Role of Carbonic Anhydrase as an Activator in Carbonate Rock Dissolution and Its Implication for Atmospheric CO_(2)Sink

The conversion of CO2 into H+ and is a relatively slow reaction. Hence, its kinetics may be rate determining in carbonate rock dissolution. Carbonic anhydrase (CA), which is widespread in nature, was used to catalyze the CO2 conversion process in dissolution experiments of limestone and dolomite. It was found that the rate of dissolution increases by a factor of about 10 after the addition of CA at a high CO2 partial pressure (Pco2) for limestone and about 3 at low Pco2 for dolomite. This shows that reappraisal is necessary for the importance of chemical weathering (including carbonate rock dissolution and silicate weathering) in the atmospheric CO2 sink and the mysterious missing sink in carbon cycling. It is doubtless that previous studies of weathering underestimated weathering rates due to the ignorance of CA as an activator in weathering, thus the contribution of weathering to the atmospheric CO2 sink is also underestimated. This finding also shows the need to examine the situ distribution and activity of CA in different waters and to investigate the role of CA in weathering.

Immobilization of carbonic anhydrase for facilitated CO2 capture and separation

Carbonic anhydrase(CA)as a typical metalloenzyme in biological system can accelerate the hydration/dehydration of carbon dioxide(CO2,the major components of greenhouse gases),which performer with high selectivity,environmental friendliness and superior efficiency.However,the free form of CA is quite expensive(~RMB 3000/100 mg),unstable,and non-reusable as the free form of CA is not easy for recovery from the reaction environment,which severely limits its large-scale industrial applications.The immobilization may solve these problems at the same time.In this context,many efforts have been devoted to improving the chemical and thermal stabilities of CA through immobilization strategy.Very recently,a wide range of available inorganic,organic and hybrid compounds have been explored as carrier materials for CA immobilization,which could not only improve the tolerance of CA in hazardous environments,but also improve the efficiency and recovery to reduce the cost of large-scale application of CA.Several excellent reviews about immobilization methods and application potential of CA have been published.By contrast,in our review,we stressed on the way to better retain the biocatalytic activity of immobilized CA system based on different carrier materials and to solve the problems facing in practical operations well.The concluding remarks are presented with a perspective on constructing efficient CO2 conversion systems through rational combining CA and advanced carrier materials.

Cloning and Bioinformation Analysis of Carbonic Anhydrase Gene FsCA1 of Tibet Wild Buckwheat

A carbonic anhydrase( CA) transcript was obtained from the Contig library according to the published sequencing information of the buckwheat transcripts. The full length of the CA gene was amplified by reverse transcription PCR( RT-PCR). The bioinformatics analysis showed that the full length of Fs CA1 gene was 1233 bp and open reading frame was 978 bp,and encoding 325 amino acids. The molecular weight was 35. 11 ku and the isoelectric point was 7. 59; there were 9 α helices,6 β folds,many randon coil and extension chain,containing one signal peptide and one transmembrane region,having a 2 amino acid conserved domains with typical beta-type carbonic anhydrase. Subcellular localization showed that the protein is most likely to appear in the chloroplast. The three-dimensional structure model of Fs CA1 was built by homologous modeling method,indicating that the homo-octamer of buckwheat CA and pea CA could match well,so it can be inferred that buckwheat CA is also homo-octamer. Real-time quantitative PCR was used to detect the expression of Fs CA1 in different organs of buckwheat.The results showed that Fs CA1 had the highest expression level in leaves,then in the stems,and the lowest in roots.

Effect of Modified HIF-1<i>α</i>Linked to Carbonic Anhydrase IX Inhibitor and Glycosylated Cisplatin on Solid Tumors: Short Review

Many cancer cells in solid tumors are hypoxic or pseudohypoxic and create acidic environment for malignancy progression. Under low oxygen conditions (hypoxia), hypoxia-inducible factors (HIFs) play pathological roles in cancer cell survival and spreading. HIF regulates several genes such as genes of glucose transporters that enhance anaerobic glycolysis, angiogenesis, erythropoiesis and carbonic anhydrase IX (CA-IX). CA-IX is a cell-surface glycoprotein that catalyzes the hydration of CO2 to protons and bicarbonate ions (respiratory acidification). This process is involved in adaptation to acidosis and implicated in cancer progression. Therefore, CA-IX inhibitors (such as sulfonamide-based compounds) showed hoping results in reduction malignancy progression. The article aims to reversal the malignant hypoxic environment in solid tumors to create a condition of weakness within the cancer for further focused cisplatin potency. This article suggests the use of modified synthesized HIF as a drug delivery molecule for both carbonic anhydrase IX inhibitor and glycosylated cisplatin that damages the DNA of malignant cell. HIF molecule has high affinity to bind with CA IX-expressing malignant cells, which is followed by cell entrance via endocytosis. Once the HIF-Cisplatin-CA-inhibitor complex enters the cell, the carbonic anhydrase inhibitor will improve the cellular pH that makes the environment unsuitable for HIF 1α function and it may be ubiquitinated. So, the raise in target genes transcription will be arrested. On the other hand, once the synthetized HIF is degraded, the cisplatin molecules will be released inside the malignant cell and start to damage its DNA. This approach may be a good solution for many solid tumors.

Overexpression of carbonic anhydrase 1 in pterygium

Carbonic anhydrase （CA） is a kind of zinc enzyme that reversibly catalyzes hydration of CO2. Sixteen CAisoenzymes have been described in mammals . These isozymes play physiological roles in erythrocytes, including CO2 transport, ion secretion, pH regulation and so forth. CA1 and 2 are both cytosolic enzymes that are found in large quantities in erythrocytes. Excluding hemoglobin, CA I is the most abundant protein in erythrocytes

Effect of Hyperglycemia on Erythrocyte Carbonic Anhydrase and Lactic Acid in Type II Diabetic Subjects

Background: Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to and H+. CA plays a crucial role in CO2 transport, acid-base balance, and in linking local acidosis to O2 unloading from hemoglobin and also facilitates lactate shuttling across the monocarboxylate transporters (MCT). The study aimed to investigate the influence of hyperglycemia on erythrocyte carbonic anhydrase activity and lactic acid in type II diabetic patients. Method: Red blood cell carbonic anhydrase activity was determined in washed lysed-hemolysate by the action of the enzyme on the substrate p-nitrophenyl acetate. The absorbance of released p-nitrophenol was recorded at 345 nm. Glycated Hemoglobin was determined by ion exchange method (Spectrum Diagnostic Kit). Blood glucose, lactate, cholesterol and triglyceride were determined using Accutrend GCT meter (Roche, Germany) with cobias® test strips. Results: The present study showed that hyperglycemia significantly (p < 0.05) increased both erythrocyte carbonic anhydrase activity and blood lactate level in type II diabetic patients. Conclusion: We may conclude that hyperglycemia may be responsible for the increased activity of carbonic anhydrase activity and blood lactate concentration.

Prognostic significance of carbonic anhydrase IX expression in clear cell renal cell carcinoma

Objective To evaluate the prognostic significance of carbonic anhydrase IX ( CA IX) expression in patients with clear cell renal cell carcinoma ( ccRCC) . Methods CA IX excression in a cohort of 120 patients with ccRCC was evaluated by P-V immunohistochemistry

Single-Molecule Measurement of Carbonic Anhydrase in Cation Coordinated Environment Using MspA Nanopore

Carbonic anhydrase accounts for catalytic reaction of CO_(2)/HCO_(3)^(–) transformation, thus resulting in neutralization and acidification of the cellular environment, thereby favoring tumor development. Hence, it is a classical protein model of greatly biocatalytic significance as well as a highly expressed biomarker with renal tumor. We herein proposed a single-molecule measurement on carbonic anhydrase using MspA nanopore, in [BMIM+] and asymmetric K^(+)/Ca^(2+) cationic coordinated environment, instead of usual symmetric KCl/NaCl electrolyte. Significantly, our empirical analysis showed that asymmetric K^(+)/Ca^(2+) cationic environment contributes to distinguishable current modulations, thus yielding better resolution for carbonic anhydrase measurement, which is independent of applied voltage and more importantly, is stable enough at varied pH conditions and for very low concentration test in urine sample. Our results provide a classical model for nanopore protein analysis, and may also permit biocatalytic measurement at single-molecule level.

Impacts of Elevated CO2 Concentration on Biochemical Composition, Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations, Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2 concentrations within the range 3-186 ümol/L and the biochemical composition, carbonic anhydrase (CA), and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate, and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186 ümol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 ümol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2 enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.

Plant Carbonic Anhydrases： Structures, Locations, Evolution, and Physiological Roles

Carbonic anhydrases （CAs） are zinc metalloenzymes that catalyze the interconversion of CO2 and HCO3- and are ubiquitous in nature. Higher plants contain three evolutionarily distinct CA families, αCAs, 13CAs, and γCAs, where each family is represented by multiple isoforms in all species. Alternative splicing of CA transcripts appears common; consequently, the number of functional CA isoforms in a species may exceed the number of genes. CAs are expressed in numerous plant tissues and in different cellular locations. The most prevalent CAs are those in the chloroplast, cytosol, and mitochondria. This diversity in location is paralleled in the many physiological and biochemical roles that CAs play in plants. In this review, the number and types of CAs in C3, C4, and crassulacean acid metabolism （CAM） plants are considered, and the roles of the α and yCAs are briefly discussed. The remainder of the review focuses on plant βCAs and includes the identification of homologs between species using phylogenetic approaches, a consideration of the inter- and intracellular localization of the proteins, along with the evidence for alternative splice forms. Current understanding of βCA tissue-specific expression patterns and what controls them are reviewed, and the physiological roles for which βCAs have been implicated are presented.

Full-length mRNA sequencing in Saccharina japonica and identification of carbonic anhydrase genes

The carbonic anhydrases(CAs)are a group of enzymes that play an important role in the absorption and transportation of CO_(2) in Saccharina japonica.They are encoded by a superfamily of genes with seven subtypes that are unrelated in sequence but share conserved function in catalyzing the reversible conversion of CO_(2) and HCO_(3)^(-).Here we have characterized the CA members in the transcriptome of S.japonica using Single-molecule real-time(SMRT)sequencing technology.Approximately 9830.4 megabases from 5,028,003 quality subreads were generated,and they were assembled into 326,512 full-length non-chimeric(FLNC)reads,with an average flnc read length of 2181 bp.After removing redundant sequences,79,010 unique transcripts were obtained of which 38,039 transcripts were successfully annotated.From the full-length transcriptome,we have identified 7 full-length cDNA sequences for CA genes(4α-CAs,1β-CAs and 2γ-CAs)and assessed for their potential functions based on phylogenetic analysis.Characterizations of CAs will provide the ground for future studies to determine the involvement of CAs in inorganic carbon absorption and transportation in S.japonica.

Quantitative Secretome Analysis Reveals Clinical Values of Carbonic Anhydrase Ⅱ in Hepatocellular Carcinoma

Early detection and intervention are key strategies to reduce mortality,increase longterm survival,and improve the therapeutic effects of hepatocellular carcinoma(HCC)patients.Herein,the isobaric tag for relative and absolute quantitation(iTRAQ)-based quantitative proteomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues,surrounding noncancerous tissues,and distal noncancerous tissues to identify diagnostic and prognostic biomarkers for HCC.In total,22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues,respectively,compared with the distal noncancerous tissues.Among these proteins,carbonic anhydrase II(CA2)was identified to be significantly upregulated in the secretome of cancerous tissues;correspondingly,the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations.Interestingly,a significant increase of serum CA2 in recurrent HCC patients after radical resection was also confirmed compared with HCC patients without recurrence,and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall survival.Regarding the mechanism,the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway.Taken together,this study identified a novel biomarker for HCC diagnosis and prognosis,and provided a valuable resource of HCC secretome for investigating serological biomarkers.

Comparative study of carbonic anhydrase activity in waters among different geological eco-environments of Yangtze River basin and its ecological significance

This study provides the presence of carbonic anhydrase（CA） activity in waters of the Yangtze River basin, China, as well as the correlation of CA activity with HCO-3 concentration and CO2 sink flux. Different degrees of CA activity could be detected in almost all of the water samples from different geological eco-environments in all four seasons. The CA activity of water samples from karst areas was significantly higher than from non-karst areas（PP3-concentration（r = 0.672, P2 sink flux（r = 0.602, P = 0.076） in karst areas. This suggests that CA in waters might have a promoting effect on carbon sinks for atmospheric CO2 in karst river basins. In conditions of similar geological type, higher CA activity was generally detected in water samples taken from areas that exhibited better eco-environments, implying that the CA activity index of waters could be used as an indicator for monitoring ecological environments and protection of river basins. These findings suggest that the role of CA in waters in the karst carbon sink potential of river basins is worthy of further in-depth studies.

Surface display of carbonic anhydrase on Escherichia coli for CO_(2) capture and mineralization

Mineralization catalyzed by carbonic anhydrase(CA)is one of the most promising technologies for capturing CO_(2).In this work,Escherichia coli BL21(DE3)was used as the host,and the N-terminus of ice nucleation protein(INPN)was used as the carrier protein.Different fusion patterns and vectors were used to construct CA surface display systems forα-carbonic anhydrase(HPCA)from Helicobacter pylori 26695 andα-carbonic anhydrase(SazCA)from Sulfurihydrogenibium azorense.The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity,while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system.The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA.Cell frac-tionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell’s outer membrane as a fusion protein.The enzyme activity of the surface display strain E-22b-I RL S(11.43 U⋅mL^(−1) OD 600−1)was significantly higher than that of the intracellular expression strain E-22b-S(8.355 U⋅mL^(−1) OD 600−1)under optimized induction conditions.Compared with free SazCA,E-22b-I RL S had higher thermal and pH stability.The long-term stability of SazCA was also significantly improved by surface display.When the engineered strain and free enzyme were used for CO_(2) mineralization,the amount of CaCO_(3) deposition catalyzed by the strain E-22b-I RL S on the surface(241 mg)was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S(173 mg).These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO_(2) capture and mineralization.

Effects of inorganic carbon concentration and pH on carbonic anhydrase activity of gametophytes of Saccharina japonica

Carbonic anhydrase(CA)was considered to be an important component of carbon concentrating mechanism(CCM)of algae.It was an inducible enzyme.Environmental factors,especially dissolved inorganic carbon and pH,were known to affect CA activity.Effects of inorganic carbon(CO_(2)and HCO_(3)^(-))and pH on CA activity of gametophytes of Saccharina japonica were evaluated in this study.Under high-CO_(2)condition(3%CO_(2)),the activity of external CA(C_(Aext))was significantly decreased(P<0.05)from 33.92 REA/g FW to 27.69 REA/g FW.In contrast,the internal CA(CA_(int))and total CA activities were elevated significantly(p<0.01)from 36.83 REA/g FW to 48.80 REA/g FW,and from 70.75 REA/g FW to 76.49 REA/g FW,respectively.Addition of an appropriate concentration of HCO_(3)^(-)to the medium,CA_(int)and total CA activities were promoted significantly,although the activity of C_(Aext)was inhibited significantly(P<0.05).In higher HCO_(3)^(-)concentration(up to 1680 mg/L),the activity of CA_(int)was promoted to 60.81REA/g FW,which was twice that of the control.Higher activities of CA_(int)and total CA were induced at low pH.At pH 6.0,the highest activities of CA_(int)(110.85 REA/g FW)and total CA(128.17 REA/g FW)were induced.However,at pH 9.0,the activities of CA_(int)and total CA were reduced to the lowest of 23.31REA/g FW and 42.19 REA/g FW,respectively.This is the first report about the successful detection of C_(Aext)activity of gametophytes of S.japonica.The results would provide data for the analysis of Ci acquisition and transport mechanism in S.japonica gametophytes.

Inhibitory Effect of Furosemide on Carbonic Anhydrase

This study investigated the inhibitory effect of a high efficiency diuretic, furosemide, on carbonic anhydrase （CA）. First, comparing the inhibitory effect of acetazolamide, a low efficiency diuretic, on CA, shows that furosemide or acetazolamide can quickly make CA inactive when its concentration is close to the enzyme concentration, different from the usual inhibitory kinetics in which the concentration of the inhibitor is far higher than the enzyme concentration. Secondly, the reaction of the enzyme indicates that the inhibitory effect of furosemide or acetazolamide on carbonic anhydrase is quickly reversible. Finally, the degree of the inhibitory effect of furosemide and of acetazolamide on CA are compared. The results show that furosemide inhibits CA less than acetazolamide.

Combination of Dezincification Carbon Anhydrase with Dansylamide for Zinc Biosensing

This work demonstrated quantitatively that DSA （dansylamide） and apo CA （dezincified carbon anhydrase） interacted with each other in the presence of Zn2＋ （zinc ion）. The fluorescence emission of DSA in the presence of Zn2＋ and the intensity of fluorescence, which was proportional to the concentration of Zn2＋, could be used in the measurement of Zn2＋ concentration. Considering that Zn2＋ was the active center of enzymes like CA （carbon anhydrase）, two dezincification reagents were compared to ensure the validity of our method. A certain range concentration of Zn2＋ could be measured by the reaction product of apo CA, DSA and Zn2＋, where the measurement limitation was about 60 nmol/L. Interaction of CA with DSA was also studied with infrared spectrometry.

Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality

Carbon neutrality is widely concerned and highly valued by many countries.Biosphere has always maintained the balance between oxidized organic substances and assimilated organic matter,resulting in netzero carbon dioxide(CO_(2)) emissions and maintaining its own carbon neutrality.Nature has set a good example for human beings to coordinate oxygen(O_(2)) balance and CO_(2)balance,and achieve carbon neutrality.How does photosynthetic oxygen evolution initiate carbon and water neutrality?My synthesis shows that photo system Ⅱ functions as carbonic anhydrase to catalyze the reaction of CO_(2)hydration under physiological conditions,and CO_(2)hydration coupled with chemical equilibrium,H^(+)+HCO_(3)^(-)→1/2O_(2)+2e^(-)+2H^(+)+CO_(2),occurs in a photosystem Ⅱ corecomplex.Meanwhile,I focused on the revisiting of four classical heavy oxygen(O^(18)) labeling experiments and found that bicarbonate can promote photo synthetic oxygen evolution,and that photo synthetic oxygen evolution can alternately come from bicarbonate and water,not only water.Bicarbonate photolysis and water photolysis account for half of the photo synthetic oxygen evolution respectively,which can well explain the bicarbonate effect,Dole effect and plants’ environmental adaptability.Photosynthetic oxygen evolution initiated the journey of water metabolism and carbon metabolism in nature,which led to the coupling as 1:1(mol/mol) stoichiometric relationship between the reduction of CO_(2)and oxidation of organic carbon,coordinated the evolution of the atmosphere,hydrosphere,lithosphere and biosphere,and realized "carbon neutrality" in the whole Earth system.

Blood Oxygenation Level-dependent Magnetic Resonance Imaging of Breast Cancer： Correlation with Carbonic Anhydrase IX and Vascular Endothelial Growth Factor

Background： Blood oxygenation level-dependent magnetic resonance imaging （BOLD-MRI） is a functional MRI technique which involves using the paramagnetic properties of deoxyhemoglobin to image the local tissue oxygen concentration. The purpose of this study was to investigate whether BOLD-MRI could evaluate hypoxia and angiogenesis of breast invasive ductal carcinoma （IDC）. Methods： Ninety-eight female patients with IDC were retrospectively included in this research. All patients underwent breast BOLD-MRI at 3.0 T before surgery. R2＊ values of BOLD-MR1 were measured. The expression of carbonic anhydrase IX （CA IX） and vascular endothelial growth factor （VEGF） was analyzed by immunohistochemistry. Spearman＇s correlation analysis was used to correlate R2＊ value with CA IX and VEGF levels. Results： Heterogeneous intensity on BOLD-MRI images was the main finding of IDCs. The mean R2＊ value was 52.8 ± 18.6 Hz. The R2＊ values in patients with axillary lymph node metastasis were significantly higher than the R2＊ values in patients without axillary lymph node metastasis （t = 2.882, P = 0.005）. R2＊ values increased with CA IX level and positively correlated with the level of CA 1X （r = 0.616, P 〈 0.001）; however, R2＊ value had no significantly correlation with the level of VEGF （r = 0.110, P = 0.281）. Conclusion： B OLD-MRI could noninvasively evaluate chronic hypoxia of IDC, but not angiogenesis.

Nitrogen in Relation to Photosynthetic Capacity and Accumulation of Osmoprotectant and Nutrients in Brassica Genotypes Grown Under Salt Stress

Different strategies of the application of nutrients are required to overcome the adverse effects of mustard （Brassica juncea L.） in response to NaCl stress. The objective of the present study was to determine if different added levels of nitrogen （N） in growth medium could alleviate the adverse effects of salt stress on photosynthetic capacity and accumulation of osmoprotectants and nutrients. 14 days mustard seedlings of salt-sensitive （cv. Chuutki） and salt-tolerant （cv. Radha） genotypes were fed with： （i） 0 mmol L^-1 NaCl ＋ 0 mg N kg^-1 sand （control）, （ii） 90 mmol L^-1 NaCl ＋ 30 mg N kg^-1 sand, （iii） 90 mmol L^-1 NaCl ＋60 mg N kg^-1 sand, （iv） 90 mmol L^-1 NaCl ＋90 mg N kg^-1 sand and （v） 90 mmol L^-1NaCl＋ 120 mg N kg^-1 sand. Under the condition of salinity stress, N application caused a significant ameliorative effect on both genotypes with respect to growth attributes [fresh weight （FW） and dry weight （DW）] and physio-biochemical parameters [percent water content （WC）, net photosynthetic rate （PN）, stomatal conductance （gs）, total chlorophyll （Ch1）, carbonic anhydrase （CA） activity and malondialdehyde （MDA）, nitrogen （N）, potassium （K） and sodium （Na） contents, and K/Na ratio] and yield attributes （number of pods/plant, seeds/pod and seed yield/plant）. The salt-tolerant genotype exhibited maximum value for growth, physio-biochemical and yield attributes at 60 mg N kg 1 sand than that of salt-sensitive genotype. These results suggest that application of N may ameliorate most of the attributes and prove to be a physiological remedy to increase the tolerance against the ill effects of salt stress in Brassicas.