RESUMEN
Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30°C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4±15.7U/g(cells)) > freezing-thawing (54.3 ± 1.9U/g(cells))>Triton X-100 (23.5 ± 0.0U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 ± 0.1 U/g(cells).
Asunto(s)
Aldehído Reductasa/aislamiento & purificación , Candida/enzimología , Fraccionamiento Celular/métodos , Compuestos de Cetrimonio/química , D-Xilulosa Reductasa/aislamiento & purificación , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Octoxinol/química , Aldehído Reductasa/química , Candida/aislamiento & purificación , Cetrimonio , D-Xilulosa Reductasa/química , Congelación , Glucosafosfato Deshidrogenasa/química , PermeabilidadRESUMEN
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.
Asunto(s)
Aldehído Reductasa/aislamiento & purificación , Fraccionamiento Químico/métodos , Debaryomyces/enzimología , Proteínas Fúngicas/aislamiento & purificación , Modelos Químicos , Aldehído Reductasa/metabolismo , Debaryomyces/metabolismo , Proteínas Fúngicas/metabolismo , Modelos Lineales , Compuestos de Litio/química , Modelos Estadísticos , Fosfatos/química , Polietilenglicoles/química , Compuestos de Potasio/química , Sulfatos/química , TemperaturaRESUMEN
A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes.
Asunto(s)
Aldehído Reductasa/aislamiento & purificación , Compuestos de Cetrimonio/química , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Micelas , Solventes/química , Candida/enzimología , Cetrimonio , FermentaciónRESUMEN
A central composite experimental design leading to a set of 16 experiments with different combinations of pH and temperature was performed to attain the optimal activities of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes from Candida mogii cell extract. Under optimized conditions (pH 6.5 and 38 degrees C), the XR and XDH activities were found to be 0.48 U/ml and 0.22 U/ml, respectively, resulting in an XR to XDH ratio of 2.2. Stability, cofactor specificity and kinetic parameters of the enzyme XR were also evaluated. XR activity remained stable for 3 h under 4 and 38 degrees C and for 4 months of storage at -18 degrees C. Studies on cofactor specificity showed that only NADPH-dependent XR was obtained under the cultivation conditions employed. The XR present in C. mogii extracts showed a superior Km value for xylose when compared with other yeast strains. Besides, this parameter was not modified after enzyme extraction by aqueous two-phase system.
Asunto(s)
Aldehído Reductasa/metabolismo , Candida/enzimología , D-Xilulosa Reductasa/metabolismo , Modelos Biológicos , Aldehído Reductasa/aislamiento & purificación , Candida/crecimiento & desarrollo , Medios de Cultivo , D-Xilulosa Reductasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , NADP/metabolismo , Temperatura , Xilitol/metabolismo , Xilosa/metabolismoRESUMEN
The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.
Asunto(s)
Aldehído Reductasa/aislamiento & purificación , Candida/enzimología , Compuestos de Cetrimonio/química , Micelas , Deshidrogenasas del Alcohol de Azúcar/aislamiento & purificación , Sistema Libre de Células , Cetrimonio , D-Xilulosa Reductasa , FermentaciónRESUMEN
Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH4)2SO4, 0.1 g/L of CaCl2 x 2H2O, and 20.0 g/L of rice bran at 35 degrees C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K(M) for XR and XD against substrates and cofactors were as follows: for XR, 6.4 x 10(-2)M (xylose) and 9.5 x 10(-3) mM (NADPH); for XD, 1.6 x 10(-1)M (xylitol) and 9.9 x 10(-2) mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is half of the Vmax, some interference on the overall xylitol production by the yeast could be expected.
Asunto(s)
Aldehído Reductasa/metabolismo , Candida/enzimología , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Xilitol/biosíntesis , Aire , Aldehído Reductasa/aislamiento & purificación , Beta vulgaris , Biomasa , Candida/crecimiento & desarrollo , D-Xilulosa Reductasa , Estabilidad de Enzimas , Fermentación , Manipulación de Alimentos , Concentración de Iones de Hidrógeno , Residuos Industriales , Cinética , Deshidrogenasas del Alcohol de Azúcar/aislamiento & purificación , TemperaturaRESUMEN
Xylose reductase (XR) activity was evaluated in extracts of Candida mogii grown in media containing different concentrations of rice straw hydrolysate. Results of XR activity were compared to xylitol production and a similar behavior was observed for these parameters. Highest values of specific production and productivity were found for xylose reductase (35 U/g of cell and 0.97 U/[g of cell x h], respectively) and for xylitol (5.63 g/g of cell and 0.13 g/[g of cell x h]) in fermentation conducted in medium containing 49.2 g of xylose/L. The maximum value of XR:XD ratio (1.82) was also calculated under this initial xylose concentration with 60 h of fermentation.
Asunto(s)
Aldehído Reductasa/metabolismo , Candida/enzimología , Aldehído Reductasa/biosíntesis , Aldehído Reductasa/aislamiento & purificación , Candida/crecimiento & desarrollo , Medios de Cultivo , Fermentación , Manipulación de Alimentos , Hidrólisis , Residuos Industriales , Cinética , Oryza , Xilitol/biosíntesisRESUMEN
Xylose reductase enzyme (EC 1.1.1.21) produced by Candida guilliermondii in sugarcane bagasse was extracted by reversed micelles of N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride cationic surfactant. An experimental design was employed to evaluate the influences of the following factors on the enzyme extraction: temperature, cosolvent, and surfactant concentration. A model was used to represent the enzyme recovery and fit of the experimental data. The extraction yielded a total recovery of 130%, and the purity increased 4.8-fold. This study demonstrates that liquid-liquid extraction by reversed micelles is a process able to recover and increase the enzymatic activity and purity of XR produced by C. guilliermondii.