PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON IN THE ECONOMIC BROWN ALGA,HIZIKIA FUSIFORME (SARGASSACEAE) FROM THE SOUTH CHINA SEA1

The mechanism of inorganic carbon (C_i) acquisition by the economic brown macroalga, Hizikia fusiforme (Harv.) Okamura (Sargassaceae), was investigated to characterize its photosynthetic physiology. Both intracellular and extracellular carbonic anhydrase (CA) were detected, with the external CA activity accounting for about 5% of the total. Hizikia fusiforme showed higher rates of photosynthetic oxygen evolution at alkaline pH than those theoretically derived from the rates of uncatalyzed CO₂ production from bicarbonate and exhibited a high pH compensation point (pH 9.66). The external CA inhibitor, acetazolamide, significantly depressed the photosynthetic oxygen evolution, whereas the anion‐exchanger inhibitor 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulfonate had no inhibitory effect on it, implying the alga was capable of using HCO₃^? as a source of C_i for its photosynthesis via the mediation of the external CA. CO₂ concentrations in the culture media affected its photosynthetic properties. A high level of CO₂ (10,000 ppmv) resulted in a decrease in the external CA activity; however, a low CO₂ level (20 ppmv) led to no changes in the external CA activity but raised the intracellular CA activity. Parallel to the reduction in the external CA activity at the high CO₂ was a reduction in the photosynthetic CO₂ affinity. Decreased activity of the external CA in the high CO₂ grown samples led to reduced sensitiveness of photosynthesis to the addition of acetazolamide at alkaline pH. It was clearly indicated that H. fusiforme, which showed CO₂‐limited photosynthesis with the half‐saturating concentration of C_i exceeding that of seawater, did not operate active HCO₃^? uptake but used it via the extracellular CA for its photosynthetic carbon fixation.     

Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.     

Characterization of functionally active immobilized carbonic anhydrase purified from sheep blood lysates

Carbonic anhydrase (CA) catalyzes the reversible reaction of hydration of CO₂ to bicarbonate and the dehydration of bicarbonate back to CO₂. Sequestration of CO₂ from industrial processes or breathing air may require a large amount of highly active and stable CA. Therefore, the objectives of the present study were to purify large amounts of CA from a cheap and easily accessible source of the enzyme and to characterize the enzymatic and kinetic properties of soluble and immobilized enzyme. We recovered 80% of pure enzyme with a specific activity of 4870 EU/mg protein in a single step using sheep blood lysates from slaughter house waste products and CA specific inhibitor affinity chromatography. Since affinity pure CA showed both anhydrase and esterase activities, we measured the esterase activities for enzymology. The Michaelis–Menten constant, K_M, pH optimum, activation energy, and thermal stability of soluble enzymes were 8 × 10^?2 M, 7.3 pH, 7.3 kcal/mol and 70 °C, respectively.The immobilization of the enzyme to Affigel-10 was very efficient and 83% of purified enzyme was immobilized. The immobilized enzyme showed a K_M of 5 × 10^?2 M and activation energy of 8.9 kcal/mol, suggesting a better preference of substrate for immobilized enzyme in comparison to soluble enzyme. In contrast to soluble enzyme, immobilized enzyme showed relatively higher activity at pH 6–8. From these results, we concluded that a shift in pH profile toward acidic pH is due to modification of lysine residues involved in the immobilization process. The immobilized enzyme was stable at higher temperatures and showed highest activity at 80 °C. The activity of immobilized enzyme in a flow reactor at 0.5–2.2 ml/min flow rate was unaffected. Collectively, results from the present study suggested the application of blood lysate waste from animal slaughterhouses for purification of homogeneous enzyme for CO₂ capture in a flow reactor.     

Carbonic anhydrase and C4 photosynthesis: a transgenic analysis

Carbonic anhydrase (CA, EC 4.2.1.1) catalyses the first reaction in the C₄ photosynthetic pathway, the conversion of atmospheric CO₂ to bicarbonate in the mesophyll cytosol. To examine the importance of the enzyme to the functioning of the C₄ photosynthetic pathway, Flaveria bidentis (L.) Kuntze, a C₄ dicot, was genetically transformed with an antisense construct in which the cDNA encoding a putative cytosolic CA (CA3) was placed under the control of a constitutive promoter. Some of the primary transformants had impaired CO₂ assimilation rates and required high CO₂ for growth. The T₁ progeny of four primary transformants were used to examine the quantitative relationship between leaf CA activity and CO₂ assimilation rate. CA activity was determined in leaf extracts with a mass spectrometric technique that measured the rate of ¹⁸O exchange from doubly labelled ¹³C¹⁸O₂. Steady‐state CO₂ assimilation rates were unaffected by a decrease in CA activity until CA activity was less than 20% of wild type when they decreased steeply. Transformants with less than 10% of wild‐type CA activity had very low CO₂ assimilation rates and grew poorly at ambient CO₂ partial pressure. Reduction in CA activity also increased the CO₂ partial pressure required to saturate CO₂ assimilation rates. The present data show that CA activity is essential for the functioning of the C₄ photosynthetic pathway.     

Characterization and inhibition effects of some metal ions on carbonic anhydrase enzyme from Kangal Akkaraman sheep

In this work, the carbonic anhydrase (CA) enzyme was purified from Kangal Akkaraman sheep in Sivas, Turkey with specific activity value of 6681.57 EU/mg and yield of 14.90% with using affinity column chromatography. For designating the subunit molecular mass and enzyme purity, sodium dodecyl sulfate polyacrylamide gel electrophoresis method was used and single band for this procedure was obtained. The molecular mass of CA enzyme was found as 28.89 kDa. In this study, the optimum temperature and optimum pH were obtained from 30 and 7.5. V_max and K_m values for p‐nitrophenylacetate substrate of the CA were determined from Lineweaver–Burk graphs. Additionally, the inhibitory results of diverse heavy metal ions (Hg⁺, Fe²⁺, Pb²⁺, Co²⁺, Ag⁺, and Cu²⁺) on sheep were studied. Indeed, CA enzyme activities of Kangal sheep were investigated with using esterase procedure under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC₅₀) and K_i values were obtained.     

The toxicological effects of some avermectins on goat liver carbonic anhydrase enzyme

Avermectins are used worldwide as antiparasitic drugs in the field of veterinary medicine and as agricultural pesticides and insecticides. Carbonic anhydrase (CA, E.C. 4.2.1.1) is a zinc‐containing metalloenzyme that catalyzes the reversible hydration of carbon dioxide (CO₂) to yield protons (H⁺) and bicarbonate (HCO₃^?). In this study, some avermectins, including abamectin, doramectin, eprinomectin, and moxidectin, were investigated for in vitro inhibitory effects on the CA enzyme purified from goat liver, which was purified (125.00‐fold) using sepharose 4B‐l ‐tyrosine‐sulfanilamide affinity chromatography, with a yield of 68.27% and a specific activity of 21765.31 EU/mg proteins. The inhibition results obtained from this study showed K_i values of 0.283, 0.153, 0.232, and 0.317 nM for abamectin, doramectin, eprinomectin, and moxidectin, respectively. On the other hand, acetazolamide, well‐known clinically established CA inhibitor, possessed a K_i value of 0.707 nM against goat liver CA.     

CO2‐CONCENTRATING MECHANISMS OF THE POTENTIALLY TOXIC DINOFLAGELLATE PROTOCERATIUM RETICULATUM (DINOPHYCEAE,GONYAULACALES)1

The low CO₂ concentration in seawater poses severe restrictions on photosynthesis, especially on those species with form II RUBISCO. We found that the potentially toxic dinoflagellate Protoceratium reticulatum Clap. et J. Lachm. possesses a form II RUBISCO. To cast some light on the mechanisms this organism undergoes to cope with low CO₂ availability, we compared cells grown at atmospheric (370 ppm) and high (5000 ppm) CO₂ concentrations, with respect to a number of physiological parameters related to dissolved inorganic carbon (DIC) acquisition and assimilation. The photosynthetic affinity for DIC was about one order of magnitude lower in cells cultivated at high [CO₂]. End‐point pH‐drift experiments suggest that P. reticulatum was not able to efficiently use HCO₃^? under our growth conditions. Only internal carbonic anhydrase (CA) activity was detected, and its activity decreased by about 60% in cells cultured at high [CO₂]. Antibodies raised against a variety of algal CAs were used for Western blot analysis: P. reticulatum extracts only cross‐reacted with anti‐ß‐CA sera, and the amount of immunoreactive protein decreased in cells grown at high [CO₂]. No pyrenoids were observed under all growth conditions. Our data indicate that P. reticulatum has an inducible carbon‐concentrating mechanism (CCM) that operates in the absence of pyrenoids and with little intracellular CO₂ accumulation. Calculations on the impact of the CA activity to photosynthetic growth [CO₂] suggest that it is an essential component of the CCM of P. reticulatum and is necessary to sustain the photosynthetic rates observed at ambient CO₂.     

Engineering the genetic components of a whole-cell catalyst for improved enzymatic CO2 capture and utilization

Carbonic anhydrase (CA) is a diffusion-limited enzyme that rapidly catalyzes the hydration of carbon dioxide (CO₂). CA has been proposed as an eco-friendly yet powerful catalyst for CO₂ capture and utilization. A bacterial whole-cell biocatalyst equipped with periplasmic CA provides an option for a cost-effective CO₂-capturing system. However, further utilization of the previously constructed periplasmic system has been limited by its relatively low activity and stability. Herein, we engineered three genetic components of the periplasmic system for the construction of a highly efficient whole-cell catalyst: a CA-coding gene, a signal sequence, and a ribosome-binding site (RBS). A stable and halotolerant CA (hmCA) from the marine bacterium Hydrogenovibrio marinus was employed to improve both the activity and stability of the system. The improved secretion and folding of hmCA and increased membrane permeability were achieved by translocation via the Sec-dependent pathway. The engineering of RBS strength further enhanced whole-cell activity by improving both the secretion and folding of hmCA. The newly engineered biocatalyst displayed 5.7-fold higher activity and 780-fold higher stability at 60°C compared with those of the previously constructed periplasmic system, providing new opportunities for applications in CO₂ capture and utilization.     

Analysis of expression and chitin‐binding activity of the wing disc cuticle protein BmWCP4 in the silkworm,Bombyx mori

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin‐binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic “R&R” chitin‐binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc‐containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full‐length BmWCP4 protein, “R&R” CBD peptide (CBD), non‐CBD peptide (BmWCP4‐CBD^?), four single site‐directed mutated peptides (M₁, M₂, M₃ and M₄) and four‐sites‐mutated peptide (M_F) were generated and purified, respectively, for in vitro chitin‐binding assay. The results indicated that both the full‐length protein and the “R&R” CBD peptide could bind with chitin, whereas the BmWCP4‐CBD^? could not bind with chitin. The single residue mutants M₁, M₂, M₃ and M₄ reduced but did not completely abolish the chitin‐binding activity, while four‐sites‐mutated protein M_F completely lost the chitin‐binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva‐to‐pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.     

Effect of the cellulose-binding domain on the catalytic activity of a β-glucosidase from <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis>

Enzyme engineering was performed to link the β-glucosidase enzyme (BGL1) from Saccharomycopsis fibuligera to the cellulose-binding domain (CBD2) of Trichoderma reesei cellobiohydrolase (CBHII) to investigate the effect of a fungal CBD on the enzymatic characteristics of this non-cellulolytic yeast enzyme. Recombinant enzymes were constructed with single and double copies of CBD2 fused at the N-terminus of BGL1 to mimic the two-domain organization displayed by cellulolytic enzymes in nature. The engineered S. fibuligera β-glucosidases were expressed in Saccharomyces cerevisiae under the control of phosphoglycerate-kinase-1 promoter (PGK1 _P ) and terminator (PGK1 _T ) and yeast mating pheromone α-factor secretion signal (MFα1 _S ). The secreted enzymes were purified and characterized using a range of cellulosic and non-cellulosic substrates to illustrate the effect of the CBD on their enzymatic activity. The results indicated that the recombinant enzymes of BGL1 displayed a 2–4-fold increase in their hydrolytic activity toward cellulosic substrates like avicel, amorphous cellulose, bacterial microcrystalline cellulose, and carboxy methyl cellulose in comparison with the native enzyme. The organization of the CBD in these recombinant enzymes also resulted in enhanced substrate affinity, molecular flexibility and synergistic activity, thereby improving the ability of the enzymes to act on and hydrolyze cellulosic substrates, as characterized by adsorption, kinetics, thermal stability, and scanning electron microscopic analyses.     

Inhibitory effects of some drugs on carbonic anhydrase enzyme purified from Kangal Akkaraman sheep in Sivas,Turkey

In this study, carbonic anhydrase (CA) enzyme was purified and characterized from blood samples of Kangal Akkaraman sheep and inhibitory properties on certain antibiotics were examined. CA purification was composed of preparation of the hemolysate and conducting the Sepharose‐4B‐tyrosine‐sulfanilamide affinity gel chromatography in having specific activity of 11626 EU mg^?1, yield of 14.40%, and 242.76‐fold purification. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was performed to assess the enzyme purity and a single band was observed. Some antibiotics were exhibited in vitro inhibition on the CA activity. IC₅₀ values of these inhibitors were calculated by plotting activity percentage. IC₅₀ values of certain drugs (dexamethasone; caffeine; metamizole sodium; tetramisol; ceftiofur HCl; ivermectin; tavilin 50; penokain G; neosym; and sulfamezathine) were found as 0.38, 8.24, 285.53, 114.77, 5.33, 2.76, 27.58, 213.50, 208.28, and 36.60 μM, respectively. K_i values of different drugs on Kangal Akkaraman sheep blood CA activity were found in the range of 0.21 ± 0.038–266.64 ± 37.11 μM.     

The effect of ethanol on erythrocyte carbonic anhydrase isoenzymes activity: An in vitro and in vivo study

The ethanol is a widely consumed as sedative-hypnotic drug throughout the world. In this study, the effects of ethanol were investigated on carbonic anhydrase (CA) enzyme activities both in vitro in human erythrocyte and in vivo in Sprague-Dawley rat erythrocyte. For in vitro study, the human carbonic anhydrase-I (HCA-I) and -II (HCA-II) are purified by Sepharose 4B–L-tyrosine-sulphanilamide affinity chromatography. In vivo CA enzyme activity was determined colorimetrically by using CO₂-hydration method of Wilbur and Anderson. Rat blood samples were taken from each rat before and after the ethanol administration at different times (1 h, 3 h, and 5 h). Rat erythrocyte CA activity was significantly inhibited by pharmacological dosage of the ethanol (2 mL.kg^{? 1}) for up to 3 h (p < 0.001) following intraperitoneally administration. The ethanol showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity, determined by colorimetrically using the CO₂-hydratase method. The inhibitor concentrations causing up to 50% inhibition (IC₅₀) were 2.09 M for HCA-I (r²:0.9273) and 1.83 M for HCA-II (r²:9749). In conclusion, it was demonstrated that carbonic anhydrase enzyme in erythrocytes was significantly inhibited by the ethanol both in in vitro and in vivo.     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

Immobilization and characterization of carbonic anhydrase purified from E. coli MO1 and its influence on CO2 sequestration

The present investigation entails the immobilisation and characterisation of Escherichia coli MO1-derived carbonic anhydrase (CA) and its influence on the transformation of CO₂ to CaCO₃. CA was purified from MO1 using a combination of Sephadex G-75 and DEAE cellulose column chromatography, resulting in 4.64-fold purification. The purified CA was immobilised in chitosan-alginate polyelectrolyte complex (C-A PEC) with an immobilisation potential of 94.5 %. Both the immobilised and free forms of the enzyme were most active and stable at pH 8.2 and at 37 °C. The K _m and V _max of the immobilised enzyme were found to be 19.12 mM and 416.66 μmol min^?1 mg^?1, respectively; whereas, the K _m and V _max of free enzyme were 18.26 mM and 434.78 μmol min^?1 mg^?1, respectively. The presence of metal ions such as Cu²⁺, Fe²⁺, and Mg²⁺ stimulated the enzyme activity. Immobilised CA showed higher storage stability and maintained its catalytic efficiency after repeated operational cycles. Furthermore, both forms of the enzyme were tested for targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The amounts of CaCO₃ precipitated over free and immobilised CA were 267 and 253 mg/mg of enzyme, respectively. The results of this study show that immobilised CA in chitosan-alginate beads can be useful for CO₂ sequestration by the biomimetic route.     

CO2‐concentrating mechanisms in three southern hemisphere strains of Emiliania huxleyi

Rising global CO₂ is changing the carbonate chemistry of seawater, which is expected to influence the way phytoplankton acquire inorganic carbon. All phytoplankton rely on ribulose‐bisphosphate carboxylase oxygenase (RUBISCO) for assimilation of inorganic carbon in photosynthesis, but this enzyme is inefficient at present day CO₂ levels. Many algae have developed a range of energy demanding mechanisms, referred to as carbon concentrating mechanisms (CCMs), which increase the efficiency of carbon acquisition. We investigated CCM activity in three southern hemisphere strains of the coccolithophorid Emiliania huxleyi W. W. Hay & H. P. Mohler. Both calcifying and non‐calcifying strains showed strong CCM activity, with HCO₃^? as a preferred source of photosynthetic carbon in the non‐calcifying strain, but a higher preference for CO₂ in the calcifying strains. All three strains were characterized by the presence of pyrenoids, external carbonic anhydrase (CA) and high affinity for CO₂ in photosynthesis, indicative of active CCMs. We postulate that under higher CO₂ levels cocco‐lithophorids will be able to down‐regulate their CCMs, and re‐direct some of the metabolic energy to processes such as calcification. Due to the expected rise in CO₂ levels, photosynthesis in calcifying strains is expected to benefit most, due to their use of CO₂ for carbon uptake. The non‐calcifying strain, on the other hand, will experience only a 10% increase in HCO₃^?, thus making it less responsive to changes in carbonate chemistry of water.     

Active transport of CO2 by three species of marine microalgae

The occurrence of an active CO₂ transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H¹³CO₃^? caused a rapid initial drop, followed by a slower decline in the extracellular CO₂ concentration. Addition of bovine CA to the medium raised the CO₂ concentration by restoring the HCO₃^?–CO₂ equilibrium, indicating that cells were taking up CO₂ and were maintaining the CO₂ concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO₂ concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of ¹⁸O from ¹³C¹⁸O₂ into water. Exchange of ¹⁸O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO₂ uptake by active transport for use as a carbon source for photosynthesis.     

N‐terminal fusion of a hyperthermophilic chitin‐binding domain to xylose isomerase from Thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes

Immobilization of a thermostable D ‐xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N‐terminal fusion with a chitin‐binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD‐TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD‐TNXI were greater than the wild‐type enzyme: k_cat (min^?1) was ～1,000, 3,800, and 5,800 at 80°C compared to 1,140, 10,350, and 7,000 at 90°C, for the wild‐type, unbound, and immobilized enzymes, respectively. These k_cat values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi‐phasic inactivation model, showed that the CBD‐TNXI bound to chitin had a half‐life approximately three times longer than the soluble wild‐type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD‐TNXI had a significantly longer half‐life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N‐terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD‐TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate‐binding affinities and generating higher turnover rates in the unbound fusion protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Carbonic anhydrase activity and inorganic carbon fluxes in low- and high-C1 cells of Chlamydomonas reinhardtü and Scenedesmus obliquus

Carbonic anhydrase (CA) activity associated with high- and low-dissolved inorganic carbon (C₁) grown cells was examined in whole cells by measuring ¹⁸O exchange from doubly labeled CO₂ (¹³C¹⁸O¹⁸O). Both algal species showed the presence of extracellular (periplasmic) as well as intracellular CA activity, which were both greatly increased in low-C₁ cells. The periplasmic CA activity was at least 40-fold higher in lowcompared to high-C₁ cells in both C. reinhardtii and S. obliquus. while low-C₁ cells of S. obliquus showed the highest activity of internal CA. The CA inhibitor ethoxyzolamide showed a strong inhibition of the C₁ uptake process in both C. reinhardtii and S. obliquus as in cyanobacteria. which may indicate that the nature of the primary uptake process is similar in both green algae and cyanobacteria. By using a mass spectrometnc disequilibrium technique it was possible to separate the C₁ fluxes of net HCO^?₃-uptake and net CO₂-uptake during steady-state photosynthesis in high- and Sow-C₁ grown cells of Chlamydomonas reinhardtii (WT. 2137+) and Scenedesmus obliquus (WT. D3). It was found that both high- and low-C₁ cells of the two algae can utilize both CO₂ and HCO^?₃ for photosynthesis, although low-C₁ cells have a higher affinity for the uptake of both C₁ species. Induction at low-C₁ causes an increase in the affinity of both species for HCO^?₃ and CO₂; changes in net CO₂-uptake were, however, significantly greater.     

Carbonic anhydrase,a respiratory enzyme in the gills of the shore crabCarcinus maenas

This paper summarizes investigations on the enzyme carbonic anhydrase (CA) in the gills of the osmoregulating shore crabCarcinus maenas. Carbonic anhydrase, an enzyme catalyzing the reversible hydration of CO₂ to HCO₃ ⁻ and H⁺, is localized with highest activities in the posterior salt-transporting gills of the shore crab- and here CA activity is strongly dependent on salinity. Contrary to the earlier hypothesis established for the blue crabCallinectes sapidus that cytoplasmic branchial CA provides the counter ions HCO₃ ⁻ and H⁺ for apical exchange against Na⁺ and Cl⁻, the involvement of CA in NaCl uptake mechanisms can be excluded inCarcinus. Differential and density gradient centrifugations indicate that branchial CA is a predominantly membrane-associated protein. Branchial CA was greatly inhibited by the sulfonamide acetazolamide (AZ) K_i=2.4·10⁻⁸ mol/l). Using the preparation of the isolated perfused gill, application of 10⁻⁴ mol/l AZ resulted in an 80% decrease of CO₂/HCO₃ ⁻ excretion. Thus we conclude that CA is localized in plasma membranes, maintaining the CO₂ gradient by accelerating adjustment of the pH-dependent CO₂/HCO₃ ⁻ equilibrium.     

Purification and Characterization of Carbonic Anhydrase from Ağrı Balık Lake Trout Gill (Salmo trutta labrax) and Effects of Sulfonamides on Enzyme Activity

Carbonic anhydrase (CA) was purified from A?r? Bal?k Lake trout gill (fCA) by affinity chromatography on a sepharose 4B‐tyrosine‐sulfanilamide column. The fCA enzyme was purified with about a 303.9 purification factor, a specific activity 4130.4 EU (mg‐protein)^–1, and a yield of 79.3 by using sepharose‐4B‐l tyrosine‐sulfanilamide affinity gel chromatography. The molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was found to be about 29.9 kDa. The kinetic parameters, K_M and V_max were determined for the 4‐nitrophenyl acetate hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CA enzymes. The K_i constants for mafenide ( 1 ), p‐toluenesulfonamide ( 2 ), 2‐bromo‐benzene sulfonamide ( 3 ), 4‐chlorobenzene sulfonamide ( 4 ), 4‐amino‐6‐chloro‐1–3 benzenedisulfonamide ( 5 ), sulfamethazine ( 6 ), sulfaguanidine ( 7 ), sulfadiazine ( 8 ), and acetozazolamide ( 9 ) were in the range of 7.5–108.75 μM.