Caracterização da xilose redutase extraída por micelas reversas-CTAB a partir de homogenato de Candida guilliermondii / Characterization of xylose reductase extracted by CTAB-reversed micelles from Candida guilliermondii homogenate

Xylosereductase (XR) (E.C.1.1.1.21), produced by Candida guilliermondii, grown in sugar cane bagasse hydrolysate, was separated directly from the cell homogenate by reversed micelles of cetyl trimethyl ammonium bromide (CTAB), attaining a recovery yield of 100% and enrichment factor of 5.6 fold. The extraction conditions were: pH=7.0, electrical conductivity= 14 mS/cm, T=5 degrees C, 5% (w/w) of hexanol, 22% (w/w) of butanol and 0.15 M CTAB. The XR after extraction was stable in pH interval of 6.0-6.5 and its heat inactivation constant was about 6.5 fold higher than that before extraction. The 'V IND. max' values against both xylose and NADPH for XR before and after extraction by reversed-micelles differed about 6%, whereas the difference on KM values were more pronounced. The ('K IND. m') 'IND. xylose' for XR after extraction was about 35% higher than before extraction, meanwhile ('K IND. m') 'IND. NADPH' was about 30% lower after than before extraction. As the KM variations indirectly signaled, the XR affinity simultaneously diminishes for xylose and increases for NADPH. This could probably explain why the 'V IND. max' values for XR before and after extraction were quite similar

A xilose redutase (XR) (E.C.1.1.1.21), produzida por Candida guilliermondii cultivada em hidrolisado de bagaço de cana de açúcar, foi separada diretamente do homogenato livre de células através da técnica de micelas reversas feitas com cetil trimetil brometo de amônio (CTAB). Obteve-se um rendimento de recuperação da enzima de 100% e um fator de enriquecimento de 5,6 vezes. As condições de extração foram: pH=7,0, condutividade elétrica = 14 mS/cm, T= 5 ºC, 5% (w/w) de hexanol, 22% (w/w) de butanol e 0.15M CTAB. A XR após a extração manteve-se estável no intervalo de pH entre 6.0 e 6.5, sendo a constante de inativação térmica cerca de 6,5 vezes maior do que aquela antes da extração. Os valores de Vmax da XR frente à xilose e NADPH antes e após a extração por micelas reversas diferiram cerca de 6%, enquanto que as diferenças nos valores de KM foram mais pronunciadas. O (KM)xilose para a XR após a extração foi cerca de 35% maior do que antes da extração, enquanto que (KM)NADPH foi 30% menor após do que antes da extração. As variações nos valores de KM indicam, indiretamente, que a afinidade da XR simultaneamente diminui para a xilose e aumenta para o NADPH. Este resultado poderia explicar a razão pela qual os valores de Vmax antes e após a extração terem sido praticamente iguais

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate.

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.

Liquid-liquid extraction of xylitol dehydrogenase from Candida guilliermondii homogenate by reversed micelles.

The intracellular enzyme xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugarcane bagasse hydrolysate, was separated by reversed micelles of BDBAC [N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride] cationic surfactant. An experimental design was employed to evaluate the influence of the following factors on the enzyme separation: temperature, co-solvent concentration and surfactant concentration. The results showed that just the temperature did not show significant effect on XD recovery. A model was used to represent the activity recovery and fit the experimental data. Under optimized conditions, the recovery of total activity was about 121%, and the purity increased 2.3-fold.

Extraçäo líquido-líquido de xilose redutase e xilitol desidrogenase por micelas reversas / Xylose reductase and xylitol dehydrogenase extraction liquid-liquid by reverse micelles

Células de Candida guilliermondii, obtidas de cultivos feitos en hidrolisado de bagaço de cana-de-açúcar, foram rompidas por sonicaçäo e os fragmentos celulares removidos por centrifugaçäo. As enzimas xilose redutase (XR) e xilitol desidrogenase (XD) foram separadas por extraçäo com micelas reversas, usando-se os tensoativos BDBAC, CTAB e AOT. A XR foi recuperada significativamente na fase aquosa de reextraçäo (FAII) em presença do BDBAC (fator de purificaçäo 4,8) ou CTAB (fator de purificaçäo 5,6). A XR, no entanto, ficou na fase aquosa de extraçäo (FAI) para ambos os tensoativos. Na presença do AOT näo se observou extraçäo significativa tanto da XR quanto da XD. Observou-se variaçäo desprezível nos valores...