RESUMO
The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of β-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD+-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery.
Assuntos
/análise , /genética , /isolamento & purificação , Extratos Vegetais/isolamento & purificação , Pseudomonas fluorescens/químicaRESUMO
Las bacterias responden a los cambios ambientales modificando su composición, para evitar el daño que dichos cambios podrían ejercer. Una de las modificaciones más importantes es la variación de la composición de los ácidos grasos de las membranas celulares, que le permite mantener la homeoviscosidad ante situaciones de estrés. Trabajos previos han estudiado la acción de la temperatura, presión hidrostática y diferentes solventes sobre cepas de Pseudomonas putida. En este trabajo se estudió la acción conjunta de la temperatura y la salinidad sobre la composición de ácidos grasos de membranas celulares de Pseudomonas fluorescens GNP-OHP-3, una cepa bacteriana aislada de un hábitat contaminado con petróleo. Pseudomonas fluorescens GNP-OHP-3 respondió a las variaciones de temperatura modificando los ácidos grasos de sus membranas de manera similar a lo descripto en otros integrantes de su género: ante el aumento de temperatura se observó un incremento de ácidos grasos saturados y una disminución de los ácidos grasos insaturados. En el rango de concentraciones salinas ensayadas las variaciones de los ácidos grasos mayoritarios fueron en general erráticas. La respuesta de los ácidos grasos ciclo propano pudo expresarse con ecuaciones matemáticas que permitieron predecir el porcentaje de estos ácidos en relación a la concentración de cloruro de sodio.
The bacteria respond to environmental changes modifying their composition. One of the most important modifications is the variation on fatty acid composition of cellular membranes to maintain the homeoviscosity. The action of temperature, hydrostatic pressure and solvents on Pseudomonas putida has been thoroughly studied. In this paper, the combined action of the temperature and salinity on fatty acid composition of cellular membranes of Pseudomonas fluorescens GNP-OHP-3, a bacterial strain isolated from a petroleum contaminated habitat, was studied. The modifications in the fatty acid composition of Pseudomonas fluorescens GNP-OHP-3 membrane were similar to those described for other members of Pseudomonas: an increase in saturated fatty acids and a decrease in unsaturated fatty acids were observed with the increase of the temperature. Variations of main fatty acids were in general erratic in the range of assayed saline concentrations. The variation of cycle propane fatty acids could be expressed with mathematic equations that allowed to predict their percentage in relation to sodium chloride concentration.