Extracellular 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.

Effect of different mixed light-emitting diode light wavelengths on CO2 absorption from biogas and nutrient removal from biogas slurry by microalgae and fungi induced using strigolactone and endophytic bacteria.

In this study, biogas and biogas slurry were simultaneously treated using two symbiotic systems: Chlorella vulgaris-Ganoderma lucidum-S395-2 (endophytic bacteria) and Scenedesmus obliquus-G. lucidum-S395-2. The influence of different mixed illumination (red and blue) intensity ratios on the algal symbionts' extracellular carbonic anhydrase activities was investigated, as well as the rates of microalgal growth and photosynthesis. The treatment performance was simultaneously assessed in terms of the efficiency of organic matter or nutrient removal and the level of CO2 absorption. The results indicated that red-blue light combinations with an intensity ratio of 5:5 were optimal. When comparing the performance of the two symbiotic systems, the C. vulgaris-G. lucidum-S395-2 symbiont co-culture system achieved significantly improved photosynthetic rates, biomass growth, and treatment effects. Under the optimal treatment conditions, the organic matter and nutrient removal rates were 81.06% ± 7.06% for chemical oxygen demand, 82.32% ± 7.18% for total nitrogen, and 82.98% ± 7.26% for total phosphorus. In addition, the rate of CO2 removal from biogas was 63.38% ± 5.35%. PRACTITIONER POINTS: The red and blue light intensity ratio of 5:5 showed the best removal performance. C. vulgaris-G. lucidum-S395-2 system obtained the best photosynthetic performance. The carbonic anhydrase activity had positive effects on CO2 removal performance.

Effects of nutrients on the utilization pattern of photosynthetic inorganic carbon of the tropical <italic>Thalassia hemprichii</italic>

<p indent="0mm">Studies on the utilization strategy of photosynthetic inorganic carbon of seagrass can reveal its ecological adaptation mechanism. <italic>Thalassia hemprichii</italic>, a widely distributed seagrass species in the Indo-Pacific and Atlantic coastal seas, needs to evolve several utilization patterns of inorganic carbon to adapt different environments and meet its growth, development and reproduction. However, the effect of nutrient on the photosynthetic inorganic carbon utilization strategy in seagrass is still unknown and further research is urgently needed. Therefore, using oxygen electrode technology, chlorophyll fluorescence technology, and non-invasive micro-test technology, this study examined the similarity and difference in the utilization patterns of photosynthetic inorganic carbon of <italic>T</italic>.<italic> hemprichii </italic>from two typical tropical seagrass beds (Tanmen, Hainan Island, and Yongxing Island, Xisha), that were affected by varying degrees of human activity. Experiments showed that the photosynthetic oxygen release rate, maximum relative electron transfer rate, and half-saturated light intensity of <italic>T</italic>.<italic> hemprichii</italic> in Yongxing Island waters were 24.95%, 24.54%, and 28.73% higher than the corresponding in Tanmen, Hainan Island. This indicated that the growth status of <italic>T</italic>.<italic> hemprichii</italic> in the waters of Yongxing Island was better with higher photosynthetic efficiency. The addition of acetazolamide (AZ) and ethoxyzolamide (EZ) significantly reduced the photosynthetic oxygen release rate of <italic>T</italic>.<italic> hemprichii</italic>, with an insignificant difference in the inhibition between AZ and EZ. This indicated that it mainly absorbed HCO₃⁻ via extracellular carbonic anhydrase catalysis. Meanwhile, hydroxymethyl (Tris) inhibited the photosynthetic oxygen release of <italic>T</italic>.<italic> hemprichii</italic> in Tanmen by up to 100% and decreased the maximum relative electron transfer rate and the H⁺ inward flow rate, implying a synergistic H⁺/HCO₃⁻ transport mode. On the other hand, the rate of photosynthetic oxygen release and the maximum electron transfer rate of <italic>T</italic>.<italic> hemprichii</italic> in Xisha did not change significantly with the addition of AZ and EZ, while Tris buffer inhibited photosynthetic oxygen release by up to 100% and significantly reduced the maximum electron transfer rate. This suggested <italic>T</italic>.<italic> hemprichii </italic>in Xisha mainly relied on the H⁺/HCO₃⁻ synergistic transport. Therefore, <italic>T</italic>.<italic> hemprichii </italic>could uptake inorganic carbon in an environment with greater anthropogenic activity, larger nutrient load and lower light availability on Hainan Island by catalyzing HCO₃⁻ into CO₂ with extracellular carbonic anhydrase and the H⁺/HCO₃⁻ synergistic transport. In contrast, <italic>T</italic>.<italic> hemprichii</italic> in an environment with less anthropogenic activity, lower nutrient loading, and greater light availability, relied chiefly on H⁺/HCO₃⁻ synergistic transport. Facing a relatively unfavorable high-nutrient and low-light growth environment, <italic>T</italic>.<italic> hemprichii</italic> could increase the photosynthetic inorganic carbon utilization strategy (such as the activity of extracellular carbonic anhydrase) to improve its utilization ability of inorganic carbon to overcome stress and growth limitation. This suggested that <italic>T</italic>.<italic> hemprichii</italic> could adjust its inorganic carbon utilization patterns according to its geographical environment. With the continuous development of technology, it is urgent to study the utilization mechanism of inorganic carbon of seagrass by applying seagrass omics technology and stable isotope tracing to deepen the understanding of the carbon sink function of seagrass.

Extracellular carbonic anhydrase activity promotes a carbon concentration mechanism in metazoan calcifying cells

Many calcifying organisms utilize metabolic CO2 to generate CaCO3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H+-selective electrodes were used to demonstrate eCA-catalyzed CO2 hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pHi regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO3- uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO2 by Cara7 coupled to HCO3- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO2 to generate a versatile construction material.

A leader peptide of the extracellular cyanobacterial carbonic anhydrase ensures the efficient secretion of recombinant proteins in Escherichia coli

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli.

The effects of two sized polystyrene nanoplastics on the growth, physiological functions, and toxin production of Alexandrium tamarense

Micro- and nano-plastics (MNPs) are increasingly prevalent pollutants in marine ecosystems and result in various deleterious effects on marine organisms. There have been studies evaluated the toxic effects of MNPs on marine microalgae, but few of them focused on the effects of MNPs on dinoflagellate species and their toxins production, which could have significant implications on human health and ecological safety in coastal areas. In this study, the common harmful algal blooms-causing dinoflagellate Alexandrium tamarense was exposed to 0.1 and 1 μm sized polystyrene nanoplastics (NPs) to investigate the responding patterns of population growth, multiple physiological functions, as well as the intracellular paralytic shellfish toxins (PSTs) productions. The results indicated the population growth, photosynthetic parameters, nutrients (nitrate and phosphate) uptake rates and extracellular carbonic anhydrase activities (CAext) were all inhibited by the two sized NPs, accompanied by the prolonged and more aggregated microalgal cells under the observation of scanning electron microscope (SEM), and the inhibition effects were more severe under 1 μm sized NPs than 0.1 μm sized NPs. Finally, we found the intracellular PSTs contents increased 73.59% exposed to 0.1 μm sized NPs while decreased 85.50% exposed to 1 μm sized NPs comparing the controls at 96 h, without significant changes of relative compositions. These results provided evidence that MNPs were toxic to A. tamarense and affected their intracellular PSTs productions within 96 h, which is critical to consider when evaluating the potential risks of MNPs in marine ecosystems.

Effects of exogenous GR24 on biogas upgrading and nutrient removal by co-culturing microalgae with fungi under mixed LED light wavelengths

To realize the synchronous purification of raw biogas and biogas slurry, the algal-fungal symbiont pellets were cultivated by supplementing strigolactone (GR24) under different mixed LED light wavelengths. The optimal light intensity was proved to be red and blue in the ratio of 5:5. The symbionts treated with 10−9 M GR24 had the highest growth rate and mean daily productivity. The extracellular carbonic anhydrase activity and the content of chlorophyll were also affected by GR24 concentrations and mixed light wavelengths. With the induction of 10−9 M GR24, the maximum removal efficiency of chemical oxygen demand, total nitrogen, and total phosphorus reached 76.35 ± 6.87%, 78.77 ± 7.13% and 79.49 ± 7.43%, respectively. Besides, the CO2 removal efficiency reached 59.32 ± 5.19% when the concentration of GR24 was 10−7 M. This work will be beneficial for large-scale biogas slurry purification and biogas upgrading using co-cultivation of microalgae and fungi.

Ultraviolet Radiation Stimulates Activity of CO2 Concentrating Mechanisms in a Bloom-Forming Diatom Under Reduced CO2 Availability.

The diatom Skeletonema costatum is cosmopolitan and forms algal blooms in coastal waters, being exposed to varying levels of solar UV radiation (UVR) and reduced levels of carbon dioxide (CO2). While reduced CO2 availability is known to enhance CO2 concentrating mechanisms (CCMs) in this diatom and others, little is known on the effects of UV on microalgal CCMs, especially when CO2 levels fluctuate in coastal waters. Here, we show that S. costatum upregulated its CCMs in response to UVR (295–395 nm), especially to UVA (320–395 nm) in the presence and absence of photosynthetically active radiation (PAR). The intensity rise of UVA and/or UVR alone resulted in an increase of the activity of extracellular carbonic anhydrase (CAe); and the addition of UVA enhanced the activity of CCMs-related CAe by 23–27% when PAR levels were low. Such UV-stimulated CCMs activity was only significant at the reduced CO2 level (3.4 μmol L−1). In addition, UVA alone drove active HCO3− uptake although it was not as obvious as CAe activity, another evidence for its role in enhancing CCMs activity. In parallel, the addition of UVA enhanced photosynthetic carbon fixation only at the lower CO2 level compared to PAR alone. In the absence of PAR, carbon fixation increased linearly with increased intensities of UVA or UVR regardless of the CO2 levels. These findings imply that during S. costatum blooming period when CO2 and PAR availability becomes lower, solar UVR (mainly UVA) helps to upregulate its CCMs and thus carbon fixation, enabling its success of frequent algal blooms.

Blood and Gill Carbonic Anhydrase in the Context of a Chondrichthyan Model of CO2 Excretion.

Pacific spiny dogfish (Squalus suckleyi) have been widely used as a representative species for chondrichthyan CO2 excretion. Pacific spiny dogfish have a slower red blood cell (RBC) carbonic anhydrase (CA) isoform than teleost fishes, extracellular CA activity, no endogenous plasma CA inhibitor, and plasma-accessible CA IV at the gills. Thus, both the RBC and plasma compartments contribute to bicarbonate ion () dehydration at the gills for CO2 excretion in contrast to teleost fishes, in which dehydration is restricted to RBCs. We compared CA activity levels, subcellular localization, and presence of plasma CA inhibitors in the blood and gills of 13 chondrichthyans to examine the hypothesis that the dogfish model of CO2 excretion applies broadly to chondrichthyans. In general, blood samples from the 12 other chondrichthyans examined had lower RBC CA activity than teleosts, some extracellular CA activity, and no endogenous plasma CA inhibitor. While type IV-like membrane-associated CA was found in the gills in all four of the chondrichthyans examined, S. suckleyi had three times more CA activity ( μmol CO2 min-1 mg protein-1) in the microsomal (membrane) fraction of gills than the other three. In addition, unexpected variation in CA characteristics was observed between chondrichthyan species. Thus, in general, it appears that the pattern of CA distribution in fishes can be generally categorized as either chondrichthyan or teleost models. However, further studies should examine the functional significance of the within-chondrichthyan differences we observed and investigate whether CO2 excretion patterns exist along a continuum or in discrete groups.

Marine ammonification and carbonic anhydrase activity induce rapid calcium carbonate precipitation

During Earth’s history, precipitation of calcium carbonate by heterotrophic microbes has substantially contributed to the genesis of copious amounts of carbonate sediment and its subsequent lithification. Previous work identified the microbial sulfur and nitrogen cycle as principal pathways involved in the formation of marine calcium carbonate deposits. While substantial knowledge exists for the importance of the sulfur cycle, specifically sulfate reduction, with regard to carbonate formation, information about carbonate genesis connected to the microbial nitrogen cycle is dissatisfactory. In addition to the established pathways for carbonate mineral formation, also the potential of microbial carbonic anhydrase, a carbonate-relevant, zinc-containing enzyme, is receiving currently increased attention. However, also in this field knowledge is scarce and fragmentary. Here we demonstrate microbial carbonate precipitation as a direct result of the interplay between the microbial nitrogen cycle and a microbially produced enzyme. Using Alcanivorax borkumensis as a model organism, our experiments depict precipitation of a peloidal carbonate matrix within days to weeks, induced by simultaneous ammonification and extracellular carbonic anhydrase activity. The precipitates show similar morphology, mineralogy, δ44/40Ca, and δ88/86Sr to analogs of modern carbonate peloids. The obtained Sr/Ca partition coefficient DSr showed no clear deviation from inorganic carbonate phases, indicating that microbially mediated carbonate precipitation, indeed, follows the principles of physico-chemical precipitation. The observed relative enrichment of the precipitates in zinc might help to constrain zinc variations in natural carbonate archives. Our study demonstrates that ammonification, due to intense microbial organic matter degradation, and carbonic anhydrase may play a substantial role for calcium carbonate precipitation in paleo- and recent shallow marine environments.

Regulation of inorganic carbon acquisition in a red tide alga (&amp;lt;i&amp;gt;Skeletonema costatum&amp;lt;/i&amp;gt;): the importance of phosphorus availability

Abstract. Skeletonema costatum is a common bloom-forming diatom and encounters eutrophication and severe carbon dioxide (CO2) limitation during red tides. However, little is known regarding the role of phosphorus (P) in modulating inorganic carbon acquisition in S. costatum, particularly under CO2 limitation conditions. We cultured S. costatum under five phosphate levels (0.05, 0.25, 1, 4, 10 µmol L−1) and then treated it with two CO2 conditions (2.8 and 12.6 µmol L−1) for 2 h. The lower CO2 reduced net photosynthetic rate at lower phosphate levels (&lt; 4 µmol L−1) but did not affect it at higher phosphate levels (4 and 10 µmol L−1). In contrast, the lower CO2 induced a higher dark respiration rate at lower phosphate levels (0.05 and 0.25 µmol L−1) and did not affect it at higher phosphate levels (&gt; 1 µmol L−1). The lower CO2 did not change relative electron transport rate (rETR) at lower phosphate levels (0.05 and 0.25 µmol L−1) and increased it at higher phosphate levels (&gt; 1 µmol L−1). Photosynthetic CO2 affinity (1/K0.5) increased with phosphate levels. The lower CO2 did not affect photosynthetic CO2 affinity at 0.05 µmol L−1 phosphate but enhanced it at the other phosphate levels. Activity of extracellular carbonic anhydrase was dramatically induced by the lower CO2 in phosphate-replete conditions (&gt; 0.25 µmol L−1) and the same pattern also occurred for redox activity of the plasma membrane. Direct bicarbonate (HCO3-) use was induced when phosphate concentration was more than 1 µmol L−1. These findings indicate P enrichment could enhance inorganic carbon acquisition and thus maintain the photosynthesis rate in S. costatum grown under CO2-limiting conditions via increasing activity of extracellular carbonic anhydrase and facilitating direct HCO3- use. This study sheds light on how bloom-forming algae cope with carbon limitation during the development of red tides.

Induction of calcite precipitation through heightened production of extracellular carbonic anhydrase by CO2 sequestering bacteria

The thermo-alkalotolerant bacterium exhibiting heightened extracellular carbonic anhydrase (CA) activity, survived at 100 mM sodium bicarbonateand 5% gaseous CO2 was identified as Bacillus sp. by 16S rRNA sequencing. Extracellular carbonic anhydrase was purified by ammonium sulfate precipitation, gel filtration chromatography and affinity chromatography with a yield of 46.61% and specific activity of 481.66 U/mg. The size of purified carbonic anhydrase was approximately 28 kDa in SDS-PAGE gel filtration and further their role in calcium carbonate production was correlated. The purified enzyme was stable with half-life of 25.36 min at 90 °C and pH 8. KM and Vmax values of the enzyme were 1.77 mg/mL and 385.69 U/mg respectively. The production of calcite was confirmed by Scanning Electron Microscopy (SEM) analysis, FTIR, and Energy-Dispersive X-ray (EDX) analysis. Carbonic anhydrase and calcite deposition coupled with CO2 fixingbacteria is a significant approach for CO2 sequestration.

Impact of extracellular alkalinization on the survival of human CD24-/CD44+ breast cancer stem cells associated with cellular metabolic shifts.

Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.

Changes in growth, carbon and nitrogen enzyme activity and mRNA accumulation in the halophilic microalga Dunaliella viridis in response to NaCl stress

Many species of microalga Dunaliella exhibit a remarkable tolerance to salinity and are therefore ideal for probing the effects of salinity. In this work, we assessed the effects of NaCl stress on the growth, activity and mRNA level of carbon and nitrogen metabolism enzymes of D. viridis. The alga could grow over a salinity range of 0.44 mol L−1 to 3.00 mol L−1 NaCl, but the most rapid growth was observed at 1.00 mol L−1 NaCl, followed by 2.00 mol L−1 NaCl. Paralleling these growth patterns, the highest initial and total Rubisco activities were detected in the presence of 1.00 mol L−1 NaCl, decreasing to 37.33% and 26.39% of those values, respectively, in the presence of 3.00 mol L−1 NaCl, respectively. However, the highest extracellular carbonic anhydrase (CA) activity was measured in the presence of 2.00 mol L−1 NaCl, followed by 1.00 mol L−1 NaCl. Different from the two carbon enzymes, nitrate reductase (NR) activity showed a slight change under different NaCl concentrations. At the transcriptional level, the mRNAs of Rubisco large subunit (rbcL), and small subunit (rbcS), attained their highest abundances in the presence of 1.00 and 2.00 mol L−1 NaCl, respectively. The CA mRNA accumulation was induced from 0.44 mol L−1 to 3.00 mol L−1 NaCl, but the NR mRNA showed the decreasing tendency with the increasing salinity. In conclusion, the growth and carbon fixation enzyme of Rubisco displayed similar tendency in response to NaCl stress, CA was proved be salt-inducible within a certain salinity range and NR showed the least effect by NaCl in D. viridis.

Growth, photosynthesis and nutrient uptake by Grateloupia livida (Halymeniales, Rhodophyta) in response to different carbon levels

:Grateloupia livida is a commercially important red macroalgal species. In the present study, thalli of G. livida, collected from Nan'ao Island, China, were cultured at different levels of CO2 (c. 1000, 390 and 50 ppm) to examine the growth and physiology and how its plasticity may affect photosynthesis at different carbon levels. The pH-drift experiments showed that G. livida could reach a final pH value of 9.75. Carbonic anhydrase (CA) activity was detected potentiometrically with both thalli and homogenates of G. livida. These implied that G. livida used HCO3− as a source of inorganic carbon for photosynthesis and that this was mediated by extracellular CA activity. Thalli of G. livida grown at low CO2 reduced the relative growth rate (RGR), both the PO43− and NO3− uptake rates, nitrate reductase activity and photochemical activity compared with the algae grown at ambient CO2. Moreover, the RGR and the initial slope of the rapid light curves (α) were inhibited by the high CO2 level in the seawater. The results indicated that either raised or lowered CO2 in culture were unfavourable environmental factors for growth and photosynthesis of G. livida.

Phosphate Chemical Probes Designed for Location Specific Inhibition of Intracellular Carbonic Anhydrases.

Chemical probes are small molecules designed to bind to a specific protein and disrupt the proteins function. Although many inhibitors are reported for human carbonic anhydrase (CA) enzymes, few may be considered useful as chemical probes as they exhibit broad action against the 12 catalytically active CA isozymes. In addition, most do not possess an appropriate physicochemical profile to discriminate intracellular CA activity from either global or extracellular CA activity. We report herein the synthesis of three monophosphate CA proinhibitors (compounds 2, 3, and 5) that are derived from cyclosaligenyl (cycloSal) phosphate and S-acyl-2-thioethyl (SATE) phosphate as protecting groups. The proinhibitors are designed as neutral, membrane-permeable compounds that once inside the cell may be hydrolyzed by pH-driven or enzymatic-driven mechanisms to release a negatively charged monophosphate. The resulting monophosphate compound is trapped intracellularly and available for locality specific inhibition of intracellular CAs.

Size scaling of extracellular carbonic anhydrase activity in centric marine diatoms.

Many microalgae have a surface-associated extracellular carbonic anhydrase (eCA) that converts HCO3 (-) to CO2 for uptake and subsequent photosynthetic fixation. We investigated eCA activity and assessed its importance for photosynthetic CO2 supply in six centric diatom species spanning nearly the full range of cell sizes for centric diatoms (equivalent spherical radius 3-67 μm). Since larger cells are more susceptible to diffusion limitation, we hypothesized that eCA activity would increase with cell size as would its importance for CO2 supply. eCA activity did increase with cell size, increasing with cell radius by a size-scaling exponent of 2.6 ± 0.3. The rapid increase in eCA activity with cell radius keeps the absolute CO2 concentration difference between bulk seawater and the cell surface very low (<~0.2 μM) allowing high rates of CO2 uptake even for large diatoms. Although inhibiting eCA did reduce photosynthesis in the diatoms, there was no overall relationship between the extent of inhibition of photosynthesis and cell size. The only indication that eCA may be more important for larger diatoms was that photosynthesis in the smallest diatoms (<4 μm radius) was only affected by eCA inhibition when CO2 concentrations were very low, while photosynthesis in some larger diatoms was affected even at typical seawater CO2 concentrations. eCA is ubiquitous in centric marine diatoms, in contrast to other taxa where its presence is irregularly distributed among different species, and plays an important role in supplying CO2 for photosynthesis across the size spectrum.

CO2 biofixation and carbonic anhydrase activity in Scenedesmus obliquus SA1 cultivated in large scale open system

The present study deals with the large scale open system cultivation of the novel microalga: Scenedesmus obliquus SA1 (KC733762) previously isolated in our laboratory. SA1 strain was cultivated in open system at varying CO2 levels ranging from 0.03% to 35% (v/v) and subsequently the carbonic anhydrase activity (CA) and the biochemical properties were monitored. Maximum biomass concentration (1.39 ± 0.023 g L(-1)), CO2 fixation rate (97.65 ± 1.03 mg L(-1)d(-1)) and total CA activity (166.86 ± 3.30 E.U./mg chla) were obtained at 35% CO2. CA inhibitors: acetazolamide and ethoxyzolamide inhibited the external and internal enzyme activity in SA1. High CO2 levels were favorable for the accumulation of lipids and chlorophyll. The present results suggested that SA1 possessed high CO2 tolerance and high carbohydrate, lipid and chlorophyll content when cultivated in open system thus being suitable for CO2 mitigation in outdoor ponds and subsequent generation of value added products.

The impact of iron limitation on the physiology of the Antarctic diatom Chaetoceros simplex.

Iron availability strongly governs the growth of Southern Ocean phytoplankton. To investigate how iron limitation affects photosynthesis as well as the uptake of carbon and iron in the Antarctic diatom Chaetoceros simplex, a combination of chlorophyll a fluorescence measurements and radiotracer incubations in the presence and absence of chemical inhibitors was conducted. Iron limitation in C. simplex led to a decline in growth rates, photochemical efficiency and structural changes in photosystem II (PSII), including a reorganisation of photosynthetic units in PSII and an increase in size of the functional absorption cross section of PSII. Iron-limited cells further exhibited a reduced plastoquinone pool and decreased photosynthetic electron transport rate, while non-photochemical quenching and relative xanthophyll pigment content were strongly increased, suggesting a photoprotective response. Additionally, iron limitation resulted in a strong decline in carbon fixation and thus the particulate organic carbon quotas. Inhibitor studies demonstrated that, independent of the iron supply, carbon fixation was dependent on internal, but not on extracellular carbonic anhydrase activity. Orthovanadate more strongly inhibited iron uptake in iron-limited cells, indicating that P-type ATPase transporters are involved in iron uptake. The stronger reduction in iron uptake by ascorbate in iron-limited cells suggests that the re-oxidation of iron is required before it can be taken up and further supports the presence of a high-affinity iron transport pathway. The measured changes to photosystem architecture and shifts in carbon and iron uptake strategies in C. simplex as a result of iron limitation provide evidence for a complex interaction of these processes to balance the iron requirements for photosynthesis and carbon demand for sustained growth in iron-limited waters.

Quantification of Extracellular Carbonic Anhydrase Activity in Two Marine Diatoms and Investigation of Its Role

Many microalgae induce an extracellular carbonic anhydrase (eCA), associated with the cell surface, at low carbon dioxide (CO2) concentrations. This enzyme is thought to aid inorganic carbon uptake by generating CO2 at the cell surface, but alternative roles have been proposed. We developed a new approach to quantify eCA activity in which a reaction-diffusion model is fit to data on (18)O removal from inorganic carbon. In contrast to previous methods, eCA activity is treated as a surface process, allowing the effects of eCA on cell boundary-layer chemistry to be assessed. Using this approach, we measured eCA activity in two marine diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii), characterized the kinetics of this enzyme, and studied its regulation as a function of culture pH and CO2 concentration. In support of a role for eCA in CO2 supply, eCA activity specifically responded to low CO2 rather than to changes in pH or HCO3(-), and the rates of eCA activity are nearly optimal for maintaining cell surface CO2 concentrations near those in the bulk solution. Although the CO2 gradients abolished by eCA are small (less than 0.5 μm concentration difference between bulk and cell surface), CO2 uptake in these diatoms is a passive process driven by small concentration gradients. Analysis of the effects of short-term and long-term eCA inhibition on photosynthesis and growth indicates that eCA provides a small energetic benefit by reducing the surface-to-bulk CO2 gradient. Alternative roles for eCA in CO2 recovery as HCO3(-) and surface pH regulation were investigated, but eCA was found to have minimal effects on these processes.