A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO₂ hydration reaction (k_cat?=?9.35?×?10⁵?s^?1 and k_cat/K_m?=?1.1?×?10⁸?M^?1?s^?1) which was maintained after heating the enzyme at 80?°C for 3?h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe₃O₄ nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.     

Direct Electrochemical Immunoassay Based on Immobilization of Protein-Magnetic Nanoparticle Composites on to Magnetic Electrode Surfaces by Sterically Enhanced Magnetic Field Force

A direct electrochemical immunoassay system based on the immobilization of α-1-fetoprotein antibody (anti-AFP), as a model system, on the surface of core-shell Fe₂O₃/Au magnetic nanoparticles (MNP) has been demonstrated. To fabricate such an assay system, anti-AFP was initially covalently immobilized on to the surface of core-shell Fe₂O₃/Au MNP. Anti-AFP-modified MNP (bio-nanoparticles) were then attached to the surface of carbon paste electrode with the aid of a permanent magnet. The performance and factors influencing the performance of the resulting immunosensor were studied. α-1-Fetoprotein antigen was directly determined by the change in current or potential before and after the antigen–antibody reaction versus saturated calomel electrode. The electrochemical immunoassay system reached 95% of steady-state potential within 2 min and had a sensitivity of 25.8 mV. The linear range for AFP determination was from 1 to 80 ng AFP ml⁻¹ with a detection limit of 0.5 ng AFP ml⁻¹. Moreover, the direct electrochemical immunoassay system, based on a functional MNP, can be developed further for DNA sensor and enzyme biosensor. Revisions requested 2 November 2005; Revisions received 17 January 2006     

Carbonic anhydrase — a universal enzyme of the carbon-based life

Carbonic anhydrase (CA) is a metalloenzyme that performs interconversion between CO₂ and the bicarbonate ion (HCO₃ ^?). CAs appear among all taxonomic groups of three domains of life. Wide spreading of CAs in nature is explained by the fact that carbon, which is the major constituent of the enzyme’s substrates, is a key element of life on the Earth. Despite the diversity of CAs, they all carry out the same reaction of CO₂/HCO₃ ^? interconversion. Thus, CA obviously represents a universal enzyme of the carbon-based life. Within the classification of CAs, here we proposed the existence of an extensive family of CA-related proteins (γCA-RPs)–the inactive forms of γ-CAs, which are widespread among the Archaea, Bacteria, and, to a lesser extent, in Eukarya. This review focuses on the history of CAs discovery and integrates the most recent data on their classification, catalytic mechanisms, and physiological roles at various organisms.     

IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO2 SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co²⁺, Cu²⁺, and Fe³⁺, increased the enzyme activity, whereas CA activity was inhibited by Pb²⁺, Hg²⁺, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The maximum CO₂ sequestration potential was achieved with immobilized CA (480 mg CaCO₃/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO₂ sequestration.     

Fe2O3 Nanoparticle Seed Catalysts Enhance Cyclability on Deep (Dis)charge in Aprotic LiO2 Batteries

Although the high energy density of Li?O₂ chemistry is promising for vehicle electrification, the poor stability and parasitic reactions associated with carbon‐based cathodes and the insulating nature of discharge products limit their rechargeability and energy density. In this study, a cathode material consisting of α‐Fe₂O₃ nanoseeds and carbon nanotubes (CNT) is presented, which achieves excellent cycling stability on deep (dis)charge with high capacity. The initial capacity of Fe₂O₃/CNT electrode reaches 805 mA h g^?1 (0.7 mA h cm^?2) at 0.2 mA cm^?2, while maintaining a capacity of 1098 mA h g^?1 (0.95 mA h cm^?2) after 50 cycles. The operando structural, spectroscopic, and morphological analysis on the evolution of Li₂O₂ indicates preferential Li₂O₂ growth on the Fe₂O₃. The similar d‐spacing of the (100) Li₂O₂ and (104) Fe₂O₃ planes suggest that the latter epitaxially induces Li₂O₂ nucleation. This results in larger Li₂O₂ primary crystallites and smaller secondary particles compared to that deposited on CNT, which enhances the reversibility of the Li₂O₂ formation and leads to more stable interfaces within the electrode. The mechanistic insights into dual‐functional materials that act both as stable host substrates and promote redox reactions in Li?O₂ batteries represent new opportunities for optimizing the discharge product morphology, leading to high cycling stability and coulombic efficiency.     

Fe2O3 Nanoparticle Seed Catalysts Enhance Cyclability on Deep (Dis)charge in Aprotic Li?O2 Batteries

Although the high energy density of Li? O₂ chemistry is promising for vehicle electrification, the poor stability and parasitic reactions associated with carbon‐based cathodes and the insulating nature of discharge products limit their rechargeability and energy density. In this study, a cathode material consisting of α‐Fe₂O₃ nanoseeds and carbon nanotubes (CNT) is presented, which achieves excellent cycling stability on deep (dis)charge with high capacity. The initial capacity of Fe₂O₃/CNT electrode reaches 805 mA h g^?1 (0.7 mA h cm^?2) at 0.2 mA cm^?2, while maintaining a capacity of 1098 mA h g^?1 (0.95 mA h cm^?2) after 50 cycles. The operando structural, spectroscopic, and morphological analysis on the evolution of Li₂O₂ indicates preferential Li₂O₂ growth on the Fe₂O₃. The similar d‐spacing of the (100) Li₂O₂ and (104) Fe₂O₃ planes suggest that the latter epitaxially induces Li₂O₂ nucleation. This results in larger Li₂O₂ primary crystallites and smaller secondary particles compared to that deposited on CNT, which enhances the reversibility of the Li₂O₂ formation and leads to more stable interfaces within the electrode. The mechanistic insights into dual‐functional materials that act both as stable host substrates and promote redox reactions in Li? O₂ batteries represent new opportunities for optimizing the discharge product morphology, leading to high cycling stability and coulombic efficiency.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.     

Biochemical characterization of recombinant β-carbonic anhydrase (PgiCAb) identified in the genome of the oral pathogenic bacterium Porphyromonas gingivalis

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β-, γ-, δ- and ζ-CAs are ubiquitous metalloenzymes present in prokaryotes and eukaryotes. CAs started to be investigated in detail only recently in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many such organisms they are essential for the life cycle of the organism. CA inhibition leads to growth impairment or growth defects in several pathogenic bacteria. The microbiota of the human oral mucosa consists of a myriad of bacterial species, Porphyromonas gingivalis being one of them and the major pathogen responsible for the development of chronic periodontitis. The genome of P. gingivalis encodes for a β- and a γ-CAs. Recently, our group purified the recombinant γ-CA (named PgiCA) which was shown to possess a significant catalytic activity for the reaction that converts CO₂ to bicarbonate and protons, with a k_cat of 4.1?×?10^5?s^?1 and a k_cat/K_m of 5.4?×?10^7?M^?1?×?s^?1. We have also investigated its inhibition profile with a range of inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate. Here, we describe the cloning, purification and kinetic parameters of the other class of CA identified in the genome of P. gingivalis, the β-CA, named PgiCAb. This enzyme has a good catalytic activity, with a k_cat of 2.8?×?10^5?s^?1 and a k_cat/K_m of 1.5?×?10^7?M^?1?×?s^?1. PgiCAb was also inhibited by the clinically used sulfonamide acetazolamide, with an inhibition constant of 214?nM. The role of CAs as possible virulence factors of P. gingivalis is poorly understood at the moment but their good catalytic activity and the fact that they might be inhibited by a large number of compounds, which may pave the way for finding inhibitors with antibacterial activity that may elucidate these phenomena and lead to novel antibiotics.     

Catecholamines Enhance Dihydrolipoamide Dehydrogenase Inactivation by the Copper Fenton System. Enzyme Protection by Copper Chelators

Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II) /H₂O₂ (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H₂O₂ and CA concentrations. Similar inhibitions were obtained wit! the assayed CAs and o-diphenols. CAs enhanced HO radical production by Cu(II) /H₂O₂, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipo amide, dihydrolipoic acid, DL-dithiothreitol, GSSG, try-panothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H₂O₂/CAs systems. Dena tured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH in creased and Captopril inhibited epinephrine oxidation by Cu(II)/H₂O₂ and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO^* from H₂O₂ by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the: enzyme active site by site-specifically generated HO^* radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation.     

Rationally Designed Hierarchical TiO2@Fe2O3 Hollow Nanostructures for Improved Lithium Ion Storage

Hollow and hierarchical nanostructures have received wide attention in new‐generation, high‐performance, lithium ion battery (LIB) applications. Both TiO₂ and Fe₂O₃ are under current investigation because of their high structural stability (TiO₂) and high capacity (Fe₂O₃), and their low cost. Here, we demonstrate a simple strategy for the fabrication of hierarchical hollow TiO₂@Fe₂O₃ nanostructures for the application as LIB anodes. Using atomic layer deposition (ALD) and sacrificial template‐assisted hydrolysis, the resulting nanostructure combines a large surface area with a hollow interior and robust structure. As a result, such rationally designed LIB anodes exhibit a high reversible capacity (initial value 840 mAh g^?1), improved cycle stability (530 mAh g^?1 after 200 cycles at the current density of 200 mA g^?1), as well as outstanding rate capability. This ALD‐assisted fabrication strategy can be extended to other hierarchical hollow metal oxide nanostructures for favorable applications in electrochemical and optoelectronic devices.     

Phenotypic analysis of the ccp1Δ and ccp1Δ-ccp1 mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN^?) ligands. Hydrogen (H₂) and oxygen (O₂) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. ⁵⁷Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fe_d in a [4Fe4S-2Fe]^3 ? complex with the net spin on Fe_d whereas Hox-CO shows an apical CN^? at Fe_d in a [4Fe4S-2Fe(CO)]^3 ? complex with net spin sharing among Fe_p and Fe_d (proximal or distal iron ions in [2Fe]). At ambient O₂ pressure, a novel H-cluster species (Hox-O₂) accumulated in C169A, assigned to a [4Fe4S-2Fe(O₂)]^3 ? complex with an apical superoxide (O₂^?) carrying the net spin bound at Fe_d. H₂ exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]^3 ? complex with the net spin on the reduced cubane, an apical hydride at Fe_d, and a proton at a cysteine ligand. Hox-O₂ and Hhyd are stabilized by impaired O₂^– protonation or proton release after H₂ cleavage due to interruption of the proton path towards and out of the active site.     

Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense YO3AOP1

A new carbonic anhydrase (CA, EC 4.2.1.1) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 was identified and characterized. The bacterial carbonic anhydrase gene was expressed in Escherichia coli yielding an active enzyme, which was purified in large amounts. The recombinant protein (SspCA) was found to belong to the α-CA class and displays esterase activity. The kinetic parameters were determined by using CO₂ and p-nitrophenylacetate (p-NpA) as substrates. The bacterial enzyme presented specific activity comparable to that of bovine carbonic anhydrase (bCA II) but it showed biochemical properties never observed for the mammalian enzyme. The thermophilic enzyme, in fact, was endowed with high thermostability and with unaltered residual activity after prolonged exposure to heat up to 100°C. SspCA and the bovine carbonic anhydrase (bCA II) were immobilized within a polyurethane (PU) foam. The immobilized bacterial enzyme was found to be active and stable at 100°C up to 50?h.     

Interactions of tropospheric CO2 and O3 enrichments and moisture variations on microbial biomass and respiration in soil

Soil microbial biomass C (C_mic) is a sensitive indicator of trends in organic matter dynamics in terrestrial ecosystems. This study was conducted to determine the effects of tropospheric CO₂ or O₃ enrichments and moisture variations on total soil organic C (C_org), mineralizable C fraction (C_Min), C_mic, maintenance respiratory (qCO₂) or C_mic death (qD) quotients, and their relationship with basal respiration (BR) rates and field respiration (FR) fluxes in wheat‐soybean agroecosystems. Wheat (Triticum aestivum L.) and soybean (Glycine max. L. Merr) plants were grown to maturity in 3‐m dia open‐top field chambers and exposed to charcoal‐filtered (CF) air at 350 μL CO₂ L^?1; CF air + 150 μL CO₂ L^?1; nonfiltered (NF) air + 35 nL O₃ L^?1; and NF air + 35 nL O₃ L^?1 + 150 μL CO₂ L^?1 at optimum (? 0.05 MPa) and restricted soil moisture (? 1.0 ± 0.05 MPa) regimes. The + 150 μL CO₂ L^?1 additions were 18 h d^?1 and the + 35 nL O₃ L^?1 treatments were 7 h d^?1 from April until late October. While C_org did not vary consistently, C_Min, C_mic and C_mic fractions increased in soils under tropospheric CO₂ enrichment (500 μL CO₂ L^?1) and decreased under high O₃ exposures (55 ± 6 nL O₃ L^?1 for wheat; 60 ± 5 nL O₃ L^?1 for soybean) compared to the CF treatments (25 ± 5 nL O₃ L^?1). The qCO₂ or qD quotients of C_mic were also significantly decreased in soils under high CO₂ but increased under high O₃ exposures compared to the CF control. The BR rates did not vary consistently but they were higher in well‐watered soils. The FR fluxes were lower under high O₃ exposures compared to soils under the CF control. An increase in C_mic or C_mic fractions and decrease in qCO₂ or qD observed under high CO₂ treatment suggest that these soils were acting as C sinks whereas, reductions in C_mic or C_mic fractions and increase in qCO₂ or qD in soils under elevated tropospheric O₃ exposures suggest the soils were serving as a source of CO₂.     

The first activation study of a bacterial carbonic anhydrase (CA). The thermostable α-CA from Sulfurihydrogenibium yellowstonense YO3AOP1 is highly activated by amino acids and amines

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) was investigated for its activation with a series of amino acids and amines. d-His, l-Phe, l-Tyr, l- and d-Trp were the most effective SspCA activators, with activation constants in the range of 1–12 nM, whereas l-His, l/d-DOPA, d-Tyr, and several biogenic amines/catecholamines were slightly less effective activators (K_A in the range of 37 nM–0.97 μM). The least effective SspCA activator was d-Phe (K_A of 5.13 μM). The thermal stability, robustness and very high catalytic activity of SspCA make this enzyme an ideal candidate for biomimetic CO₂ capture processes.     

Soil nitrogen transformations under Populus tremuloides, Betula papyrifera and Acer saccharum following 3 years exposure to elevated CO2 and O3

Increases in atmospheric CO₂ and tropospheric O₃ may affect forest N cycling by altering plant litter production and the availability of substrates for microbial metabolism. Three years following the establishment of our free‐air CO₂–O₃ enrichment experiment, plant growth has been stimulated by elevated CO₂ resulting in greater substrate input to soil; elevated O₃ has counteracted this effect. We hypothesized that rates of soil N cycling would be enhanced by greater plant productivity under elevated CO₂, and that CO₂ effects would be dampened by O₃. We found that elevated CO₂ did not alter gross N transformation rates. Elevated O₃ significantly reduced gross N mineralization and microbial biomass N, and effects were consistent among species. We also observed significant interactions between CO₂ and O₃: (i) gross N mineralization was greater under elevated CO₂ (1.0 mg N kg^?1 day^?1) than in the presence of both CO₂ and O₃ (0.5 mg N kg^?1 day^?1) and (ii) gross NH₄⁺ immobilization was also greater under elevated CO₂ (0.8 mg N kg^?1 day^?1) than under CO₂ plus O₃ (0.4 mg N kg^?1 day^?1). We used a laboratory ¹⁵N tracer method to quantify transfer of inorganic N to organic pools. Elevated CO₂ led to greater recovery of NH₄⁺‐¹⁵N in microbial biomass and corresponding lower recovery in the extractable NO₃^? pool. Elevated CO₂ resulted in a substantial increase in NO₃^?‐¹⁵N recovery in soil organic matter. We observed no O₃ main effect and no CO₂ by O₃ interaction effect on ¹⁵N recovery in any soil pool. All of the above responses were most pronounced beneath Betula papyrifera and Populus tremuloides, which have grown more rapidly than Acer saccharum. Although elevated CO₂ has increased plant productivity, the resulting increase in plant litter production has yet to overcome the influence of the pre‐existing pool of soil organic matter on soil microbial activity and rates of N cycling. Ozone reduces plant litter inputs and also appears to affect the composition of plant litter in a way that reduces microbial biomass and activity.     

The biotechnological potential of subtilisin‐like fibrinolytic enzyme from a newly isolated Lactobacillus plantarum KSK‐II in blood destaining and antimicrobials

An antimicrobial oxidative‐ and SDS‐stable fibrinolytic alkaline protease designated as KSK‐II was produced by Lactobacillus plantarum KSK‐II isolated from kishk, a traditional Egyptian food. Maximum enzyme productivity was obtained in medium containing 1% lactose and 0.5% soybean flour as carbon and nitrogen sources, respectively. Purification of enzyme increased its specific activity to 1,140‐fold with a recovery of 33% and molecular weight of 43.6 kDa. Enzyme activity was totally lost in the presence of ethylenediaminetetraacetic acid and was restored after addition of Fe²⁺ suggesting that KSK‐II is a metalloprotease and Fe²⁺ acts as cofactor. Enzyme hydrolyzed not only the natural proteins but also synthetic substrates, particularly Suc‐Ala‐Ala‐Pro‐Phe‐pNA. KSK‐II can hydrolyze the Lys‐X easier than Arg‐X; thus, it was considered as a subtilisin‐family protease. Its apparent K_m, V_max, and K_cat were 0.41 mM, 6.4 µmol mg^?1 min^?1, and 28.0 s^?1, respectively. KSK‐II is industrially important from the perspectives of its maximal activity at 50°C (stable up to 70°C), ability to function at alkaline pH (10.0), stability at broad pH ranges (7.5–12.0) in addition to its stability toward SDS, H₂O₂, organic solvents, and detergents. We emphasize for the first time the potential of fibrinolytic activity for alkaline proteases used in detergents especially in blood destaining. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:316–324, 2015