Carbonic Anhydrase Activity Research Articles

Effects of HCO3− and CO2 conversion rates on carbon assimilation strategies in marine microalgae: Implication by stable carbon isotope analysis of fatty acids

Marine microalgae are an essential component of marine plankton and critical primary producers, playing a vital role in marine ecosystems. The seawater carbonate system is a dynamic equilibrium system, and changes in any component can alter the carbonate balance. In CO2-concentrating mechanisms (CCMs), carbonic anhydrase (CA) regulates CO2 concentration by catalyzing the interconversion between CO2 and HCO3−. Therefore, limiting the activity of extracellular carbonic anhydrase (exCA) alters the rate at which carbonate equilibrium is reached and further affects the carbon assimilation process in microalgae. In this study, two different microalgae, Phaeodactylum tricornutum and Nannochloropsis oceanica, were selected to investigate the effects of changes in the carbonate system on photosynthetic carbon assimilation in microalgae by inhibiting exCA activity with acetazolamide (AZ). Inhibition of exCA activity reduces specific growth rates and photosynthetic efficiency of microalgae. The total alkalinity, HCO3− concentration, and CO2 concentration of the cultures increased with the decrease of pH, but the changes of the ribulose 1,5- bisphosphate carboxylase/oxygenase (Rubisco) activities of the two microalgae were different. In addition, the two microalgae possessed different lipid and carbohydrate synthesis strategies, but both restricted triacylglycerol (TAG) synthesis. Meanwhile, the microalgal cells had to utilize more 13CO2 when HCO3− and CO2 conversion rates were limited and restricted. This led to the continuous accumulation of 13C in fatty acids and the elevation of δ13CFAs. In conclusion, our study provides a new perspective on the role of microalgae in correlating carbonate changes with photosynthetic carbon assimilation strategies under mechanistic constraints on inorganic carbon utilization.

Morphometric and Enzymatic Changes in Gills of Rainbow Trout after Exposure to Elevated Temperature-Indications for Gill Remodeling.

Seven-month-old rainbow trout acclimated to 9 °C were used. The fish were gradually adapted to a water temperature of 20 °C over a period of seven days and then exposed to this temperature for 32 days. Changes in gill morphometry and histology and in enzyme activities in comparison to fish kept at 9 °C were investigated. No histopathological abnormalities were discerned at the heightened temperature. The gill epithelium thickened by approximately 40%, suggesting an increase in the branchial diffusion barrier for ions, water, and gases. Concurrently, there was a significant decrease in the activities of gill H+-ATPase and Na+/K+-ATPase, indicative of a reduction in osmoregulation under elevated temperatures. Carbonic anhydrase activity exhibited an increase following the 32-day exposure to 20 °C, potentially mitigating the adverse effects of increased gill epithelium thickness on gaseous exchange. There were no indications of gill surface enlargement as the measurements of the length of the primary and secondary lamellae, as well as of the distances between them, were similar at 9 and 20 °C. The activities of the gill enzymes associated with glycolysis and the citric acid cycle displayed a varied response following the 32-day exposure of rainbow trout to 20 °C. Pyruvate kinase decreased, while lactate dehydrogenase increased, and malate dehydrogenase remained constant. This might suggest a decrease in the glycolytic rate, a greater reliance on anaerobic pathways at 20 °C compared to 9 °C, and the consistent efficiency of the citric acid cycle in the gills of rainbow trout in response to elevated temperatures. In summation, the data suggest a remodeling of rainbow trout gills in response to elevated temperatures, affecting both morphometric and metabolic aspects.

RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring

BackgroundRibulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity.ResultsWe simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO2 and NaHCO3, and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO2 were 5–7 times higher than the ones grown with 20 mM NaHCO3, at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO3− to CO2. The highest RuBisCO activity of 2.13 nmol of NAD+/ µg of Chl-a/ min was observed with 50 µg of protein and 5% CO2. Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited ~ 3.4-fold slower reaction rate (0.37 min−1) than the biochemical assay when using 5% CO2. We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO3 as compared to the cells that were grown without NaHCO3 supplementation.ConclusionsOverall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

An Evidence of Carbonic Anhydrase Activity in Native Microalgae for CO2 Capture Application.

A promising alternative for effective carbon capture has been found in microalgae because of their high photosynthetic capacity and quick growth. The carbon concentration mechanism of many microalgae is heavily reliant on the enzyme carbonic anhydrase (CA), which catalyze the production of bicarbonate from carbon dioxide. In this study, microalgal samples were collected, characterized, and cultured under controlled conditions for their optimal growth of cultures I-IX. The CA activity was investigated using a standard method; the Wilbur-Anderson assay was used to calculate CA activity in microalgal cultures. The comparative study was then used to measure the activity rate of the collected microalgae. Among the tested, culture I, VI, and IX showed a high enzyme activity rate of 4.15, 4.0, and 4.2µg·mL-1, respectively. To determine the rate of carbon dioxide hydration, the method involved tracking the pH change in a reaction mixture. In addition, genetic analysis facilitates the identification of key genes involved in CA activity and other metabolic processes, which enhance the knowledge of microalgal physiology, and enables genetic engineering efforts in the future studies. Overall, this investigation emphasizes the significance of studying unknown microalgal culture and their potential CA activity for industrial and bio-energy applications.

Synergistic bimetallic nanozymes of Ni/ZIF-8 and Cu/ZIF-8 as carbonic anhydrase mimics

The remarkable diversity of metal nodes and ligands, coupled with the extensive array of connection modes, enables the precise design and assembly of metal-organic frameworks (MOFs), which have garnered significant attention in the field of nanozyme. In this study, the synthesis of zeolite imidazole framework materials (ZIFs) with varying Zn2+ and Ni2+ or Cu2+ concentrations were investigated and regulated by methanol, aiming to mimic the CO2 hydration activity of carbonic anhydrase (CA). The materials were comprehensively characterized using TEM, XRD, FTIR, UV-Vis DRS, XPS, and subsequently the catalytic performance of the materials was assessed. The results demonstrated that the structural characteristics of bimetallic Ni/ZIF-8 and Cu/ZIF-8 were influenced by varying concentrations of metal ions. Notably, ZIF-8 doped with lower metal content exhibited enhanced stability, while a precursor containing nickel and copper ions exceeding 50% hindered the formation of the desired ZIFs, resulting in an unstable cluster structure. However, methanol can counteract the inhibitory effect of metal ions on the structure. The bimetallic synergies of doped metals in ZIFs CA mimics can simultaneously expedite both two rate-limiting steps in the CO2 hydration process. The esterase activity of ZIF-8 doped with a small quantity of Ni2+ or Cu2+ can be achieved at a level comparable to that of human carbonic anhydrase (hCA II) at 80 °C, along with remarkable catalytic performance and hydrothermal stability. Specifically, Cu66%/ZIF-8 exhibited an activity of 0.31 U·mg−1 at 25 °C, which was 2.31 times higher than that of ZIF-8, moreover, the esterase activity at 80 °C was approximately 2.21 times greater than that of ZIF-8 under the same condition. While the relative activity of Ni50%/ZIF-8 and Cu50%/ZIF-8 still exceeding 50% even after five repeated utilization cycles. The bimetallic Ni/ZIF-8 and Cu/ZIF-8 can be easily synthesized using a simple method, resulting in enhanced catalytic activity. Therefore, it holds great potential for future applications in CO2 capture.

Unraveling non-linear dynamics of biomass, photosynthesis efficiency, and lipid accumulation in Chlorella vulgaris under mixotrophic cultivation

In recent years, the potential of microalgae-based biofuels as a renewable energy source has garnered significant attention due to their inherent ability to sequester carbon dioxide (CO2) efficiently. This study investigates Chlorella vulgaris FACHB-8's growth, photosynthesis, and lipid accumulation under different carbon source and CO2 concentration conditions with Central composite design. Results reveal that a 10% CO2 concentration enhances biomass and cell count, yielding 0.94 g L−1 and 106.8 × 106 cells·ml−1, respectively, after 14 days of autotrophic cultivation. However, excessive CO2 inhibits growth, notably observed with 20% CO2 yielding 0.86 g L−1 biomass. Pigment content positively correlates with biomass under autotrophic conditions, reaching 42.55 mg g−1 in the 10% CO2 group. Under photosynthetic mixotrophy, carbonic anhydrase activity decreases with higher CO2 concentrations, suggesting reduced enzyme demand. Glucose exhibits the highest lipid content (31.87%), surpassing autotrophic conditions (24.80%). Glycerol presents favorable lipid content (31.78%) and settling efficiency (85.07%), indicating potential for self-flocculation harvesting. Chlorophyll fluorescence data highlight active photosynthesis in mixotrophic conditions, with glucose and sucrose affecting energy conversion. This highlights the critical role of microalgae biofuels in the pursuit of cleaner and more sustainable energy solutions.

Molecular characterization of a carbon dioxide-dependent Proteus mirabilis small-colony variant isolated from a clinical specimen

IntroductionCarbon dioxide-dependent Proteus mirabilis has been isolated from clinical specimens. It is not clear whether mutations in carbonic anhydrase are responsible for the carbon dioxide dependence of P. mirabilis. The pathogenicity of carbon dioxide-dependent P. mirabilis also remains unclear. The purpose of this study was to determine the cause carbon dioxide dependence of P. mirabilis and its pathogenicity. MethodsThe DNA sequence of can encoding carbonic anhydrase of a carbon dioxide-dependent P. mirabilis small colony variant (SCV) isolate was analyzed. To confirm that impaired carbonic anhydrase activity is responsible for the formation of the carbon dioxide-dependent SCV phenotype of P. mirabilis, we performed complementation experiments using plasmids with intact can. Additionally, mouse infection experiments were performed to confirm the change in virulence due to the mutation of carbonic anhydrase. ResultsWe found that the can gene of the carbon dioxide-dependent P. mirabilis SCV isolate showed had a frameshift mutation with a deletion of 1 bp (c. 173delC). The can of P. mirabilis encodes carbonic anhydrase was also found to function in Escherichia coli. The cause of the carbon dioxide-dependent SCV phenotype of P. mirabilis was an abnormality in carbonic anhydrase. Nevertheless, no changes were observed in virulence due to the mutation of carbonic anhydrase in mouse infection experiments. ConclusionsThe can gene is essential for the growth of P. mirabilis in ambient air. The mechanisms underlying this fitness advantage in terms of infection warrant further investigation.

Synthesis and in vitro carbonic anhydrase and acetylcholinesterase inhibitory activities of novel hydrazide–hydrazone compounds containing 1,2,4-triazole ring

In this work, 1,2,4-triazole derived hydrazide–hydrazones were synthesized with a five-step synthesis pathway. The novel derivatives were characterized by various analytical methods. Acetylcholinesterase (AChE), and human carbonic anhydrase (hCA) inhibitory qualifications of these thirteen original compounds (4, 5a–l) were also determined. The overall biological activity results were very interesting. The reported compounds were found to be very effective inhibitors of hCA I, hCA II, and AChE enzymes with IC50 values in the range of 17.33−77.00 nM for hCA I (Acetazolamide IC50: 62.80 nM), 13.07−46.20 nM for hCA II (Acetazolamide IC50: 57.75 nM) and 4.91−36.47 nM for AChE (Tacrine IC50: 28.88 nM). Within the new compounds, 5i displayed the best hCA I, hCA II and AChE inhibitory activities with IC50 values of 17.33 nM, 13.07 nM, and 4.91 nM, respectively.

Terrestrial algae: pioneer organisms of carbonate rock solutional weathering in South China karst.

The formation of soil in karst ecosystem has always been a scientific problem of great concern to human beings. Algae can grow on the exposed and non-nutrition carbonate surface, inducing and accelerating weathering of rock substrates, thus promoting soil formation. Yet the actual contribution of algae to solutional weathering intensity remains unclear. In this study, we performed weathering simulation experiment on two algae species (Klebsormidium dissectum (F.Gay) H.Ettl & G.Gärtner and Chlorella vulgaris Beijerinck), which were screened from carbonated rock surfaces from a typical karst region in South China. The results showed: (1) both algae have solutional weathering effect on carbonate rock, (2) there is no difference of solutional intensity observed, yet the solutional modes are different, suggesting different ecological adaptative strategies, (3) algae on carbonate rocks have higher carbonic anhydrase activity (CAA) and secrete more extracellular polysaccharide (EPS), accelerating rock weathering. (4) The absolute dissolution amount of carbonate rock with algae participation is 3 times of that of without algae. These results indicate the significant impact of terrestrial algae on carbonate rock solutional weathering and provides quantitative evidence that terrestrial algae are pioneer species. It also contributes to our further understanding of soil formation in karst ecosystems in South China.

Hedgehog Pathway Inhibition by Novel Small Molecules Impairs Melanoma Cell Migration and Invasion under Hypoxia.

Melanoma is the principal cause of death in skin cancer due to its ability to invade and cause metastasis. Hypoxia, which characterises the tumour microenvironment (TME), plays an important role in melanoma development, as cancer cells can adapt and acquire a more aggressive phenotype. Carbonic anhydrases (CA) activity, involved in pH regulation, is related to melanoma cell migration and invasion. Furthermore, the Hedgehog (Hh) pathway, already known for its role in physiological processes, is a pivotal character in cancer cell growth and can represent a promising pharmacological target. In this study, we targeted Hh pathway components with cyclopamine, glabrescione B and C22 in order to observe their effect on carbonic anhydrase XII (CAXII) expression especially under hypoxia. We then performed a migration and invasion assay on two melanoma cell lines (SK-MEL-28 and A375) where Smoothened, the upstream protein involved in Hh regulation, and GLI1, the main transcription factor that determines Hh pathway activation, were chemically inhibited. Data suggest the existence of a relationship between CAXII, hypoxia and the Hedgehog pathway demonstrating that the chemical inhibition of the Hh pathway and CAXII reduction resulted in melanoma migration and invasion impairment especially under hypoxia. As in recent years drug resistance to small molecules has arisen, the development of new chemical compounds is crucial. The multitarget Hh inhibitor C22 proved to be effective without signs of cytotoxicity and, for this reason, it can represent a promising compound for future studies, with the aim to reach a better melanoma disease management.

Strigolactone and nitric oxide collaborate synergistically to boost tomato seedling resilience to arsenic toxicity via modulating physiology and antioxidant system

Arsenic (As) poses a significant environmental threat as a metalloid toxin, adversely affecting the health of both plants and animals. Strigolactones (SL) and nitric oxide (NO) are known to play crucial roles in plant physiology. Therefore, the present experiment was designed to investigate the potential cumulative role of SL (GR24–0.20 μM) and NO (100 μM) in mitigating the adverse effect of AsV (53 μM) by modulating physiological mechanisms in two genotypes of tomato (Riogrand and Super Strain 8). A sample randomized design with four replicates was used to arrange the experimental pots in the growth chamber. 45-d old both tomato cultivars under AsV toxicity exhibited reduced morphological attributes (root and shoot length, root and shoot fresh weight, and root and shoot dry weight) and physiological and biochemical characteristics [chlorophyll (Chl) a and b content, activity of δ-aminolevulinic acid dehydratase activity (an enzyme responsible for Chl biosynthesis), and carbonic anhydrase activity (an enzyme responsible for photosynthesis), and enhanced Chl degradation, overproduction of reactive oxygen species (ROS) and lipid peroxidation due to enhanced malondialdehyde (MDA) content. However, the combined application of SL and NO was more effective in enhancing the tolerance of both varieties to AsV toxicity compared to individual application. The combined application of SL and NO improved growth parameters, biosynthesis of Chls, NO and proline. However, the combined application significantly suppressed cellular damage by inhibiting MDA and overproduction of ROS in leaves and roots, as confirmed by the fluorescent microscopy study and markedly upregulated the antioxidant enzymes (catalase, peroxidase, superoxide dismutase, ascorbate dismutase and glutathione reductase) activity. This study provides clear evidence that the combined application of SL and NO supplementation significantly improves the resilience of tomato seedlings against AsV toxicity. The synergistic effect of SL and NO was confirmed by the application of cPTIO (an NO scavenger) with SL and NO. However, further molecular studies could be imperative to conclusively validate the simultaneous role of SL and NO in enhancing plant tolerance to abiotic stress.

Optimizing light regimes for neutral lipid accumulation in Dunaliella salina MCC 43: a study on physiological status and carbon allocation.

Dunaliella salina is a favourable source of high lipid feedstock for biofuel and medicinal chemicals. Low biomass output from microalgae is a significant barrier to industrial-scale commercialisation. The current study aimed to determine how photosynthetic efficiency, carbon fixation, macromolecular synthesis, accumulation of neutral lipids, and antioxidative defence (ROS scavenging enzyme activities) of D. salina cells were affected by different light intensities (LI) (50, 100, 200, and 400µmolm-2s-1). The cells when exposed to strong light (400µmolm-2s-1) led to reduction in chlorophyll a but the carotenoid content increased by 19% in comparison to the control (LI 100). The amount of carbohydrate changed significantly under high light and in spite of stress inflicted on the cells by high irradiation, a considerable increase in activity of carbonic anhydrase and fixation rate of CO2 were recorded, thus, preserving the biomass content. The high light exposed biomass when subjected to nitrogen-deficient medium led to increase in lipid content (59.92% of the dry cell weight). However, neutral lipid made up 78.26% of the total lipid while other lipids like phospholipid and glycolipid content decreased, showing that the lipid was redistributed in these cells under nitrogen deprivation, making the organism more appropriate for biodiesel/jet fuel use. Although D. salina cells had a relatively longer generation time (3.5 d) than other microalgal cells, an economic analysis concluded that the amount of carotenoid they produced and the quality of their lipids made them more suited for commercialization.

Bacterial carbonic anhydrase-induced carbonates mitigate soil erosion in biological soil crusts

Soil erosion is a significant environmental issue worldwide, particularly in island regions where land resources are exceedingly scarce. Biological soil crusts play a crucial role in mitigating soil erosion, yet the precise effect and mechanism of biological soil crusts against erosion remain ambiguous. In this study, biological soil crusts at various developmental stages from a tropical coral island in the South China Sea were chosen to investigate the role of carbonic anhydrase in mitigating erosion. A cohesive strength meter, real-time quantitative PCR, and 16S rRNA gene high-throughput sequencing were employed to assess variations in soil antiscouribility as well as bacterial abundance and composition during the formation and development of biological soil crusts. Scanning electron microscopy was utilized to detect carbonates induced by bacterial carbonic anhydrase and elucidate their role in the solidification of sand particles. The findings indicate that the formation and development of biological soil crusts significantly enhance anti-scouribility. Comparison to those of bare coral sand, the shear stress increased from 0.35 to 1.11 N/m2 in the dark biocrusts. Moreover, significantly elevated carbonic anhydrase activity was observed in biological soil crusts, demonstrating a positive correlation with antiscouribility. In addition, there was a significant increase in bacterial abundance within the biological soil crusts. The enrichment of Cyanobacteriales and Chloroflexales potentially contributed to the increased carbonic anhydrase activity and antiscouribility. Furthermore, three cyanobacterial strains with carbonic anhydrase activity were isolated from biological soil crusts and subsequently confirmed to enhance sand solidification through microbial carbonate precipitation. This study presents initial evidence for the role of microbial carbonic anhydrase in enhancing the antiscouribility of biological soil crusts during their formation and development. These findings offer novel insights into the functional and mechanistic dimensions underlying the mitigation of soil erosion facilitated by biological soil crusts, which are valuable for implementing sustainable biorestoration and environmental management technologies to prevent soil erosion.

CAH3 from Chlamydomonas reinhardtii: Unique Carbonic Anhydrase of the Thylakoid Lumen.

CAH3 is the only carbonic anhydrase (CA) present in the thylakoid lumen of the green algae Chlamydomonas reinhardtii. The monomer of the enzyme has a molecular weight of ~29.5 kDa with high CA activity. Through its dehydration activity, CAH3 can be involved either in the carbon-concentrating mechanism supplying CO2 for RuBisCO in the pyrenoid or in supporting the maximal photosynthetic activity of photosystem II (PSII) by accelerating the removal of protons from the active center of the water-oxidizing complex. Both proposed roles are considered in this review, together with a description of the enzymatic parameters of native and recombinant CAH3, the crystal structure of the protein, and the possible use of lumenal CA as a tool for increasing biomass production in higher plants. The identified involvement of lumenal CAH3 in the function of PSII is still unique among green algae and higher plants and can be used to understand the mechanism(s) of the functional interconnection between PSII and the proposed CA(s) of the thylakoid lumen in other organisms.

Foliar application of cyanobacterial formulations stimulates plant growth and fruit quality in tomato under protected cultivation

ABSTRACT Cyanobacterial biofertilizers enhance plant and soil productivity across crops, through soil-seed application at the field level; however, their use as foliar treatment is less investigated. This study evaluated the role of foliar sprays, initiated 4 weeks after transplantation of nursery-raised seedlings in beds, and effected at weekly intervals for a period of eight weeks. towards enhancing plant growth, yield and quality of tomato grown in a climate-controlled polyhouse. Sprays were composed of aqueous formulations of carrier alone, and those amended with the formulations of plant-growth-promoting cyanobacterium Anabaena laxa (A. laxa) or biofilm Anabaena torulosa -Trichoderma viride (An-Tr) or suspension of 100 µM Fe EDTA. A. laxa brought about a significant increase in nitrate reductase and glutamine synthetase activity in leaves by 13%–17%, along with 3-fold stimulation in carbonic anhydrase activity. Cyanobacterium-amended foliar agents significantly enhanced soil dehydrogenase, urease activity, along with increments in organic carbon, Fe, Zn in soil and stimulated yield, fresh weight and quality of fruits. Both cyanobacterial formulations were promising in enhancing plant growth, fruit yield and quality, however, A. laxa also significantly improved microbiological activity and nutrient enrichment in soil. Future research is focused towards optimising the dose and storage life of such foliar agents for protected horticulture.

Mn2+ recycling in hypersaline wastewater: unnoticed intracellular biomineralization and pre-cultivation of immobilized bacteria.

Microbially induced manganese carbonate precipitation has been utilized for the treatment of wastewater containing manganese. In this study, Virgibacillus dokdonensis was used to remove manganese ions from an environment containing 5% NaCl. The results showed a significant decrease in carbonic anhydrase activity and concentrations of carbonate and bicarbonate ions with increasing manganese ion concentrations. However, the levels of humic acid analogues, polysaccharides, proteins, and DNA in EPS were significantly elevated compared to those in a manganese-free environment. The rhodochrosite exhibited a preferred growth orientation, abundant morphological features, organic elements including nitrogen, phosphorus, and sulfur, diverse protein secondary structures, as well as stable carbon isotopes displaying a stronger negative bias. The presence of manganese ions was found to enhance the levels of chemical bonds O-C=O and N-C=O in rhodochrosite. Additionally, manganese in rhodochrosite exhibited both + 2 and + 3 valence states. Rhodochrosite forms not only on the cell surface but also intracellularly. After being treated with free bacteria for 20days, the removal efficiency of manganese ions ranged from 88.4 to 93.2%, and reached a remarkable 100% on the 10th day when using bacteria immobilized on activated carbon fiber that had been pre-cultured for three days. The removal efficiency of manganese ions was significantly enhanced under the action of pre-cultured immobilized bacteria compared to non-pre-cultured immobilized bacteria. This study contributes to a comprehensive understanding of the mineralization mechanism of rhodochrosite, thereby providing an economically and environmentally sustainable biological approach for treating wastewater containing manganese.

Bacterial ι-CAs.

Recent research has identified a novel class of carbonic anhydrases (CAs), designated ι-CA, predominantly found in marine diatoms, eukaryotic algae, cyanobacteria, bacteria, and archaea genomes. This class has garnered attention owing to its unique biochemical properties and evolutionary significance. Through bioinformatic analyses, LCIP63, a protein initially annotated with an unknown function, was identified as a potential ι-CA in the marine diatom Thalassiosira pseudonana. Subsequent biochemical characterization revealed that LCIP63 has CA activity and its preference for manganese ions over zinc, indicative of evolutionary adaptation to marine environments. Further exploration of bacterial ι-CAs, exemplified by Burkholderia territorii ι-CA (BteCAι), demonstrated catalytic efficiency and sensitivity to sulfonamide and inorganic anion inhibitors, the classical CA inhibitors (CAIs). The classification of ι-CAs into two variant types based on their sequences, distinguished by the COG4875 and COG4337 domains, marks a significant advancement in our understanding of these enzymes. Structural analyses of COG4337 ι-CAs from eukaryotic microalgae and cyanobacteria thereafter revealed a distinctive structural arrangement and a novel catalytic mechanism involving specific residues facilitating CO2 hydration in the absence of metal ion cofactors, deviating from canonical CA behavior. These findings underscore the biochemical diversity within the ι-CA class and highlight its potential as a target for novel antimicrobial agents. Overall, the elucidation of ι-CA properties and mechanisms advances our knowledge of carbon metabolism in diverse organisms and underscores the complexity of CA evolution and function.

Insight into the activation mechanism of carbonic anhydrase(II) through 2-(2-aminoethyl)-pyridine: a promising pathway for enhanced enzymatic activity.

Activation of human carbonic anhydrase II (hCA II) holds great promise for treating memory loss symptoms associated with Alzheimer's disease. Despite its importance, the activation mechanism of hCA II has been largely overlooked in favor of the well-studied inhibition mechanism. To address this unexplored realm, we use first-principles calculations to tease out the activation mechanism of hCA II using 2-(2-aminoethyl)-pyridine (2-2AEPy), a promising in vitro activator. We explored both stepwise and concerted mechanisms via both available nitrogen sites of 2-2AEPy: (i) aminoethyl group (Nα) and (ii) pyridine ring (Nβ). Our results show that a concerted mechanism via Nα holds the key to hCA II activation. The activation process of the concerted mechanism exhibits the characteristics of an exergonic reaction, wherein the transition state resembles the reactant with a notably low imaginary frequency of 452.4i cm-1 and barrier height of 5.2 kcal mol-1. Such meager transition barriers propel the activation of hCA II at in vivo temperatures. These findings initiate future research into hCA II activation mechanisms and the development of efficient activators, which may lead to promising therapeutic interventions for Alzheimer's disease.

Osmoregulatory Plasticity in Oreochromis niloticus-Nile Tilapia, and it’s Possible Estuary Use for Rivers Dispersion

Nile tilapia is a widely farmed species globally, due to its resistance to environmental challenges. Additionally, it is a generalist consumer and a prolific breeder. In Brazil, this fish is recognized as an invasive species because of its high dispersion and establishment rates, which compromise native biota. Estuarine waters (0.06 - 32‰) are being used in tilapia aquaculture, which is a promisor fish farming for euryahalinity species. The tilapia’s euryhalinity is well-know, but most protocols are based on gradual exposure. This research showed the tolerance of Nile tilapia on abrupt exposure to 0‰ (FW), and 10, 20, 30, 35 or 40‰, over in 6, 12 and 24 hours. An abrupt exposure resistance demonstrate that this fish can use estuaries to disperse for other watersheds. After experimental design the blood sample was taken after anesthesia was administered. Subsequently, fish was euthanized by spinal section, for muscle and gills samples collections. All samples were keep freezing until ionic, dehydration and enzyme measurement. Nile tilapia survived 24h until 30‰, 12h at 35‰ and 9h at 40‰. Osmolality increase and muscle dehydration decrease both above 20‰. While sodium levels keep regulated in all salinities, chloride, potassium and magnesium levels also increased above 20‰. These results prove that tilapia can easily tolerate abrupt exposure until 20‰ without an osmotic stressor. Gill carbonic anhydrase (C.A.) activity decreased at 30‰, and NKA did not change along salinities and times. These results complements plasma and dehydration averages, showing the large tolerance of this specie to salinity challenges, even in abrupt exposure. Then, these results can be used for discuss about the estuary fish farming system for tilapia, as this species may use estuarine waters to nearby rivers and also can harm the native ecosystem.

Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase.

The conversion of CO2 by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, in situ methods following the microscopic details of CO2 conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO2, water, bicarbonate, and other reactants with β-carbonic anhydrase from Escherichia coli (EcCA) and crotonyl-CoA carboxylase/reductase from Kitasatospora setae (KsCcr), two of the fastest CO2-converting enzymes in nature. Our data reveal that KsCcr possesses a so far unknown metal-independent CA-like activity. Site-directed mutagenesis of conserved active site residues combined with molecular dynamics simulations tracing CO2 distributions in the active site of KsCCr identify an 'activated' water molecule forming the hydroxyl anion that attacks CO2 and yields bicarbonate (HCO3-). Computer simulations also explain why substrate binding inhibits the anhydrase activity. Altogether, we demonstrate how in situ infrared spectroscopy combined with molecular dynamics simulations provides a simple yet powerful new approach to investigate the atomistic reaction mechanisms of different enzymes with CO2.