Heterologous expression of carbonic anhydrase in Acinetobacter sp. Tol 5 for whole-cell biocatalysis.

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

Procedures for isolating carbonic anhydrase (EC 4.2.1.1) enzymes from the erythrocytes and the mucosae of the gastrointestinal tract of guinea pigs are described. From a haemolysate, haemoglobin was removed by the addition of ammonium sulphate, and also by two other methods, namely by gel filtration or by adsorption on DEAE-Sephadex. The crude enzyme thus obtained was resolved into the different isoenzymes by chromatography with DEAE-cellulose. From particle-free supernatants of homogenates of some gastrointestinal tissues, carbonic anhydrases were purified by ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography with DEAE-cellulose. The major isoenzymes from blood, stomach, proximal colonic mucosa and caecal mucosa were homogeneous during ion-exchange chromatography, acrylamide-gel electrophoresis, and centrifugal examination. From these tissues, carbonic anhydrase was isolated as two major isoenzymes. They resemble the pairs of isoenzymes discovered in the bloods of other species. The carbon dioxide hydratase activity of one isoenzyme (;high activity' carbonic anhydrase) was 40 times that of the other isoenzyme (;low activity' carbonic anhydrase), as measured at a single substrate concentration. Two other minor components of the enzyme are also found in guinea-pig erythrocytes. All of the enzymes isolated had molecular weights of nearly 30000 (sedimentation equilibrium). ;High activity' carbonic anhydrases from blood and gastrointestinal tissues were indistinguishable according to some chemical, physical and kinetic measurements; similarly ;low activity' carbonic anhydrases from those tissues were indistinguishable. ;High activity' carbonic anhydrase was markedly different from the ;low activity' carbonic anhydrase with respect to its amino acid composition, chromatographic behaviour and isoelectric pH value. Marked differences were also found in the tissue concentrations of the major isoenzymes. It is suggested that the characteristic and selective distribution of the different forms of carbonic anhydrase in the guinea-pig tissues is related to the specific and different physiological functions of the enzymes.

Summary1. Carbonic anhydrases from vertebrates, plants and bacteria have molecular weights of 30,000 (or multiples thereof), contain one zinc atom per 30,000 molecular weight, and are inhibited by acetazolamide and related compounds.2. In mammals, there are two major isoenzymes of carbonic anhydrase. The so‐called ‘high activity’ carbonic anhydrase possesses a carbon dioxide‐hydratase activity many times (in the case of the guinea‐pig isoenzymes, 18 times) that of the ‘low activity’ isoenzyme.3. The mammalian isoenzymes differ from one another in their amino‐acid compositions (the difference in serine contents being a consistent finding in a number of species), in their physical properties (isoelectric pH, retention by ion‐exchange resins, electrophoretic mobility) and in their kinetic properties.4. Mammalian carbonic anhydrases also catalyse the hydrolysis of some esters and the hydration of aldehydes. Their relative activities with these other substrates may be substantially different from their relative activities with carbon dioxide as substrate. The discovery that carbonic anhydrase may also catalyse other reactions raises the possibility that the enzyme may have other roles in metabolic pathways (e.g., in certain dehydrogenation reactions).5. In plants, the role of carbonic anhydrase may be to catalyse the inter‐conversion of bicarbonate and carbon dioxide, to provide ‘carbon dioxide’ in the form appropriate for carbon‐fixing reactions. A similar role has been suggested for the carbonic anhydrase found in Neissariae.6. The carbonic anhydrase of vertebrates (the ‘high activity’ isoenzyme of mammals) is found in many ion‐transporting epithelia, but its role in them is still uncertain. It occurs in acid‐transporting epithelia such as the stomach and kidney, and also in certain bicarbonate‐transporting epithelia like the large intestine. There is, however, a poor correlation between the presence of the enzyme and the occurrence of acid or bicarbonate secretion. Thus there is little or no carbonic anhydrase in some tissues noted for their ability to secrete bicarbonate ions, such as the pancreas and ileum. Conversely, carbonic anhydrase‐containing tissues like the avian salt gland and the elasmobranch rectal gland form concentrated sodium chloride solutions which are of nearly neutral pH. The correlation between the distribution of carbonic anhydrase and the occurrence of active chloride transport appears similar to that between carbonic anhydrase and bicarbonate ion transport.7. While the red cells of most mammalian species so far studied contain both ‘high activity’ and ‘low activity’ isoenzymes, the latter is reported to be absent from the erythrocytes of sheep, ox and dog. Presumably the ‘low activity’ isoenzyme is not necessary for an adequate rate of carbon dioxide exchange to occur between tissues and lungs in these species.8. The ‘low activity’ isoenzyme is present in tissues other than blood, for example, the colon, caecum and ox rumen, and probably also, the gall bladder and kidney medulla. Its distribution differs markedly from that of the ‘high activity’ isoenzyme and presumably it has a particular functional importance of its own. It is not possible to define the role of the ‘low activity’ isoenzyme at present, but attention is drawn to the possibility that it is concerned in the handling of the products of microbial fermentation, such as ammonia and organic acids, by the large intestines and ruminant forestomach.My work on the isoenzymes of carbonic anhydrase was supported by a Medical Research Council Scholarship, and by an equipment grant to Dr D. S. Parsons. I am also grateful to Dr D. S. Parsons for his comments on the manuscript.

Carbonic anhydrase is a valuable and efficient catalyst for CO(2) hydration. Most often the free enzyme is employed which complicates catalyst recycling, and can increase cost due to the need for protein purification. Immobilization of the enzyme may address these shortcomings. Here we report the development of whole-cell biocatalysts for CO(2) hydration via periplasmic expression of two forms of carbonic anhydrase in Escherichia coli using two different targeting sequences. The enzymatic turnover numbers (kcat ) and catalytic efficiencies (k(cat)/K(M)) were decreased by an order of magnitude as compared to the free soluble enzyme, indicating the introduction of transport limitations. However, the thermal stabilities were improved for most configurations (>88% activity retention up to 95°C for three of four whole-cell biocatalysts), operational stabilities were more than satisfactory (100% retention after 24 h of use for all four whole-cell biocatalysts), and CO(2) hydration was significantly enhanced relative to the uncatalyzed reaction (~50-70% increase in CaCO(3) precipitate formed). A significant advantage of the whole-cell approach is that protein purification is no longer necessary, and the cells can be easily separated and recycled in future applications including biofuel production, biosensors, and carbon capture and storage.

1. Details are given of an electrometric method for measuring the activity of isoenzymes of carbonic anhydrase (EC 4.2.1.1) in catalysing the hydration of carbon dioxide under different conditions at 0 degrees C. In the method, a measured volume of water saturated with carbon dioxide at a known partial pressure and appropriate temperature is introduced into a buffered solution. Using a sensitive electrometer and recording instrument, the subsequent change in hydrogen ion concentration is recorded as a function of time. Under the conditions of assay, the pH change induced in the presence of substrate is very small (DeltapH < 0.05 units) and the period of observation need not exceed 10 sec.2. For enzymes isolated from guinea-pig tissues, it is found that the specific activity of the ;high activity' isoenzyme (carbonic anhydrase C, carbonic anhydrase II, HACA) is about eighteen times that of the ;low activity' counterpart (carbonic anhydrase B, carbonic anhydrase I, LACA) when measured at 0 degrees C, pH 7.2, and ionic strength 0.19. Under the same conditions, the K(m) was found to be 10 mM for the ;high activity' isoenzyme and 23 mM for the ;low activity' isoenzyme. No differences were found between the equivalent kinetic parameters of the corresponding isoenzymes isolated from different tissues.3. The isoenzymes isolated from guinea-pig tissues are found to be inhibited by acetazolamide in a non-competitive manner. It is also found that the ;high activity' isoenzyme is many times more sensitive to this inhibitor than is the ;low activity' isoenzyme. Evidence is presented which indicates that one acetazolamide binding site is present on each molecule of either isoenzyme.4. While chloride ions specifically inhibit the ;low activity' component of guinea-pig carbonic anhydrase (I(0.5) = 40 mM), acetate, butyrate and pyruvate inhibit both isoenzymes. Under the conditions employed, acetate and pyruvate are more strongly inhibitory to the ;low activity' isoenzyme than to the ;high activity' isoenzyme, while butyrate is more strongly inhibitory to the ;high activity' isoenzyme.5. The findings are discussed with particular reference to the physiological significance of the presence of the isoenzymes in the gastro-intestinal tract. Also considered are possible relationships between the distribution of the ;low activity' isoenzyme in these tissues and the transport and metabolism of products of fermentation occurring in the intestinal lumen.

Carbonic anhydrase (CA) is a diffusion-controlled enzyme that rapidly catalyzes carbon dioxide (CO2) hydration. CA has been considered as a powerful and green catalyst for bioinspired CO2 capture and utilization (CCU). For successful industrial applications, it is necessary to expand the pool of thermostable CAs to meet the stability requirement under various operational conditions. In addition, high-level expression of thermostable CA is desirable for the economical production of the enzyme. In this study, a thermostable CA (tdCA) of Thermosulfurimonas dismutans isolated from a deep-sea hydrothermal vent was expressed in Escherichia coli and characterized in terms of expression level, solubility, activity and stability. tdCA showed higher solubility, activity, and stability compared to those of CA from Thermovibrio ammonificans, one of the most thermostable CAs, under low-salt aqueous conditions. tdCA was engineered for high-level expression by the introduction of a point mutation and periplasmic expression via the Sec-dependent pathway. The combined strategy resulted in a variant showing at least an 8.3-fold higher expression level compared to that of wild-type tdCA. The E. coli cells with the periplasmic tdCA variant were also investigated as an ultra-efficient whole-cell biocatalyst. The engineered bacterium displayed an 11.9-fold higher activity compared to that of the recently reported system with a halophilic CA. Collectively these results demonstrate that the highly expressed periplasmic tdCA variant, either in an isolated form or within a whole-cell platform, is a promising biocatalyst with high activity and stability for CCU applications.

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

1. The total carbonic anhydrase activity in some guinea-pig tissues has been measured using a pH-stat procedure. Stomach, gall bladder, proximal colon and caecum all possess more carbonic anhydrase activity per unit amount of protein than does whole blood.2. The carbonic anhydrase activity of the small intestine is low. Reasons are given for supposing that activity found there is not entirely due to contamination by whole blood, and it is suggested that in this tissue the enzyme may be localized in some cell type other than the columnar absorbing cells.3. Evidence is presented which indicates that heavy metals interfere with the activity of the enzyme as measured in tissue homogenates.4. The distribution and concentration of the two major isoenzymes of carbonic anhydrase have been measured in different tissues. Blood and proximal colon contain both isoenzymes in comparable concentrations, the ratio of the concentration of the ;low activity' isoenzyme to that of the ;high activity' being about 2. The gastric mucosa contains much ;high activity' carbonic anhydrase, but only a negligible amount of the ;low activity' isoenzyme. In the caecal mucosa, the ;low activity' isoenzyme is predominant, the ratio of its concentration to that of the ;high activity' isoenzyme being about 9. It is also found that more than 1.5% of the protein in the caecal mucosa is accounted for as carbonic anhydrase enzymes.5. It is found that some 45% of the total carbonic anhydrase activity of sucrose homogenates of the guinea-pig colon is bound to particles. The activity is located mainly in the nuclear and microvillous fraction and in the ;high-speed supernatant' fraction. The form of enzyme bound is largely of the ;high activity' variety. When the tissue is homogenized in potassium chloride solutions less than 4% of the total activity is recovered in particulate fractions. The amount of activity which is bound to particulate fractions increases as the ionic strength or pH of the homogenate is lowered.6. The findings are discussed in relation to the possible physiological roles of the isoenzymes in tissues other than blood. Possible relationships between the presence of the enzymes and the metabolism and transport of ammonium and fatty acids are considered.

Two major components of carbonic anhydrase were purified from porcine red cells by column chromatography and electrofocusing techniques. Both forms behaved as single components in sedimentation velocity experiments and during starch gel electrophoresis. The observed molecular weight of both forms was about 3 x 104. On the basis of their specific CO2 hydrase activities and amino acid compositions, these two carbonic anhydrase isozymes were designated as high activity (carbonic anhydrase C) or low activity (carbonic anhydrase B) forms which appear to be homologous to the high and low activity carbonic anhydrases, respectively, of other mammals. When these pig B and C isozymes were compared with the red cell carbonic anhydrases of other ungulates (cattle and horse), several interesting features were observed. In contrast to the electrophoretic gel patterns of the horse B and C isozymes in which the C form is markedly more basic than the B form, the high activity C form of pig was observed to be more acidic than the low activity B form. The tryptic peptide map of bovine carbonic anhydrase appears to be more similar to that of porcine carbonic anhydrase C than to B, indicating that they are probably homologous proteins. Neoprontosil binding by the pig and horse B isozymes give rise to essentially identical spectra in the 425- to 600-mµ region, whereas the C isozymes, from these two sources, generate quite different spectra.

Carbonic anhydrase: chemistry, physiology, and inhibition.

Enhanced carbon capture and utilization (CCU) using heterologous carbonic anhydrase in Chlamydomonas reinhardtii for lutein and lipid production

The urgent need to address global warming necessitates the development of advanced technologies for carbon capture and storage (CCS). Although carbon dioxide absorption into alkanolamines or carbonate solutions is among the most common CCS processes, these approaches harbor some limitations. Carbonic anhydrase enzymes can significantly increase the efficacy of CO2 absorption into capture solvent solutions. Carbonic anhydrase from Sulfurihydrogenibium yellowstonense (SspCA) is a well-known enzyme with favorable properties for CO2 absorption into capture solutions. Here, using computational tools, strategic proline substitutions were designed to enhance the thermal stability of SspCA. Compared to the wild type, the engineered mutants, E145P and N153P, showed an increase of 1.6-4.3 °C in the melting temperature. After 14 h at 80 °C, the wild type retained only 6% ± 1% of its initial activity, while N153P and E145P retained 33% ± 3% and 44% ± 1%, respectively. The E145P and N153P mutants in aqueous potassium carbonate medium at 60 °C outperformed the wild type in retention of CO2 hydration activity. In addition, an increase in the catalytic efficiency of the E145P mutant, along with a decrease in its Km value, indicated that proline substitution facilitates substrate binding. Molecular dynamics simulations exhibited the proline-induced structural changes, particularly reduced terminal fluctuations. Structural studies unveiled the formation of a new salt bridge connecting the C- and N-terminal regions of carbonic anhydrase, contributing to reduced fluctuations and enhanced stability. This study underscores the success of introducing proline substitutions in fortifying carbonic anhydrase stability and catalytic efficacy, which is vital for enzymatic carbon capture and storage technologies.

Mineralization has emerged as a promising strategy for long-term carbon sequestration. These processes involve carbon dioxide hydration followed by mineral precipitation. We have explored the production of whole-cell biocatalysts engineered with carbonic anhydrase (CA) activity to accelerate the CO₂ hydration reaction. In this study, short polypeptides were displayed on the surface of E. coli cells and whole-cell biocatalysts containing periplasmically expressed CAs in an attempt to enhance calcite mineral formation. It was found that cells coexpressing recombinant periplasmic CA and surface-displayed GPA peptide (PEVPEGAFDTAI) outperformed other peptide-expressing biocatalysts evaluated in terms of the amount of precipitate formed, as well as the overall formation rate of solids. Cells expressing the Cab CA isoform (BLR-pCab) and Cam isoform (BLR-pCam) with the surface-displayed GPA peptide exhibited 36 and 59% improvements in precipitation amounts, as well as 18 and 60% improvements in overall formation rates, respectively, over similar biocatalysts without GPA expression. The biocatalyst with the best performance was BLR-pCam/GPA, which generated 0.15 g of CaCO₃, while BLR cells generated only 0.08 g of CaCO₃ under the same small batch reaction conditions. The BLR-pCam/GPA cells also exhibited the fastest formation rates, achieving the maximum change in solution turbidity after only 2.2 min, as opposed to 6.3 min for BLR cells. These results demonstrate that synthetic biology approaches can be used to create novel biocatalysts with the ability to enhance both catalysis and precipitation activities.

Carbonic anhydrase isonenzymes in infants with respiratory distress syndrome

Characterisation of carbonic anhydrases from tissues of the cat