Influence of activation on the multipoint immobilization of penicillin G acylase on macroporous silica

Penicillin G acylase is the second most important enzyme used by industry in an immobilized form. Penicillin hydrolysis is its main application. This reaction is used to produce 6-aminopenicillanic acid (6-APA), an intermediate in the synthesis of semisynthetic antibiotics. This work aims to compare catalytic properties of different penicillin G acylase (PGA) derivatives obtained by multipoint immobilization of the enzyme on macroporous silica. Enzyme amino groups react with different aldehyde groups produced in the support using either glutaraldehyde or glyoxyl activation. In the former method, silica reacts with g-aminopropyltriethoxysilane (g-APTS) and glutaraldehyde; in the latter, a reaction with glycidoxypropyltrimethoxysilane (GPTMS) is followed by acid hydrolysis and oxidation using sodium periodate. This work determines the influence of degree of activation, using glutaraldehyde, on immobilization parameters. PGA was immobilized on these two different supports. Maximum enzyme load, immobilized enzyme activity (derivative activity), rate of immobilization and thermal stability were checked for both cases. For glutaraldehyde activation, the results showed that 0.5% of the g-APTS is sufficient for all the hydroxyl groups in the silica to react. They also showed that degree of activation only affects immobilization yield and reaction velocity and that reduction of the glutaraldehyde derivatives with sodium borohydride does not affect their thermal stability. In comparing the derivatives obtained using glyoxyl and glutaraldehyde activation, it was observed that the glyoxyl derivatives presented better immobilization parameters, with a maximum enzyme load of 264 IU/g silica and a half-life of 20 minutes at 60 °C.

Antiulcer activity and subacute toxicity of trans-dehydrocrotonin from Croton cajucara

The antiulcerogenic activity of trans-dehydrocrotonin (DHC), a nor-clerodane diterpene isolated from Croton cajucara Benth. ( Euphorbiaceae), and its subacute ( 35 days) toxicity were studied in mice and rats, respectively. For the antiulcerogenic tests, models of gastric ulcers induced in mice by ethanol/HCl or stress were used. In both models, an oral dose of DHC ( 100 mg/kg) significantly reduced (P< 0.01) the formation of gastric lesions. DHC was also tested for its ability to scavenge free radicals, but no such action was observed in rat liver mitochondria. To assess the subacute toxicity, rats were treated orally with DHC (25, 50 and 100 mg/kg) for 5 weeks. A significant increase in liver weight was observed in male and female rats at highest doses, whereas a significant reduction in plasma alkaline phosphatase and cholesterol levels and an increase in gamma glutamyl transpeptidase were observed only at the highest dose ( 100 mg/kg) in female rats. DHC caused histopathological alterations in the liver that included a turbid tumefaction, microvacuolar degeneration and nuclear alterations. Despite the beneficial antiulcerogenic activity of DHC, our results suggest that the long-term use of this compound may induce liver damage.

Variable influence of ferric and cupric ions on Saccharomyces cerevisiae strains used in asymmetric organic synthesis.

Fe (III) and Cu (II) each at 50 mu M in four commercial strains of Saccharomyces cerevisiae induced an increase of NAD(P)(+) reduction in one strain (Turkish), but two others (Chilean and Brazilian), the presence of Fe(III) and/or Cu(II) diminished NAD(P)(+) reduction presumably due to free radicals formation inside these living cells. Suprisingly, in the American strain, Fe(III) induced a decrease and Cu (II) an increase of NAD(P)(+) reduction.

Multimethod study of the degree of humification of humic substances extracted from different tropical soil profiles in Brazil's Amazonian region

This work consisted of determining the degree of humification of humic substances (HS) extracted from six different Amazonian soils collected from flooded and unflooded regions at different depths (0-10, 10-20, 20-40, and 60 cm). The humic substances were extracted according to procedures recommended by the International Humic Substances Society and characterized using elemental analysis, electron paramagnetic resonance (EPR), and fluorescence spectroscopy. The findings on semiquinone-type free radical concentrations in HS showed variations of 0.10-7.55x10(18) spins g(-1) of carbon (g C)(-1), indicating considerable differences between the humification levels of HS extracted from Amazonian soils. The results showed an average of 1.71 +/- 0.04 x 10(18) spins (g C)(-1), which is congruent with other data reported in the literature on Tropical soils. It was found that, on average, HS extracted from flooded soil contained higher semiquinone-type free radical concentrations than HS extracted from unflooded soils, indicating the influence of humidity in the humification process of organic matter. The humification process varies according to the profile, and the 10-20- and 0-10-cm profiles generally showed more humified HS. The degree of humification of the HS studied here displayed a similar behavior when exposed to fluorescence (excitation at 465 nm) and EPR (R=0.85). However, the low correlation between the C/H, C/O, and C/N atomic ratios and the semiquinone-type free radical concentration/fluorescence intensities indicated that data obtained by these techniques with regard to the degree of humification of HS may lead to different conclusions. (c) 2004 Elsevier B.V. All rights reserved.

Prediction of cassava starch edible film properties by chemometric analysis of infrared spectra

Cassava starch has been shown to make transparent and colorless flexible films without any previous chemical treatment. The functional properties of edible films are influenced by starch properties, including chain conformation, molecular bonding, crystallinity, and water content. Fourier-transform infrared (FTIR) spectroscopy in combination with attenuated total reflectance (ATR) has been applied for the elucidation of the structure and conformation of carbohydrates. This technique associated with chemometric data processing could indicate the relationship between the structural parameters and the functional properties of cassava starch-based edible films. Successful prediction of the functional properties values of the starch-based films was achieved by partial least squares regression data. The results showed that presence of the hydroxyl group on carbon 6 of the cyclic part of glucose is directly correlated with the functional properties of cassava starch films.

Thermal degradation of a composite solid propellant examined by DSC - Kinetic study

The thermal decomposition of ammonium perchlorate (AP)/hydroxyl-terminated-polybutadiene (HTPB), the AP/HTPB solid propellant, was studied at different heating rates in dynamic nitrogen atmosphere. The exothermic reaction kinetics was studied by differential scanning calorimetry (DSC) in non-isothermal conditions. The Arrhenius Parameters were estimated according to the Ozawa method. The calculated activation energy was 134.5 W mol(-1), the pre-exponential factor, A, was 2.04.10(10) min(-1) and the reaction order for the global composite decomposition was estimated in 0.7 by the kinetic Shimadzu software based on the Ozawa method. The Kissinger method for obtaining the activation energy value was also used for comparison. These results are discussed here.

Response of L-alanine and 2-methylalanine minidosimeters for K-Band (24 GHz) EPR dosimetry

Minidosimeters of L-alanine and 2-methylalanine (2MA) were prepared and tested as potential candidates for small radiation field dosimetry. To quantify the free radicals created by radiation a K-Band (24 GHz) EPR spectrometer was used. X-rays provided by a 6 MV clinical linear accelerator were used to irradiate the minidosimeters in the dose range of 0.5-30 Gy. The dose-response curves for both radiation sensitive materials displayed a good linear behavior in the dose range indicated with 2MA being more radiation sensitive than L-alanine. Moreover, 2MA showed a smaller LLD (lower limit detection) value. The proposed system minidosimeter/K-Band spectrometer was able to detect 10 Gy EPR spectra with good signal-to-noise ratio (S/N). The overall uncertainty indicates that this system shows a good performance for the detection of dose values of 20 Gy and above, which are dose values typically used in radiosurgery treatments. (c) 2007 Elsevier B.V. All rights reserved.

The effect of alpha-tocopherol on the oxidative cleavage of beta-carotene

Two cleavage pathways of beta-carotene have been proposed, one by central cleavage and the other by random (excentric) cleavage. The central cleavage pathway involves the metabolism of beta-carotene at the central double bond (15, 15') to produce retinal by beta-carotene 15, 15'-dioxygenase (E.C.888990988). The random cleavage of beta-carotene produces beta-apo-carotenoids, but the mechanism is not clear. To understand the various mechanisms of beta-carotene cleavage, beta-carotene was incubated with the intestinal postmitochondrial fractions of 10-week-old male rats for 1 h and cleavage products of beta-carotene were analyzed using reverse-phase, high-performance liquid chromatography (HPLC). We also studied the effects of alpha-tocopherol and NAD(+)/NADH on beta-carotene cleavage. In addition to beta-carotene, we used retinal and beta-apo-14'-carotenoic acid as substrates in these incubations. Beta-apo-14'-carotenoic acid is the two-carbon longer homologue of retinoic acid. In the presence of alpha-tocopherol, beta-carotene was converted exclusively to retinal, whereas in the absence of alpha-tocopherol, both retinal and beta-apo-carotenoids were formed. Retinoic acid was produced from both retinal and beta-apo-14'-carotenoic acid incubations only in the presence of NAD(+). Our data suggest that in the presence of an antioxidant such as alpha-tocopherol, beta-carotene is converted exclusively to retinal by central cleavage. In the absence of an antioxidant, beta-carotene is cleaved randomly by enzyme-related radicals to produce beta-apo-carotenoids, and these beta-apo-carotenoids can be oxidized further to retinoic acid via retinal. (C) 2000 Elsevier B.V.

Shikimate kinase: A potential target for development of novel antitubercular agents

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

Active and exo-site inhibition of human factor Xa: Structure of des-Gla factor Xa inhibited by NAP5, a potent nematode anticoagulant protein from Ancylostoma caninum

Hookworms are hematophagous nematodes capable of growth, development and subsistence in living host systems such as humans and other mammals. Approximately one billion, or one in six, people worldwide are infected by hookworms causing gastrointestinal blood loss and iron deficiency anemia. The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75-84 amino acid residues). One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of factor Xa (fXa) with K-i = 43 pM, and is the most potent natural fXa inhibitor identified thus far. The crystal structure of NAP5 bound at the active site of gamma-carboxyglutamic acid domainless factor Xa (des-fXa) has been determined at 3.1 angstrom resolution, which indicates that Asp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar to that of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39 of NAP5 additionally forms a hydrogen bond (2.5 angstrom) with His57 NE2 of the catalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in the native structure, resulting in an interaction that has not been observed before. Furthermore, the C-terminal extension of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a short intermolecular beta-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, or the exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does not inhibit fVIIa of the fVIIa/TF complex. (c) 2007 Elsevier Ltd. All rights reserved.

Differential/combined effect of water contamination with cadmium and nickel on tissues of rats

The contamination of water by metal compounds is a worldwide environmental problem. Concentrations of metals are widely related to biochemical values which are used in disease diagnosis due to environmental toxicity. The acute combined effects of cadmium and nickel on biochemical parameters were determined and compared with those of Cd2+ or Ni2+ alone in rats. Male adult rats were given drinking solutions of CdCl2 [Cd(II) cation, 100 mg/liter] or NiSO4 [Ni(II) cation, 100 mg/liter]. For the combined treatment, the animals (Ni+Cd) received both Ni(II)) cation (100 mg/liter) and Cd(II) cation (100 mg/liter). Nickel treatment induced increased alanine transaminase (ALT) activity and hepatotoxicity, but not renal injury. In contrast, cadmium exposure produced hepatic, renal and myocardial damage, characterized by increased creatinine, total and direct bilirubin concentrations and increased ALT and lactate dehydrogenase (LDH) activities. The combined effect Ni-Cd is less toxic than cadmium alone, suggesting antagonism between these toxicants. The toxicity of nickel and cadmium, alone and in combination, decreased Cu-Zn superoxide dismutase (SOD) activity and increased lipoperoxide formation. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

A nonisothermal study of the kinetics of the nanoporosity elimination in sonogels-derived silica xerogels

A nonisothermal study of the kinetics of the nanoporosity elimination in monolithic silica xerogels, prepared from acid and ultrasound catalyzed hydrolysis of tetraethylortosilicate (TEOS), has been carried out by means of in situ linear shrinkage measurements performed with different heating rates. The study could be applied up to almost alpha similar to 0.6 of the volume fraction alpha of eliminated pores. The activation energy was found increasing from about 3.2 x 10(2) kJ/mol for alpha similar to 0.06 up to about 4.4 x 10(2) kJ/mol for alpha. similar to 0.44. The sintering process accompanying the nanopore elimination in this set of xerogels is in agreement with a viscous flux sintering process with the hydroxyl content diminishing with the volume fraction of eliminated pores. All the volume fraction of eliminated pores versus temperature (T) curves can be matched onto a unique curve with an appropriate rescaling of the T axis, independent of the heating rate. This scaling property suggests that the path of sintering seems the same, regardless of the heating rate; the difference is that the rate is faster at higher temperature.

An investigation of cadmium(II) and nickel(II) adsorption by chitin graft covolymer

Chitin hydrogels of poly(vinylpyrrolidone) (VP) were prepared by means of the hydrogen peroxide graft copolymerization process. The effect of the VP grafted chain on water diffusion through the biopolymer was studied. Fourier transform infrared spectra of the VP-g-Ch showed an increase in the intensities of the hydroxyl and carbonyl stretching bands indicating a reduction in the hydrogen bonding of chitin. An investigation was undertaken regarding the adsorption of nickel(II) and cadmium(II) ions from aqueous solutions by the VP grafted chitin and the effects of the grafting degree on the Cd2+ and Ni2+ sorption were studied. The Cd2+ and Ni2+ adsorption equilibrium data correlate well with the Freundlich equation. The results indicate that the Ch-g-VP graft copolymer under investigation is a potentially powerful chelating material that can be employed for Ni2+ and Cd2+ ion removal from wastewater effluents. (C) 2004 Wiley Periodicals, Inc.

TOXIC EFFECTS OF WATER EUTROPHICATION ON PANCREATIC, HEPATIC AND OSTEOGENIC TISSUES OF RATS

Pollution, industrial solvents, concentrations of metals and other environmental agents are widely related to biochemicals values which are used in disease diagnosis of environmental toxicity. A rat bioassay validated for the identification of toxic effects of eutrophication revealed increased serum activities of amylase, alanine transaminase (BLT) and alkaline phosphatase (ALP) in rats that received algae, filtered water and nickel or cadmium from drinking water. Serum Cu-Zn superoxide dismutase activity decreased from its basal level of 40.8 +/- 2.3 to 26.4 U/mg protein, at 7 days of algae and at 48 hr of nickel and cadmium water ingestion. The observation that lipoperoxide concentration was not altered in rats treated with filtered water, while amylase, ALT and ALP were increased in these rats and in those treated with nickel or cadmium, indicated that pancreatic, hepatic and osteogenic lesions by eutrophication were not related to superoxide radicals, and might be due to a novel toxic environmental agent found in filtered and non-filtered algae water.

Structural and phenomenological characterization of the thermoreversible sol-gel transition of a zirconyl aqueous precursor modified by sulfuric acid

The thermoreversible sol-gel transition is well-known in biological and organic polymeric systems but has not been reported for inorganic systems. In this paper we put in evidence a thermoreversible sol-gel transition for zirconyl chloride aqueous solutions modified by sulfuric acid in the ratio 3:1 Zr:SO4. The synthesis conditions are detailed and a variety of experimental techniques (turbidimetry, dynamic rheology, and EXAFS) have been employed for investigating the thermal reversibility and the chemical structure of this new material. Turbidimetric measurements performed for solutions containing different concentrations of precursor have evidenced that the sol-gel transformation temperature increases from 50 to 80 degrees C as the concentration of zirconyl chloride decreases from 0.22 to 0.018 mol L-1. A more detailed study has been done for the sample with [Zr] = 0.156 mol L-1, in which the sol-gel-sol transformation has been repeated several times by a cyclic variation of the temperature. The mechanical properties of this sample, evaluated by measuring the storage and the loss moduli, show a change from liquid like to viscoelastic to elastic behavior during the sol-gel transition and vice versa during the gel-sol one. In situ EXAFS measurements performed at the Zr K-edge show that no change of the local order around Zr occurs during the sol-gel-sol transition, in agreement with the concept of physical gel formation. We have proposed for the structure of the precursor an inner core made of hydroxyl and oxo groups bridging together zirconium atoms surrounded in surface by complexing sulfate ligands, the sulfate groups act as a protective layer, playing a key role in the linking propagation among primary particles during sol-gel-sol transition.