Bovine Carbonic Anhydrase Research Articles

Enhanced sequestration of carbon dioxide into calcium carbonate using pressure and a carbonic anhydrase from alkaliphilic Coleofasciculus chthonoplastes.

CO2 in the atmosphere is a major contributor to global warming but at the same time it has the potential to be a carbon source for advanced biomanufacturing. To utilize CO2, carbonic anhydrase has been identified as a key enzyme. Furthermore, attempts have been made to accelerate the sequestration via pressure. This study aims to combine both approaches to achieve high sequestration rates. The carbonic anhydrase of the alkaliphilic cyanobacterium Coleofasciculus chthonoplastes (cahB1) and bovine carbonic anhydrase (BCA) are introduced into a high‐pressure reactor to catalyze the hydration of CO2 at up to 20 bar. The reactor is filled with a CaCl2 solution. Due to the presence of Ca2+, the hydrated CO2 precipitates as CaCO3. The impact of the carbonic anhydrase is clearly visible at all pressures tested. At ambient pressure a CO2 sequestration rate of 243.68 kgCaCO3/m3 h for cahB1 was achieved compared to 150.41 kgCaCO3/m3 h without enzymes. At 20 bar the rates were 2682.88 and 2267.88 kgCaCO3/m3 h, respectively. The study shows the benefit of a combined CO2 sequestration process. To examinate the influence of the enzymes on the product formation, the precipitated CaCO3 was analyzed regarding the crystalline phase and morphology. An interchange of the crystalline phase from vaterite to calcite was observed and discussed.

Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions.

A versatile approach to entrap relatively small enzymes in hydrogels allows their diverse biotechnological applications. In the present work, bovine carbonic anhydrase (BCA) was efficiently entrapped in calcium alginate beads with the help of liposomes. A mixture of sodium alginate (3 wt %) and carbonic anhydrase–liposome conjugates (BCALs) was dripped into a Tris-HCl buffer solution (pH = 7.5) containing 0.4 M CaCl2 to induce the gelation and curing of the dispersed alginate-rich droplets. The entrapment efficiency of BCALs, which was defined as the amount of catalysts entrapped in alginate beads relative to that initially charged, was 98.7 ± 0.2% as determined through quantifying BCALs in the filtrate being separated from the beads. When free BCA was employed, on the other hand, a significantly lower entrapment efficiency of 27.2 ± 4.1% was obtained because free BCA could pass through alginate matrices. Because the volume of a cured alginate bead (10 μL) entrapped with BCALs was about 2.5 times smaller than that of an original droplet, BCALs were densely present in the beads to give the concentrations of lipids and BCA of 4.6–8.3 mM and 1.1–1.8 mg/mL, respectively. Alginate beads entrapped with BCALs were used to catalyze the hydrolysis of 1.0 mM p-nitrophenyl acetate (p-NA) at pH = 7.5 using the wells of a microplate or 10 mL glass beakers as batch reactors. Furthermore, the beads were confined in a column for continuous-flow hydrolysis of 1.0 mM p-NA for 1 h at a mean residence time of 8.5 or 4.3 min. The results obtained demonstrate that the conjugation of BCA to liposomes gave an opportunity to achieve efficient and stable entrapment of BCA in alginate hydrogels for applying to catalytic reactions in bioreactors.

Uncovering the structure-function aspects of an archaeal CsaA protein

CsaA is known to function as a protein secretion chaperone in bacteria. Homologs of CsaA are also found in archaea while they are absent in eukaryotes. This paper presents the biophysical, biochemical analysis and crystallographic structure determination of CsaA from a thermoacidophilic archaeon Picrophilus torridus (PtCsaA). The PtCsaA appears to prevent the aggregation of heat denatured Bovine Carbonic Anhydrase II (BCAII). Differential denaturation of PtCsaA by guanidine hydrochloride (Gdn-HCl) and urea indicates the stabilization of the protein via salt bridges. Denaturant mediated decrease in 8-Anilinonaphthalene-1-sulfonic acid (ANS) binding and shift in wavelength signifies the partial unfolding of the protein molecule and exposure of hydrophobic patches to solvent on denaturation. The crystal structure of PtCsaA was solved to a resolution of 1.7 Å. The structure of PtCsaA appears to be similar to bacterial CsaA in architecture. Docking of a six amino acid peptide in the substrate binding pocket of PtCsaA suggests conservation in the substrate binding cavity. Residues involved in the formation of the binding cavity and hydrogen bonds responsible for the dimerization of PtCsaA were compared with those observed in the structure of Bacillus subtilis CsaA. The similarities and differences in electrostatic surface potential of the substrate binding cavities in bacterial CsaA and PtCsaA are discussed.

Nitrous anhydrase activity of carbonic anhydrase II: cysteine is required for nitric oxide (NO) dependent phosphorylation of VASP in human platelets

The carbonic anhydrase (CA) family does not only catalyse the reversible hydration of CO2 to bicarbonate, but it also possesses esterase and phosphatase activity. Recently, bovine CA II and human CA II have been reported to convert inorganic nitrite (O=N-O−) to nitric oxide (NO) and nitrous anhydride (N2O3). Given the ability of NO to mediate vasodilation and inhibit platelet aggregation, this CA II activity would represent a bioactivation of nitrite. There are contradictory reports in the literature and the physiological role of CA II nitrite bioactivation is still disputed. Here, we provide new experimental data in support of the nitrous anhydrase activity of CA II and the key role L-cysteine in the bioactivation of nitrite by CA II. Using washed human platelets and by measuring VASP phosphorylation we provide evidence that exogenous nitrite (10 µM) is bioactivated to NO in a manner strongly depending on L-cysteine (100 and 200 µM). The process is not inhibitable by acetazolamide, a potent CA inhibitor. The contradictory results of recently published studies in this area are thoroughly discussed.

Protein-caged zinc porphyrin as a carbonic anhydrase mimic for carbon dioxide capture

Zinc tetraphenylporphyrin (Zn-TPP) solubilized by GroEL protein cage was prepared as a supramolecular mimic of carbonic anhydrase (CA) for CO2 capture. It is shown that the soluble Zn-TPP-GroEL complex can be formed easily by detergent dialysis. The Zn-TPP/GroEL binding ratio was found to increase with their dialysis ratio until reaching the maximum of about 30 porphyrins per protein cage. Moreover, the complex showed hydrase activity that catalyzes the CO2 hydration in HCO3− and H+. It is further seen that the catalytic activity of Zn-TPP-GroEL was about one-half of that of a bovine CA at 25 °C. On the other hand, as the temperature was increased to 60 °C close to an industrial CO2 absorption temperature, the natural enzyme lost function while Zn-TPP-GroEL exhibited better catalytic performance indicative of a higher thermal stability. Finally, we demonstrate that the GroEL-solubilized Zn-TPP is able to accelerate the precipitation of CO2 in the form of CaCO3 and has better long-term performance than the bovine CA. Thus a new type of nano-caged system mimicking natural CAs for potential applications in carbon capture has been established.

PH swing adsorption process for ambient carbon dioxide capture using activated carbon black adsorbents and immobilized carbonic anhydrase biocatalysts

Adsorbent-based carbon capture is only feasible if adsorption-desorption cycles are both fully regenerating and efficient. This work proposes a regenerative pH swing process and a pH swing regenerative adsorbent that are inspired by natural CO2 conversion by carbonic anhydrase biocatalysts found in mammalian red blood cells. The main objective is to develop, test and analyze a synthetic pH Swing Adsorption (pHSA) system as well as a pHSA compatible solid adsorbent to capture CO2 from a simulated ambient air gas stream. The lead developed adsorbent is a carbon black co-activated with potassium carbonate and nitrogenous copolymer that is impregnated with immobilized bovine carbonic anhydrase and thereby deemed “BCA/KN-CB”. BCA/KN-CB has preliminarily demonstrated both competitive CO2 adsorption capacity and limited regenerative ability under experimental pHSA conditions. In addition, BCA-based adsorbents achieved higher adsorption capacities than non-BCA adsorbent counterparts. The BCA/KN-CB adsorbent displayed both large point of zero charge (PZC) swings and regenerative stability. The proposed pHSA system requires essentially zero energy expenditure to achieve intended environments for capture and regeneration. With 1 kg of adsorbent, pHSA has the ability to capture 1 kg CO2 in less than 4 h of cycling. The tested pHSA adsorbent can also capture more than 96% of total CO2 in a given raw gas stream flowing through the capture chamber. This proof-of-concept study of a pH swing adsorption/biocatalytic adsorbent system suggests the potential to effectively operate under ambient conditions and exhibit advantageous operational efficiencies to other high-profile CO2 capture systems.

Investigation into reversed phase chromatography peptide separation systems part III: Establishing a column characterisation database

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape.

The presence of cross-β-structure as a key determinant of carbonic anhydrase amyloid fibrils cytotoxicity

In most cases high cytotoxicity is characteristic of aggregates formed during lag phase of amyloid formation, whereas mature fibrils represent the depot of protein molecules incapable of damaging cell membranes. However, new experimental data show that in cases of some proteins the fibrils are the most toxic type of aggregates. Meanwhile, structural characteristics of cytotoxic fibrils and mechanisms of their cell damaging action are insufficiently explored. This work is dedicated to studying amyloid aggregation of bovine carbonic anhydrase (BCA) and effect of aggregates formed at different stages of amyloid formation on viability of the cells. Here we demonstrate that oligomers formed during lag phase do not decrease cell viability, whereas protofibrils and amyloids of BCA are cytotoxic. Obtained results allow concluding that toxicity of BCA aggregates is associated with the presence of amyloid cross-β-structure, which signature is absorbance peak at low wavenumbers at FTIR spectra (1615-1630 cm−1). Our data suppose that cross-β-core of ВСА amyloid fibrils is responsible for their cytotoxicity.

Stabilized and Immobilized Carbonic Anhydrase on Electrospun Nanofibers for Enzymatic CO2 Conversion and Utilization in Expedited Microalgal Growth.

Carbonic anhydrases convert CO2 to bicarbonate at a high turnover rate up to 106 s-1, but their actual applications in CO2 conversion processes are hampered by their poor stability. This study reports highly loaded and stabilized bovine carbonic anhydrase (bCA) upon being immobilized onto electrospun polymer nanofibers in the form of enzyme precipitate coating (EPC). The EPC protocol, consisting of enzyme covalent attachment, precipitation, and cross-linking, maintained 65.3% of initial activity even after being incubated in aqueous solution at room temperature under shaking at 200 rpm for 868 days. EPC also showed strong resistance to the treatment of the metal chelation agent, ethylenediaminetetraacetic acid, and molecular dynamic simulation was carried out to elucidate the prevention of metal leaching from the active site of bCA upon being cross-linked in the form of EPC. Highly stable EPC with high bCA loading was employed for the conversion of bubbling CO2 to bicarbonate, and the bicarbonate solution was utilized as a carbon source for expedited microalgae growth in a separate bioreactor. The addition of EPC in the bubbling CO2 reactor resulted in 134 and 231% accelerated microalgae growths compared to the controls with and without 25 mM sodium bicarbonate, respectively. EPC with high enzyme loading and unprecedentedly successful stabilization of enzyme stability has a great potential to be used for the development of various enzyme-mediated CO2 conversion and utilization technologies.

A two-enzyme cascade reaction consisting of two reaction pathways. Studies in bulk solution for understanding the performance of a flow-through device with immobilised enzymes.

Enzyme-catalysed cascade reactions in flow-through systems with immobilised enzymes currently are of great interest for exploring their potential for biosynthetic and bioanalytical applications. Basic studies in this field often aim at understanding the stability of the immobilised enzymes and their catalytic performance, for example, in terms of yield of a desired reaction product, analyte detection limit, enzyme stability or reaction reproducibility. In the work presented, a cascade reaction involving the two enzymes bovine carbonic anhydrase (BCA) and horseradish peroxidase (HRP) – with hydrogen peroxide (H2O2) as HRP “activator” – was first investigated in great detail in bulk solution at pH = 7.2. The reaction studied is the hydrolysis and oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA) to 2′,7′-dichlorofluorescein (DCF), which was found to proceed along two reaction pathways. This two-enzyme cascade reaction was then applied for analysing the performance of BCA and HRP immobilised in glass fiber filters which were placed inside a filter holder device through which a DCFH2-DA/H2O2 substrate solution was pumped. Comparison was made between (i) co-immobilised and (ii) sequentially immobilised enzymes (BCA first, HRP second). Significant differences for the two arrangements in terms of measured product yield (DCF) could be explained based on quantitative UV/vis absorption measurements carried out in bulk solution. We found that the lower DCF yield observed for sequentially immobilised enzymes originates from a change in one of the two possible reaction pathways due to enzyme separation, which was not the case for enzymes that were co-immobilised (or simultaneously present in the bulk solution experiments). The higher DCF yield observed for co-immobilised enzymes did not originate from a molecular proximity effect (no increased oxidation compared to sequential immobilisation).

생물 유기분자 유도 실리카 합성실험을 통한 생체내 유무기 복합체 형성이해

The discovery of the principle molecules involved in biosilicification both in diatoms and sponges brings out a new paradigm for silica synthesis at environmentally benign conditions. Here we introduce the biosynthetic method of silica using organic molecules for the undergraduate students. First, the biosilica synthesized was assessed through both a colorimetric method for quantification of silica and structural analysis by SEM image observation. Next, facile immobilization of an enzyme was demonstrated. Specifically, bovine carbonic anhydrase II (bCAII) was efficiently immobilized within silica matrix by simply adding itself to the reaction mixture containing polyamines and tetramethyl orthosilicate in phosphate buffer, obtaining 97% immobilization to initial amount of bCAII. The immobilized bCAII retained 40% of initial activity at 90℃ whereas free form did less than 10% of initial activity at 70℃ and the total yield from the 5 times reuses of immobilized bCAII was twice that of free bCAII. This experimental content that is hard to find in general biology or biochemistry lab could contribute to students’ interest in knowledge expansion and application of new materials.

Carbonic anhydrase II does not regulate nitrite-dependent nitric oxide formation and vasodilation.

Although it has been reported that bovine carbonic anhydrase CAII is capable of generating NO from nitrite, the function and mechanism of CAII in nitrite-dependent NO formation and vascular responses remain controversial. We tested the hypothesis that CAII catalyses NO formation from nitrite and contributes to nitrite-dependent inhibition of platelet activation and vasodilation. The role of CAII in enzymatic NO generation was investigated by measuring NO formation from the reaction of isolated human and bovine CAII with nitrite using NO photolysis-chemiluminescence. A CAII-deficient mouse model was used to determine the role of CAII in red blood cell mediated nitrite reduction and vasodilation. We found that the commercially available purified bovine CAII exhibited limited and non-enzymatic NO-generating reactivity in the presence of nitrite with or without addition of the CA inhibitor dorzolamide; the NO formation was eliminated with purification of the enzyme. There was no significant detectable NO production from the reaction of nitrite with recombinant human CAII. Using a CAII-deficient mouse model, there were no measurable changes in nitrite-dependent vasodilation in isolated aorta rings and in vivo in CAII-/- , CAII+/- , and wild-type mice. Moreover, deletion of the CAII gene in mice did not block nitrite reduction by red blood cells and the nitrite-NO-dependent inhibition of platelet activation. These studies suggest that human, bovine and mouse CAII are not responsible for nitrite-dependent NO formation in red blood cells, aorta, or the systemic circulation.

Screening of benzenesulfonamide in combination with chemically diverse fragments against carbonic anhydrase by differential scanning fluorimetry

The differential scanning fluorimetry (DSF) screening of 5.692 fragments in combination with benzenesulfonamide (BSA) against bovine carbonic anhydrase (bCA) delivered >100 hits that either caused, on their own, a significant thermal shift (ΔTm , °C) in the protein melting temperature or significantly influenced the thermal shift observed for BSA alone. Three hits based on 1,2,3-triazole moiety represent the periphery of the recently reported potent inhibitors of hCA II, IX and XII which were efficacious in vivo. Such a re-discovery of suitable BSA periphery essentially validates the new fragment-based approach to the discovery of future CAIs. Structures of other validated fragment hits are reported.

Further validation of strecker-type α-aminonitriles as a new class of potent human carbonic anhydrase II inhibitors: hit expansion within the public domain using differential scanning fluorimetry leads to chemotype refinement

Testing of an expanded, 800-compound set of analogues of the earlier described Strecker-type α-aminonitriles (selected from publicly available Enamine Ltd. Screening Collection) in thermal shift assay against bovine carbonic anhydrase (bCA) led to further validation of this new class of inhibitors and identification a new, refined chemotype represented by inhibitors with 10-improved potency.

The copper(II) binding centres of carbonic anhydrase are differently affected by reductants that ensure the redox intracellular environment

Copper is involved in several biological processes. The static and labile copper pools are controlled by means of a network of influx and efflux transporters, storage proteins, chaperones, transcription factors and small molecules as glutathione (GSH), which contributes to the cell reducing environment. To follow the fate of intracellular copper labile pool, a variant of human apocarbonic anhydrase has been proposed as fluorescent probe to monitor cytoplasmic Cu2+. Aware that in this cellular compartment copper ion is present as Cu+, electron spin resonance technique (ESR) was used to ascertain whether (bovine or human) carbonic anhydrase (CA) was able to accommodate Cu+ in the same sites occupied by Cu2+, in the presence of naturally occurring reducing agents such as ascorbate and GSH. Our ESR results on Cu2+ complexes with CA allow for a complete characterization of the two metal binding sites of the protein in solution. The use of the reported affinity constants of zinc in the catalytic site and of Cu2+ in the peripheral and catalytic site, allow us to obtain the speciation of copper species mimicking the spectroscopic study conditions. The different Cu2+ coordination features in the catalytic and the peripheral (the N-terminus cleft mouth) binding sites influence the chemical reduction effect of the two main naturally occurring reductants. Ascorbate reversibly reduces the Cu2+ complex with CA, while glutathione irreversibly induces the formation of Cu2+ complex with its oxidized form (GSSG). Our results questioned the use of CA as intracellular Cu2+ sensor. Furthermore, translating these findings to intracellular environment, the conversion of GSH in GSSG can significantly alter the metallostasis.

Direct immobilization and recovery of recombinant proteins from cell lysates by using EctP1-peptide as a short fusion tag for silica and titania supports

Immobilization of protein, compared to the use of free protein, offers improved stability, easy separation and continuous reusability. However, the classic routes for protein immobilization, based on non-specific adsorption, often negatively affect protein functionality. In this study, EctP1 peptide was explored as a novel short fusion tag for non-covalent adsorption on unmodified solid surfaces, silica and titania. A fusion of EctP1 with bovine carbonic anhydrase (BCA) was employed to investigate the optimal binding conditions that could diminish the nonspecific adsorption of Escherichia coli proteins. The stable binding of BCA-EctP1 on titania was observed in the pH range of 2–9, while the stable binding on silica was in the pH range 6–9. Moreover, the immobilized BCA-EctP1 on silica and titania particles showed enhanced thermal and storage stability and retained 95% of its residual activity after 5 uses. We further demonstrated the merits of the noncovalent immobilization of EctP1 fusion proteins to silica and titania in the recovery of the bound proteins. Interestingly, monomeric arginine showed better recovery yield of EctP1 fusion proteins (about 78–84%), compared to the recovery yield by the salts, NaCl and MgCl2 (about 30–51%). Using BCA and monomeric red fluorescent protein (mRFP) as model proteins, the EctP1 fusion proteins were released in a biologically active form with approximately 80% recovery and 93% purity. Our approach is a simple and reproducible technique for direct immobilization of recombinant proteins from E. coli lysates on solid supports, with the potential high-purity recovery of recombinant proteins.

Enzyme-Immobilized Chitosan Nanoparticles as Environmentally Friendly and Highly Effective Antimicrobial Agents.

Highly effective and minimally toxic antimicrobial agents have been prepared by immobilizing glucose oxidase (GOx) onto biocompatible chitosan nanoparticles (CS-NPs). CS-NPs were prepared via ionotropic gelation and used for the immobilization of GOx via approaches of covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetic nanoparticle-incorporated EPC (Mag-EPC). EPC represents an approach consisting of enzyme covalent attachment, precipitation, and cross-linking, with CA and EC being control samples while Mag-EPC was prepared by mixing magnetic nanoparticles (Mag) with enzymes during the preparation of EPC. The GOx activities of CA, EC, EPC, and Mag-EPC were 8.57, 17.7, 219, and 247 units/mg CS-NPs, respectively, representing 26 and 12 times higher activity of EPC than those of CA and EC, respectively. EPC improved the activity and stability of GOx and led to good dispersion of CS-NPs, while Mag-EPC enabled facile magnetic separation. To demonstrate the expandability of the EPC approach to other enzymes, bovine carbonic anhydrase was also employed to prepare EPC and Mag-EPC samples for their characterizations. In the presence of glucose, EPC of GOx generated H2O2 in situ, which effectively inhibited the proliferation of Staphylococcus aureus in both suspended cultures and biofilms, thereby demonstrating the potential of EPC-GOx as environmentally friendly and highly effective antimicrobial materials.

Effect of Aquaporin 5 and Carbonic Anhydrase II on the Diffusion of CO2 in Xenopus Oocytes

Transport of gases across the plasma membrane is not only dependent on the permeability, but also the concentration gradient. Carbon dioxide (CO2) is one of the most important physiological gases in the human body, but undergoes slow hydration/dehydration reactions. As a gas channel, human (h) AQP5 can accelerate the diffusion of CO2 by increasing membrane permeability to CO2. Carbonic anhydrases (CAs) also can accelerate the transport of CO2 across the membrane by maximizing the CO2 gradient via the consumption/production of CO2. Here, by measuring the maximal extracellular‐surface pH increase (DpHS) caused by the introduction of extracellular CO2/HCO3− at constant extracellular pH (pHo), we obtain a semiquantitative index of the CO2 influx into Xenopus oocytes. On Day 1, we injected oocytes with the cRNA encoding hAQP5 (or H2O as a control). On Day 4, we injected purified bovine CA II protein in Tris buffer (or Tris buffer as a control). In agreement with previous reports, both hAQP5 and CA II individually increase DpHS vs. H2O/Tris‐injected control oocytes. However, the effect of CA II alone in raising DpHS even at very high CA II concentrations (1000ng/oocyte), is not as effective as AQP5 alone. Most likely, the native CO2 permeability of Xenopus oocytes is so low that it limits transmembrane CO2 diffusion. Interestingly, injection of CA II—even at CA II levels so low that they barely increase DpHS in oocytes not expressing AQP5—into oocytes expressing AQP5 causes DpHS to increase substantially vs. AQP5 oocytes without CA II. Our data suggest that when membrane CO2 permeability is low, the effects of AQP5 and CA on CO2 fluxes are supra‐additive.Support or Funding InformationNIH U01‐GM111251, N00014‐15‐1‐2060, N00014‐16‐1‐2535This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Experimental approach to study the effect of mutations on the protein folding pathway.

Is it possible to compare the physicochemical properties of a wild-type protein and its mutant form under the same conditions? Provided the mutation has destabilized the protein, it may be more correct to compare the mutant protein under native conditions to the wild-type protein destabilized with a small amount of the denaturant. In general, is it appropriate to compare the properties of proteins destabilized by different treatments: mutations, pH, temperature, and denaturants like urea? These issues have compelled us to search for methods and ways of presentation of experimental results that would allow a comparison of mutant forms of proteins under different conditions and lead to conclusions on the effect of mutations on the protein folding/unfolding pathway. We have studied equilibrium unfolding of wild-type bovine carbonic anhydrase II (BCA II) and its six mutant forms using different urea concentrations. BCA II has been already studied in detail and is a good model object for validating new techniques. In this case, time-resolved fluorescence spectroscopy was chosen as the basic research method. The main features of this experimental method allowed us to compare different stages of unfolding of studied proteins and prove experimentally that a single substitution of the amino acid in three mutant forms of BCA II affected the native state of the protein but did not change its unfolding pathway. On the contrary, the inserted disulfide bridge in three other mutant forms of BCA II affected the protein unfolding pathway. An important result of this research is that we have validated the new approach allowing investigation of the effect of mutations on the folding of globular proteins, because in this way it is possible to compare proteins in the same structural states rather than under identical conditions.

Novel bovine carbonic anhydrase encapsulated in a metal-organic framework: a new platform for biomimetic sequestration of CO2.

In this work, maximizing the utilization of CO2 and its precipitation as CaCO3 by using immobilized bovine carbonic anhydrase (BCA) was evaluated. In this way, selection of suitable carriers which have a gas adsorption function would enhance the CO2 sequestration efficiency of the carbonic anhydrase (CA). So a metal–organic framework (MOF), an excellent material for gas adsorption and enzyme immobilization was used. In this manner, BCA was encapsulated into the microporous zeolite imidazolate framework, ZIF-8, for the first time, using a bottle-around-a-ship method. Systematic characterization including powder X-ray diffraction (PXRD), UV-vis, and Fourier transform infrared (FT-IR) spectroscopies, BET, field emission scanning electron microscopy (FE-SEM) and energy dispersive X-ray (EDX) confirmed that the entrapment of BCA molecules was successfully achieved during the crystal growth of ZIF-8 with an enzyme loading of ca. 100 ± 1.2 mg g−1 of BCA–ZIF-8. Optimization of the matrix for increasing the stability of the enzyme in an encapsulated form is the main aim of the present study. The de novo approach was proposed because this method provides better enzyme protection from degradation, minimizes enzyme leaching and enables multiple reuse. Then, the influence of different parameters, including pH, temperature, storage and reusability, was evaluated for enzyme@MOF composites versus free enzymes. The prepared biocatalyst exhibited outstanding activity in a wide pH and temperature range and demonstrates high storage stability up to 37 days. This efficient and simple association procedure seems well-adapted to produce an enzymatic bio-catalyst for biocatalytic hydration of CO2. The FT-IR analysis revealed that the structure of BCA was well maintained during the encapsulation process. The thermal stability and reusability of the BCA–ZIF-8 increased noticeably due to the structural rigidity and confinement of the ZIF-8 scaffolds. These two parameters are very important for practical applications.