RESUMO
Xylose reductase (XR) from Debaryomyces hansenii was extracted by partitioning in aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) 4000 in the presence of different salts, specifically sodium sulfate, lithium sulfate and potassium phosphate. Batch extractions were carried out under different conditions of temperature (25-45 degrees C) and tie-line length (TLL) for each system, according to a central composite design face-centered of 36 tests, and the response surface methodology was used to evaluate the results. Quadratic polynomial models were adjusted to the data to predict the behavior of four responses, namely the XR partition coefficient (K(XR)), the selectivity (S), the purification factor (PF(T)) and the activity yield (Y(T)) in the top phase. The optimal extraction conditions were found using the PEG 4000/sodium sulfate system at 45 degrees C and TLL=25.1, which ensured PF(T)=3.1 and Y(T)=131%. The ATPS proved effective for partial purification of D. hansenii xylose reductase in cell-free crude extract, and the response surface methodology revealed to be an appropriate and powerful tool to determine the best dominion of temperature and ATPS composition.
Assuntos
Aldeído Redutase/isolamento & purificação , Fracionamento Químico/métodos , Debaryomyces/enzimologia , Proteínas Fúngicas/isolamento & purificação , Modelos Químicos , Aldeído Redutase/metabolismo , Debaryomyces/metabolismo , Proteínas Fúngicas/metabolismo , Modelos Lineares , Compostos de Lítio/química , Modelos Estatísticos , Fosfatos/química , Polietilenoglicóis/química , Compostos de Potássio/química , Sulfatos/química , TemperaturaRESUMO
To develop a new enzymatic xylose-to-xylitol conversion, deeper knowledge on the regulation of xylose reductase (XR) is needed. To this purpose, a new strain of Debaryomyces hansenii (UFV-170), which proved a promising xylitol producer, was cultivated in semi-synthetic media containing different carbon sources, specifically three aldo-hexoses (D-glucose, D-galactose and D-mannose), a keto-hexose (D-fructose), a keto-pentose (D-xylose), three aldo-pentoses (D-arabinose, L-arabinose and D-ribose), three disaccharides (maltose, lactose and sucrose) and a pentitol (xylitol). The best substrate was lactose on which cell concentration reached about 20 g l(-1) dry weight (DW), while the highest specific growth rates (0.58-0.61 h(-1)) were detected on lactose, D-mannose, D-glucose and D-galactose. The highest specific activity of XR (0.24 U mg(-1)) was obtained in raw extracts of cells grown on D-xylose and harvested in the stationary growth phase. When grown on cotton husk hemicellulose hydrolyzates, cells exhibited XR activities five to seven times higher than on semi-synthetic media.