Acid Pullulanase from Bacillus polymyxa MIR-23

Within the frame of a screening program aimed at the isolation of amylolytic sporeformers, one strain with high amylolytic activity designated MIR-23 was selected. The microbial characterization was carried out by morphological and biochemical tests and, by means of statistical treatment, was identified asBacillus polymyxa. The organism could grow in acidic conditions (pH 5.0) on a starch medium and produce α-amylase, pullulanase, and α-glucosidase. Batch cultures showed the highest enzyme activities in the stationary phase. Pullulanase activity exhibited an optimal temperature of 52–57°C at pH 4.5–5.5. These properties would allow its use in the saccharification processes in the starch industries.     

Flow injection amperometric detection of ascorbic acid using a Prussian Blue film-modified electrode

The PB film-modified electrode was used as an amperometric detector for flow injection analysis of ascorbic acid. The modified electrode detector showed good sensitivity, stability and reproducibility. The calibration curve for ascorbic acid was linear over the concentration range from 5.0x10(-6) to 1.0x10(-3) mol l(-1) with a slope of 19.9 mA mol(-1) per litre and a correlation coefficient of 0.999. The detection limit of this method was 2.49x10(-6) mol l(-1). The relative standard deviation of six replicate injections of 2.5x10(-4) mol l(-1) ascorbic acid was 2.5%. The results obtained for ascorbic acid determination in pharmaceutical products are in good agreement with those obtained by using the procedure involving the reaction between triiodide and ascorbic acid.     

Effect of pH on the interfacial adsorption activity of pulmonary surfactant

The effect of pH on the interfacial adsorption activity of pulmonary surfactant was examined. Measurements of the surface tension were made in a Wilhelmy-like surface microbalance specially designed to assay small volumes of hypophase in thermostatically controlled conditions. Alkaline pH caused a significant decrease of the surface activity of both pulmonary surfactant and a lipid extract from surfactant (LES) (containing all of the lipids and surfactant protein-B (SP-B) and surfactant protein-C (SP-C) hydrophobic surfactant proteins, but lacking surfactant protein-A). The pK calculated from the change of surface activity versus pH was 9.18±0.26 and 9.27±0.31 for pulmonary surfactant and LES, respectively. The results from this study support the idea that electrostatic interactions between basic residues of SP-B and SP-C and negatively charged surfactant phospholipids could be important for the interfacial adsorption activity of pulmonary surfactant.     

A sensitive, rapid and specific determination of midazolam in human plasma and saliva by liquid chromatography/electrospray mass spectrometry

A rapid, sensitive and selective liquid chromatography/electrospray mass spectrometry (LC/ES-MS) method was developed for the quantitative determination of the anaesthetic benzodiazepine midazolam (MID) in human saliva and plasma from patients undergoing anesthesia procedures. Biological samples spiked with diazepam-d5, the internal standard, were extracted into diethyl ether. Compounds were separated on a Xterra RP18 column using a mobile phase of acetonitrile/formic acid 0.1% at a flow rate of 0.25 mL/min under a linear gradient. Column effluents were analyzed using MS with an ES source in the positive ionization mode. Calibration curves were linear in the concentration ranges of 1-250 and 0.2-25 ng/mL in plasma and saliva, respectively. The limits of detection were 0.5 ng/mL in plasma and 0.1 ng/mL in saliva, using a 0.5-mL sample volume. The recoveries of the spiked samples were above 65%. The method was applied to ten real samples from patients undergoing midazolam treatment.     

Effects of ultrasonic irradiation in flow-injection systems

The effects of ultrasonic irradiation on transport and reactions in several flow-injection manifolds are described. The influences of ultrasound on the physical dispersion of the injected plug is considered. Its effects on systems involving homogeneous (catalytic and non-catalytic) and heterogeneous (liquid/liquid extraction, precipitation) reactions are critically evaluated. In general, greater sensitivity is obtained, especially for heterogeneous systems, slow reactions and low analyte concentrations. Finally, practical considerations and potential uses of the combination of ultrasonic irradiation with flow-injection analysis are discussed.     

Flow injection determination of oxalate in urine based on an inhibitory effect

Summary Two methods based on the use of the normal and stopped-flow injection modes were developed for the determination of oxalate based on its inhibitory effect on the catalytic action of Fe(III) on the 2,4-diaminophenol/hydrogen peroxide system. The linear determination ranges achieved were between 0.2 and 12.0 g ml^–1 and between 0.2 and 40.0 g ml^–1, the precision was ±5.4%, and ±3.5%, and the sampling rate was 30 and 20 samples h^–1 for the normal and stopped-flow method, respectively. Both methods have been applied to the determination of oxalate in urine with excellent results.     

Kinetic investigation of the reactions of S-4-nitrophenyl 4-substituted thiobenzoates with secondary alicyclic amines in aqueous ethanol

The reactions of S-4-nitrophenyl 4-X-substituted thiobenzoates (X = H, Cl, and NO(2): 1, 2, and 3, respectively) with a series of secondary alicyclic amines (SAA) were subjected to a kinetic investigation in 44 wt % ethanol-water, at 25.0 degrees C and an ionic strength of 0.2 M (KCl). The reactions were followed spectrophotometrically by monitoring the release of 4-nitrobenzenethiolate anion at 420-425 nm. Under excess amine, pseudo-first-order rate constants (k(obsd)) are obtained for all reactions. The plots of k(obsd) vs [SAA] at constant pH are linear with the slope (k(N)) independent of pH. The statistically corrected Br?nsted-type plots (log k(N)/q vs pK(a) + log p/q) for the reactions of 1 and 2 are nonlinear with slopes at high pK(a), beta(1) = 0.27 and 0.10, respectively, and slopes at low pK(a), beta(2) = 0.86 and 0.84, respectively. The Br?nsted curvature is centered at pK(a) (pK(a)(0)) 10.0 and 10.4, respectively. The reactions of SAA with 3 exhibit a linear Br?nsted-type plot of slope 0.81. These results are consistent with a stepwise mechanism, through a zwitterionic tetrahedral intermediate (T(+/-)). For the reactions of 1 and 2, there is a change in rate-determining step with amine basicity, from T(+/-) breakdown to products at low pK(a), to T(+/-) formation at high pK(a). For the reactions of 3, breakdown to products of T(+/-) is rate limiting for all the SAA series (pK(a)(0) > 11). The increasing pK(a)(0) value as the substituent in the acyl group becomes more electron withdrawing is attributed to an increasing nucleofugality of SAA from T(+/-). The greater pK(a)(0) value for the reactions of SAA with 1, relative to that found in the pyridinolysis of 2,4-dinitrophenyl benzoate (pK(a)(0) = 9.5), is explained by the greater nucleofugality from T(+/-) of the former amines, compared to isobasic pyridines, and the greater leaving ability from T(+/-) of 2,4-dinitrophenoxide relative to 4-nitrobenzenethiolate.     

Kinetics and mechanism of the aminolysis of O-aryl S-methyl thiocarbonates

[reaction: see text] The reactions of secondary alicyclic (SA) amines and quinuclidines (QUI) with 4-nitrophenyl and 2,4-dinitrophenyl S-methyl thiocarbonates (1 and 2, respectively) and those of SA amines with 2,3,4,5,6-pentafluorophenyl S-methyl thiocarbonate (3) are subjected to a kinetic study in aqueous solution, at 25.0 degrees C, and an ionic strength of 0.2 M (KCl). The reactions of thiocarbonates 1, 2, and 3 were followed spectrophotometrically at 400, 360, and 220 nm, respectively. Under amine excess, pseudo-first-order rate coefficients (k(obsd)) are found. Plots of k(obsd) vs amine concentration at constant pH are linear, with the slope (kN) independent of pH. The Br?nsted-type plots (log kN vs pKa of aminium ions) are linear for all the reactions, with slopes beta = 0.9 for those of 1 with SA amines and QUI, beta = 0.36 and 0.57 for the reactions of 2 with SA amines and QUI, respectively, and beta = 0.39 for the reactions of SA amines with 3. The magnitude of the slopes indicates that both aminolyses of 1 are governed by stepwise mechanisms, through a zwitterionic tetrahedral intermediate (T+/-), where expulsion of the nucleofuge from T+/- is the rate-determining step. The values of the Br?nsted slopes found for the aminolyses of thiocarbonates 2 and 3 suggest that these reactions are concerted. By comparison of the reactions under investigation between them and with similar aminolyses, the following conclusions arise: (i) Thiocarbonate 2 is more reactive than 1 toward the two amine series. (ii) The change of the nonleaving group from MeO in 4-nitrophenyl methyl carbonate to MeS in thiocarbonate 1 results in lower kN values. (iii) The greater reactivity of this carbonate than thiocarbonate 1 is attributed to steric hindrance of the MeS group, compared to MeO toward amine attack. (iv) The change of a pyridine to an isobasic SA amine or QUI destabilizes the T+/- intermediate formed in the aminolyses of 2. (v) The change of 4-nitrophenoxy to 2,3,4,5,6-pentafluorphenoxy or 2,4-dinitrophenoxy as the leaving group destabilizes the tetrahedral intermediate formed in the reactions with SA amines, changing the mechanism from a stepwise process to a concerted reaction.     

Supramolecular phthalocyanine dimers based on the secondary dialkylammonium cation/dibenzo-24-crown-8 recognition motif

[formula: see text] New unsymmetrically substituted DB24C8-phthalocyanines, which are able to form complexes with suitable dialkylammonium cations, have been prepared. These complexes most probably have a pseudorotaxane geometry.     

Kinetics and mechanism of the reactions of S-2,4-dinitrophenyl 4-substituted thiobenzoates with secondary alicyclic amines

[reaction: see text] The title reactions, in 44 wt % ethanol-water at 25.0 degrees C, exhibit slightly curved Br?nsted-type plots (log kN versus pKa of amines) with slopes beta1 = 0.1-0.44 (at high pKa) and beta2 ca. 0.7 (at low pKa). The magnitude of some of these slopes, together with the fact that the curvature center (pKa(0) = 9.5-10.8) does not change with the electronic effects of the benzoyl substituent, suggests that these reactions are not stepwise, but concerted.