Purification and characterization of a beta-xylosidase from potatoes (Solanum tuberosum).
Potatoes are a cheap and easily available source for the preparation of beta 1,2-xylosidase. The soluble enzyme was purified from potato tubers by ammonium sulfate precipitation, hydrophobic interaction chromatography, affinity gel blue chromatography, ion exchange and size exclusion chromatography yielding a glycoprotein with a molecular weight of 39-40 kDa, an isoelectric point of 5.1 and a typical plant N-glycosylation pattern. The enzyme releases xylose residues beta1,2-linked to the beta-mannose of an N-glycan core, if the 3-position of this mannose is not occupied. It showed an optimal enzymatic activity at pH 4.0-4.5 and at a temperature of 50 degrees C. The activity was reduced in the presence of Ni(2+) and Cu (2+) and slightly increased by the addition of Mn(2+) or Ca(2+). At 37 degrees C the cleavage of xylose from p-nitrophenyl-beta-xylopyranoside or appropriate pyridylaminated N-glycans was proportional to the time of incubation over a period of 8 h and increased with time for at least 24 h. N-Methoxycarbonylpentyl-1,5-dideoxy-1,5-iminoxylitol inhibits the enzyme effectively. Sequencing of the N-terminus showed a high homology to a number of isoforms of patatin, the main protein of potato tubers. This enzyme will be an important tool for the analysis of N-glycans and in the modification of N-glycans for immunological studies.
Peyer
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C
Department of Chemistry, BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Bonay
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Staudacher
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D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011198:Solanum tuberosum / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D014995:Xylosidases / Q000302:isolation & purification* / Q000378:metabolism
D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase