Carbonic Anhydrase Activity Research Articles

Novel three-stage microalgal cultivation system for lipid production utilizing nutrients derived from refinery waste

The pollution and transformation of refineries are receiving increasing attention. The carbonic anhydrase in Tetradesmus obliquus was found exhibiting a hysteresis phenomenon in response to periodic changes in the composition of external carbon sources, with a surge in inorganic carbon concentration stressing the carbonic anhydrase activity to increase by 6–9 times. On this basis, a novel three-stage culture system of T. obliquus was proposed, which mainly uses refinery waste as the nutrients. By controlling the nutrient content in the environment, especially the composition of carbon sources, microalgae could sequentially complete rapid biomass accumulation, efficient inorganic carbon assimilation, and oil production. Compared to a single-environment culture system, the biomass yield increased by 1.34 times, the oil content increased by more than 6%, and the oil productivity increased by 2.08 times. Above findings may lay a partial theoretical foundation for the future evolution of traditional refineries towards “fossil–algal-biomass” hybrid refineries.

Response of nitrifiers to gradually increasing pH conditions in a membrane nitrification bioreactor: Microbial dynamics and alkali-resistant mechanism

Nitrification and nitrifiers are pH-sensitive especially under the alkaline environment in the activated sludge system. However, it is unclear how nitrifiers and nitrification respond to long-term alkaline environment. This study employed a continuous flow membrane nitrification bioreactor to investigate the dynamics of nitrification efficiency and microbial community adaptation under a 320-day alkaline operation. Results showed that activated sludge adapted remarkably to a progressive increase in pH from 7.5 to 10.0, achieving robust nitrification with average ammonia removal efficiencies of 96.6 ± 2.2%. Subsequently, an integrated alkali-resistant mechanism of nitrifiers was proposed. Specifically, under the long-term operation of pH 10.0, certain bacteria secreted enhanced extracellular acidic polysaccharides (i.e., up to 10.95 ± 0.27 mg·g−1 MLVSS in soluble extracellular polymeric substances (EPS)) and acidic organic compounds (e.g., humic acids increased by 1.47-fold in tightly bounded EPS) to neutralize external alkalinity. Moreover, significant enrichments in both the ammonia oxidizing bacteria Nitrosomonas (by 1.3%) and the nitrite oxidizing bacteria Nitrospira (by 5.4%) were observed in a 170-day operation of pH 10.0 condition. Meanwhile, norank_f__JG30-KF-CM45 (2.0%) and Rhodobacter (0.9%) also contributed to ammonia removal at pH 10.0. On the cellular-level, bacteria enabled to maintain intracellular pH stabilization primarily through cation/proton antiporters, evidenced by significant increases in NhaA, TrkA and KefB activities by 98.0%, 151.7% and 115.2%, respectively. A 43.1% increase in carbonic anhydrase activity also facilitated consumption of aqueous OH− ions through biomineralization, leading to CaCO3 deposition on microbial surface. These findings further enhanced understandings of physiological adaptation of nitrifiers in the long-term alkaline activated sludge system.

The physiological significance of plasma-accessible carbonic anhydrase in the respiratory systems of fishes.

Carbonic anhydrase (CA) activity is ubiquitously found in all vertebrate species, tissues and cellular compartments. Most species have plasma-accessible CA (paCA) isoforms at the respiratory surfaces, where the enzyme catalyzes the conversion of plasma bicarbonate to carbon dioxide (CO2) that can be excreted by diffusion. A notable exception are the teleost fishes that appear to lack paCA at their gills. The present review: (i) recapitulates the significance of CA activity and distribution in vertebrates; (ii) summarizes the current evidence for the presence or absence of paCA at the gills of fishes, from the basal cyclostomes to the derived teleosts and extremophiles such as the Antarctic icefishes; (iii) explores the contribution of paCA to organismal CO2 excretion in fishes; and (iv) the functional significance of its absence at the gills, for the specialized system of O2 transport in most teleosts; (v) outlines the multiplicity and isoform distribution of membrane-associated CAs in fishes and methodologies to determine their plasma-accessible orientation; and (vi) sketches a tentative time line for the evolutionary dynamics of branchial paCA distribution in the major groups of fishes. Finally, this review highlights current gaps in the knowledge on branchial paCA function and provides recommendations for future work.

Enhanced Recognition Memory through Dual Modulation of Brain Carbonic Anhydrases and Cholinesterases.

This study introduces a novel multitargeting strategy that combines carbonic anhydrase (CA) activators and cholinesterase (ChE) inhibitors to enhance cognitive functions. A series of tacrine-based derivatives with amine/amino acid moieties were synthesized and evaluated for their dual activity on brain CA isoforms and ChEs (AChE and BChE). Several derivatives, notably compounds 26, 30, 34, and 40, demonstrated potent CA activation, particularly of hCA II and VII, and strong ChE inhibition with subnanomolar to low nanomolar IC50 values. In vivo studies using a mouse model of social recognition memory showed that these derivatives significantly improved memory consolidation at doses 10-100 times lower than the reference compounds (either alone or in combination). Molecular modeling and ADMET predictions elucidated the compound binding modes and confirmed favorable pharmacokinetic and safety profiles. The findings suggest that dual modulation of CA and ChE activities is a promising strategy for treating cognitive deficits associated with neurodegenerative and psychiatric disorders.

Comparative study of stability and activity of wild-type and mutant human carbonic anhydrase II enzymes using molecular dynamics and docking simulations

The human carbonic anhydrase II (HCA II) enzyme is a cytosolic protein that catalyzes the reversible hydration of carbon dioxide (CO2). Considering the critical role of the HCA II and the effects of some mutations on the activity and stability of the enzyme in humans, several computational methods are used to study the structure and dynamics of the wild-type and the mutant enzymes with three ligands, CO2, 4-nitrophenyl acetate and acetazolamide. Our results of MD simulation of a wild-type enzyme with 4-nitrophenyl acetate show that it created essential effects on the fluctuation of this enzyme and made it more unstable and less compact than the same enzyme without ligand. In the MD of the mutant enzyme with 4-nitrophenyl acetate ligand, no significant difference is observed between with and without ligand. The affinity of the wild-type enzyme to the 4-nitrophenyl acetate is notably higher than the mutant enzyme with the same ligand. Furthermore, results showed that wild-type and mutant enzymes with CO2 are more favorable in stability and flexibility than the same enzymes without ligand. The MD results of wild-type with acetazolamide indicate instability compare without ligand, but in MD of mutant enzyme with acetazolamide show that it more stable and compact than the same enzyme without ligand. Finally, Comparing protein trajectories to assess the impact of ligands on the stability and activity of HCA II enzymes can have medical applications and can in the engineering and design of new variants of carbonic anhydrase enzyme.

Structure based exploration of mitochondrial alpha carbonic anhydrase inhibitors as potential leads for anti-obesity drug development.

Obesity has emerged as a major health challenge globally in the last two decades. Dysregulated fatty acid metabolism and de novo lipogenesis are prime causes for obesity development which ultimately trigger other co-morbid pathological conditions thereby risking life longevity. Fatty acid metabolism and de novo lipogenesis involve several biochemical steps both in cytosol and mitochondria. Reportedly, the high catalytically active mitochondrial carbonic anhydrases (CAVA/CAVB) regulate the intercellular depot of bicarbonate ions and catalyze the rapid carboxylation of pyruvate and acetyl-co-A to acetyl-co-A and malonate respectively, which are the precursors of fatty acid synthesis and lipogenesis. Several in vitro and in vivo investigations indicate inhibition of mitochondrial carbonic anhydrase isoforms interfere in the functioning of pyruvate, fatty acid and succinate pathways. Targeting of mitochondrial carbonic anhydrase isoforms (CAVA/CAVB) could thereby modulate gluconeogenetic as well as lipogenetic pathways and pave way for designing of novel leads in the development pipeline of anti-obesity medications. The present review unveils a diverse chemical space including synthetic sulphonamides, sulphamates, sulfamides and many natural bioactive molecules which selectively inhibit the mitochondrial isoform CAVA/CAVB with an emphasis on major state-of-art drug design strategies. More than 60% similarity in the structural framework of the carbonic anhydrase isoforms has converged the drug design methods towards the development of isoform selective chemotypes. While the benzene sulphonamide derivatives selectively inhibit CAVA/CAVB in low nanomolar ranges depending on the substitutions on the phenyl ring, the sulpamates and sulpamides potently inhibit CAVB. The virtual screening and drug repurposing methods have also explored many non-sulphonamide chemical scaffolds which can potently inhibit CAVA. The review could pave way for the development of novel and effective anti-obesity drugs which can modulate the energy metabolism.

Preparation and characterization of chitosan nanoparticles with extracts of Rheum ribes, evaluation of biological activities of extracts and extract loaded nanoparticles

The biological activities of different parts of the Rheum ribes plant were evaluated comparatively. Extracts showing strong biological activity were identified and it was determined which of the extract-loaded nanoparticles showed stronger activity. Cytotoxic activity of R. ribes extracts was calculated on glial (C6) and fibroblast (NIH 3T3) cells using XTT assay. Spectrophotometry was used to evaluate the impact of these compounds on the enzyme activities of human carbonic anhydrase I and II (hCA I and hCA II). The findings showed that chitosan NPs with extracts loaded on them have a lower IC50 value and more cytotoxic activity in C6 cells than chitosan NPs with only extracts. R. ribes young shoots ultrasonic methanol extract (RYU) was shown to have the strongest antiproliferative efficacy against C6 cells. Results showed that RYU and ultrasonic methanol extract of R. ribes radix (RRU) were determined as the best carbonic anhydrase inhibitors. According to results of particle size, encapsulation efficiency, and release studies of chitosan NPs, it has been observed that they are suitable for application. At a concentration of 10 µg/mL, it was found that none of the R. ribes extracts exhibited cytotoxic action toward the NIH 3T3 cell line. According to results of particle size, encapsulation efficiency, and release studies of chitosan NPs, it has been observed that they are suitable for application. It was observed that none of the extracts of R. ribes at a concentration of 10 µg/mL showed cytotoxic activity in the NIH 3T3 cell line.

Interaction of Micro- and Nanoplastics with Enzymes: The Case of Carbonic Anhydrase.

Microplastics (MPs) and nanoplastics (NPs) have emerged as significant environmental pollutants with potential detrimental effects on ecosystems and human health. Several studies indicate their interaction with enzymes; this topic represents a multifaceted research field encompassing several areas of interest from the toxicological and ecotoxicological impact of MPs and NPs on humans and wildlife to the biodegradation of plastics by microbial enzymes. This review aims to provide a critical analysis of the state-of-the-art knowledge of the interaction of MPs and NPs on the enzyme carbonic anhydrase (CA), providing recent insights, analyzing the knowledge gaps in the field, and drawing future perspectives of the research and its application. CA is a widespread and crucial enzyme in various organisms; it is critical for various physiological processes in animals, plants, and bacteria. It catalyzes the reversible hydration of CO2, which is essential for respiration, acid-base balance, pH homeostasis, ion transport, calcification, and photosynthesis. Studies demonstrate that MPs and NPs can inhibit CA activity with mechanisms including adsorption to the enzyme surface and subsequent conformational changes. In vitro and in silico studies highlight the role of electrostatic and hydrophobic interactions in these processes. In vivo studies present mixed results, which are influenced by factors like particle type, size, concentration, and organism type. Moreover, the potentiality of the esterase activity of CA for plastic degradation is discussed. The complexity of the interaction between CA and MPs/NPs underscores the need for further research to fully understand the ecological and health impacts of MPs and NPs on CA activity and expression and glimpses of the potentiality and perspectives in this field.

Investigating Active Site Binding of Ligands to High and Low Activity Carbonic Anhydrase Enzymes Using Native Mass Spectrometry.

Carbonic anhydrases (CAs) are a family of enzymes that play an important pH regulatory role in health and disease. While different CA isozymes have a high degree of structural similarity, they have variable enzymatic activity, with CA III being the least active and having less than 1% of the activity of CA II, the most active. Furthermore, ligand binding studies for CA III are limited, and a resulting lack of chemical probes impedes understanding of this CA isozyme in comparison to other CA family members where studies are abundant. Therefore, we employed native mass spectrometry (nMS), also known as intact mass spectrometry, to assess ligand binding to CA II and CA III and discovered two novel compounds that for the first time display strong binding to CA III. We present a new data visualization and quantification tool developed to display native mass spectra as an intuitive stacked heat map representation for rapidly interpreting the results of ligand-protein binding from nMS screening.

Assessing the potential of a genetically modified Parachlorella kessleri-I with low CO2 inducible proteins for enhanced biomass and biofuel productivity

The biological process is one of the promising approaches for CO2 capture and storage. Recently, biological sequestration using microalgae has gained much interest due to its capability to utilize CO2 as a carbon source to generate valorizable biomass. This algal biomass further can be used as a feedstock for bioenergy and different value-added products. Here, we have heterogeneously overexpressed the low CO2 inducible proteins (LCIA & LCIB) from Chlamydomonas reinhardtii in the marine alga Parachlorella kessleri-I. Incorporation of these two genes boosted the endogenous CCM leading to a hybrid system and improved overall carbon acquisition. The combination of these CCM proteins enhanced the supply of inorganic carbon to RuBisCO with positive effects on growth and biomass productivity. Biochemical analyses revealed that the hybrid CCM in P. kessleri-I was characterised by 2- fold higher carbonic anhydrase activity, increased starch, and 2X more lipid yield in comparison to wild type (WT) strain. Moreover, the semi-continuous cultivation of GM strain with supply of 1 % CO2 led to around 60 % rise in the overall biomass productivity. These traits also persisted in scale-up studies leading to 2X more biomass productivity in the GM strain than WT in 100 L PBR. A comparative life-cycle assessment underlines the sustainability of the process of carbon capture and both biofuel and biochar production from P. kessleri-I with a hybrid CCM.

Enhancing Photosynthesis and Plant Productivity through Genetic Modification.

Enhancing crop photosynthesis through genetic engineering technologies offers numerous opportunities to increase plant productivity. Key approaches include optimizing light utilization, increasing cytochrome b6f complex levels, and improving carbon fixation. Modifications to Rubisco and the photosynthetic electron transport chain are central to these strategies. Introducing alternative photorespiratory pathways and enhancing carbonic anhydrase activity can further increase the internal CO2 concentration, thereby improving photosynthetic efficiency. The efficient translocation of photosynthetically produced sugars, which are managed by sucrose transporters, is also critical for plant growth. Additionally, incorporating genes from C4 plants, such as phosphoenolpyruvate carboxylase and NADP-malic enzymes, enhances the CO2 concentration around Rubisco, reducing photorespiration. Targeting microRNAs and transcription factors is vital for increasing photosynthesis and plant productivity, especially under stress conditions. This review highlights potential biological targets, the genetic modifications of which are aimed at improving photosynthesis and increasing plant productivity, thereby determining key areas for future research and development.

Carbonic anhydrase encapsulation using bamboo cellulose scaffolds for efficient CO2 capture and conversion

Utilizing carbonic anhydrase (CA) to catalyze CO2 hydration offers a sustainable and potent approach for carbon capture and utilization. To enhance CA's reusability and stability for successful industrial applications, enzyme immobilization is essential. In this study, delignified bamboo cellulose served as a renewable porous scaffold for immobilizing CA through oxidation-induced cellulose aldehydation followed by Schiff base linkage. The catalytic performance of the resulting immobilized CA was evaluated using both p-NPA hydrolysis and CO2 hydration models. Compared to free CA, immobilization onto the bamboo scaffold increased CA's optimal temperature and pH to approximately 45 °C and 9.0, respectively. Post-immobilization, CA activity demonstrated effective retention (>60 %), with larger scaffold sizes (i.e., 8 mm diameter and 5 mm height) positively impacting this aspect, even surpassing the activity of free CA. Furthermore, immobilized CA exhibited sustained reusability and high stability under thermal treatment and pH fluctuation, retaining >80 % activity even after 5 catalytic cycles. When introduced to microalgae culture, the immobilized CA improved biomass production by ∼16 %, accompanied by enhanced synthesis of essential biomolecules in microalgae. Collectively, the facile and green construction of immobilized CA onto bamboo cellulose block demonstrates great potential for the development of various CA-catalyzed CO2 conversion and utilization technologies.

Genetically engineered whole-cell biocatalyst for efficient CO2 capture by cell surface display of carbonic anhydrase from Bacillus cereus GLRT202 on Escherichia coli

CO2 sequestration is important for reducing greenhouse effects. Carbonic anhydrase (CA) from bacteria has a promising role because it can be modified by genetic techniques and bioengineering. In this study, the CA from B. cereus GLRT202 (Bc-CA) was genetically engineered and anchored on the surface of E. coli by using the N-domain of the ice nucleation protein from P. syringae (INPN). Both surface-displayed and cytosolic Bc-CA yielded high expression levels of CA when induced with 0.5 mM IPTG. It exhibited no adverse influence on the host cell growth. Additionally, surface-displayed Bc-CA enhanced its stability and specificity compared to cytosolic expressed Bc-CA. The CA activity of whole-cell surface-displayed cells was 1.66-fold higher (5.19 U/mL) than that of the cytosolic form. Besides the advantages of higher activity, the whole-cell displaying CA was comparatively stable, with better storage (at 4 ℃) and resting culture stability (at 37 ℃). The whole-cell biocatalyst induced the calcite precipitation, which indicated that the cell facilitated the CO2 capture. XRD, FTIR, and FESEM characterized calcite precipitates thus obtained. This study demonstrates that Bc-CA can be correctly expressed on the E. coli surface through fusion with the INPN. This leads to an effective whole-cell biocatalyst with enhanced stability and specificity of the enzyme for efficient CO2 capture applications.

Zinc-based organophyllosilicate nanosheets as novel carbonic anhydrase-like nanozyme for efficient CO2 fixation

The unparalleled catalytic activity of carbonic anhydrases (CAs) towards CO2 hydrolysis can potentially contribute to carbon capture, utilization, and storage technology aimed at global decarbonization; however, the high cost and low stability of CAs significantly limit their practical applications. Recently, the development of robust and effective nanozymes with CA-mimicking activity has been proposed as a promising solution to this challenge. Herein, a novel zinc-based organophyllosilicate (ZnAC) nanozyme with CA-like catalytic activity was synthesized by a facile one-pot sol–gel method for the first time, leading to a Zn-O coordination structure that mimicked the catalytic center fragments of natural CAs, with surface amino groups acting as the affinity sites and proton shuttles. The synergistic effect of the two components on the open surface of the layered structure resulted in excellent CA-like activity with a Michaelis–Menten constant (Km) of 4.071 mM, maximum velocity (Vmax) of 0.220 mM/s, and turnover number (kcat) of 11.213 /s at 25 °C and pH=7.5, which were superior to those of most previously reported CA-like nanozymes. The catalytic performance of ZnAC significantly improved with increasing pH and temperature; furthermore, ZnAC exhibited excellent stability, maintaining high catalytic activity after the exposure to extreme temperatures or pH values, eight reaction cycles, and 30 d of storage. Additionally, ZnAC significantly increased the rate of CO2 hydration and the extent of CO2 mineralization and precipitation. This work proposes a new strategy for designing and constructing efficient CA-like nanozymes, providing valuable insights into the structure–activity relationship and new perspectives for the implementation of CO2 fixation.

New Thiazole Derivatives: Carbonic Anhydrase I–II and Cholinesterase Inhibition Profiles, Molecular Docking Studies

AbstractThiosemicarbazone derivative was obtained by the addition of thiosemicarbazide to 5‐nitro‐thiophene‐2‐carbaldehyde. The addition–cyclization of the 2‐bromoacetophenone derivative to thiosemicarbazone derivative gave the new thiazole derivatives (2 a‐k). Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), antioxidant, and human carbonic anhydrase (hCA) I and II isoform inhibitory activities of the thiazole derivatives 2 a‐k were investigated. The effects of thiazole derivatives on carbonic anhydrase I and II (hCA I–II) isoenzymes were examined in vitro using the esterase method. IC50 values for enzyme inhibition were found to be 2.661–22.712 μM for hCA I and 5.372–26.813 μM for hCA II. All derivatives reduced the activities of carbonic anhydrase I and II isoenzymes and were new potential inhibitor molecule candidates. These compounds were found to have minimal effects on AChE and BChE. The antioxidant properties of the target compounds were determined using DPPH and ferric ion‐chelating assays. In particular, compounds 2 k and 2 h had the highest antioxidant effect in the series with IC50 values of 30.11±0.008 μM and 30.21±0.006 μM, respectively. ADMET properties of the compounds found to be effective were evaluated and their interactions with the enzyme were determined by molecular docking.

A novel heat-inducible plasmid-driven T7 system for enhancing carbonic anhydrase in engineered Escherichia coli strains

Over the past decades, the heat-inducible (HI) promoter, derived from cI857 phage system has been widely utilized. However, the developments of alternative temperature-controlled promoters are limited. In this study, we explored a novel HI promoter through PCR amplification using the degenerate primer design. BLAST analysis identified this sequence as a partial plsB on Escherichia coli chromosome. Antisense RNAs targeting distinct regions of HI promoter were applied to examine the mechanism. The fluorescence of super-folder green fluorescent protein (sfGFP) driven by the HI promoter showed 5.5-fold increase from 30 to 42 °C, whereas the truncated HI yielded a trace amount of sfGFP. Among various E. coli strains, BL21 exhibited the highest fluorescence, reaching 3976 a.u. at 42 °C. Subsequently, the novel HI promoter was employed to drive T7RNA polymerase within a plasmid-driven T7 (PDT7) plasmid, serving its capability to express sfGFP and carbonic anhydrases (CA), respectively. The maximum intensity of sfGFP reached 38,702 a.u., and CA activity surged to 14286 WAU/mL in W3110 among other strains. Finally, the highest CA activity was 27,521 WAU/mL at pH 9. The promising results from the novel heat-inducible promoter-driven T7RNAP, incorporating the T7 terminator as HI-PDT7-TT, demonstrate potential for expressing more heterogeneous proteins across various chassis in the future.

A study of bacteria producing carbonic anhydrase enzyme for CaCO3 precipitation and soil biocementation

We study the carbonic anhydrase (CA) pathway using autochthonous CA-producing bacteria as a means of inducing calcite precipitation, which acts as a biocement to improve the engineering soil properties. Forty different microbial strains producing CA were isolated from the foundation soil of a railway embankment in Prickwillow, UK. Three of the best CA-producing strains were selected and identified by DNA sequencing as Bacillus licheniformis, Bacillus toyonensis and Bacillus pumilus with CA activity values respectively of 1.79 U/ml, 1.42 U/ml and 1.55 U/ml. To optimise the treatments, we investigated the effect of pH, temperature, zinc co-factor and cementation solution molarity on the growth and CA activity and bioprecipitates, with CO2 added in the form of bicarbonate. Scanning electron microscope (SEM) analysis of the bioprecipitates showed that these had characteristic morphologies of calcite and vaterite crystals. The formation of calcite was further corroborated by FT-IR and Raman analysis of bioprecipitates. The precultured bacteria were injected into the fine-grained soil together with cementation solution. Unconfined compressive strength in treated soil increased up to 1 MPa, and its calcium carbonate content increased by 2.78%. This, as well as the stability of the treated soil upon water immersion, proved the biocementation of the fine-grained soil. These findings suggest the potential of employing the CA biocementation route for soil stabilisation pending further development of the technique.

Carbonyl Sulfide (COS) in Terrestrial Ecosystem: What We Know and What We Do Not

Over the past six decades, carbonyl sulfide (COS) in terrestrial ecosystems has been extensively studied, with research focusing on exploring its ecological and environmental effects, estimating source–sink volume, and identifying influencing factors. The global terrestrial COS sink has been estimated to be about 1.194–1.721 Tg a−1, with the terrestrial sink induced by plants and soils 0.50–1.20 Tg a−1, accounting for 41%–69% of the total. Hence, the role of plants and soils as COS sinks has been extensively explored. Now we know that factors such as the activity of carbonic anhydrase (CA), leaf structural traits, soil microbial activity, and environmental factors play significant roles in the COS budget. Developments in observational techniques have also made important contributions to the COS budget. This paper provides an overview of the research progress made on COS based on a comprehensive review of the literature. Then, it highlights the current research hotspots and issues requiring further exploration. For instance, it has been demonstrated that there are still significant uncertainties in the estimation of COS sources and sinks, emphasizing the need for further exploration of COS measuring techniques. This review aims to provide comprehensive guidance for COS research in terrestrial ecosystems.

Elucidating the role of primary and secondary sphere Zn2+ ligands in the cyanobacterial CO2 uptake complex NDH-14: The essentiality of arginine in zinc coordination and catalysis

Inorganic carbon uptake in cyanobacteria is facilitated by an energetically intensive CO2-concentrating mechanism (CCM). Specialized Type-1 NDH complexes function as a part of this mechanism to couple photosynthetic energy generated by redox reactions of the electron transport chain (ETC) to CO2 hydration. This active site of CO2 hydration incorporates an arginine side chain as a Zn ligand, diverging from the typical histidine and/or cysteine residues found in standard CAs. In this study, we focused on mutating three amino acids in the active site of the constitutively expressed NDH-14 CO2 hydration complex in Synechococcus sp. PCC7942: CupB-R91, which acts as a zinc ligand, and CupB-E95 and CupB-H89, both of which closely interact with the arginine ligand. These mutations aimed to explore how they affect the unusual metal ligation by CupB-R91 and potentially influence the unusual catalytic process. The most severe defects in activity among the targeted residues are due to a substitution of CupB-R91 and the ionically interacting E95 since both proved essential for the structural stability of the CupB protein. On the other hand, CupB-H89 mutations show a range of catalytic phenotypes indicating a role of this residue in the catalytic mechanism of CO2-hydration, but no evidence was obtained for aberrant carbonic anhydrase activity that would have indicated uncoupling of the CO2-hydration activity from proton pumping. The results are discussed in terms of possible alternative CO2 hydration mechanisms.

Co-removal and recycling of Ba2+ and Ca2+ in hypersaline wastewater based on the microbially induced carbonate precipitation technique: Overlooked Ba2+ in extracellular and intracellular vaterite

This study investigates the co-precipitation of calcium and barium ions in hypersaline wastewater under the action of Bacillus licheniformis using microbially induced carbonate precipitation (MICP) technology, as well as the bactericidal properties of the biomineralized product vaterite. The changes in carbonic anhydrase activity, pH, carbonate and bicarbonate concentrations in different biomineralization systems were negatively correlated with variations in metal ion concentrations, while the changes in polysaccharides and protein contents in bacterial extracellular polymers were positively correlated with variations in barium concentrations. In the mixed calcium and barium systems, the harvested minerals were vaterite containing barium. The increasing concentrations of calcium promoted the incorporation and adsorption of barium onto vaterite. The presence of barium significantly increased the contents of O-CO, N-CO, and Ba-O in vaterite. Calcium promoted barium precipitation, but barium inhibited calcium precipitation. After being treated by immobilized bacteria, the concentrations of calcium and barium ions decreased from 400 and 274 to 1.72 and 0 mg/L (GB/T15454–2009 and GB8978–1996). Intracellular minerals were also vaterite containing barium. Extracellular vaterite exhibited bactericidal properties. This research presents a promising technique for simultaneously removing and recycling hazardous heavy metals and calcium in hypersaline wastewater.