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     

Control of CO2 fixation during the induction period. The role of thiol-mediated enzyme activation in the alga,Dunaliella

(1) Illumination of the unicellular green alga, Dunaliella, produced a 2–3-fold enhancement of ATPase activity in subsequently lysed algae. Using the inhibitor, tentoxin, it was shown that this light-induced activity, but not the light-independent activity, was attributable to the chloroplast coupling factor, CF₁. (1) A 4–5-fold increase in fructose-1,6-bisphosphatase activity was measured in Dunaliella lysed subsequent to illumination. (3) Experiments with methyl viologen demonstrated that both light-induced CF₁-ATPase and fructose-1,6-bisphosphatase activities were due to thiol-modulation of the enzymes by the algal thioredoxin system. (4) The light-induced increase in fructose-1,6-bisphosphatase activity could be simulated by incubation of intact algae in the dark with dithiothreitol. This thiol-induced increase in enzyme activity was accompanied by a decrease in the induction period of CO₂-dependent O₂ evolution upon subsequent measurement. (5) The kinetics of induction of both enzyme activities were very similar to the kinetics of induction of CO₂-dependent O₂ evolution in Dunaliella. As the light intensity was increased to 180 W · m² the steady-state enzyme activities increased in parallel with the rate of CO₂-dependent O₂ evolution. (6) The results are consistent with the imposition of a kinetic restraint on CO₂ fixation by the extent of enzyme activation under certain conditions in Dunaliella.     

Absorption characteristics and kinetics of CO2 capture into N-methyldiethanolamine aqueous solution catalyzed by the immobilized carbonic anhydrase

Abstract

Carbonic anhydrase (CA) is the most effective CO₂ hydratase catalyst, but the poor storage stability and repeatability of CA limit its development. Therefore, CA was immobilized on the epoxy magnetic composite microspheres to enhance the CO₂ absorption into N-methyldiethanolamine (MDEA) aqueous solution in this work. In the presence of immobilized CA, the CO₂ absorption rate of MDEA solution (10?wt%) (0.63?mmol·min^?1) was greatly improved by almost 40%, and their reaction equilibrium time was shortened from 150?min to 90?min compared with that into MDEA solution. The results indicated that the absorption of CO₂ into MDEA solution had been significantly enhanced by using CA. After the 7th reuse recycle, the activity of the immobilized CA was still closed to its initial value at 313.15?K. Moreover, enzyme catalytic kinetics of immobilized CA was investigated using the p-nitrophenyl acetate (p-NPA) as substrate. The values of Michaelis–Menten constant (K_m) and the maximum velocity (V_max) of the immobilized CA were calculated to be 27.61?mmol/L and 20.14?×?10^?3?mmol·min^?1·mL^?1, respectively. Besides, the kinetics of CO₂ reaction into MDEA with or without CA were also compared. The results showed that CO₂ absorption into CA/MDEA aqueous solution obeyed the pseudo first order regime and the second order kinetics rate constant (k₂) was calculated to be 929?m³·kmol^?1·s^?1, which was twice higher than that of MDEA aqueous solution without immobilized CA (k₂=414 m³·kmol^?1·s^?1) at 313.15?K.     

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO₂). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO₂, a major greenhouse gas, making this a promising alternative for chemical CO₂ mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO₂ sequestration by mineral carbonation, a process with the potential to store large quantities of CO₂. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO₂ compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO₃) formation and exerted a striking impact on the maximal amount of CaCO₃ produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO₂ sequestration.     

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.     

CRISPRi-mediated programming essential gene can as a Direct Enzymatic Performance Evaluation & Determination (DEPEND) system

Harnessing enzyme expression for production of target chemicals is a critical and multifarious process, where screening of different genes by inspection of enzymatic activity plays an imperative role. Here, we conceived an idea to improve the time-consuming and labor-intensive process of enzyme screening. Controlling cell growth was achieved by the Cluster Regularly Interspaced Short Palindromic Repeat (CRISPRi) system with different single guide RNA targeting the essential gene can (CRISPRi::CA) that encodes a carbonic anhydrase for CO₂ uptake. CRISPRi::CA comprises a whole-cell biosensor to monitor CO₂ concentration, ranging from 1% to 5%. On the basis of CRISPRi::CA, an effective and simple Direct Enzymatic Performance Evaluation & Determination (DEPEND) system was developed by a single step of plasmid transformation for targeted enzymes. As a result, the activity of different carbonic anhydrases corresponded to the colony-forming units. Furthermore, the enzymatic performance of 5-aminolevulinic acid synthetase (ALAS), which converts glycine and succinate-CoA to release a molecule of CO₂, has also been distinguished, and the effect of the chaperone GroELS on ALAS enzyme folding was successfully identified in the DEPEND system. We provide a highly feasible, time-saving, and flexible technology for the screening and inspection of high-performance enzymes, which may accelerate protein engineering in the future.     

CO2 bioconversion using carbonic anhydrase (CA): Effects of PEG rigidity on the structure of bio-mineralized crystal composites

The combined effect of both carbonic anhydrase (CA) and the rigidity of polyethylene glycol (PEG) were found to assist the bio-mineralized crystallization behavior of CO₂ differentially. In this study, different forms of magnetically responsive calcium carbonate (CaCO₃) crystal composites were successfully formed from gaseous CO₂ by using the different forms of polyethylene glycols (PEGs) in a constant CO₂ pressure controlled chamber. Polygonal particles were produced with more rigid polymer chains (branched PEG), whereas less rigid polymer chains (PEG) induced the formation of ellipsoidal particles. However, no morphological changes occurred without the presence of CA.     

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.     

Immobilization of carbonic anhydrase in alginate and its influence on transformation of CO2 to calcite

Present investigation entails carbonic anhydrase (CA) immobilization and its influence on transformation of CO₂ to calcite. CA enzyme was immobilized in alginate beads, subsequently maintained its catalytic efficiency after sequential operational cycles. The immobilized beads showed better operational stability by retaining nearly 67% of its initial activity even after six cycles. Batch scale studies with free and immobilized enzyme revealed that the entrapped CA hydrates CO₂ to bicarbonate and/or carbonate which was then made to react with Ca²⁺ ions to transform into calcite. Calcite was characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The entrapped CA was employed for the performance evaluation with respect to several operational parameters including the influence of enzyme concentration in free and immobilized condition. It was concluded that immobilized CA in alginate beads would have the potential for CO₂ sequestration by biomimetic route.     

Measurement of the Concentration of the Active Centers of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo

Methods for in vivo measurement of the concentration of the reactive centers of ribulose-1,5,-bisphosphate carboxylase/oxygenase (Rubisco) are suggested that are based on saturation of the active centers with RuBP and determination of the concentration of the Rubisco–RuBP complex. The total concentration of potentially reactive centers is calculated from the dependence of the concentration of this complex on CO₂ concentration at a steady-state photosynthetic rate with further extrapolation of the carbon dioxide dependence curve to a zero CO₂ concentration. The concentration of centers that possessed a catalytic activity under given environmental conditions was measured after transferring leaves having a steady-state photosynthetic rate into a medium devoid of CO₂ and O₂. This procedure ensured the saturation of the carboxylation centers with RuBP. The carboxylation rates were measured during a short-term exposure to ¹⁴CO₂, and the concentration of the complex was calculated using the values of CO₂ concentration during the exposure time, as well as the carboxylation rate and constant. Rubisco activity was found to decrease at elevated CO₂ concentrations due to a lower concentration of catalytically active enzyme centers.     

<Emphasis Type="Italic">Symbiobacterium</Emphasis> Lost Carbonic Anhydrase in the Course of Evolution

Recent genetic studies have elucidated that carbonic anhydrase (CA; EC 4.2.1.1), a ubiquitous enzyme catalyzing interconversion between CO₂ and bicarbonate, is essential for microbial growth under ambient air but not under high-CO₂ air. The irregular distribution of the phylogenetically distinct types of CA in the prokaryotic genome suggests its complex evolutionary history in prokaryotes. This paper deals with the genetic defect of CA in Symbiobacterium thermophilum, a syntrophic bacterium that effectively grows on CO₂ generated by other bacteria. Phylogenetic analysis based on 31 ribosomal protein sequences demonstrated the affiliation of Symbiobacterium with the class Clostridia with 100% bootstrap support. The phylogeny of β- and γ-type CA distributed among Clostridia supported the view that S. thermophilum and several related organisms lost this enzyme during the course of evolution. The loss of CA could be based on the availability of a high level of CO₂ in their living environments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A field portable gas-exchange system for measuring carbon dioxide and water vapour exchange rates of leaves during fumigation with SO2

Abstract A field portable, steady-state gas-exchange system which measures both CO₂ and water vapour exchange of single intact leaves during fumigations with SO₂ is described. Within the leaf cuvette temperature, light, humidity and both CO₂ and SO₂ concentrations are controlled to preset levels. Gas flow and concentrations are controlled by mass flow controllers. Photosynthetic uptake of CO₂ can be determined either by differential depletion or null balance measurement. Water vapour exchange is measured differentially and transpiration and conductance to water vapour determined. Sulphur dioxide is measured directly within the cuvette exhaust gas line by UV-pulse fluorescence. The performance of this system under field conditions is described and the physiological measurements compared with those obtained with other systems.     

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO₂ assimilation in Chlamydomonas has been developed in order to evaluate whether a CO₂-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO₂, low photorespiration, little O₂ inhibition of photosynthesis, and low CO₂ compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO₂-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO₂, and active transport of HCO ₃ ^- . Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO₂ diffusion or reversal of HCO ₃ ^- transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO₂-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO₂-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO₂-concentrating system. These calculations indicate that the system may be no more energy-costly than C₄ photosynthesis.Abbreviations Chl chlorophyll - RuBPC/O ribulose 1,5-bisphosphate carboxylase-oxygenase - CA carbonic anhydrase     

Control of steady-state photosynthesis in sunflowers growing in enhanced CO2

Control coefficients were used to describe the degree to which ribulose bisphosphate carboxylase/oxygenase (Rubisco) limits the steady-state rate of CO₂ assimilation in sunflower leaves from plants grown at high (800 μmol mol⁻¹) and low (350 μmol mol⁻¹) CO₂. The magnitude of a control coefficient is approximately the percentage change in the flux that would result from a 1% rise in enzyme active site concentration. In plants grown at low CO₂, leaves of different ages varied considerably in their photosynthetic capacities. In a saturating light flux and an ambient CO₂ concentration of 350 μmol mol⁻¹, the Rubisco control coefficient was about 0.7 in all leaves, indicating that Rubisco activity largely limited the assimilation flux. The Rubisco control coefficient for leaves grown at 350 μmol mol⁻¹ CO₂ dropped to about zero when the ambient CO₂ concentration was raised to 800 μmol mol⁻¹. In relatively young, fully expanded leaves of plants grown at high CO₂, the Rubisco control coefficient was also about 0.7 at a saturating light flux and at the CO₂ concentration at which the plants were grown (800 μmol mol⁻¹). This apparently resulted from a decrease in the concentration of Rubisco active sites. In older leaves, however, the control coefficient was about 0.2. Because, on the whole, Rubisco activity still largely limits the assimilation flux in plants grown at high CO₂, the kinetics of this enzyme can still be used to model photosynthesis under these conditions. The relatively high Rubisco control coefficient under enhanced CO₂ indicates that the young sunflower leaves have the capacity to acclimate their photosynthetic biochemistry in a way consistent with an optimal use of protein resources.     

Visualization of Cavitated Vessels in Winter and Refilled Vessels in Spring in Diffuse-Porous Trees by Cryo-Scanning Electron Microscopy

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 μmol quanta m⁻² s⁻¹ was followed, the slow increase in CO₂ assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO₂ assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO₂ assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.     

An improvement of the method for calibrating measurements of photosynthetic CO2 flux

Measurements of rapid changes in concentrations and fluxes of gaseous compounds relating to photosynthetic gas exchange are commonly performed using flow-through cuvettes in connection with infrared gas analysers. The accuracy and repeatability of these measurements relies ultimately upon the design of the system as a whole, rather than upon each of its components, and therefore the calibration and testing of the system should be performed keeping this in mind. We present here a simple and efficient method for the calibration of such a measurement system using a precisely determined CO₂ flow. This method gives us the opportunity to take into account any disturbing effects caused by undesired properties of the chamber or tubing materials. With the proposed calibration method, the accuracy of the CO₂ flux measurement is improved from 8% up to the level of 2%, determined mainly by the accuracy of the control gas used for calibration of the CO₂ analyser.     

Mathematische Analyse des Respirationssystems

The respiratory system is described as a control system. The controller consists of the peripheral and central chemoreceptors, the respiratory centre in the medulla oblongata and the controlling signal “alveolar ventilation”. The controlled system comprise three compartments (lung, brain, tissue) connected by the circulating blood. The controlled values of the system are explicit the arterial O₂-pressure and the CO₂-pressure of the brain-compartment. Hypoxia, hyperoxia and hypercapnia are the disturbing signals, which are caused by changing concentrations in the inspired gas. In this research both dynamic and steady-state behavior are studied. The steady-state and transient data of the model generally approach the findings of the experiments. The analysis of the efficiency of the regulation states the quality of the control system. In the on-and off-transients the CO₂-fractions of the alveolar gas, and in the off-transient the alveolar ventilation deviate from the experimental results in hypercapnic disturbances. Reasons for these differences and others existing between simulation and experiment are discussed.

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Continuous Measurements of the Free Dissolved CO(2) Concentration during Photosynthesis of Marine Plants: Evidence for HCO(3) Use in Chondrus crispus

An experimental system consisting of a gas exchange column linked to an assimilation chamber has been developed to record continuously the free dissolved CO₂ concentration in seawater containing marine plants. From experiments performed on the red macroalga Chondrus crispus (Rhodophyta, Gigartinales), this measurement is in agreement with the free CO₂ concentration calculated from the resistance to CO₂ exchanges in a biphasic system (gas and liquid) as earlier reported. The response time of this apparatus is short enough to detect, in conditions of constant pH, a photosynthesis-caused gradient between free CO₂ and HCO₃⁻ pools which half-equilibrates in 25 seconds. Abolished by carbonic anhydrase, the magnitude of this gradient increases with decreasing time of seawater transit from the chamber to the column apparatus. But its maximum magnitude (0.35 micromolar CO₂) is negligible compared to the difference between air and free CO₂ (11.4 micromolar CO₂). This illustrates the extent of the physical limiting-step occurring at the air-water interface when inorganic carbon consumption in seawater is balanced by dissolving gaseous CO₂. The direction of this small free CO₂/HCO₃⁻ gradient indicates that HCO₃⁻ is consumed during photosynthesis.     

Carbonic Anhydrase Activity Associated with the Cyanobacterium Synechococcus PCC7942

Intact cells and crude homogenates of high (1% CO₂) and low dissolved inorganic carbon (C_i) (30-50 microliters per liter of CO₂) grown Synechococcus PCC7942 have carbonic anhydrase (CA)-like activity, which enables them to catalyze the exchange of ¹⁸O from CO₂ to H₂O. This activity was studied using a mass spectrometer coupled to a cuvette with a membrane inlet system. Intact high and low C_i cells were found to contain CA activity, separated from the medium by a membrane which is preferentially permeable to CO₂. This activity is most apparent in the light, where ¹⁸O-labeled CO₂ species are being taken up by the cells but the effluxing CO₂ has lost most of its label to water. In the dark, low C_i cells catalyze the depletion of the ¹⁸O enrichment of CO₂ and this activity is inhibited by both ethoxyzolamide and 2-(trifluoromethoxy)carbonyl cyanide. This may occur via a common inhibition of the C_i pump and the C_i pump is proposed as a potential site for the exchange of ¹⁸O. CA activity was measurable in homogenates of both cell types but was 5- to 10-fold higher in low C_i cells. This was inhibited by ethoxyzolamide with an I₅₀ of 50 to 100 micromolar in both low and high C_i cells. A large proportion of the internal CA activity appears to be pelletable in nature. This pelletability is increased by the presence of Mg²⁺ in a manner similar to that of ribulose bisphosphate carboxylase-oxygenase activity and chlorophyll (thylakoids) and may be the result of nonspecific aggregation. Separation of crude homogenates on sucrose gradients is consistent with the notion that CA and ribulose bisphosphate carboxylase-oxygenase activity may be associated with the same pelletable fraction. However, we cannot unequivocally establish that CA is located within the carboxysome. The sucrose gradients show the presence of separate soluble and pelletable CA activity. This may be due to the presence of separate forms of the enzyme or may arise from the same pelletable association which is unstable during extraction.