title: Development of a new method for d-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus.
abstract: The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD(+) and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250days both at 4°C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150days of incubation at 4°C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568mg/dL and 1.89mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.
authors.affiliation: Venter Pharma S.L., Azalea 1, 28109, Alcobendas, Madrid, Spain. Electronic address: isanchez@venterpharma.com.
authors.forename: Israel
authors.initials: I
authors.lastname: Sánchez-Moreno
authors.affiliation: Departamento de Química Bioorgánica, Instituto de Química Orgánica General (IQOG-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. Electronic address: eduardo.junceda@iqog.csic.es.
authors.forename: Eduardo
authors.initials: E
authors.lastname: García-Junceda
authors.affiliation: Venter Pharma S.L., Azalea 1, 28109, Alcobendas, Madrid, Spain. Electronic address: chermida@venterpharma.com.
authors.forename: Carmen
authors.initials: C
authors.lastname: Hermida
authors.affiliation: Departamento de Química Bioorgánica, Instituto de Química Orgánica General (IQOG-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. Electronic address: alfonso.mayoralas@csic.es.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Fernández-Mayoralas
mesh_terms: D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D057230:Limit of Detection; D013058:Mass Spectrometry; D009243:NAD / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D014994:Xylose / Q000652:urine*
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D009243:NAD; D014994:Xylose; D000429:Alcohol Oxidoreductases; C035795:D-xylose dehydrogenase
keywords: Enzymatic detection; Gaxilose; Hypolactasia; Intestinal lactase activity; Xylose dehydrogenase; Xylose quantification