A beta-glucosidase from Sclerotinia sclerotiorum: biochemical characterization and use in oligosaccharide synthesis

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one beta-glucosidase (beta-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that beta-glu x may be a homodimer. For p-nitrophenyl beta-d-glucopyranoside hydrolysis, apparent Km and Vmax values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55-60 degrees C and pH 5.0, respectively. beta-Glu x was strongly inhibited by Fe2+ and activated about 35% by Ca2+. beta-Glu x possesses strong transglucosylation activity in comparison with commercially available beta-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50 degrees C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 3C nuclear magnetic resonance spectroscopy.     

Synthesis of 2′-deoxyguanosine containing a N2-polyamine

The synthesis of benzylated N²-(4,7,10,13-tetraazatridec-1-yl)-2′-deoxyguanosines 4 was accomplished by a key nucleophilic reaction of the novel unsymmetrical polyamine 2 , with 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2-chloro-2′-deoxyinosine ( 1 ).     

Unique hydrophobic interactions of pyrene in aqueous solution as effected by polyelectrolytes and surfactants

Pyrene substituents covalently bounded to polyelectrolytes show not only excited-state interactions but also unique ground-state interactions in aqueous solution. The pyrene moieties in pyrenesubstituted ionic molecules also show these interactions when aqueous solutions of these molecules are treated with polyelectrolytes or surfactants well below their critical micelle concentrations. These hydrophobic interactions are revealed by changes in absorption, fluorescence, and excitation spectra. The well-resolved vibrational bands in the absorption and excitation spectra of pyrene become somewhat diffuse, whereas monomer fluorescence is reduced and replaced by excimer fluorescence. The rationale for these results is that the pyrene moieties in these ionic solutions seek out hydrophobic locations on the polyelectrolytes or surfactants, where pyrene aggregation is responsible for these interactions and the corresponding changes in spectra.     

Synthesis and elaboration of functionalised carbohydrate-derived spiroketals

The scope of a stereoselective three-step approach for the synthesis of sugar derived spiroketals is presented. The methodology consists of Grignard addition of vinyl- or allylmagnesium bromide to a carbohydrate lactone, followed by K-10 clay mediated glycosidation with a terminal alkenol and subsequent ring-closing metathesis of the resulting diene. The generality of this procedure is demonstrated by the synthesis of various pyranose- and furanose-derived spiroketals, as well as more advanced tricyclic spiroketal derivatives. It is shown that functionalisation of the double bond in the resulting spiroketals leads to fused polycyclic ethers.     

The solvent extraction of iron from steel solutions with 2-hexylpyridine prior to the determination of trace elements

The distribution of iron(III) between aqueous hydrochloric acid and 0.1M 2-hexylpyridine in benzene was examined as a function of acid concentration in the aqueous solution, the metal concentration being kept constant, and as a function of increasing ferric chloride concentration at a constant acidity of hydrochloric acid. The distribution coefficient of Fe(III) (tracer) is dependent on the square of the 2-hexylpyridine concentration in the benzene phase. Negatively charged complexes of the type FeCl(2-)(5) may be the species extracted. The formation of a mixture of 1:1 and 1:2 complexes with 2-hexylpyridine is indicated. Salting-out effects of a number of salts have been investigated. Separation factors of several metal ions relative to iron(III) in 7M hydrochloric acid are also reported. The results indicate that iron(III) can be selectively separated from a large number of elements, and the method has been utilized for the preconcentration of non-ferrous metal ions in mild steels by selective separation of iron, before their subsequent determination by emission spectrometry.     

Solvent-free microwave extraction of essential oil from aromatic herbs: comparison with conventional hydro-distillation

Solvent-free microwave extraction (SFME) is a combination of microwave heating and dry distillation, performed at atmospheric pressure without added any solvent or water. Isolation and concentration of volatile compounds are performed by a single stage. SFME has been compared with a conventional technique, hydro-distillation (HD), for the extraction of essential oil from three aromatic herbs: basil (Ocimum basilicum L.), garden mint (Mentha crispa L.), and thyme (Thymus vulgaris L.). The essential oils extracted by SFME for 30min were quantitatively (yield) and qualitatively (aromatic profile) similar to those obtained by conventional hydro-distillation for 4.5 h. The SFME method yields an essential oil with higher amounts of more valuable oxygenated compounds, and allows substantial savings of costs, in terms of time, energy and plant material. SFME is a green technology and appears as a good alternative for the extraction of essential oils from aromatic plants.     

Reactions with 2-aminonicotinic acid,I Some 8-aza analogs of quinazolinones and derived tricyclic compounds

The fusion of 2-acetamidonicotinic acid witho-toluidine,p-bromoaniline oro-chloroaniline afforded the corresponding 3-aryl-2-methyl-pyrido-[2,3-d]pyrimidin-4(3H)-ones (4), the 8-aza analogs of 3-aryl-2-methyl-4-quinazolinones, alongside 2-aminonicotinic acid. 2-Methyl-3-2(2-methylphenyl)-pyrido[2,3-d]pyrimidin-4(3H)-one (4a), the 8-aza analog of methaqualone, was converted to the 2-substituted styryl derivatives6 by condensation with some aromatic aldehydes and to the tricyclic system, 10-aza-5,6-dihydro-3-hydroxy-5-(2-methylphenyl)-2-substituted-1H-pyrido [1,2-a] quinazoline-1,6-diones (8) by reaction with monosubstituted bis-2,4,6-trichlorophenyl malonates.     

Fast folding of a four-helical bundle protein

The FK506-FKBP12 binding-domain of the kinase FRAP (FRB) forms a classic up-down four-helical bundle. The folding pathway of this protein has been investigated using a combination of equilibrium and kinetic studies. The native state of the protein is stable with respect to the unfolded state by some 7 kcal mol(-1) at pH 6.0, 10 degrees C. A kinetic analysis of unfolding and refolding rate constants as a function of chemical denaturant concentration suggests that an intermediate state may be populated during folding at low concentrations of denaturant. The presence of this intermediate state is confirmed by refolding experiments performed in the presence of the hydrophobic dye 8-anilinonaphthalene-1 sulfonate (ANS). ANS binds to the partially folded intermediate state populated during the folding of FRB and undergoes a large change in fluorescence that can be detected using stopped-flow techniques. Analysis of the kinetic data suggests that the intermediate state is compact and it may even be a misfolded species that has to partially unfold before it can reach the transition state. Folding and unfolding rate constants in water are approximately 150-200 s(-1) and 0.005-0.06 s(-1), respectively, at neutral pH and 10 degrees C. The folding of FRB is somewhat slower than for other all-helical proteins, probably as a consequence of the formation of a metastable intermediate state. The folding rate constant in the absence of any populated intermediate can be estimated to be 8800 s(-1). Despite the presence of an intermediate state, which effectively slows folding, the protein still folds rapidly with a half-life of 5 ms at 10 degrees C. The dependence of the rate constants on denaturant concentration indicates that the transition state for folding is compact with some 80% of the surface area exposed in the unfolded state buried in the transition state. Data presented for FRB is compared with kinetic data obtained for other all-helical proteins.     

New double negative and positive temperature coefficients of conductive EPDM rubber TiC ceramic composites

In this paper, we have successfully prepared ethylene-propylene-diene monomer (EPDM)/TiC composites as thermistors, with new double negative and positive temperature coefficients of conductivity (NTCC/PTCC). EPDM composites loaded with 50 phr HAF carbon black and different concentrations of TiC were prepared. This study focuses on the effect of TiC content on the vulcanization process, the network structure and the electrical and thermal properties of EPDM/TiC composites. The effect of TiC on the network structure was evaluated e.g. the curing process, the characteristic time constant during vulcanization, the volume fraction of rubber, gel fraction, interparticle distance between conductive particles, the extent of TiC reinforcement in the rubber matrix and molecular weight between cross-linking through experimental and affine-phantom models. The effects of TiC content on the percolation theory, electrical conductivity, conducting mechanism of conductivity, conducting hysteresis and I-V characteristics were also studied, as well as its TiC on the (NTCC/PTCC), thermoelectric power, dielectric constant and thermal conductivity. Stability and reproducibility of the thermal cycles for heating element applications was tested. Specific heat and the amount of heat transfer by radiation and convection as a function of TiC content was calculated using both the calorimetric technique and a theoretical model. It was proved that TiC improves the network structure, electrical and thermal properties of EPDM composites for practical applications.