title: Purification and biochemical characterization of a moderately halotolerant NADPH dependent xylose reductase from Debaryomyces nepalensis NCYC 3413.
abstract: A Xylose reductase (XR) from the halotolerant yeast, Debaryomyces nepalensis NCYC 3413 was purified to apparent homogeneity. The enzyme has a molecular mass of 74 kDa with monomeric subunit of 36.4 kDa (MALDI-TOF/MS) and pI of 6.0. The enzyme exhibited its maximum activity at pH 7.0 and 45 °C (21.2U/mg). In situ gel digestion and peptide mass fingerprinting analysis showed 12-22% sequence homology with XR from other yeasts. Inhibition of the enzyme by DEPC (diethylpyrocarbonate) confirmed the presence of histidine residue in its active site. The enzyme exhibited high preference for pentoses over hexoses with greater catalytic efficiency for arabinose than xylose. The enzyme also showed absolute specificity with NADPH over NADH. The enzyme retained 90% activity with 100 mM of NaCl or KCl and 40% activity with 1 M KCl which suggest that the enzyme is moderately halotolerant and can be utilized for commercial production of xylitol under conditions where salts are present.
authors.affiliation: Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India.
authors.forename: Sawan
authors.initials: S
authors.lastname: Kumar
authors.forename: Sathyanarayana N
authors.initials: SN
authors.lastname: Gummadi
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D015003:Yeasts / Q000201:enzymology*
chemical_list: D009249:NADP; D000449:Aldehyde Reductase