engineering of CA

FIGURE 5.2 Design concept for the C02 mitigation by genetic engineering of CA.

Directed Evolution

Another strategy is the directed evolution, which causes changes in the catalytic site to give enzyme variants with catalysing capabilities, and the creation of highly stable p-CA originating from Desulfovibrio vulgaris is the example, which accelerates the C02 absorption above 100°C in alkaline solvents (Alvizo et al., 2014). The structure and active site of many CAs like a-CA from 5. azorense can alter the proton transfer rate of enzyme. A disulphide bonding is conserved in human carbonic anhydrases (hCAs) (IV, VI, IX, XII, and XIV) along with P-CA from many microbes like N. gononhoeae and is responsible for the stability of CA (Aggarwal et ah, 2013). The engineering of disulphide bond present in the microbial CA shows an enhancement of thermostability and a resistance to denaturation as compared to native CA (Martensson et ah, 2002). Introduction of the designed, de novo disulphide bond into CA originating from N. gonorrhoeae (ngCA), which later expressed in E. coli, shows an improved kinetic and thermodynamic stability (Dombkowski et ah, 2013). Gould and Tawfik (2005) suggested that two mutations of human CA (Ala65Val and Thr200Ala) through the directed evolution can increase a promising function of CA in the industrial applications. CA obtained from D. vulgaris (DvCA) accelerates C02 absorption at 100°C and in the presence of alkaline solvent (Fox et ah, 2007; Alvizo et ah, 2014; Kumar et ah, 2018c; 2019; Sharma et ah, 2019). Directed evolution can be carried out in different ways; for example, in vitro recombination-based directed evolutions focused on the generation of gene libraries and the process of increasing the enzyme activity should be developed. The directed evolution computational methods are also rapidly used for improving and creating new enzymes, thus enhancing their catalytic function (Hibbert and Dalby, 2005). The substitutions of the hydrophobic residues into hydrophilic substitutes in CA retain the high catalytic efficiency and stabilize the denaturing temperature (Boone et al., 2013). Parra-Cruz et al. (2018) used molecular dynamics simulations to study the association between thermostability and structural flexibility of the bacterial a-CA and designed the disulphide bonds and mutants with increased stability. Random, targeted mutagenesis, and recombination strategies are mainly used for the directed evolution of protein by producing sequence libraries. Random mutagenesis has taken the place of amino acid substitutions to explore beneficial mutations. However, targeted mutagenesis has taken the place of conformational changes in protein for the randomization of amino acids. Recombination introduces a wide number of changes in a simultaneous sequence of the targeted protein (Bloom et ah, 2005).

CA Immobilization and Chemical Modification

The immobilization of CA onto the solid surface and the chemical modification of CA are eco-friendly and effective approaches successfully used to increase its catalytic property for various industrial and biotechnological applications. Chemically modified and immobilized CA can be capable of enhancing carbon dioxide absorption from a gas stream and enhancing carbon dioxide sequestration and mineral formation (Cowan and Fernandez-Lafuente, 2011). The incorporation of aldehyde can lead to the best protein modifications; various studies on the incorporation of aldehyde in in vivo and in vitro conditions were carried out because aldehyde can easily react witli lysine residues (Pant et ah, 2017; Giri and Pant, 2019b). Bootorabi et ah (2008) studied the reaction of CA with acetaldehyde after monitoring its activity and found that acetaldehyde binding with CA may affect the hydrogen-bonding patterns, may result in conformational changes of CA, and may change the acid-base properties within the active site and the surface of an enzyme, which in turn leads to the decreased CA activity. The various cross-linking agents such as bis(N-hydroxysuccinimide) ester, dialdehyde, bis-imidate, diacid chloride, malondialdehyde, glutaraldehyde, and dimethyl suberimidate are useful for the preparation and modification of a-, (3-, and y-CA polypeptides and exhibit improved catalytic properties under carbon-capture process conditions (Novick et ah, 2013). The modification of cysteine residue of CA leads to an enhancement in the proton transfer, whereas unmodified cysteine residue shows threefold higher CO, hydration rate (Elder et ah, 2004).

The binding of different ligands within the active site of CA depends on (i) coordination sphere of CA active site, including Zn+ atoms; (ii) hydrophobic sites of targeted CA; (iii) hydrophilic sites of CA; and (iv) nucleophilicity (Giri and Pant, 2019b). Cysteine residues of /7-СА can be modified by the thiol group as a nucleophilic reagent (Kim et ah, 2008). The catalytic rate of CA in CO, hydration process can be enhanced by various cross-linking agents (Tu et ah, 1990). Stefanucci et ah (2018) studied the activation of /]- and y-CA from the pathogenic bacteria M. tuberculosis, V. cholera, and Burkholderia pseudomallei by the incorporation of acidic amino acids as tripeptide. CA from T weissflogii is also significantly activated by different amines and amino acids (Angeli et ah, 2018).

CA immobilization provides an effective way to improve the operational stability of enzymatic technology in carbon capture or sequestration process. Based on a large number of studies, enzyme immobilization can be done by different methods using supports such as solid surface, polymeric materials, and on or within soft poly molecular assemblies (Cao, 2005; Mateo et al., 2007; Wu et al„ 2016; Kumar et al., 2019). Currently, CA immobilization is an active research area due to its large number of applications. The simplest CA immobilization method is the physical adsorption on the soft or solid surface like colloidal gold sols used for the adsorption of CA molecule and retention of its enzymatic activity (Jesionowski et al., 2014; Drozdov et al., 2016). The CA was successfully immobilized on spherical SBA-15 silica particles by different approaches, such as adsorption, cross-linking, and covalent binding, and studied for the sequestration of CO, (Vinoba et al., 2011). The results demonstrated that the silver nanoparticle-conjugated hCA showed ~25-fold higher СО,- capture efficiency and the highest operational stability than the free hCA (Vinoba et al., 2012). Chitosan, polyurethane (PU) foam, glass, activated carbon, hydrogels, and silica beads are the supports that have been used in the immobilization of CA (Zhang et al., 2011; Wanjari et al., 2011; Forsyth et al., 2013). Recently, bioinspired silica has been used for enzyme immobilization as a green method due to its mild processing conditions, improved stability, high activity, and excellent immobilization efficiency (Patwardhan, 2011) (Table 5.1).

Overall, the immobilized CA acts as a green catalyst for reducing carbon dioxide emissions compared to other alkali or a soluble biocatalyst process. Other than these methods, the recent development of CA engineering is via chemical modifications involving (i) the modification of natural or unnatural amino acids (i.e. site-selective


Example of Microbial CA Immobilization and Function

Sr. No.

CA Used

Support Material and Methods




Microbial CA

Silica or SiO,-ZrO: composite nanoparticles

Improved CA stability

Zhang et al. (2013)


Bacillus pumilus TS1 CA

Surfactant-modified (SDS) silylated chitosan

Longer storage stability, increased K,„ and Vm

Yadav et al. (2010)


CA from Bacillus pumilus, Pseudomonas fragi, and Micrococcus fylae

Surfactant-modified silylated chitosan

Improved storage stability

Prabhu et al. (2011)


CA from Bacillus pumilus

Chitosan beads

Increased K„, value

Wanjari et al. (2011)






Magnetic Fe,04 nanoparticles (MNP)

Enhanced stability and storage of CA

Perfetto et al. (2017)


a-CA from Sulfurihydrogenibium yellowstonense

PU foam

Long-term stability

Migliardini et al. (2014)

incorporation) and (ii) the recognition-driven modification, which also improves the catalytic activity of microbial CA (Sakamoto and Hamachi, 2018).

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