Separation of isomeric cereal-derived arabinoxylan-oligosaccharides by collision induced dissociation-travelling wave ion mobility spectrometry-tandem mass spectrometry (CID-TWIMS-MS/MS).

The potential of travelling wave ion mobility spectroscopy in combination with collision induced dissociation tandem mass spectrometry (CID-TWIMS-MS/MS) to separate cereal-derived isomeric arabinoxylan-oligosaccharides (A)XOS was investigated. Three trisaccharide, four tetrasaccharide, and four pentasaccharide (A)XOS isomers were analyzed by positive and negative ionization TWIMS-MS and CID-TWIMS-MS/MS. The tri- and pentasaccharide isomers were distinguishable by the ATDs of the precursor ions. The CID-TWIMS-MS/MS could separate most of the isomeric fragment ions produced from tetra- and pentasaccharide (A)XOS. Finally, the base peak mobility spectrum is introduced as a practical tool for (A)XOS fingerprinting.

Identification and structural analysis of cereal arabinoxylan-derived oligosaccharides by negative ionization HILIC-MS/MS.

Recent works provide evidence of the prebiotic potential of arabinoxylan-derived oligosaccharides (A)XOS. In this study, we developed a structural analysis for cereal-derived (A)XOS by negative ionization HILIC-MS/MS. Initially, we assessed twelve (A)XOS samples of known structures with different linkage positions and branching points by direct-infusion negative ESI-MSn. We subsequently developed the negative ion HILIC-MS/MS with a post-column addition of ammonium chloride. The selected (A)XOS represented both linear (arabinofuranosyl residue linked to the non-reducing end of xylooligosaccharide) and branched structures. Each (A)XOS sample produced a specific spectrum in negative ion ESI-MSn. By analyzing cross-ring fragment ions, we determined the linkage positions of linear (A)XOS. The presence or absence of diagnostic ions in the MS3 allowed us to detect different branches (O-2- or/and O-3-linked arabinofuranosyl with/or without O-4-linked xylopyranosyl at the non-reducing end). Furthermore, we could identify all analyzed samples by HILIC-MS/MS, based on the formed spectral library and chromatographic retention times.

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family.

We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.-) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family.IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing.

Optimization of Isomaltooligosaccharide Size Distribution by Acceptor Reaction of Weissella confusa Dextransucrase and Characterization of Novel α-(1→2)-Branched Isomaltooligosaccharides.

Long-chain isomaltooligosaccharides (IMOs) are promising prebiotics. IMOs were produced by a Weissella confusa dextransucrase via maltose acceptor reaction. The inputs of substrates (i.e., sucrose and maltose, 0.15-1 M) and dextransucrase (1-10 U/g sucrose) were used to control IMO yield and profile. According to response surface modeling, 1 M sucrose and 0.5 M maltose were optimal for the synthesis of longer IMOs, whereas the dextransucrase dosage showed no significant effect. In addition to the principal linear IMOs, a homologous series of minor IMOs were also produced from maltose. As identified by MS(n) and NMR spectroscopy, the minor trisaccharide contained an α-(1→2)-linked glucosyl residue on the reducing residue of maltose and thus was α-d-glucopyranosyl-(1→2)-[α-d-glucopyranosyl-(1→4)]-d-glucopyranose (centose). The higher members of the series were probably formed by the attachment of a single unit branch to linear IMOs. This is the first report of such α-(1→2)-branched IMOs produced from maltose by a dextransucrase.

Lactose- and cellobiose-derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase.

Dextran-producing Weissella have received significant attention. However, except for maltose, the acceptor reactions of Weissella dextransucrases with different sugars have not been investigated. The action of recombinant Weissella confusa VTT E-90392 dextransucrase was tested with several potential acceptors, particularly, analogs lactose and cellobiose. The major acceptor products of both disaccharides were identified as branched trisaccharides, with a glucosyl residue α-(1 → 2)-linked to the acceptor's reducing end. An additional product, isomelezitose (6(Fru)-α-Glcp-sucrose), was also produced when using lactose as an acceptor. This is the first report of the synthesis of isomelezitose by a dextransucrase. The NMR spectra of the three trisaccharides were fully assigned, and their structures were confirmed by selective enzymatic hydrolysis. The trisaccharides prepared from (13)C6(glc) sucrose and lactose were analyzed by ESI-MS(n), and the fragmentation patterns of these compounds were characterized.

Glucuronic acid in Arabidopsis thaliana xylans carries a novel pentose substituent.

Glucuronic acids in Arabidopsis thaliana xylans exist in 4-O-methylated (MeGlcA) and non-methylated (GlcA) forms at a ratio of about 3:2. The matrix-assisted laser desorption/ionization mass spectrometry analysis of the endoxylanase liberated acidic oligosaccharides from the Arabidopsis inflorescence stem showed that two peaks with GlcA (GlcA-Xyl4Ac1 and GlcA-Xyl5Ac2) had abnormally high intensities, as well as different tandem mass spectra, than their 4-O-methylated counterparts. These peaks were interestingly enriched in the xylan biosynthesis mutant irx7 and irx9-1. Multi-stages fragmentation analysis using negative ion electrospray-ion trap mass spectrometry indicated that this GlcA was further carrying a pentose residue in the glucuronoxylan-derived oligosaccharide from irx9-1. The structure was also identified in Arabidopsis wild type. The results prove evidence of a new pentose substitution on the GlcA residue of Arabidopsis GX, which is likely present in the primary walls.

Supercritical water treatment for cello-oligosaccharide production from microcrystalline cellulose.

Microcrystalline cellulose was treated in supercritical water at 380 °C and at a pressure of 250 bar for 0.2, 0.4, and 0.6s. The yield of the ambient-water-insoluble precipitate and its average molar mass decreased with an extended treatment time. The highest yield of 42 wt% for DP2-9 cello-oligosaccharides was achieved after the 0.4s treatment. The reaction products included also 11 wt% ambient-water-insoluble precipitate with a DP(w) of 16, and 6.1 wt% monomeric sugars, and 37 wt% unidentified degradation products. Oligo- and monosaccharide-derived dehydration and retro-aldol fragmentation products were analyzed via a combination of HPAEC-PAD-MS, ESI-MS/MS, and GC-MS techniques. The total amount of degradation products increased with treatment time, and fragmented (glucosyl(n)-erythrose, glucosyl(n)-glycolaldehyde), and dehydrated (glucosyl(n)-levoglucosan) were identified as the main oligomeric degradation products from the cello-oligosaccharides.

O-acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants.

O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 → 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.

Weissella confusa Cab3 dextransucrase: properties and in vitro synthesis of dextran and glucooligosaccharides.

Food-derived Weissella spp. have gained attention during recent years as efficient dextran producers. Weissella confusa Cab3 dextransucrase (WcCab3-DSR) was isolated applying PEG fractionation and used for in vitro synthesis of dextran and glucooligosaccharides. WcCab3-DSR had a molar mass of 178 kDa and was activated by Co(2+) and Ca(2+) ions. Glycerol and Tween 80 enhanced enzyme stability, and its half-life at 30°C increased from 10h to 74 h and 59 h, respectively. The (1)H and (13)C NMR spectral analysis of the produced dextran confirmed the presence of main chain α-(1→6) linkages with only 3.0% of α-(1→3) branching, of which some were elongated. An HPSEC analysis in DMSO revealed a high molecular weight of 1.8 × 10(7)g/mol. Glucooligosaccarides produced through the acceptor reaction with maltose, were analyzed with HPAEC-PAD and ESI-MS/MS. They were a homologous series of isomaltooligosaccharides with reducing end maltose units. To the best of our knowledge, this is a first report on native W. confusa dextransucrase.

Xylo- and cello-oligosaccharide oxidation by gluco-oligosaccharide oxidase from Sarocladium strictum and variants with reduced substrate inhibition.

BACKGROUND: The oxidation of carbohydrates from lignocellulose can facilitate the synthesis of new biopolymers and biochemicals, and also reduce sugar metabolism by lignocellulolytic microorganisms, reserving aldonates for fermentation to biofuels. Although oxidoreductases that oxidize cellulosic hydrolysates have been well characterized, none have been reported to oxidize substituted or branched xylo-oligosaccharides. Moreover, this is the first report that identifies amino acid substitutions leading to GOOX variants with reduced substrate inhibition. RESULTS: The recombinant wild type gluco-oligosaccharide oxidase (GOOX) from the fungus Sarocladium strictum, along with variants that were generated by site-directed mutagenesis, retained the FAD cofactor, and showed high activity on cello-oligosaccharide and xylo-oligosaccharides, including substituted and branched xylo-oligosaccharides. Mass spectrometric analyses confirmed that GOOX introduces one oxygen atom to oxidized products, and 1H NMR and tandem mass spectrometry analysis confirmed that oxidation was restricted to the anomeric carbon. The A38V mutation, which is close to a predicted divalent ion-binding site in the FAD-binding domain of GOOX but 30 Å away from the active site, significantly increased the kcat and catalytic efficiency of the enzyme on all oligosaccharides. Eight amino acid substitutions were separately introduced to the substrate-binding domain of GOOX-VN (at positions Y72, E247, W351, Q353 and Q384). In all cases, the Km of the enzyme variant was higher than that of GOOX, supporting the role of corresponding residues in substrate binding. Most notably, W351A increased Km values by up to two orders of magnitude while also increasing kcat up to 3-fold on cello- and xylo-oligosaccharides and showing no substrate inhibition. CONCLUSIONS: This study provides further evidence that S. strictum GOOX has broader substrate specificity than the enzyme name implies, and that substrate inhibition can be reduced by removing aromatic side chains in the -2 binding subsite. Of the enzyme variants, W351A might be particularly advantageous when oxidizing oligosaccharides present at high substrate concentrations often experienced in industrial processes.

Structural analysis of linear mixed-linkage glucooligosaccharides by tandem mass spectrometry.

Dextrans and glucooligosaccharides (GLOS) are produced by lactic acid bacteria (LAB) during sourdough fermentation. The dextrans can act as hydrocolloids in sourdough bread, while the GLOS may have antistaling and prebiotic properties, depending on their structure. Development of high-throughput methods for screening the structural properties of dextrans and GLOS produced by different LAB in varying fermentation conditions is therefore of interest. In this study we explored the possibility of using electrospray ionisation tandem mass spectroscopy (ESI-MS/MS) to unequivocally determine the structures of underivatised GLOS. The emphasis was on linear mixed linked model GLOS, especially those containing (1→3) linkages that are common in dextrans. After evaluation of the model GLOS, the ESI-MS/MS method was used to determine the linkage positions of two mixed-linked tetrasaccharides obtained by hydrolysis of Weissella confusa and Leuconostoc citreum dextrans. In positive mode, only the reducing end linkage could be determined because isomeric fragment ions, present in subsequent MS(n) cycles, hindered assignment of the remaining linkages. By contrast, it was possible to unambiguously assign all the linkages in each GLOS using the negative mode spectra. The present study thus shows that negative mode is the preferred method for ESI-MS/MS structural analysis of underivatised GLOS. In combination with liquid chromatography this method will enable rapid profiling of the structural variation of dextrans and prebiotic GLOS.