Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas.

Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a ß-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.

Role of Trp19 and Tyr200 in catalysis by the γ-class carbonic anhydrase from Methanosarcina thermophila.

Although widely distributed in Nature, only two γ class carbonic anhydrases are reported besides the founding member (Cam). Although roles for active-site residues important for catalysis have been identified in Cam, second shell residues have not been investigated. Two residues (Trp19 and Tyr200), positioned distant from the catalytic metal, were investigated by structural and kinetic analyses of replacement variants. Steady-state k(cat)/K(m) and k(cat) values decreased 3- to 10-fold for the Trp19 variants whereas the Y200 variants showed up to a 5-fold increase in k(cat). Rate constants for proton transfer decreased up to 10-fold for the Trp19 variants, and an increase of ~2-fold for Y200F. The pK(a) values for the proton donor decreased 1-2 pH units for Trp19 and Y200 variants. The variant structures revealed a loop composed of residues 62-64 that occupies a different conformation than previously reported. The results show that, although Trp19 and Y200 are non-essential, they contribute to an extended active-site structure distant from the catalytic metal that fine tunes catalysis. Trp19 is important for both CO(2)/bicarbonate interconversion, and the proton transfer step of catalysis.

Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the {gamma} class of carbonic anhydrases.

The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the gamma class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from M. thermophila (Mt-CamH) was overproduced in Escherichia coli and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from E. coli cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had k(cat) and k(cat)/K(m) values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from E. coli cultured with supplemental iron (Fe-Mt-CamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective k(cat) (k(cat)(eff)) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the k(cat)/K(m)(eff) was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the k(cat)(eff) of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H(2)O(2), which supports the hypothesis that Fe(2+) has a role in the active site. The k(cat) for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a "ping-pong" mechanism accepting a proton. The k(cat)/K(m) was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO(2) and HCO(3)(-) is separate from intermolecular proton transfer.

The archetype gamma-class carbonic anhydrase (Cam) contains iron when synthesized in vivo.

A recombinant protein overproduction system was developed in Methanosarcina acetivorans to facilitate biochemical characterization of oxygen-sensitive metalloenzymes from strictly anaerobic species in the Archaea domain. The system was used to overproduce the archetype of the independently evolved gamma-class carbonic anhydrase. The overproduced enzyme was oxygen sensitive and had full incorporation of iron instead of zinc observed when overproduced in Escherichia coli. This, the first report of in vivo iron incorporation for any carbonic anhydrase, supports the need to reevaluate the role of iron in all classes of carbonic anhydrases derived from anaerobic environments.

Carbonic anhydrase activators: activation of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases with amino acids and amines.

Activation of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases (CAs, EC 4.2.1.1) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. Cab, Zn-Cam and Co-Cam showed an activation profile with natural, L- and D-amino acids very different of those of the alpha-class enzymes CA I, II and III. Most of these compounds showed medium efficacy as archaeal CA activators, except for D-Phe and L-Tyr which were effective Cab activators (K(A)s of 10.3-10.5 microM), 2-pyridylmethylamine and 1-(2-aminoethyl)-piperazine which effectively activated Zn-Cam (K(A)s of 10.1-11.4 microM) and serotonin, L-adrenaline and 2-pyridylmethylamine which were the best Co-Cam activators (K(A)s of 0.97-8.9 microM). We prove here that the activation mechanisms of the alpha-, beta-, and gamma-class CAs are similar, although the activation profiles with various compounds differ dramatically between these diverse enzymes.

Influence of current or former smoking on asthma management and control.

BACKGROUND: In patients with asthma, smoking has been associated with accelerated decline in pulmonary function, poor disease control and reduced responsiveness to corticosteroids. OBJECTIVE: To assess the influence of current and former smoking on self-reported asthma control and health care use in a large population of asthma patients. METHODS: The present analysis was conducted following a telephone survey of adult Canadians aged 18 to 54 years who had physician-diagnosed asthma and a smoking history of less than 20 pack-years. RESULTS: Of 893 patients, 268 were former smokers and 108 were current smokers. Daytime and nighttime symptoms, absenteeism from work or school, emergency care use for asthma in the past year, and use of a short-acting bronchodilator without controller medication were reported more frequently by current smokers than nonsmokers and former smokers. Former smokers were not significantly different from nonsmokers with respect to most asthma outcomes. CONCLUSIONS: Current smokers with asthma show evidence of poorer asthma control and greater acute care needs than lifelong nonsmokers or former smokers. These observations stress the importance of smoking cessation to help achieve asthma control.

Asthma control in Canada: no improvement since we last looked in 1999.

OBJECTIVE: To determine whether asthma control in Canada had improved since the last major survey in 1999 by exploring how well patients' asthma was controlled, how much they knew about asthma control, and how they used health care resources. DESIGN: National telephone survey of patients between April and August 2004. SETTING: Canada. PARTICIPANTS: Eight hundred ninety-three adults 18 to 54 years old diagnosed with asthma more than 6 months before the survey. MAIN OUTCOME MEASURES: Patients' control of their asthma, patients' knowledge about asthma, the frequency and duration of periods of worsening asthma, and patients' use of health care resources to manage those periods. RESULTS: In total, 26,210 households listed in a consumer database were contacted. Excluding ineligible households and households with a language barrier, a member of 13% of the households completed the 35-minute survey. Based on definitions in Canadian guidelines, 53% of patients had symptomatic uncontrolled asthma. In the previous year, almost all asthma patients had experienced worsening of symptoms that lasted on average 13.6 days for patients with uncontrolled asthma and 8.0 days for patients with controlled asthma (P < .02). Markedly more patients with uncontrolled asthma used health care resources for episodes of asthma than patients with controlled asthma did (72% vs 15% for urgent office visits, P < .01; 32% vs 3% for emergency department visits, P < .01; and 7% vs 0% for hospitalizations, P < .01) in the year before the survey. Patients were confused about the differences between reliever and controller medications. One third of patients claimed that no one had taught them about asthma medications, and one quarter said they had received no training on how to recognize the early signs of asthma worsening. CONCLUSION: Asthma control and management remained suboptimal in Canada and relatively unchanged since the previous major survey in 1999.

Asthma control in Canada remains suboptimal: the Reality of Asthma Control (TRAC) study.

BACKGROUND: Two Canadian studies showed that 55% of patients with asthma had daily symptoms (in 1996) and that 57% of patients suffered from poorly controlled asthma (in 1999). OBJECTIVES: To assess the state of asthma control of adult Canadians, and asthma knowledge and practices of Canadian physicians actively involved in the care of patients with asthma. METHODS: Telephone interviews were conducted with adults 18 to 54 years of age who had been diagnosed with asthma at least six months before the survey, who did not have chronic obstructive pulmonary disease and who had a smoking history of fewer than 20 pack-years. Physicians were surveyed by telephone and mail. The surveys took place between April and August 2004. RESULTS: Almost all (97%) of the 893 patients believed that they had controlled asthma; however, only 47% had controlled disease according to symptom-based guideline criteria. Just 39% of 463 physicians based their treatment recommendations on the Canadian asthma guidelines most or all of the time, despite having a high awareness of them. Only 11% of patients had written action plans, and one-half of patients with action plans did not use them regularly. Almost three-quarters of patients expressed concerns about taking inhaled corticosteroids. CONCLUSIONS: Since the last major national survey, guideline implementation has not resulted in significant changes in asthma-related morbidity. Effective means of knowledge transfer should be developed and implemented to improve the translation of guideline recommendations into care.

Lignocellulose-derived thin stillage composition and efficient biological treatment with a high-rate hybrid anaerobic bioreactor system.

BACKGROUND: This study aims to chemically characterize thin stillage derived from lignocellulosic biomass distillation residues in terms of organic strength, nutrient, and mineral content. The feasibility of performing anaerobic digestion on these stillages at mesophilic (40 °C) and thermophilic (55 °C) temperatures to produce methane was demonstrated. The microbial communities involved were further characterized. RESULTS: Energy and sugar cane stillage have a high chemical oxygen demand (COD of 43 and 30 g/L, respectively) and low pH (pH 4.3). Furthermore, the acetate concentration in sugar cane stillage was high (45 mM) but was not detected in energy cane stillage. There was also a high amount of lactate in both types of stillage (35-37 mM). The amount of sugars was 200 times higher in energy cane stillage compared to sugar cane stillage. Although there was a high concentration of sulfate (18 and 23 mM in sugar and energy cane stillage, respectively), both thin stillages were efficiently digested anaerobically with high COD removal under mesophilic and thermophilic temperature conditions and with an organic loading rate of 15-21 g COD/L/d. The methane production rate was 0.2 L/g COD, with a methane percentage of 60 and 64, and 92 and 94 % soluble COD removed, respectively, by the mesophilic and thermophilic reactors. Although both treatment processes were equally efficient, there were different microbial communities involved possibly arising from the differences in the composition of energy cane and sugar cane stillage. There was more acetic acid in sugar cane stillage which may have promoted the occurrence of aceticlastic methanogens to perform a direct conversion of acetate to methane in reactors treating sugar cane stillage. CONCLUSIONS: Results showed that thin stillage contains easily degradable compounds suitable for anaerobic digestion and that hybrid reactors can efficiently convert thin stillage to methane under mesophilic and thermophilic conditions. Furthermore, we found that optimal conditions for biological treatment of thin stillage were similar for both mesophilic and thermophilic reactors. Bar-coded pyrosequencing of the 16S rRNA gene identified different microbial communities in mesophilic and thermophilic reactors and these differences in the microbial communities could be linked to the composition of the thin stillage.

Inhibition of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases.

Five independently evolved classes (alpha-, beta-, gamma-, delta-, zeta-) of carbonic anhydrases facilitate the reversible hydration of carbon dioxide to bicarbonate of which the alpha-class is the most extensively studied. Detailed inhibition studies of the alpha-class with the two main classes of inhibitors, sulfonamides and metal-complexing anions, revealed many inhibitors that are used as therapeutic agents to prevent and treat many diseases. Recent inhibitor studies of the archaeal beta-class (Cab) and the gamma-class (Cam) carbonic anhydrases show differences in inhibition response to sulfonamides and metal-complexing anions, when compared to the alpha-class carbonic anhydrases. In addition, inhibition between Cab and Cam differ. These inhibition patterns are consistent with the idea that although, alpha-, beta-, and gamma-class carbonic anhydrases participate in the same two-step isomechanism, diverse active site architecture among these classes predicts variations on the catalytic mechanism. These inhibitor studies of the archaeal beta- and gamma-class carbonic anhydrases give insight to new applications of current day carbonic anhydrase inhibitors, as well as direct research to develop new compounds that may be specific inhibitors of prokaryotic carbonic anhydrases.

Proposal for a hydrogen bond network in the active site of the prototypic gamma-class carbonic anhydrase.

The crystal structure of Cam, the prototypic gamma-class carbonic anhydrase, reveals active site residues Gln75, Asn73, and Asn 202 previously hypothesized to participate in catalysis. These potential roles were investigated for the first time by kinetic analyses of site-specific replacement variants of the zinc and cobalt forms of Cam. Gln75 replacement variants showed large decreases in k(cat)/K(m) relative to wild-type. Further, the Gln75 variants showed a loss of the pK(a) in pH versus k(cat)/K(m) profiles previously attributed to ionization of the metal-bound water yielding the hydroxyl group attacking CO(2). These results support the previously proposed role for Gln75 in hydrogen bonding with the catalytic hydroxyl orienting it for attack on CO(2). Kinetic analyses of Asn73 variants were consistent with a role in hydrogen bonding with Gln75 to position it for optimal interaction with the catalytic hydroxyl. Kinetic analyses of Asn202 variants showed substantial decreases in k(cat)/K(m) relative to the wild-type enzyme supporting the previously hypothesized role in polarizing CO(2) and facilitating attack from the metal-bound hydroxyl. On the basis of results presented here, and previously reported structural analyses, we present a catalytic mechanism involving Gln75, Asn73, and Asn202 that also suggests a role for Glu62 not previously recognized. Finally, the results suggest that the gamma-, beta-, and alpha-class carbonic anhydrases each independently evolved variations of a fundamental hydrogen bond network essential for catalysis.

Women and asthma: lessons from a gender analysis of the asthma in Canada survey.

OBJECTIVE: To compare the degree of asthma control achieved by men and women with asthma in Canada and to explore differences in patient perspectives, treatments used, and health care resources used between men and women with asthma. DESIGN: Population-based cross-sectional telephone interview survey of Canadians with doctor-diagnosed asthma. SUBJECTS AND METHODS: Random digit dialing was used to identify a representative sample of Canadians with asthma. A total of 801 adults were interviewed over the telephone. Analysis was performed on the data from a subgroup of 20- to 50-year-old patients with asthma who participated in the original survey (329 women and 183 men). RESULTS: Women and men were equally likely to be poorly controlled (58% vs. 56%, p > 0.05) as defined by failing to meet two or more of six symptom-based criteria listed by the 1996 Canadian Asthma Consensus (CAC) Guidelines as appropriate treatment targets. However, there were significant differences in medication used; women were more likely than men to use an inhaled corticosteroid in the treatment of their asthma (59% vs. 45%, p < 0.05) and were more knowledgeable about their appropriate use (62% vs. 46%, p < 0.05), and were more likely to be satisfied with their physicians' care. Despite this, women were more likely than men to have required urgent care for their asthma in the year preceding the survey (50% vs. 36%, p < 0.05). CONCLUSION: Women report greater need for urgent asthma care despite more frequent use of inhaled corticosteroids and better asthma knowledge scores than men.

Carbonic anhydrase inhibitors. Inhibition of the prokariotic beta and gamma-class enzymes from Archaea with sulfonamides.

A detailed inhibition study of carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the beta- and gamma-families from Archaea with sulfonamides has been performed. Compounds included in this study were the clinically used sulfonamide CA inhibitors, such as acetazolamide, methazolamide, ethoxzolamide, topiramate, valdecoxib, celecoxib, dorzolamide, sulfanilamide, dichlorophanamide, as well as sulfanilamide analogs, halogenated sulfanilamides, and some 1,3-benzenedisulfonamide derivatives. The two gamma-CAs from Methanosarcina thermophila (Zn-Cam and Co-Cam) showed very different inhibitory properties with these compounds, as compared to the alpha-CA isozymes hCA I, II, and IX, and the beta-CA from Methanobacterium thermoautotrophicum (Cab). The best Zn-Cam inhibitors were sulfamic acid and acetazolamide, with inhibition constants in the range of 63-96 nM, whereas other investigated aromatic/heterocylic sulfonamides showed a rather levelled behavior, with KIs in the range of 0.12-1.70 microM. The best Co-Cam inhibitors were topiramate and p-aminoethyl-benzenesulfonamide, with KIs in the range of 0.12-0.13 microM, whereas the worst one was homosulfanilamide (KI of 8.50 microM). In the case of Cab, the inhibitory power of these compounds varied to a much larger extent, with sulfamic acid and sulfamide showing millimolar affinities (KIs in the range of 44-103 mM), whereas the best inhibitor was ethoxzolamide, with a KI of 5.35 microM. Most of these sulfonamides showed inhibition constants in the range of 12-100 microM against Cab. Thus, the three CA families investigated up to now possess a very diverse affinity for sulfonamides, the inhibitors with important medicinal, and environmental applications.

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions.

The first inhibition study of the beta-class carbonic anhydrase (CA, EC 4.2.1.1) from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV and the gamma-class enzyme from another archaeon, Methanosarcina thermophila (Cam) with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, sulfate, etc., are also provided for comparison. The best Cab anion inhibitors were thiocyanate and hydrogen sulfide, with inhibition constants in the range of 0.52-0.70 mM, whereas cyanate, iodide, carbonate, and nitrate were weaker inhibitors (Ki's in the range of 7.8-13.2 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably, whereas the CA substrate bicarbonate, or other anions, such as bromide, nitrite, bisulfite, or sulfamate behave as weak inhibitors (Ki in the range of 40-45 mM). It is interesting to note that the metal poison, coordinating anions cyanide and azide are also rather weak Cab inhibitors (Ki in the range of 27-55 mM), whereas sulfamide is a very weak Cab inhibitor (Ki of 103 mM), although it strongly inhibits Cam (Ki of 70 microM). Surprisingly, phenylboronic and phenylarsonic acids, which have been investigated for the inhibition of all these CAs for the first time, showed very weak activity against the alpha-CA isozymes, but were effective Cab and Cam inhibitors. The best Cab inhibitors were just these two compounds (Ki's of 0.20-0.33 mM), whereas the best Cam inhibitor was sulfamic acid (Ki of 96 nM). These major differences of behavior between the diverse CAs investigated here toward anion inhibitors can be difficultly explained considering the convergent evolution of so diverse enzymes for the binding and turnover of small molecules such as carbon dioxide and anions.

Carbonic anhydrase inhibitors. Inhibition of the zinc and cobalt gamma-class enzyme from the archaeon Methanosarcina thermophila with anions.

Anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), in addition to sulfonamides, which possess clinical applications. The first inhibition study of the zinc and cobalt gamma-class enzyme from the archaeon Methanosarcina thermophila (Cam) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, and sulfate, etc., are also provided for comparison. The best Zn-Cam anion inhibitors were hydrogen sulfide and cyanate, with inhibition constants in the range of 50-90 microM, whereas thiocyanate, azide, carbonate, nitrite, and bisulfite were weaker inhibitors (K(I)s in the range of 5.8-11.7 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably up to concentrations of 200 mM, whereas the substrate bicarbonate behaves as a weak inhibitor (K(I)s of 42 mM). The best Co-Cam inhibitor was carbonate, with an inhibition constant of 9 microM, followed by nitrate and bicarbonate (K(I)s in the range of 90-100 microM). The metal poisons were much more ineffective inhibitors of this enzyme, with cyanide possessing an inhibition constant of 51.5mM, whereas cyanate, thiocyanate, azide, iodide, and hydrogen sulfide showed K(I)s in the range of 2.0-6.1mM. As for Zn-Cam, fluoride, chloride, and sulfate are not inhibitors of Co-Cam. These major differences between the two gamma-CAs investigated here can be explained only in part by the different geometries of the metal ions present within their active sites.