Inoculum studies in production of penicillin g acylase by Bacillus megaterium ATCC 14945

This article reports studies concerning the production of penicillin G acylase (PGA) by Bacillus megaterium. This enzyme has industrial use in the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid, an essential intermediate for the production of semisynthetic β-lactam antibiotics. Although most microorganisms produce the enzyme intracellularly, B. megaterium provides extracellular PGA. The enzyme production by microorganisms involves several steps, resulting in a many operational variables to be studied. The study of the inoculum is an important step to be accomplished, before addressing other issues such as culture optimization and downstream processing. In this study, using a standard inoculum as reference, several runs were performed aiming at the definition of operational conditions in the PGA production. Cell concentration and PGA activity in the production medium were measured after 24, 48, and 72 h of the beginning of the production phase. This study encompasses the duration of the inoculum germination phase and the concentration of cells used to startup the germination. Based on these results, PGA productivity during the production phase was maximized. The selected values for these variables were 1.5 × 10⁷ spores/mL of germination medium, germination during 24 h, and 72 h for the production phase.     

A new tripodal receptor for molecular recognition of monosaccharides. A paradigm for assessing glycoside binding affinities and selectivities by 1H NMR spectroscopy

A new tripodal receptor for the recognition of monosaccharides is described. The prototypical host 1 features a 1,3,5-substituted 2,4,6-triethylbenzene scaffold bearing three convergent H-bonding units. The binding ability of the t-octyl derivative 1a toward a set of octylglycosides of biologically relevant monosaccharides, including Glc, Gal, Man, and GlcNAc, was investigated by 1H NMR in CDCl3. A protocol for the correct evaluation of binding affinities was established, which can be generally applied for the recognition of monosaccharides by 1H NMR spectroscopy. A three-constant equilibrium model, including 1:1 and 2:1 host-guest association and dimerization of the receptor, was ascertained for the interaction of 1a with all the investigated glycosides. An affinity index, which we defined median binding concentration BC50 in analogy to the IC50 parameter, intended to address the general issue of comparing dimensionally heterogeneous binding data, and a limiting BC0(50)quantity describing intrinsic binding affinities were developed for evaluating the results. BC0(50) values for 1a range from 1 to 6 mM, indicating an intrinsic binding affinity in the millimolar range and a selectivity factor of 5 toward the investigated glycosides. The treatment has been extended to include any generic host-guest system involved in single or multiple binding equilibria.     

Conformation of bicyclo [n.1.0] Derivatives: Part 3. Dioxa- and trioxa-bicyclo[5.1.0] octanes

The conformational geometries and possible interconversion paths for some oxa derivatives of bicyclo[5.1.0] octane have been studied by the molecular mechanics method. The theoretical results are compared with the experimental data for the molecular geometry of bicyclo[5.1.0] octane and 3,5,8-trioxabicyclo[5.1.0] octane, the free energy of activation for cycloheptene epoxide and 3,5-dioxabicyclo[5.1.0] octane, the dipole moments and molar Kerr constants in solution for cycloheptene epoxide, 3,5-dioxa- and 3,5,8-trioxabicyclo[5.1.0] octane.     

Thermodynamic analysis of the province of Ravenna (Italy)

This is a thermodynamic analysis of the Province of Ravenna (Italy) and its districts (Ravenna, Faenza and Lugo) and an evaluation of entropy waste production based on a balance sheet of greenhouse gases. The method used is energy analysis. The results show that the Province and Ravenna are characterized by a heavy exploitation of local non renewable resources, Faenza strikes a good balance between economic development and environment conservation and Lugo is less sustainable than the others. The greenhouse gas balance shows that the Province emits 10.5 times the quantity of greenhouse gases that it adsorbs and that the emissions reduction is required in the energy sector, which is responsible for 92% of the total.     

Interfacial adsorption of microorganisms and its effect on oxygen absorption by fermentation broths

Steady-state mass transfer experiments were done to determine whether certain strains of bacteria in homogeneous suspension will accumulate at the gas:liquid interface and thereby significantly increase the oxygen transfer rate to the suspension. In particular, with suspensions ofB. licheniformis, the measured transfer rates are as much as three times the rate expected for a uniform suspension. In contrast, suspensions ofM. luteus show no increase in oxygen flux above expected values. The fact thatB. licheniformis is motile, whereasM. luteus is not suggests that cell motility may play an important role in the accumulation process.

     

Multivariate optimization of an axially-viewed inductively coupled plasma multichannel-based emission spectrometer for the analysis of environmental samples

An experimental design procedure was applied to optimize the operating conditions of an axially-viewed inductively coupled plasma emission spectrometer instruments equipped with echelle optics with cross dispersion and charge transfer device. The multivariate effect of carrier gas flow rate and r.f. power on several analytical figures was investigated and discussed. Both ultrasonic and pneumatic nebulization were used. For the final choice of the optimum, different criteria were taken into account, mainly plasma robustness, instrumental precision, analyte and background net emission, detection limits and signal-to-background ratios. It was found that the use of moderate power (1100W) and mean carrier gas flow rate (0.75 L/min) allows to obtain sufficient plasma robustness, satisfactory precision, and excellent signal-to-background ratios and limits of detection, favorable for ultratrace element determinations in environmental matrices.     

1,3-Diaxial steric effects and intramolecular hydrogen bonding in the conformational equilibria of new cis-1,3-disubstituted cyclohexanes using low temperature NMR spectra and theoretical calculations

The conformational equilibria of 3-X-cyclohexanol [X=F (1), Cl (2), Br (3), I (4), Me (5), NMe(2) (6) and MeO (7)] and of 3-X-methoxycyclohexane [X=F (8), Cl (9), Br (10), I (11), Me (12), NMe(2) (13) and MeO (14)] cis isomers were determined from low temperature NMR spectra and PCMODEL calculated coupling constants. The energy differences between aa and ee conformers were obtained from these data (DeltaG(J)(av) and DeltaG(PC)(av), respectively) and also by the additivity principle from data for the monosubstituted cyclohexanes (DeltaG(Ad)). H-1 and H-3 hydrogen vicinal coupling constants and DeltaG(J)(av) values showed that the diequatorial conformer is predominant in the conformational equilibrium of the compounds studied at low temperature. However, DeltaG(PC)(av) data show that compounds 6 and 7 constitute an exception, since they are almost equally populated by ee and aa at room temperature, due to stabilization of their aa conformer by an intramolecular hydrogen bond. DeltaG(Ad) values, obtained according to the additivity principle, show a better agreement for compounds 2 and 3, since the 1,3-diaxial steric effect is counterbalanced by the formation of an intramolecular hydrogen bond (IAHB). For the remaining compounds, DeltaG(Ad) values underestimate the energy differences, since the 1,3-diaxial steric effect, between X and OH or OCH(3), is absent in the monosubstituted compounds used as references. Moreover, the DeltaG(PC)(av), calculated from the coupling constants, obtained through the PCMODEL program, are rather smaller than the DeltaG(J)(av) values, since the program does not have parameters for the effect, observed in this report, of a substituent at gamma position on coupling constants values for the hydrogen under consideration.     

Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.     

Phase evolution of a barium tin 1,2-ethanediolato complex to barium stannate during thermal decomposition

The thermal behaviour of [Ba(C₂H₆O₂)₄][Sn(C₂H₄O₂)₃] used as a BaSnO₃ precursor, and its phase evolution during thermal decomposition are described. The initially formed transient barium tin oxycarbonate phase disintegrates into BaCO₃ and SnO₂, reacting subsequently to BaSnO₃. The existence of the intermediate oxycarbonate phase is evidenced by Fourier transformed infrared (FT-IR), X-ray powder diffraction (XRD) and electron energy loss spectroscopy (EELS (ELNES)) investigations.