Periplasmic expression of carbonic anhydrase in Escherichia coli: A new biocatalyst for CO2 hydration

Carbonic anhydrase is a valuable and efficient catalyst for CO₂ 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 CO₂ hydration via periplasmic expression of two forms of carbonic anhydrase in Escherichia coli using two different targeting sequences. The enzymatic turnover numbers (k_cat) 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 CO₂ hydration was significantly enhanced relative to the uncatalyzed reaction (～50–70% increase in CaCO₃ 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. Biotechnol. Bioeng. 2013; 110: 1865–1873. © 2013 Wiley Periodicals, Inc.     

碳酸酐酶的生理功能、多样性及其在CO2捕集中的应用

碳酸酐酶（carbonic anhydrase）作为一种活性中心含有锌离子的金属酶,能够可逆催化CO2生成碳酸氢盐的水合反应,该反应在生物体内承担着多样的生理学功能,具有高度的生物学意义。除广泛存在于真核生物以外,该酶在淡水、海水、嗜常温、嗜热、厌氧、好氧、致病、产酸、自养、异养等多种原核微生物中也有广泛的分布,并参与光合作用、呼吸作用和以CO2作为底物的反应,维持生理pH以及离子转运等生理过程。近年来,随着温室效应的日益加剧．生物固定CO2作为该酶的一种全新应用引起了研究者的广泛关注。回顾了碳酸酐酶作为催化剂参与CO2固定过程的历史、现状和最新发现,同时展望了未来应用的趋势。     

Synthesis,characterization and carbonic anhydrase inhibitory activity of novel benzothiazole derivatives

N-protected amino acids were reacted with substituted benzothiazoles to give the corresponding N-protected amino acid-benzothiazole conjugates (60–89%). Their structures were confirmed by proton nuclear magnetic resonance (¹H NMR), carbon-13 nuclear magnetic resonance (¹³C NMR), IR and elemental analysis. Their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activities were determined against two cytosolic human isoforms (hCA I and hCA II), one membrane-associated (hCA IV) and one transmembrane (hCA XII) enzyme by a stopped-flow CO₂ hydrase assay method. The new compounds showed rather weak, micromolar inhibitory activity against most of these enzymes.     

Trends,application and future prospectives of microbial carbonic anhydrase mediated carbonation process for CCUS

Summary

Growing industrialization and the desire for a better economy in countries has accelerated the emission of greenhouse gases (GHGs), by more than the buffering capacity of the earth's atmosphere. Among the various GHGs, carbon dioxide occupies the first position in the anthroposphere and has detrimental effects on the ecosystem. For decarbonization, several non‐biological methods of carbon capture, utilization and storage (CCUS) have been in use for the past few decades, but they are suffering from narrow applicability. Recently, CO₂ emission and its disposal related problems have encouraged the implementation of bioprocessing to achieve a zero waste economy for a sustainable environment. Microbial carbonic anhydrase (CA) catalyses reversible CO₂ hydration and forms metal carbonates that mimic the natural phenomenon of weathering/carbonation and is gaining merit for CCUS. Thus, the diversity and specificity of CAs from different micro‐organisms could be explored for CCUS. In the literature, more than 50 different microbial CAs have been explored for mineral carbonation. Further, microbial CAs can be engineered for the mineral carbonation process to develop new technology. CA driven carbonation is encouraging due to its large storage capacity and favourable chemistry, allowing site‐specific sequestration and reusable product formation for other industries. Moreover, carbonation based CCUS holds five‐fold more sequestration capacity over the next 100 years. Thus, it is an eco‐friendly, feasible, viable option and believed to be the impending technology for CCUS. Here, we attempt to examine the distribution of various types of microbial CAs with their potential applications and future direction for carbon capture. Although there are few key challenges in bio‐based technology, they need to be addressed in order to commercialize the technology.     

The case for chloroplast thylakoid carbonic anhydrase

Washed thylakoid membranes and photosystem II-enriched membrane fragments from cyanobacteria, green algae, and chloroplasts from both C₃ and C₄ plants possess the ability to reversibly hydrate CO₂. That is, the membranes have an intrinsic carbonic anhydrase activity. The present review outlines the discovery of thylakoid carbonic anhydrase and presents the evidence that it is a unique isozyme, distinct from other cellular carbonic anhydrases. It appears that at least some thylakoid carbonic anhydrase is closely associated with photosystem II and may be required for electron transport. This would explain why all inhibitors of carbonic anhydrase also inhibit photosystem II. Several speculative functions of thylakoid carbonic anhydrase are discussed. These include a possible role in carbon metabolism, in the protonation of plastoquinone, and/or in oxygen evolution.     

Virtual screening-driven identification of human carbonic anhydrase inhibitors incorporating an original, new pharmacophore

Combinated ligand- and pharmacophore-based virtual screening approaches were used to discover novel potential pharmacophores acting as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). A free database of commercially available compounds was screened through drug-like filters using a four-point pharmacophore, and followed by docking calculation within the active site of an X-ray structure of isoform CA II. One compound, bearing a trifluoro-dihydroxy-propanone moiety, showed an interesting, selective inhibitory activity in low micromolar range against this isoform versus CA I. The chemical originality of this new pharmacophore can represent an important bioisosteric alternative to the sulfonamido-based functionalities, thus leading to the development of a new class of CAIs.     

Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.

Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAIV, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.     

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-l-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K_m and V_max values for the same substrate by Lineweaver–Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K_m values at optimum pH and 20°C were 0.625?mM, 0.541?mM, 0.785?mM and 0.862?mM with p-nitro phenyl acetate, for all CA localizations. The respective V_max values at optimum pH and 20°C were 0.875?μmol/L?min, 0.186?μmol/L?min, 0.214?μmol/L?min and 0.253?μmol/L?min with the same substrate. The K_i and I₅₀ values for the inhibitors sulphanilamide, KSCN, NaN₃ and acetazolamide were determined for all the CA localizations.     

Production,purification, and characterization of a fusion protein of carbonic anhydrase from Neisseria gonorrhoeae and cellulose binding domain from Clostridium thermocellum

Carbon dioxide capture technologies have the potential to become an important climate change mitigation option through sequestration of gaseous CO₂. A new concept for CO₂ capture involves use of immobilized carbonic anhydrase (CA) that catalyzes the reversible hydration of CO₂ to HCO₃^? and H⁺. Cost‐efficient production of the enzyme and an inexpensive immobilization system are critical for development of economically feasible CA‐based CO₂ capture processes. An artificial, bifunctional enzyme containing CA from Neisseria gonorrhoeae and a cellulose binding domain (CBD) from Clostridium thermocellum was constructed with a His₆ tag. The chimeric enzyme exhibited both CA activity and CBD binding affinity. This fusion enzyme is of particular interest due to its binding affinity for cellulose and retained CA activity, which could serve as the basis for improved technology to capture CO₂ from flue gasses. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.     

Discovering novel carbonic anhydrase type IX (CA IX) inhibitors from seven million compounds using virtual screening and in vitro analysis

Carbonic anhydrase type IX (CA IX) enzyme is mostly over expressed in different cancer cell lines and tumor tissues. Potent CA IX inhibitors can be effective for adjusting the pH imbalance in tumor cells. In the present work, we represented the successful application of high throughput virtual screening (HTVS) of large dataset from ZINC database included of ～7 million compounds to discover novel inhibitors of CA IX. HTVS and molecular docking were performed using consequence Glide/standard precision (SP), extra precision (XP) and induced fit docking (IFD) molecular docking protocols. For each compound, docking code calculates a set of low-energy poses and then exhaustively scans the binding pocket of the target with small compounds. Novel CA IX inhibitor candidates were suggested based on molecular modeling studies and a few of them were tested using in vitro analysis. These compounds were determined as good inhibitors against human CA IX target with K_i in the range of 0.85–1.58?μM. In order to predict the pharmaceutical properties of the selected compounds, ADME (absorption, distribution, metabolism and excretion) analysis was also carried out.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

Virtual screening,molecular dynamics,and binding free energy calculations on human carbonic anhydrase IX catalytic domain for deciphering potential leads

Carbonic anhydrase IX is a tumor-associated membrane-bound metallo-enzyme which catalyzes the reversible hydration of carbon dioxide (CO₂) to bicarbonate (HCO₃^?) and proton (H⁺) ions. It is a hypoxia-inducible enzyme and plays a critical role in tumor pH homeostasis favoring tumor cell invasiveness and drug resistance. Over expression of CAIX is documented in cancers of breast, lung, kidney, colon/rectum, etc. Chemical inhibition of CAIX activity has proven to be an effective therapeutic modality towards targeting cancer. Hence, in this study, we intend to identify potential molecules from NCI (National Cancer Institute) and Maybridge databases implementing high-throughput virtual screening. CAIX co-crystallized with acetazolamide (a known inhibitor of CAIX) (PDB ID: 3IAI) was used for reference-guided docking protocol. The potential inhibitors among the coupled data sets were finalized based on Glide docking score, Prime/MMGBSA scoring, significant intermolecular interactions, ADMET (absorption, distribution, metabolism and excretion, toxicity) prediction and stability of complex formation, molecular dynamics simulation, and comparative analysis. By this study, we propose NSC_93618, NSC_170253, NSC_93618, JFD03677, SEW06488, and BTB09372 to be highly significant, as all these compounds were found to qualify as potential leads surpassing all the stringent filtering process. However, NSC_93618 was found to be the most potential, as it featured with higher complex stability with strong bonded interactions, binding affinity synonymous to acetazolamide. Hence, these proposed compounds shall prove to be effective in targeting CAIX towards modulating carcinogenesis.     

Decreased total carbonic anhydrase esterase activity and decreased levels of carbonic anhydrase 1 isozyme in erythrocytes of type II diabetic patients

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pH_i for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes. Jehan Ornasir—these studies were undertaken as a partial requirement of her M.S. Degree, Graduate School, Howard University, Washington, DC, USA     

Role of intracellular carbonic anhydrase in inorganic-carbon assimilation by Porphyridium purpureum

Air-grown cells of Porphyridium purpurem contain appreciable carbonic-anhydrase activity, comparable to that in air-grown Chlamydomonas reinhardtii, but activity is repressed in CO₂-grown cells. Assay of carbonic-anhydrase activity in intact cells and cell extracts shows all activity to be intracellular in Porphyridium. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution shows that sodium ions increase the affinity of Porphyridium cells for HCO ₃ ^- . Acetazolamide and ethoxyzolamide were potent inhibitors of carbonic anhydrase in cell extracts but at pH 5.0 both acetazolamide and ethoxyzolamide had little effect upon the concentration of inorganic carbon required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]). At pH 8.0, where HCO ₃ ^- is the predominant species of inorganic carbon, the K_0.5 (CO₂) was increased from 50 M to 950 M in the presence of ethoxyzolamide. It is concluded that in air-grown cells of Porphyridium. HCO ₃ ^- is transported across the plasmalemma and intracellular carbonic anhydrase increases the steady-state flux of CO₂ from inside the plasmalemma to ribulose-1,5-bisphosphate carboxylase-oxygenase by catalysing the interconversion of HCO ₃ ^- and CO₂ within the cell.Abbreviations AZ acetazolamide - EZ ethoxyzolamide - K_0.5[CO₂] half-maximal rate of photosynthetic O₂ evolution     

Discovery of new potent inhibitors for carbonic anhydrase IX by structure-based virtual screening

Through structure-based virtual screening, some dozen of benzene sulfonamides with novel scaffolds are identified as potent inhibitors against carbonic anhydrase (CA) IX with IC₅₀ values ranging from 2.86 to 588.34 nM. Among them, compounds 1 and 9 show high selectivity against tumor-target CA IX over CA II (the selectivity ratios are 21.3 and 136.6, respectively). The possible binding poses of hit compounds are also explored and the selectivity is elucidated by molecular docking simulations. The hit compounds discovered in this work would provide novel scaffolds for further hit-to-lead optimization.     

Characterisation of cDNA clones for rat muscle carbonic anhydrase III

cDNA clones for rat muscle carbonic anhydrase III have been isolated from a gt-11 library and sequenced. Comparison with human CAIII cDNA showed about 90% homology to rat. The rat clones were used to estimate mRNA from liver and muscle on Northern blots and showed that the sexual dimorphism of CAIII in rat liver relates to a difference in mRNA levels.     

Selected trace elements and esterase activity of carbonic anhydrase levels in lambs with pneumonia

The levels of, zinc, copper, Fe, Zn, Cu, Mn, Mg, K, Na, and Cl and the activity of carbonic anhydrase were determined in lambs with pneumonia. A significant decrease of p<0.01 level in Zn concentration, in Cu level (p<0.001) and significant increases in K and Na levels (p<0.05) and of the Cu/Zn ratio (p<0.001) were observed in the study group. The carbonic anhydrase activity was decreased in the study group, but the decrease was not statistically significant (p>0.05). Also, nonsignificant decreases of Fe, Mg, and Cl and increase of the Mn concentration were also observed in the lambs with pneumonia (p>0.05). Our results suggest that the significant element changes reported here and the Cu/Zn ratio, but not the activity of carbonic anhydrase, can be used as indicators of pneumococcal infection.     

Synthesis and characterization of novel substituted thiophene derivatives and discovery of their carbonic anhydrase and acetylcholinesterase inhibition effects

Novel substituted thiophene derivatives ( 1, 2a‐e, 3, and 4 ) were synthesized and their structures were characterized by infrared radiation, nuclear magnetic resonance, and mass analysis. These novel substituted thiophene derivatives were effective inhibitor compounds of the carbonic anhydrase I and II isozymes (hCA I and II), and acetylcholinesterase (AChE) enzyme with K _i values in the range of 447.28 to 1004.65 nM for hCA I, 309.44 to 935.93 nM for hCA II, and 0.28 to 4.01 nM for AChE, respectively. Novel substituted thiophene derivatives can be good candidate drugs for the treatment of some diseases like neurological disorders, epilepsy, glaucoma, gastric and duodenal ulcers, mountain sickness, or osteoporosis as carbonic anhydrase isozymes inhibitors, and for the treatment of Alzheimer’s and Parkinson’s diseases as acetylcholinesterase inhibitors.