CO2-capture by engineered mammalian carbonic anhydrases

C H A P T E R 23

CO2-capture by engineered mammalian carbonic anhydrases Alessio Nocentini1, Muhammet Tanc2 1

Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Firenze, Italy; 2Department of Pharmaceutical Sciences, University of Antwerp, Campus Drie Eiken, Antwerp, Belgium

23.1 Introduction Carbonic anhydrases (CAs) are among the fastest enzymes known to date exhibiting an incredibly high conversion of CO2 molecules into bicarbonate
 þ CO2 þ H2 O/HCO3 þ H , with turnover numbers ranging between 104 and 106 per second for a single catalytic site (kcat) [1,2]. The hydration reaction catalyzed by the CAs has been proposed to be exploited to capture CO2 in various applications for different biomedical, industrial, or environmental purposes, as discussed in this chapter [3]. The employments of CAs mainly include (1) modulating the CO2 level in confined spaces, removal of CO2 from the bloodstream, creating an alternative blood supply, and treatment of analgesic overdoses as biomedical purposes; (2) calcite production for agricultural and construction materials, as well as polymer production for industrial purposes; and (3) production of alternative fuel sources, carbon separation from flue gas as environmental cleaning strategies. In particular, human (and other mammalian) CAs offer several advantages in these applications as they are remarkably efficient, easily overexpressed in bacteria or commercially available, reusable, and operate at ambient temperatures and under mild conditions [1e3]. Human CA II (hCA II) is the most studied isozyme to date and is commonly proposed to be used for biotechnologic applications [1]. However, it should be noted that some issues have limited the use of highly efficient mammalian CAs in the industrial setting, such as their chemical instability in the operating conditions (e.g., organic solvents, low pH, and high temperatures) [4]. hCA II is irreversibly denatured at approximately 58 C and is inhibited by small anions, including sulfate, cyanate, thiocyanate, and azide [1,4]. The kinetic parameters for CO2 hydration of some of the CAs discussed in this chapter are reported in Table 23.1.

Carbonic Anhydrases. https://doi.org/10.1016/B978-0-12-816476-1.00023-X Copyright © 2019 Elsevier Inc. All rights reserved.

515

516 Chapter 23 Table 23.1: Kinetic parameters for CO2 hydration reaction of some of the carbonic anhydrases (CAs) discussed in this chapter. Isoform

kcat (s¡1)

Km (mM)

kcat/Km (M¡1 s¡1)

hCA I

2.0  105

4.0

5.0  107

hCA II

1.4  106

9.3

1.5  108

bCA

8.3  105

9.0

9.2  107

HP-a-CA

2.5  105

16.6

1.5  107

SspCA

9.4  105

8.5

1.1  108

23.2 CO2-capture for biomedical purposes Reversible absorption represents the conventional process for CO2-capture and requires a high amount of energy and significant costs, thus leading to a high environmental impact [5]. A milestone in CO2 capturing for biomedical purposes has been reached with the development of effective thin liquid membranes (TLMs) [6]. This efficient and environmentally friendly separation tool consists of a packed liquid layer between semipermeable barriers (polypropylene derivatives). CO2 diffuses from a flowing gas outside into the liquid layer placed underneath, where CAs are located. The enzymes catalyze the hydration of CO2 into bicarbonate, which can be subsequently desorbed by lowered CO2 partial pressures further downstream [6,7]. TLMs provide an economic, selective, and efficient separation of CO2 from flue gas containing oxygen, hydrogen, nitrogen, water, hydrochloric acid, sulfur dioxide, and oxides of nitrogen [8]. CO2-capture through TLMs has exceptional potential in isolated spaces such as submarines, spacecrafts, and futuristic living units in space or under water. It is known that an elevated level of CO2 in the human body has deleterious effects such as acidosis, impaired judgment, coma, and death [9]. The CA-immobilized liquid membrane systems can selectively capture CO2 from a mixture of air containing nitrogen and oxygen in ratios of 1400:1 and 900:1, respectively [10e13], and thus can serve a critical role in isolated living systems. Biologic systems have evolved to improve the gas exchange depending on the habitat in which they are present. The CO2-exchange in the respiratory system of vertebrates represents a significant example. CAs are located in erythrocytes, producing the fast and selective dissolution of the CO2 generated by tissues and regeneration of the CO2 exhaled from lungs. TLM-based CO2 sequestration has been exploited to realize extracorporeal lung assistance to aid the blood gas exchange in patients suffering from respiratory failure (Fig. 23.1) [15e19]. Hollow fiber membranes (HFMs) were coated with CAs to facilitate faster gas exchange at a high blood flow rate [14,20,21] and reduce the required surface area for an effective yield [22e25]. The “bioactive” HFM containing immobilized CAs demonstrated a 75% increase in the rate of CO2 removal compared with an untreated

CO2-capture by engineered mammalian carbonic anhydrases 517 CO2 and O2

HCO3–

CO2

Blood inlet

Blood outlet

CA

CO2 HCO3–

O2

Figure 23.1 Schematic representation of an artificial lung system. From Boone CD, Robert McKenna. Engineered mammalian carbonic anhydrases for CO2 capture. In: Supuran CT, De Simone G, editors. Carbonic anhydrases as biocatalysts. Amsterdam: Elsevier; 2015.

HFM [26e29]. However, improvements in the efficiency of these systems are needed before they can become a valid alternative to mechanical ventilators [21]. Feedback-regulated drug-delivery vehicles are the most promising way to cope with the detrimental side effects of drugs, when used in high concentrations [30]. This type of vehicle has been used in diabetes treatment functioning as a regulator of insulin release depending on the glucose level in the blood [31e34]. The application of CAs in this field is connected to the treatment of analgesic overdose [30]. Drugs targeting opioid receptors, such as morphine, can lead to respiratory depression due to increased levels of somatic CO2. A successful responsive system consists of monitoring the blood opioid levels with morphine-activated enzymes that release the antidotes naltrexone and naloxone, gathered into polymeric clathrates [35]. An alternative method does not depend on the blood opioid levels and uses a hydrogel composed of N,N-dimethyaminoethyl methacrylate (DMAEMA) polymers (biocompatible, biodegradable, and nontoxic), which incorporates CAs as a “detector” to follow up disproportion of CO2 in the case of a morphine overdose [34,36,37]. The antidote (naltrexone) is released as a response to a toxicity biomarker (high CO2 level) instead of the free-drug concentration. The use of CAs in these systems is beneficial in the

518 Chapter 23 context of high levels of CO2 but showed drawbacks when the CO2 concentration range was 5%e7.5% [34].

23.3 CO2-capture for industrial and environmental purposes The latest results from the Global Atmosphere Watch (GAW) program of the World Meteorological Organization stated that globally averaged surface mole fractions for CO2, CH4, and N2O reached new highs in 2017, with 405.5  0.1 ppm, 1859  2 ppb, and 329.9  0.1 ppb, respectively [38]. These values represent values that are 146%, 257%, and 122%, respectively, of those from before 1750 (preindustrial period). Additionally, Antarctic ice analysis shows that the atmospheric CO2 concentration is higher today than in the past 800,000 years [39e42]. In addition, analysis of the boron isotope ratio in ancient planktonic foraminifer shells shows that a similar CO2 level in the atmosphere existed approximately 20 million years ago during the first and longest warming Miocene series periods [43]. Carbon dioxide is the most important human-induced greenhouse gas in the atmosphere [38,44]. Use of fossil fuels and cement production are the primary causes of greenhouse gas emissions [44]. NASA analysis shows that the Earth’s global surface temperatures in 2017 ranked as the second warmest since 1880 and the planet’s long-term warming trend is continuing [45]. Separately from NASA, analysis of the National Oceanic and Atmospheric Administration (NOAA), the Japan Meteorological Agency (JMA), and the Met Office Hadley Center (United Kingdom) also confirms the trend of warming since preindustrial times [46]. Melting of glaciers and polar ice gaps are only two results of the increasing surface temperature of the Earth, which is leading to a rise in sea levels, ocean acidification, and desalination. All these climate changes are threatening a tremendous number of plant and animal species, by disturbing or annihilating their ecologic systems [47,48]. The target of the Paris Agreement (2015) is to maintain the rise in global temperature this century to within 2 C above preindustrial levels. The required developments and obligatory political changes increase the importance of CO2-capture from fuel gases or directly from the atmosphere. In this context, it is of great importance to capture CO2 from fuel gases, that also may contain nitrogen and sulfur oxides, as well as other organic compounds, although it may be technically challenging and costly [8,49,50]. Current indirect separation methods include dissolving CO2 in amine scrubbing (also known as amine gas treating) or mineral carbonation in an aqueous phase. Such financially and environmentally challenging methods should be replaced with inexpensive, renewable, and environmentally friendly industrial protocols. Hydrated CO2, expressed either as H2CO3 or HCO3  (depending on pH), can be chemically converted into calcite (CaCO3) or other mineral derivatives (aragonite, vaterite) for industrial and agricultural purposes [51]. CAs are the most promising catalyst to

CO2-capture by engineered mammalian carbonic anhydrases 519 increase the efficiency of the hydration of CO2 [52]. Indeed, the use of CAs (mainly hCA II) offers several advantages, such as increased efficiency and specificity toward CO2, commercial availability, and functioning under mild conditions [53,54]. As reported above, the main drawbacks related to the application of hCAs in this type of process are their relative instability at low pH, in organic solvents, at high temperature, and their inhibition by a range of small inorganic anions [1,55e57]. The chemical conversion of CO2 to stable compounds such as CaCO3 (calcite) or MgCO3 (magnesite) and their subsequent burial represents a valid solution for the storage of captured CO2. Possible concerns over the effects of acid rain from these deposits can be reduced by storing the carbonates in regions specific for geosequestration, such as in wollastonite (for calcite) and serpentinite (for magnesite) deposits or directly in rain-poor regions [58,59]. However, all these methods required energy only for storage, thus the conversion of CO2 into various beneficial by-products including polycarbonates, acrylates, methane, stable carbonate storage polymers, and building materials represents a more important alternative to date [60e62]. Since 2006, the number of vehicles has increased approximately by 38% worldwide and has reached over 1.25 billion, with the parallel crude oil demand reaching 99 million barrels per day [63,64]. The long-term environmental effect of excessive consumption and limited availability of fossil fuels are motivating countries to explore alternative resources [65]. Biodiesel is one of the best options to replace diesel because it is nontoxic and emits less pollutants, including zero CO2 (because the carbon emitted during its combustion corresponds to that already present in the atmosphere and fixed by plants) and sulfur compounds [66]. However, if all of the arable land in the United States was used to grow soybean for oil production, only a small part of the US biodiesel demand would be satisfied. The current production of biofuels also displaces crop lands, which increases prices for the consumer [66e68]. Algae-based systems (cyanobacteria) allow for higher biofuel production and carbon fixation rates compared with terrestrial plants [69,70]. Moreover, algae are environmentally friendly because they require only a light source (sunlight or an artificial one) and a minimal amount of micronutrients for growth, with plenty of cultivation locations such as ponds or enclosed photobioreactors being available [65]. In addition, vitamins, minerals, proteins, fatty acids, dietary supplements, agents used in food production, pigments, and fertilizers can be harvested as by-products [71,72]. Recent studies have shown that the addition of engineered extracellular CAs to algal cultures can increase carbon flux and fixation rates, resulting in improved biomass production [73e75]. With the transformation of CO2 to bicarbonate being the rate-limiting step of biomass production for these organisms they have evolved carbon-concentrating mechanisms (carboxysomes), providing a soluble inorganic carbon source to ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) by the intervention of CAs [73,76,77]. RuBisCO catalyzes

520 Chapter 23 the carboxylation of ribulose-1,5-bisphosphate (RuBP). Carboxysomes are bacterial microcompartments, where carbon fixation occurs, composed of polyhedral protein shells including RuBisCO, CA, and active bicarbonate transporters. In carboxysomes, CA is responsible for the production of carbon dioxide by dehydration of bicarbonate, which is then fixed to RuBP by RuBisCO [73]. Further studies have demonstrated that algal and cyanobacteria cultures (Chlorella and Spirulina spp.) can also be used for the indirect production of calcite [71,72,78,79]. It has been shown that CA inhibition with acetazolamide resulted in a decrease in CO2-capture, and thus calcite production, which implies a critical function of CAs in this process [80e82]. Parameter optimization (temperature, pH, nutrient, aeration, etc.) of these systems should be undertaken to design tools for the simultaneous production of biofuels and calcite in a cost-efficient manner.

23.4 Applications of CO2-capture techniques Various techniques allow the use of CAs in the above-discussed different areas. Immobilization is the leading technique, which enables the most technologic applications of CAs and consists of attaching the enzyme to various inorganic and biopolymer surfaces, which include enriched microorganisms, in addition to adhesion onto several matrices such as chitosan, alginate, and acrylamide [14,83e92]. Free hydroxyl groups, cross-linked enzyme aggregation, and surface lysine resides facilitate the immobilization of CA onto these materials (Fig. 23.2) [93]. Immobilization of bovine CA (bCA) to inorganic surfaces (metal-based nanoparticles and mesoporous silica) was yielded by covalent linkage [85,94e96]. Iron-based nanoparticles present the advantages of reusability (up to 30 cycles), facile surface renewal (magnet separation), and duration (retaining 80% activity over 30 days) [85]. Moreover, by the measurement of calcite precipitation, it was shown that immobilized bCA operates more efficiently than the free enzyme in solution. Gold and silver nanoparticles have also been explored. The gold-immobilized bCA only loses 13% of its enzymatic activity after 30 days of storage, while it was shown that silver-immobilized bCA can be reused after 20 times, retaining full catalytic activity if stored at 25 C storage temperature [85,95]. Covalent attachment, enzyme adsorption, and cross-linked enzyme aggregation are the techniques used to immobilize bCA to mesoporous silica nanoparticles [94]. These immobilized bCA showed enhanced stability, reusability, and durability, although possessing comparable kinetic activity to the free enzyme in solution [96]. bCA immobilized onto an aluminum-based derivative of the mesoporous silicate surface also exhibited increased stability, but a decreased binding affinity toward CO2 [94]. In addition, protocols have been established to simply entrap enzymes in a porous material such as polyurethane (PU) foam [97]. The trapping protocol is very efficient and fast and consists of mixing the enzyme solution with a mixture of polyethylene glycol

CO2-capture by engineered mammalian carbonic anhydrases 521

Figure 23.2 Cartoon image of hCA II on a biopolymer surface. Immobilization is made by linkage of biopolymer hydroxyl groups and surface lysine resides of the protein, some of which are represented in yellow as balls and sticks. The proton shuttle residue H64 is shown in both in and out conformations. A bicarbonate ion and a CO2 molecule (green) are present in the active site (PDB 3U7C).

substituent with isocyanate end groups (HYPOL). Nucleophilic attack of the hydroxide on carbonyl groups triggers the polymerization of HYPOL, followed by the CO2 release which forms a spongy PU foam around the enzyme. By this mechanism, the isocyanate end groups are converted into amines that rapidly cross-link with another neighboring end group, thereby cross-linking the two polymer chains. The enzyme is thus cross-linked to HYPOL via covalent linkages by surface amine and hydroxyl groups [93]. The result is a highly stable matrix that retains all catalytic activity after 45 days of storage at room temperature, whereas the enzyme free in solution is fully denatured after the same period at 4 C [93]. Toxicity and degradability issues have motivated researchers to develop new immobilization systems. Hydrogels, that are nontoxic and biodegradable, are the results of these efforts and have been applied in several tools [34]. Hydrogels are composed primarily of polysaccharides including chitosan, a linear polysaccharide chain composed of glucosamine and N-acetylglucosamine sugars, and/or alginate, containing mannuronate and guluronate moieties. Although displaying comparable esterase activity, immobilized CAs onto chitosan show a fourfold enhanced efficacy in calcite precipitation with respect to the enzyme in solution [92]. The surface of different bacteria such as Bacillus pumilus, Pseudomonas fragi, and Micrococcus lylae can be also used for CA immobilization. A study demonstrated that immobilized CAs on various bacteria retain 50% of their initial activity after 30 days of

522 Chapter 23 storage [98]. The use of immobilized live P. fragi and B. pumilus cells to chitosan beads may be useful to avoid protein purification [84,86]. Moreover, it is possible to immobilize live bacterial cells with surface-expressed CA (such as Helicobacter pylori CA on the surface of E. coli) onto hydrogels for biomineralization of CO2 [99].

23.5 Site-directed mutagenesis of hCA II: toward enhanced activity and thermostability The main limitations of CA-based CO2-capturing systems are the stability and catalytic activity of the enzyme. As attractive candidates for these systems, several isoform hCA II variants have been produced, which show enhanced kinetics [100,101] and/or thermostability [102,103]. The kinetically boosted hCA II variants share great efficiency in extreme conditions with some other human isoforms such as hCA IV [104,105], hCA IX [106,107], an a-CA of Sulfurihydrogenibium azorense [108,109], and other enzymes [110]. A kinetically enhanced variant has been engineered to improve the proton shuttle system, which controls the rate-limiting step of the catalytic cycle [111]. As a result, hCA II variants containing Y7F and/or N67Q revealed a three- to ninefold increase in the rate in proton transfer compared with the wild-type enzyme [100,101]. To increase the thermostability of hCA II variants were engineered decreasing the hydrophobicity of the enzymatic surface or incorporating a disulfide linkage between residues 23 and 203 [102,103]. Leucine residues at positions 100, 224, and 240 were changed into hydrophilic ones that were able to heighten the denaturing temperature of the variant of 7 C with respect to the wild-type hCA II while retaining the characteristic high catalytic efficiency of the enzyme (Fig. 23.3A). These stabilizing surface variants were subsequently incorporated into the kinetically enhanced Y7F and N67Q mutants with comparable catalytic rates although a slight decrease in thermostability emerged (2e3 C). The disulfide bond between residues 23 and 203 (hCA II numbering, Fig. 23.3B) conserved among the extracellular hCAs IV, VI, IX, XII, and XIV and several bacterial CAs such as Neisseria gonorrhoeae has been associated with their relative stability [110]. A hCA II variant was engineered to display this disulfide linkage (Fig. 23.3B) and showed a twofold increased resistance to denaturation by guanidine-HCl [103] and a 13 C [102] increase in thermostability. The desirable active site geometry and catalytic activity were also retained. A disulfide linkage on the surface of hCA II engineered between residues 99 and 242 was also shown to increase the enzymatic resistance to 1.4M guanidine-HCl [112]. Recent genome sequencing enabled the discovery of novel a-CAs from halotolerant and thermotolerant microorganisms that could be useful as templates for further rational optimization of hCA II for industrial applications. For instance, the CA from the marine bacterium Hahella chejuensis was identified [113] and later overexpressed in E. coli [114], where it was shown to have a melting temperature of 60 C, maximal activity at 50 C, very

CO2-capture by engineered mammalian carbonic anhydrases 523

Figure 23.3 Cartoon images of engineered thermostabilized hCA II variants with mutation sites shown in balls and sticks and labeled for (A) L100H, L224S, and L240P (PDB 3V3F) and (B) A23C and L203C (PDB 4HBA).

high pH and salt tolerance, activity at pH 10, and calcite precipitation. An extremely thermostable a-CA has been isolated from the bacterium Sulfurihydrogenibium yellowstonense (SspCA) from a hot spring in Yellowstone National Park [111]. SspCA in solution is able to retain 100% activity after 2-h incubations at 100 C, which increase to 50 h in the case of immobilization of the bacterial enzyme onto PU foam, resulting in its application in biomimetic CO2-capture reactors [115]. SspCA shows increased surface charge networks and the lack of long, flexible surface loops, which could be responsible for its high thermostability [116].

23.6 Future perspectives and conclusions The growing interest in CA-based CO2-capture has promoted new tools for a rapid improvement of the existing or new technologies. In this context, simulations can improve HFM systems by the simultaneous analysis of various effects such as membrane wetting, operational conditions, enzyme packing, buffer acidebase constant, and flow orientation [117]. New membrane types based on poly(vinylidene fluoride) (PVDF) (pretreated with water plasma) represent a promising CA-immobilization method to increase reusability and stability. PVDF modified with 3-aminopropyltriethoxysilane groups exhibits notable performance since it retains 85% of its CA activity after 10 cycles [118]. The combination of CAs from various organisms with different materials enables the creation of a further wide variety of platforms, such as chitosanealginate polyelectrolyte complex [119], functionalized mesoporous silica [120], titania-based biocatalytic nanoparticles [121], epoxy-functionalized magnetic polymer microspheres [122], “Janus”

524 Chapter 23 membrane containing a layer of hydrophilic carbon nanotubes coated on a fluorosilanetreated superhydrophobic membrane support [123], liposomes conjugated with CA via amide bond formation between the carboxyl group compartment of liposomes and primary amines of CA molecules [124]. Varying the functionalization of these platforms, a wealth of applicative purposes can be targeted [125]. Additionally, glass micropipettes and glass fiber filters (as solid platforms) can be considered promising technology for capturing CO2 [126]. To date, a chorus of strategies have been proposed to use various CAs in different applications, which differ by their enzyme/support interactions: (1) noncovalent interactions between enzyme and support via direct enzyme adsorption [127]; (2) covalent interactions by linker/s between enzymes and support [127]; (3) self-assembled monolayers able to covalently interact with the enzyme or to adsorb it on the layer surface [128]; (4) analog amphiphile bilayers [129]; and (5) dendronized polymereenzyme conjugates [130,131]. Despite the great potential of most such biotechnologic applications, several issues still need to be tackled, pertaining to their efficiency, stability, localization control, and toxicity, especially for biologic applications. Another key challenge is the cheap mass production of enzymes [132], which appears to be partially surmountable by the mass extraction of bCA from blood derived from the meat industry [54]. The combination of genome sequencing with innovative techniques to improve CAs and platforms is in the homestretch to enable sustainable real-life applications, but this field is still in its infancy.

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