Enhanced curdlan production in <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749 by addition of low-polyphosphates

A large amount of adenosine triphosphate with high energy phosphate bonds is required for uridine triphosphate regeneration during curdlan biosynthesis by Agrobacterium sp. ATCC 31749. To supply high energy for curdlan synthesis, three low-polyphosphates (Na₄P₂O₇, Na₅P₃O₁₀, and (NaPO₃)₆) with higher energy phosphate bonds were employed to substitute for KH₂PO₄-K₂HPO₄ in fermentation medium. Two genes encoding the polyphosphate metabolizing enzymes, polyphosphate kinase and exopolyphosphatase, were amplified and showed 95% homology to those in Agrobacterium sp. C58 by sequence analysis. The curdlan yields were enhanced by 23 and 134% when phosphate concentrations 0.024 mol/L of Na₅P₃O₁₀ and 0.048 mol/L of (NaPO₃)₆ respectively, were added in the medium. The maximum curdlan yield of 30 ± 1.02 g/L was obtained with the addition of 0.048 mol/L of (NaPO₃)₆ with 5 g/L CaCO₃ in the medium. When CaCO₃ was removed from the culture and the three lowpolyphosphates were added, the pH and biomass yield dropped remarkably and little or no curdlan was produced. The culture containing 0.048 mol/L of (NaPO₃)₆ was mixed with KH₂PO₄-K₂HPO₄ and CaCO₃ in the medium, but showed no effect on curdlan production. However, curdlan yield was improved by 49 ∼ 60% when CaCO₃ was removed from the medium and KH₂PO₄-K₂HPO₄ acted as a buffer. It appears that the positive effect of (NaPO₃)₆ on curdlan production required the buffering capacity of CaCO₃ and the absence of KH₂PO₄-K₂HPO₄ competing as a phosphate supplier.     

Optimization and characterization of rhamnolipid production by Pseudomonas aeruginosa NY3 using waste frying oil as the sole carbon

Yield and cost are two major factors limiting the widespread use of rhamnolipids (RLs). In the present study, waste frying oil (WFO) was used as the sole carbon source to produce environmentally friendly RLs by Pseudomonas aeruginosa NY3. The Plackett–Burman design (PBD) and Box–Behnken design (BBD) methods were used to maximize the production yield of RL. The PBD results showed that the concentrations of NaNO₃, Na₂HPO₄, and trace elements were the key factors affecting the yield of RL. Furthermore, the BBD results showed that at NaNO₃, Na₂HPO₄, and trace elements concentrations were 4.95, 0.66, and 0.64 mL/L, respectively, the average RL yield reached 9.15 ± 0.52 g/L, 1.58-fold higher than that observed before optimization. Fourier transform infrared spectroscopy (FTIR) and liquid chromatography-ion trap-time of flight mass spectrometry (LCMS-IT-TOF) were used to elucidate the diversity of RL congeners. The results showed that, after optimization, the RL congener diversity increased, and the major RL constituent was converted from di-RLs (64.04%) to mono-RLs (60.44%). These results suggested that the concentrations of the components contained in the culture medium of P. aeruginosa NY3 influenced not only the yield of RL, but also its congener distribution.     

Optimization of extracellular alkaline protease production from species of <Emphasis Type="Italic">Bacillus</Emphasis>

Thirty-five strains capable of secreting extracellular alkaline proteases were isolated from the soil and waste water near the milk processing plant, slaughterhouse. Strain APP1 with the highest-yield alkaline proteases was identified as Bacillus sp. The cultural conditions were optimized for maximum enzyme production. When the initial pH of the medium was 9.0, the culture maintained maximum proteolytic activity for 2,560 U ml⁻¹ at 50°C for 48 h under the optimized conditions containing (g⁻¹): soyabean meal, 15; wheat flour, 30; K₂HPO₄, 4; Na₂HPO₄, 1; MgSO₄·7H₂O, 0.1; Na₂CO₃, 6. The alkaline protease showed extreme stability toward SDS and oxidizing agents, which retained its activity above 73 and 110% on treatment for 72 h with 5% SDS and 5% H₂O₂, respectively.     

Characterization and structure determination of prolyl‐tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform

Pseudomonas aeruginosa is an opportunistic multi‐drug resistant pathogen implicated as a causative agent in nosocomial and community acquired bacterial infections. The gene encoding prolyl‐tRNA synthetase (ProRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. ProRS was kinetically evaluated and the K_M values for interactions with ATP, proline, and tRNA were 154, 122, and 5.5 μM, respectively. The turn‐over numbers, k_cat^obs, for interactions with these substrates were calculated to be 5.5, 6.3, and 0.2 s^?1, respectively. The crystal structure of the α₂ form of P. aeruginosa ProRS was solved to 2.60 Å resolution. The amino acid sequence and X‐ray crystal structure of P. aeruginosa ProRS was analyzed and compared with homologs in which the crystal structures have been solved. The amino acids that interact with ATP and proline are well conserved in the active site region and overlay of the crystal structure with ProRS homologs conforms to a similar overall three‐dimensional structure. ProRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 890 chemical compounds, resulting in the identification of two inhibitory compounds, BT06A02 and BT07H05. This work confirms the utility of a screening system based on the functionality of ProRS from P. aeruginosa.     

An Endophytic <Emphasis Type="Italic">Phomopsis</Emphasis> sp. Possessing Bioactivity and Fuel Potential with its Volatile Organic Compounds

An unusual Phomopsis sp. was isolated as endophyte of Odontoglossum sp. (Orchidaceae), associated with a cloud forest in Northern Ecuador. This fungus produces a unique mixture of volatile organic compounds (VOCs) including sabinene (a monoterpene with a peppery odor) only previously known from higher plants. In addition, some of the other more abundant VOCs recorded by GC/MS in this organism were 1-butanol, 3-methyl; benzeneethanol; 1-propanol, 2-methyl and 2-propanone. The gases of Phomopsis sp. possess antifungal properties and an artificial mixture of the VOCs mimicked the antibiotic effects of this organism with the greatest bioactivity against a wide range of plant pathogenic test fungi including: Pythium, Phytophthora, Sclerotinia, Rhizoctonia, Fusarium, Botrytis, Verticillium, and Colletotrichum. The IC₅₀ values for the artificial gas mixture of Phomopsis sp. varied between 8 and 25.65 μl/mL. Proton transfer reaction-mass spectrometry monitored the concentration of VOCs emitted by Phomopsis sp. and yielded a total VOC concentration of ca. 18 ppmv in the head space at the seventh day of incubation at 23°C on PDA. As with many VOC-producing endophytes, this Phomopsis sp. did survive and grow in the presence of the inhibitory gases of Muscodor albus. A discussion is presented on the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship.     

Study of the Vapor Phase Over Fusarium Fungi Cultured on Various Substrates

The compositions of volatile organic compounds (VOCs) emitted by Fusarium fungi (F. langsethiae, F. sibiricum, F. poae, and F. sporotrichioides) grown on two nutritive substrates: potato sucrose agar (PSA) and autoclaved wheat kernels (WK) were investigated. The culturing of fungi and study of their VOC emissions were performed in chromatographic vials at room temperature (23 – 24 °C) and the VOCs were sampled by a solid‐phase microextraction on a 85 μm carboxen/polydimethylsiloxane fiber. GC/MS was performed using a 60‐m HP‐5 capillary column. Components of the VOC mixture were identified by electron impact mass spectra and chromatographic retention indices (RIs). The most abundant components of the VOC mixture emitted by Fusarium fungi are EtOH, AcOH, ⁱBuOH, 3‐methylbutan‐1‐ol, 2‐methylbutan‐1‐ol, ethyl 3‐methylbutanoate, terpenes with M 136, sesquiterpenes with M 204 (a total of about 25), and trichodiene. It was found that the strains grown on PSA emit a wider spectrum and larger amount of VOCs compared with those grown on wheat kernels. F. langsethiae strain is the most active VOC producer on both substrates. The use of SPME and GC/MS also offers the potential for differentiation of fungal species and strains.     

Different tolerances and responses to low temperature and darkness between waterbloom forming cyanobacterium <Emphasis Type="Italic">Microcystis</Emphasis> and a green alga <Emphasis Type="Italic">Scenedesmus</Emphasis>

The dynamics of planktonic cyanobacteria in eutrophicated freshwaters play an important role in formation of annual summer blooms, yet overwintering mechanisms of these water bloom forming cyanobacteria remain unknown. The responses to darkness and low temperature of three strains (unicellular Microcystis aeruginosa FACHB-905, colonial M. aeruginosa FACHB-938, and a green alga Scenedesmus quadricauda FACHB-45) were investigated in the present study. After a 30-day incubation under darkness and low temperature, cell morphology, cell numbers, chlorophyll a, photosynthetic activity (ETR_max and I _k), and malodialdehyde (MDA) content exhibited significant changes in Scenedesmus. In contrast, Microcystis aeruginosa cells did not change markedly in morphology, chlorophyll a, photosynthetic activity, and MDA content. The stress caused by low temperature and darkness resulted in an increase of the antioxidative enzyme-catalase (CAT) in all three strains. When the three strains re-grew under routine cultivated condition subjected to darkness and low temperature, specific growth rate of Scenedesmus was lower than that of Microcystis. Flow cytometry (FCM) examination indicated that two distinct types of metabolic response to darkness and low temperature existed in the three strains. The results from the present study reveal that the cyanobacterium Microcystis, especially colonial Microcystis, has greater endurance and adaptation ability to the stress of darkness and low temperature than the green alga Scenedesmus. Handling editor: D. Hamilton     

Prostaglandin E2 modulates Na+,K+-ATPase activity in rat hippocampus: implications for neurological diseases

Prostaglandin E₂ (PGE₂) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na⁺,K⁺‐ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE₂ decreases Na⁺,K⁺‐ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE₂. Na⁺,K⁺‐ATPase activity was determined by assessing ouabain‐sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE₂ (0.1–10 μM) for 30 min decreased Na⁺,K⁺‐ATPase activity in a concentration‐dependent manner. However, PGE₂ did not alter Na⁺,K⁺‐ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE₂ on Na⁺,K⁺‐ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic α subunit of Na⁺,K⁺‐ATPase. We found that the inhibitory effect of PGE₂ (1 μM) on Na⁺,K⁺‐ATPase activity was receptor‐mediated, as incubation with selective antagonists for EP1 (SC‐19220, 10 μM), EP3 (L‐826266, 1 μM) or EP4 (L‐161982, 1 μM) receptors prevented the PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1–10 μM) increased enzyme activity per se in a concentration‐dependent manner, but did not prevent the inhibitory effect of PGE₂. Incubation with a protein kinase A (PKA) inhibitor (H‐89, 1 μM) and a protein kinase C (PKC) inhibitor (GF‐109203X, 300 nM) also prevented PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. Accordingly, PGE₂ increased phosphorylation of Ser943 at the α subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE₂ decreases Na⁺,K⁺‐ATPase activity in vivo. The results presented here imply Na⁺,K⁺‐ATPase as a target for PGE₂‐mediated signaling, which may underlie PGE₂‐induced increase of brain excitability.     

<Emphasis Type="Italic">Alona alsafadii</Emphasis> n. sp. from Yemen,a primitive,groundwater-dwelling member of the <Emphasis Type="Italic">A. karua</Emphasis>-group

The effect of algae on the production of musty-smelling compounds by actinomycetes was studied. Streptomyces spp., causing intensive musty odor, were isolated from hypertrophic Lake Kasumigaura and cultured in association with algae from the same lake. Isolate E and I effectively utilized the cyanobacteria, Microcystis aeruginosa and Anabaena spiroides, and the diatom, Synedra acus, as a carbon source and produced a musty-smelling 2-methylisoborneol in the shaken sediment cultures. High populations of algae and actinomycetes, and aerobic condition in the sediment seem responsible for the occurrence of musty odor in Lake Kasumigaura.     

Elimination of volatile compounds of leaf tobacco from air emissions using biofiltration

The composition of the volatile organic compounds (VOCs) of various leaf tobacco brands and their blends has been studied. The differences in the content of nicotine, solanone, tetramethyl hexadecenol, megastigmatrienones, and other compounds, determining the specific tobacco smell, have been revealed. A microbial consortium, which is able to deodorize simulated tobacco emissions and decompose nicotine, has been formed by long-term adaptation to the VOCs of tobacco leaves in a laboratory reactor, functioning as a trickle-bed biofilter. Such a biofilter eliminates 90% of the basic toxic compound (nicotine) and odor-active compounds; the filtration efficiency does not change for tobacco brands with different VOC concentrations or in the presence of foreign substances. The main strains, isolated from the formed consortium and participating in the nicotine decomposition process, belong to the genera Pseudomonas, Bacillus, and Rhodococcus. An examination of the biofilter trickling fluid has shown full decomposition of nicotine and odor-active VOCs. The compounds, revealed in the trickling fluid, did not have any odor and were nontoxic. The obtained results make it possible to conduct scaling of the biofiltration process to eliminate odor from air emissions in the tobacco industry.     

Effects of phosphorus enrichment and grazing snails on modern stromatolitic microbial communities

1. The effects of phosphorus enrichment and grazing snails on a benthic microbial community that builds stromatolic oncolites were examined in an experiment at Rio Mesquites, Cuatro Ciénegas, Mexico. Chemical analyses of stream water samples indicated that overall atomic ratios of total nitrogen (N) to total phosphorus (P) were approximately 110, indicating a strong potential for P‐limitation of microbial growth. 2. Phosphorus enrichment involved addition of 5 μmol Na₂HPO₄ L^?1 to streamside microcosms receiving intermittent inputs of stream water while grazer manipulation involved removal of the dominant grazer, the snail Mexithauma quadripaludium. After 7 weeks, we examined responses in organic matter content, C : N : P ratios, metabolism (P removal, primary production, dark respiration, and calcification), and microbial community structure using molecular fingerprinting of 16S rRNA genes. 3. Manipulation of snails did not affect response variables measured in these treatments (organic matter, C : P ratio, P removal rate). However, P enrichment significantly decreased the C : P and N : P ratios of surficial materials in the oncolites (organic matter content was unchanged), increased net and gross photosynthesis (oxygen consumption in the dark was unchanged), increased rates of calcification, and increased diatoms relative to cyanobacteria. Heterotrophic Eubacteria and Archaea were only modestly affected. Thus, our results indicate weak grazing effects but strong impacts of P in this benthic system. 4. We hypothesise that a state of severe P‐limitation is imposed on autotrophic production in this food web due, at least in part, to co‐precipitation of phosphate during calcite deposition. This produces severe P‐limitation of the benthic algae and cyanobacteria, resulting in high C : P ratio of microbial mats relative to the biomass of photoautotrophs (phytoplankton, terrestrial foliage) in other ecosystems. In turn, this high C : P ratio is likely to generate severe stoichiometric constraints on the herbivores, thus limiting their populations and resulting in weak overall grazing impacts.     

Monovalent Cations Induce Microsporidian Spore Germination In Vitro

ABSTRACT The relative capacity of Na⁺, K⁺ and Cl^- to stimulate germination of spores of the microsporidian Nosema algerae, a pathogen of mosquitoes, was examined by ion substitution experiments. Sodium at 0.1 M was ineffective to produce the high percentage of germination that typically occurs with 0.1 M NaCl (the normal stimulation solution) if Cl^- was substituted with the usually impermeant anions SO₄^2-, HPO₄^2-, or the organic acids oxalate, cacodylate, EGTA, MES and HEPES. However, substantial concentration- and pH-dependent germination was seen with Na₂SO₄ in the 0.2-0.8 M Na⁺ range. Similar results were obtained with solutions of K⁺ accompanied by impermeant anions. In contrast, the chloride salts of usually impermeant cations, like choline and triethanolamine, failed to germinate spores even at 0.8 M unless Na⁺ or K⁺ was independently added. The presence of 0.5 M choline chloride in the medium reduced the levels of Na₂SO₄ required to produce germination down to equivalence with those of Na⁺ in the normal stimulation solution. Monensin, a Na⁺ ionophore, facilitated the germination induced by a medium-level stimulus (0.04 M NaCl) in sonicated samples. These findings indicate that N. algerae spores germinate in response to the alkali metal cations, while CI^- plays a passive role by diffusing to maintain internal electroneutrality during cation influx. A possible mechanism of cation action in spore germination is suggested on the basis of these results and observations on other systems of intracellular motility.     

H2O2 and cytosolic Ca2+ signals triggered by the PM H+‐coupled transport system mediate K+/Na+ homeostasis in NaCl‐stressed Populus euphratica cells

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H₂O₂, cytosolic Ca²⁺ ([Ca²⁺]_cyt) and the PM H⁺‐coupled transport system in K⁺/Na⁺ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na⁺/H⁺ antiport was seen in salinized cells; however, NaCl stress caused a net K⁺ efflux, because of the salt‐induced membrane depolarization. H₂O₂ levels, regulated upwards by salinity, contributed to ionic homeostasis, because H₂O₂ restrictions by DPI or DMTU caused enhanced K⁺ efflux and decreased Na⁺/H⁺ antiport activity. NaCl induced a net Ca²⁺ influx and a subsequent rise of [Ca²⁺]_cyt, which is involved in H₂O₂‐mediated K⁺/Na⁺ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na⁺/H⁺ antiport system, the NaCl‐induced elevation of H₂O₂ and [Ca²⁺]_cyt was correspondingly restricted, leading to a greater K⁺ efflux and a more pronounced reduction in Na⁺/H⁺ antiport activity. Results suggest that the PM H⁺‐coupled transport system mediates H⁺ translocation and triggers the stress signalling of H₂O₂ and Ca²⁺, which results in a K⁺/Na⁺ homeostasis via mediations of K⁺ channels and the Na⁺/H⁺ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.     

Chemical Composition and Character Impact Odorants in Volatile Oils from Edible Mushrooms

The aim of this study was to investigate the chemical composition and the odor‐active components of volatile oils from three edible mushrooms, Pleurotus ostreatus, Pleurotus eryngii, and Pleurotus abalonus, which are well‐known edible mushrooms. The volatile components in these oils were extracted by hydrodistillation and identified by GC/MS, GC‐olfactometry (GC‐O), and aroma extract dilution analysis (AEDA). The oils contained 40, 20, and 53 components, representing 83.4, 86.0, and 90.8% of the total oils in P. ostreatus, P. eryngii, and P. abalonus, respectively. Odor evaluation of the volatile oils from the three edible mushrooms was also carried out using GC‐O, AEDA, and odor activity values, by which 13, eight, and ten aroma‐active components were identified in P. ostreatus, P. eryngii, and P. abalonus, respectively. The most aroma‐active compounds were C₈‐aliphatic compounds (oct‐1‐en‐3‐ol, octan‐3‐one, and octanal) and/or C₉‐aliphatic aldehydes (nonanal and (2E)‐non‐2‐enal).     

Sodium requirement and metabolism in nitrogen-fixing cyanobacteria

Sodium affects the metabolism of eukaryotes and prokaryotes in several ways. This review collates information on the effects of Na⁺ on the metabolism of cyanobacteria with emphasis on the N₂,fixing filamentous species. Na⁺ is required for nitrogenase activity inAnabaena torulosa, Anabaena L-31 andPlectonema boryanum. The features of this requirement have been mainly studied inAnabaena torulosa. The need for Na⁺ is specific and cannot be replaced by K⁺, Li⁺, Ca 2 + or Mg²⁺. Processes crucial for expression of nitrogenase such as molybdenum uptake, protection of the enzyme from oxygen inactivation and conformational activation of the enzyme are not affected by Na⁺. Mo-Fe protein and Fe protein, the two components of nitrogenase are synthesized in the absence of Na⁺ but the enzyme complex is catalytically inactive. Photoevolution of O₂ and CO₂ fixation, which are severely inhibited in the absence of Na⁺, are quickly restored by glutamine or glutamate indicating that Na⁺ deprivation affects photosynthesis indirectly due to deficiency in the products of N₂ fixation. Na⁺ deprivation decreases phosphate uptake, nucleoside phosphate pool and nitrogenase activity. These effects are reversed by the addition of Na⁺ suggesting that a limitation of available ATP caused by reduced phosphate uptake results in loss of nitrogenase activity during Na⁺ starvation. Na⁺ influx inAnabaena torulosa andAnabaena L-31 is unaffected by low K⁺ concentration, is carrier mediated, follows Michaelis-Menten kinetics and is modulated mainly by membrane potential. Treatments which cause membrane depolarisation and hyperpolarisation inhibit and enhance Na⁺ influx respectively. These cyanobacteria exhibit rapid active efflux of Na⁺, in a manner different from the Na⁺/H⁺ antiporter mechanism found inAnacystis nidulans. Na⁺ requirement in nitrogen metabolism including nitrate assimilation, synthesis of amino acids and proteins, in respiration and oxidative phosphorylation, in transport of sugars and amino acids, cellular distribution of absorbed sodium, physiological basis of salt tolerance and prospects of reclamation of saline soils by cyanobacteria are the other aspects discussed in this review.     

Response surface methodology for optimization of medium components in submerged culture of Aspergillus flavus for enhanced heparinase production

Aims: Aim of the study was to develop a medium for optimal heparinase production with a strain of Aspergillus flavus (MTCC‐8654) by using a multidimensional statistical approach. Methods and Results: Statistical optimization of intracellular heparinase production by A. flavus, a new isolate, was investigated. Plackett–Burman design was used to evaluate the affect of medium constituents on heparinase yield. The experimental results showed that the production of heparinase was dependent upon heparin, the inducer; chitin, structurally similar to heparin and NH₄NO_3, the nitrogen source. A central composite design was applied to derive a statistical model for optimizing the composition of the fermentation medium for the production of heparinase enzyme. The optimum fermentation medium consisted of (g l^?1) Mannitol, 8·0; NH₄NO₃, 2·5; K₂HPO₄, 2·5; Na₂HPO₄, 2·5; MgSO₄.7H₂O, 0·5; Chitin, 17·1; Heparin, 0·6; trace salt solution (NaMoO₄.2H₂O, CoCl₂.6H₂O, CuSO₄.5H₂O, FeSO₄.7H₂O, CaCl₂), 10^?4 mol l^?1. Conclusions: A 2·37‐fold increase in heparinase production was achieved in economic and effective manner by the application of statistical designs in medium optimization. Significance and Impact of the Study: Heparinase production was doubled by statistical optimization in a cost‐effective manner. This heparinase can find application in pharmaceutical industry and for the generation of low‐molecular‐weight heparins, active as antithrombotic and antitumour agents.     

Gas-phase degradation of VOCs using supported bacteria biofilms

Herein we report the use of Pseudomonas putida F1 biofilms grown on carbonized cellulosic fibers to achieve biodegradation of airborne volatile organic compounds (VOCs) in the absence of any bulk aqueous-phase media. It is believed that direct exposure of gaseous VOC substrates to biomass may eliminate aqueous-phase mass transfer resistance and facilitate VOC capture and degradation. When tested with toluene vapor as a model VOC, the supported biofilm could grow optimally at 300 p.p.m. toluene and 80% relative humidity, with a specific growth rate of 0.425 day⁻¹. During long-term VOC biodegradation tests in a tubular packed bed reactor, biofilms achieved a toluene degradation rate of 2.5 mg g_DCW⁻¹ h⁻¹ during the initial growth phase. Interestingly, the P. putida F1 film kept biodegrading activity even at the stationary nongrowth phase. The supported biofilms with a biomass loading of 20% (wt) could degrade toluene at a rate of 1.9 mg g_DCW⁻¹ h⁻¹ during the stationary phase, releasing CO₂ at a rate of 6.4 mg g_DCW⁻¹ h⁻¹ at the same time (indicating 100% conversion of substrate carbon to CO₂). All of these observations promised a new type of “dry” biofilm reactors for efficient degradation of toxic VOCs without involving a large amount of water.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Ascorbate-2-phosphate inhibition of ascorbate-2-sulfate sulfohydrolase from bovine liver.

The hydrolysis of ascorbate-2-sulfate by the enzyme, ascorbate-2-sulfate sulfohydrolase, purified from bovine liver has been shown to be powerfully inhibited by ascorbate-2-phosphate. The inhibition by ascorbate phosphate is competitive with a K_I of 0.3 μM. Na₂HPO₄ also inhibits by an apparent non-competitive process. The Na₂HPO₄ concentration at 50% inhibition is 7.7 μM. A possible control role for ascorbate phosphate in ascorbate biochemistry is suggested.