Yvonne Westphal

Branched arabino-oligosaccharides isolated from sugar beet arabinan.

Sugar beet arabinan consists of an alpha-(1,5)-linked backbone of L-arabinosyl residues, which can be either single or double substituted with alpha-(1,2)- and/or alpha-(1,3)-linked L-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an alpha-(1,5)-linked backbone of L-arabinosyl residues. One series carries single substituted alpha-(1,3)-linked L-arabinosyl residues at the backbone, whereas the other series consists of a double substituted alpha-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established.

The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel α‐glucosides through reverse phosphorolysis by maltose phosphorylase

A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin‐binding protein, three maltodextrin ATP‐binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI‐GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing β‐glucose 1‐phosphate (β‐Glc 1‐P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and β‐Glc 1‐P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3‐OH and 4‐OH, such as glucose and mannose, and also reacted with 2‐deoxy glucosamine and 2‐deoxy N‐acetyl glucosamine. By contrast, none of the tested di‐ and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mmβ‐Glc 1‐P and 50 mm glucose, glucosamine, N‐acetyl glucosamine, mannose, xylose or l‐fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI‐MS to be α‐Glcp‐(1→4)‐Glcp (maltose), α‐Glcp‐(1→4)‐GlcNp (maltosamine), α‐Glcp‐(1→4)‐GlcNAcp (N‐acetyl maltosamine), α‐Glcp‐(1→4)‐Manp, α‐Glcp‐(1→4)‐Xylp and α‐Glcp‐(1→4)‐ l‐Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

Introducing porous graphitized carbon liquid chromatography with evaporative light scattering and mass spectrometry detection into cell wall oligosaccharide analysis.

Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MS(n)-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar beta-(1,4)-xylo- and beta-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of beta-(1,4)-galacto-, beta-(1,4)-manno- and alpha-(1,4)-gluco-oligosaccharides. Charged oligomers such as alpha-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides.

Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum.

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl](n)-(1→2)-D-glucopyranose, and β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl](n)-(1→3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.

Aspergillus nidulansα‐galactosidase of glycoside hydrolase family 36 catalyses the formation of α‐galacto‐oligosaccharides by transglycosylation

The α‐galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic α‐galactosidases and α‐galacto‐oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L−1 culture) as His‐tag fusion in Escherichia coli, catalysed efficient transglycosylation with α‐(1→6) regioselectivity from 40 mm 4‐nitrophenol α‐d‐galactopyranoside, melibiose or raffinose, resulting in a 37–74% yield of 4‐nitrophenol α‐d‐Galp‐(1→6)‐d‐Galp, α‐d‐Galp‐(1→6)‐α‐d‐Galp‐(1→6)‐d‐Glcp and α‐d‐Galp‐(1→6)‐α‐d‐Galp‐(1→6)‐d‐Glcp‐(α1→β2)‐d‐Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4‐nitrophenol α‐d‐galactopyranoside (40 mm), α‐(1→6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39–58%. AglC did not transglycosylate monosaccharides without the 6‐hydroxymethyl group, i.e. xylose, l‐arabinose, l‐fucose and l‐rhamnose, or with axial 3‐OH, i.e. gulose, allose, altrose and l‐rhamnose. Structural modelling using Thermotoga maritima GH36 α‐galactosidase as the template and superimposition of melibiose from the complex with human GH27 α‐galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3‐OH and Trp358 and a hydrophobic environment around the C‐6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred α‐galactosyl to 6‐OH of the terminal residue in the α‐linked melibiose, maltose, trehalose, sucrose and turanose in 6–46% yield and the β‐linked lactose, lactulose and cellobiose in 28–38% yield. The product structures were identified using NMR and ESI‐MS and five of the 13 identified products were novel, i.e. α‐d‐Galp‐(1→6)‐d‐Manp; α‐d‐Galp‐(1→6)‐β‐d‐Glcp‐(1→4)‐d‐Glcp; α‐d‐Galp‐(1→6)‐β‐d‐Galp‐(1→4)‐d‐Fruf; α‐d‐Galp‐(1→6)‐d‐Glcp‐(α1→α1)‐d‐Glcp; and α‐d‐Galp‐(1→6)‐α‐d‐Glcp‐(1→3)‐d‐Fruf.

LC/CE-MS tools for the analysis of complex arabino-oligosaccharides.

Recently, various branched arabino-oligosaccharides as present in a sugar beet arabinan digest were characterized using NMR. Although HPAEC often has been the method of choice to monitor the enzymatic degradation reactions of polysaccharides, it was shown that HPAEC was incapable to separate all known linear and branched arabino-oligosaccharides present. As this lack of resolution might result in an incorrect interpretation of the results, other separation techniques were explored for the separation of linear and branched arabino-oligosaccharides. The use of porous-graphitized carbon liquid chromatography with evaporative light scattering and mass detection as well as capillary electrophoresis with laser-induced fluorescence and mass detection demonstrated the superiority of both the techniques toward HPAEC by enabling the separation and unambiguous identification of almost all the linear and branched arabino-oligosaccharides available. The elution behavior of all arabino-oligosaccharides for the three tested separation techniques was correlated with their chemical structures and conclusions were drawn for the retention mechanisms of the arabino-oligosaccharides on the different chromatographic and electrophoretic systems. The combination of the elution/migration behavior on LC/CE and the MS fragmentation patterns of the arabino-oligosaccharides led to the prediction of structures for new DP6 arabino-oligosaccharides in complex enzyme digests.

Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase

Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (alpha/alpha)(6)-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with beta-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (alpha/alpha)(6)-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413-Glu421, His413-Ile418 and His413-Glu415 from loop 3, that include His413 and Glu415 presumably recognising the alpha-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426-Ala431 in TP and Thr419-Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (alpha1, alpha1)- and alpha-(1,2)-regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.

MALDI-TOF MS and CE-LIF Fingerprinting of Plant Cell Wall Polysaccharide Digests as a Screening Tool for Arabidopsis Cell Wall Mutants

Cell wall materials derived from leaves and hypocotyls of Arabidopsis mutant and wild type plants have been incubated with a mixture of pure and well-defined pectinases, hemicellulases, and cellulases. The resulting oligosaccharides have been subjected to MALDI-TOF MS and CE-LIF analysis. MALDI-TOF MS analysis provided a fast overview of all oligosaccharides released, whereas CE-LIF-measurements enabled separation and characterization of many oligosaccharides under investigation. Both methods have been validated with leaf material of known mutant Arabidopsis plants and were shown to be able to discriminate mutant from wild type plants. Downscaling of the MALDI-TOF MS and CE-LIF approaches toward the hypocotyl level was established, and the performance of MALDI-TOF MS and CE-LIF was shown in the successful recognition of the Arabidopsis mutant gaut13 as an interesting candidate for further analysis.

Mode of action of Chrysosporium lucknowense C1 α-l-arabinohydrolases

The mode of action of four Chrysosporium lucknowense C1 α-L-arabinohydrolases was determined to enable controlled and effective degradation of arabinan. The active site of endoarabinanase Abn1 has at least six subsites, of which the subsites -1 to +2 have to be occupied for hydrolysis. Abn1 was able to hydrolyze a branched arabinohexaose with a double substituted arabinose at subsite -2. The exo acting enzymes Abn2, Abn4 and Abf3 release arabinobiose (Abn2) and arabinose (Abn4 and Abf3) from the non-reducing end of reduced arabinose oligomers. Abn2 binds the two arabinose units only at the subsites -1 and -2. Abf3 prefers small oligomers over large oligomers. It is able to hydrolyze all linkages present in beet arabinan, including the linkages of double substituted residues. Abn4 is more active towards polymeric substrate and releases arabinose monomers from single substituted arabinose residues. Depending on the combination of the enzymes, the C1 arabinohydrolases can be used to effectively release branched arabinose oligomers and/or arabinose monomers.

Strategy to identify and quantify polysaccharide gums in gelled food concentrates.

A strategy for the unambiguous identification and selective quantification of xanthan gum and locust bean gum (LBG) in gelled food concentrates is presented. DNA detection by polymerase chain reaction (PCR) showed to be a fast, sensitive, and selective method that can be used as a first screening tool in intact gelled food concentrates. An efficient isolation procedure is described removing components that may interfere with subsequent analyses. NMR spectroscopy enabled the direct identification of xanthan gum and the discrimination between different galactomannans in the isolated polysaccharide fraction. An enzymatic fingerprinting method using endo-β-mannanase, in addition to being used to differentiate between galactomannans, was developed into a selective, quantitative method for LBG, whereas monosaccharide analysis was used to quantify xanthan gum. Recoveries for xanthan gum and LBG were 87% and 70%, respectively, with in-between day relative standard deviations below 20% for xanthan gum and below 10% for LBG.