Obtaining information about a biosynthetic pathway is a complex and laborious procedure. In this sense, this work presents a new approach for the initial analysis of the biosynthesis of fluorescent natural products using as example the violacein biosynthesis. For this, a culture of Chromobacterium violaceum was grown in a bioreactor from which aliquots were collected every 2 h for subsequent analysis by multi-wavelength fluorescence spectroscopy. The excitation-emission matrices demonstrated the dynamic behavior of the fluorophores signal that are consumed and produced by the bacterium. These signals were resolved by PARAFAC (parallel factor analysis) method totalizing six pure components. Tryptophan and violacein were identified by comparison to spectra available in the literature. The identification of other fluorophores was critical step due to the lack of a database of fluorescent natural products to compare spectra. Finally, this methodology has great potential to achieve a deeper insight into the biosynthesis of natural products.
A obtenção de informações sobre uma rota biossintética é um procedimento complexo e trabalhoso. Neste sentido, o presente trabalho apresenta uma nova abordagem para a análise inicial da biossíntese de produtos naturais fluorescentes usando a biossíntese da violaceína como exemplo. Para tanto, uma cultura da Chromobacterium violaceum foi inoculada em um biorreator de onde alíquotas eram retiradas a cada 2 h para posterior análise por espectroscopia de fluorescência 2D. As matrizes de emissão-excitação obtidas demonstraram o comportamento dinâmico dos sinais de fluoróforos que são consumidos e produzidos pela bactéria. Estes sinais foram resolvidos pelo método PARAFAC (análise paralela de fatores) perfazendo um total de seis espécies químicas. O triptofano e a violaceína foram identificados por comparação espectral. A identificação dos outros fluoróforos mostrou-se a etapa crítica devido à falta de banco de dados de produtos naturais fluorescentes para comparação. Por fim, esta metodologia apresenta um grande potencial para fornecer informações da biossíntese de produtos naturais.
In this work we first introduce the reader to the basic concepts of biology, bioenergetics and biochemistry, concerning the area of cell biology. Then we explain what diauxism is and an example of this phenomenon, applied to S. cerevisiae, is presented. Finally, thermograms obtained by microcalorimetry, from S. cerevisiae that undergo diauxism, are discussed from a biochemical point of view.
The aim of this work is to present a tutorial on Multivariate Calibration, a tool which is nowadays necessary in basically most laboratories but very often misused. The basic concepts of preprocessing, principal component analysis (PCA), principal component regression (PCR) and partial least squares (PLS) are given. The two basic steps on any calibration procedure: model building and validation are fully discussed. The concepts of cross validation (to determine the number of factors to be used in the model), leverage and studentized residuals (to detect outliers) for the validation step are given. The whole calibration procedure is illustrated using spectra recorded for ternary mixtures of 2,4,6 trinitrophenolate, 2,4 dinitrophenolate and 2,5 dinitrophenolate followed by the concentration prediction of these three chemical species during a diffusion experiment through a hydrophobic liquid membrane. MATLAB software is used for numerical calculations. Most of the commands for the analysis are provided in order to allow a non-specialist to follow step by step the analysis.
The calorimetric experiments based on technique breaking ampoule were carried out by measuring of the heat of solution of alcohol in isotonic solution (NaCl 0.10 M) and alcohol in suspension of Sc at 298 K. From these data the enthalpy of interaction alcohol with suspension of Sc (DtrsH°) was calculate by Hess law. In this study, the results indicate that the enthalpy of interaction of aliphatic alcohol (C2-C8) with suspensions of Sc is a process exothermic and becomes more exothermic with increasing of -CH2 group of alcohol in range -1,14 to -4,0 kJ.mol-1. We concluded that enthalpy of interaction shows a linear relationship with increasing of alcohol's lipophilicity, in agreement with Traube's rule.
The present study shows that with liquid nitrogen stored inocula of Saccharomyces cerevisiae, and standardized experimental procedure, flow microcalorimetry can be a valuable tool for monitoring in real time the alcoholic fermentation processes on line. The avaliation of cultural conditions contained different carbon sources for alcohol fermentation (sucrose, glucose, fructose, manose, maltose, galactose, molasses, honey and sugar cane) and their effects on the heat output recording is discussed. Some examples of diauxic growth is given, where the microcalorimeters serves to detect the temporal order of succession of alternating metabolic pathways.
Systematic study of the interactions of ionic surfactants with protein trypsin in buffer solution pH 3.5, 7.0 and 9.0, ionic strength 10 mM at 298 K was done using the microcalorimetric technique. In this study, anionic surfactant solutions of the sodium n-alkyl sulfates series (C8, C10, C12 and C14) were used. The enthalpy of interaction (ΔintHº) shows that the interaction of the surfactants C8, C10, C12 and C14 with trypsin in the solution pH 3.5 is an endothermic process with the value of ΔintHº decreasing linearly with increasing carbon chain length, which is attributed to the unfolding of the polypeptide chain. In the solution pH 7.0, we observed the same trend except for C14. In the solution pH 9.0, from C10 the enthapy of interaction didn't change with the increasing of the carbon chain length due to unfolding of the polypeptide. We concluded that when trypsin is folded, the enthalpy of interaction shows a linear relationship with the surfactant's hydrophobicity, in agreement with Traube's rule.