ABSTRACT Xylose reductase (EC 184.108.40.206) of Candida tropicalis IEC5-ITV, an indigenous xylitol-producing strain, was partially purified by reversed micelles and characterized, an 8.1 fold purification factor being obtained. The XR present in the crude extract exhibited its highest specific activity at pH 6.0 and 40℃, while in that obtained by reverse micelles, this occurs at pH 6.0 and 30℃. XR before and after extraction is stable within a range of 30 to 40℃, pH 7 after one hour of incubation under these conditions. After two months’storage at –18℃, the enzyme obtained by reverse micelles lost 76.60% specific activity. The estimated molecular weight by PAGE-SDS was 32.42 kD. KM for xylose was higher for the XR extracted by reverse micelles (0.026 M) than that obtained for the enzyme before extraction (0.0059 M), while KM for cofactor NADPH was lower after than before extraction (1.85 mM to 12.0 mM respectively). There was no activity with NADH as a cofactor. Variations in pH and temperature optima, as well as kinetic parameters before and after partial XR purification by reverse micelles are probably due to an alteration in enzyme molecule structure caused by the solvents used during extraction.
Cite this paper
Y. Cocotle-Ronzon, M. Zendejas-Zaldo, M. Castillo-Lozano and M. Aguilar-Uscanga, "Preliminary Characterization of Xylose Reductase Partially Purified by Reversed Micelles from Candida tropicalis IEC5-ITV, an Indigenous Xylitol-Producing Strain," Advances in Chemical Engineering and Science, Vol. 2 No. 1, 2012, pp. 9-14. doi: 10.4236/aces.2012.21002.
 B. V. Kilikian, M. R. Bastazin, N. M. Minami, E. M. R. Goncalves and A. Jr. Pessoa, “Liquid-Liquid Extraction by Micelles in Biotechnological Processes,” Brazilian Journal of Chemical Engineering, Vol. 17, No. 1, 2000, pp. 1-14. doi:10.1590/S0104-66322000000100003
 A. Carlson and R. Nagarajan, “Release and Recovery of Porcine Pepsin and Bovine Chymosin from Reverse Micelles: A New Technique Based on Isopropyl Alcohol Addition,” Biotechnology Progress, Vol. 8, No. 1, 1992, pp. 85-90. doi:10.1021/bp00013a013
 T. X. Zhang, H. Z. Liu and J. Y. Chen, “Extraction of Yeast Alcohol Dehydrogenase Using Reversed Micelles Formed with CTAB,” Journal of Chemical Technology and Biotechnology, Vol. 75, 2000, pp. 798-802.
 E. M. G. Rodrigues and E. B. Tambourgi, “Continuous Extraction of Xylanase from Penicillium janthinellum with Reversed Micelles Using Experimental Design Mathematical Model,” Biotechnology Letters, Vol. 23, No. 5, 2001, pp. 365-367. doi:10.1023/A:1005659618433
 F. A. Hasmann, C. D. Vieira, A. Jr. Pessoa and I. C. Roberto, “Optimization of β-Xylosidase Recovery by Reversed Micelles Using Response Surface Methodology,” Electronic Journal of Biotechnology, Vol. 6, No. 2, 2003, pp. 153-160.
 L. F. P. Ferreira, M. E. Taqueda, A. Converti, M. Vitolo and A. Jr. Pessoa, “Purification of Glucose Oxidase from Aspergillus niger by Liquid-Liquid Cationic Reversed Micelles Extraction,” Biotecnology Progress, Vol. 21, No. 3, 2005, pp. 868-874. doi:10.1021/bp049623x
 J. G. Liu, J. M. Xing, T. S. Chang and H. Z. Liu, “Purification of Nattokinase by Reverse Micelles Extraction from Fermentation Broth: Effect of Temperature and Phase Volume Ratio,” Bioprocess Biosystems Engineering, Vol. 28, No. 4, 2006, pp. 267-273.
 E. V. Cortez, A. Jr. Pessoa, M. G. Almeida-Felipe, I. C. Roberto and M. Vitolo, “Liquid-Liquid Extraction of Xylitol Dehydrogenase from Candida guilliermondii Homogenate by Reversed Micelles,” Journal Chromatography B, Vol. 807, No. 1, 2004, pp. 55-60.
 E. V. Cortez, A. Pessoa, Jr. M. G. Almeida-Felipe, I. C. Roberto and M. Vitolo, “Optimized Extraction by Cetyl Trimethyl Ammonium Bromide Reversed Micelles of Xylose Reductase and Xilitol Dehydrogenase from Can- dida guilliermondii Homogenate,” Journal Chromatography B, Vol. 807, No. 1, 2004, pp. 47-54.
 E. V. Cortez, M. G. Almeida-Felipe, I. C. Roberto, A. Jr. Pessoa and M. Vitolo, “Extraction by Reversed Micelles of the Intracelular Enzyme Xilose Reductase,” Applied Biochemical Biotechnology, Vol. 91-93, 2001, pp. 753-759. doi:10.1385/ABAB:91-93:1-9:753
 E. V. Cortez, A. Jr. Pessoa, M. G. Almeida-Felipe, I. C. Roberto and M. Vitolo, “Characterization of Xylose Reductase Extracted by CTAB-Reversed Micelles from Candida guilliermondii Homogenate,” Brazilian Journal of Pharmaceutical Sciences, Vol. 42, No. 2, 2006, pp. 251-257.
 T. B. Granstr?m, K. Izumori and M. Leisola, “A Rare Sugar Xylitol. Part I: The Biochemistry and Biosynthesis of Xylitol,” Applied Microbiology and Biotechnology, Vol. 74, No. 2, 2007, pp. 277-278.
 R. S. Prakasham, R. S. Rao and P. J. Hobbs, “Current trends in Biotechnological Production of Xylitol and Future Prospects,” Current Trends Biotechnology Pharmacy, Vol. 3, No. 1, 2009, pp. 8-36.
 E. Gastélum, “Producción de Xilitol por la Levadura IEC5-ITV a Partir de Hidrolizado de Bagazo de Ca?a en Cultivo por Lote Alimentado,” Master Dissertation, Veracruz Institute of Technology, Veracruz, 2007.
 D. M. Bollag, M. D. Rozycki and S. J. Edelstein, “Protein Methods,” 2th Edition, John Wiley & Sons, New York, 1996.
 S. I. Yokoyama, T. Suzuki, K. Kawai, H. Horitsu and K. Takamizawa, “Purification, Characterization and Structure Analysis of NADPH-Dependent D-Xylose Reductases from Candida tropicalis,” Journal of Fermentation and Bioengineering, Vol. 79, No. 3, 1995, pp. 217-223.
 L. Sene, M. G. A. Felipe, S.S. Silva and M. Vitolo, “Preliminary Kinetic Characterization of Xylose Reductase and Xylitol Dehydrogenase Extracted from Candida guilliermondii FTI 20037 Cultivated in Sugarcane Bagasse Hydrolysate for Xylitol Production,” Applied Biochemestry and Biotechnology, Vol. 91-93, No. 1-9, 2001, pp. 671-680.
 G. Dietzelmüller, C. P. Kubicek, W. W?hrer and M. R?ehr, “Xylose Metabolism in Pachysolen tannophilus: Purification and Propierties of Xylose Reductase,” Canadian Journal of Microbiology, Vol. 30, No. 11, 1984, pp. 1330-1336. doi:10.1139/m84-214
 C. Verduyn, R. Van-Kleef, J. Frank, H. Schreuder, J. P. Van-Dijken and A. W. Scheffers, “Properties of the NAD (P)H-Dependent Xylose Reductase from the Xylosefermenting Yeast Pichia stipitis,” Biochemical Journal, Vol. 226, No. 3, 1985, pp. 669-677.
 A. A. Abou Zeid, M. Z. El-Fouly, Y. A. El-Zawahry, T. M. El-Mongy and A. B. Abd El-Aziz, “Bioconversion of Rice Straw Xylose to Xylitol by a Local Strain of Candida Tropicalis,” Journal of Applied Sciences Reserche, Vol. 4, No. 8, 2008, pp. 975-986.
 X. Zhao, P. Gao and Z. Wang, “The Production and Properties of a New Xylose Reductase from Fungus Neurospora crassa,” Applied Biochemestry and Biotechnology, Vol. 70, No. 72, 1998, pp. 405-414.
 U. B. Rawat and M. B. Rao, “Purification, Kinetic Characterization and Involvement of Trypstophan Reside at the NADPH Binding Site of Xylose Reductase from Neurospora crassa,” Biochemica et Biophysica Acta, Vol. 1293, No. 2, 1996, pp. 222-230.
 P. K. Mayr, K. D. Kulbe and B. Nidetzky, “D-Xylose Metabolism by Candida intermedia: Isolation and Characterisation of Two Forms of Aldose Reductase with Different Coenzyme Specificities,” Journal of Chromatography B, Vol. 737, 2002, pp. 195-202.