Oligosaccharides obtained by enzymatic hydrolysis of birch kraft pulp xylan: analysis by capillary zone electrophoresis and mass spectrometry.

Neutral and acidic oligosaccharides were obtained from an unbleached birch kraft pulp by treatment with a Trichoderma reesei endoxylanase pI 9 and subsequently characterized using capillary zone electrophoresis (CZE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The borate complexes of unsaturated acidic oligosaccharides having a 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid (4 delta UA) residue linked to a beta-D-(1-->4)-xylooligosaccharide backbone were separated by CZE and detected by their UV absorption at 232 nm without prior derivatization. Pre-column derivatization with the chromophore 6-aminoquinoline (6-AQ) followed by CZE in alkaline borate buffer using detection based on absorption at 245 nm was used in the case of neutral xylosaccharides. Furthermore, MALDI-TOF-MS was employed to determine the molecular masses of both unsaturated and saturated acidic oligosaccharides. The acidic oligosaccharides released upon endoxylanase treatment of the birch kraft pulp were a (4 delta UA)-beta-D-xylotetraose, a (4 delta UA)-beta-D-xylopentaose, a (4-O-methyl-alpha-D-glucurono)-beta-D-xylotetraose, and a (4-O-methyl-alpha-D-glucurono)-beta-D-xylopentaose. Analysis after enzymatic hydrolysis with beta-xylosidase and alpha-glucuronidase from Trichoderma reesei strongly indicated that the uronic acid residue in these acidic oligosaccharides was linked to the D-xylose unit adjacent to the non-reducing D-xylose unit. The neutral xylosaccharides obtained after endoxylanase treatment of the pulp sample were D-xylose, beta-(1-4)-D-xylobiose and beta-(1-4)-D-xylotriose.

Efficient capillary zone electrophoretic separation of wood-derived neutral and acidic mono- and oligosaccharides.

Neutral and acidic monosaccharides, commonly present as structural units in wood-derived hemicelluloses, were derivatized by reductive amination using 6-aminoquinoline (6-AQ) and subsequently separated as their borate complexes by capillary zone electrophoresis. By using a quite concentrated (420 mmol 1(-1) alkaline borate buffer, a fused-silica capillary column with a small inner diameter (30 microns nominal I.D.) and a constant power of 1200 mW (corresponding to an applied voltage of approximately 21 kV), optimal separation was achieved. Under these conditions, the monosaccharides investigated were separated with a resolution, Rs, of 1.0-1.2 or greater. On-column UV detection at 245 nm was found to provide highly sensitive detection of the 6-AQ-derivatized monosaccharides. The minimum detectable concentrations were on the order of 10(-6) mol 1(-1) (corresponding to an estimated limit of detection of a few fmol). The linear calibration range of the method, including the 6-AQ derivatization step, was found to be about two orders of magnitude. Several neutral beta (1-4)-D-xylooligomers and acidic oligosaccharides containing 4-O-methyl-D-glucuronic acid units, which are common structural elements in hemicelluloses such as birch and spruce xylan, were also efficiently separated as 6-AQ derivatives, using the same buffer system. Finally, the usefulness of this analytical method has been demonstrated using a spruce wood xylan sample subjected to chemical and enzymatic hydrolysis.