Aldotetrauronic acid (terminal substitution, borohydride reduced)

Background: Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin-carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood. Results: Here, we show for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan. Conclusion: The data verify the enzyme’s unique ability to catalyze removal of all glucuronoxylan associated with lignin and we propose that this is a direct result of enzymatic cleavage of the ester bonds connecting glucuronoxylan to lignin via 4-O-methyl glucuronoyl-ester linkages. This function appears important for the fungal organism’s ability to effectively utilize all available carbohydrates in lignocellulosic substrates. In bioprocess perspectives, this enzyme is a clear candidate for polishing lignin for residual carbohydrates to achieve pure, native lignin fractions after minimal pretreatment.

Penicillium purpurogenum grows on a variety of natural carbon sources and secretes to the medium a large number of enzymes that degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel xylanase (XynC) belonging to family 30 of the glycoside hydrolases (GH), has been identified in the genome of the fungus. The enzyme has been expressed in Pichia pastoris and characterized. The mature XynC has 454 amino acid residues and a calculated molecular weight of 49 240. The purified protein shows a molecular weight of 67 000, and it is partially deglycosylated using EndoH. Its pH optimum is in the range of 3-5, and the optimal temperature is 45°C. It is active on both arabinoxylan and glucuronoxylan, similarly to other fungal GH 30 xylanases. It liberates a set of oligosaccharides, which have been detected by thin-layer chromatography, thus indicating that it is an endo-acting xylanase. It hydrolyzes xylooligosaccharides, releasing mainly xylobiose, in contrast to other fungal GH family 30 enzymes which generate chiefly xylose. Highest sequence identity to a characterized family 30 xylanase is found with the enzyme from the fungus Bispora sp (53%). This is the first GH 30 xylanase described from a Penicillium.

The oligosaccharides of Carignan and Merlot wines have been characterized for the first time. In a first step, this fraction was prepared after discoloration of the wines and was collected by elution on an HPSEC system. In a second step, the glycosyl composition and linkages of wine oligosaccharides were determined by several methods. High resolution MS spectra of the Carignan and Merlot oligosaccharide fractions were obtained on an AccuTOF mass spectrometer equipped with an electrospray ionization (ESI) source and a time-of-flight (TOF) mass analyser. Oligosaccharides were present at a concentration of 330 and 250 mg/L in Carignan and Merlot red wines, respectively. Glycosyl residue composition analysis showed the presence of mannose, arabinose, galactose, rhamnose, fucose, xylose, glucose, galacturonic acid, and glucuronic acid. We show that oligosaccharides are present in significant amounts in wines, that they result from the degradation of cell wall polysaccharides and that they have an extreme diversity, about 30 peaks in ESI–TOF spectra corresponding each to at least one oligosaccharidic structure. The ESI–TOF spectra in negative mode of the Carignan and Merlot oligosaccharides showed oligosaccharidic structures corresponding to oligogalacturonic acids, partially esterified by methyl group (trigalacturonic acid detected at m/z 545, m/z 559 and m/z 573) or to the repetition of the basic unit [→4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→] two (m/z 661), or three (m/z 983) times. These units can be substituted either by a hexose, or by a pentose, or by both, but also by deoxyhexose or uronic acid. The identification of [4-OMe-GlcA-[Xyl]₂-Xylitol] and [4-OMe-GlcA-[Xyl]₃-Xylitol] by MSⁿfragmentation performed on a mass spectrometer equipped with an ESI source and an ion trap mass analyser makes it possible to explain the presence of xylose in wines.