Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate
Cortez, E.Vieira.; Pessoa, A.; das Graças de Almeida Felipe, M.; Roberto, Iês.Conceição.; Vitolo, M.
Journal of Chromatography. B Analytical Technologies in the Biomedical and Life Sciences 807(1): 47-54
ISSN/ISBN: 1570-0232 PMID: 15177159 DOI: 10.1016/j.jchromb.2004.02.011
The intracellular enzymes xylose reductase (XR, EC 184.108.40.206) and xylitol dehydrogenase (XD, EC 220.127.116.11) 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 degreeC, hexanol: isooctane proportion = 5% (v/v), butanol concentation = 22%, surfactant concentration = 0.15 M, pH = 7.0 and electrical conductivity = 14 mS cm-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 KM 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 Vmax 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.