Enzymatic synthesis of a beta-D-galactopyranosyl cyclic tetrasaccharide by beta-galactosidases

The galactosyl transfer reaction to cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS) was examined using lactose as a donor and beta-galactosidases from Aspergillus oryzae and Bacillus circulans. The A. oryzae beta-galactosidase produced three galactosyl derivatives of CTS. The main galactosyl derivative produced by the A. oryzae enzyme was identified as 6-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-[beta-D-Galp-(1-->6)]-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. The B. circulans beta-galactosidase also synthesized three galactosyl-transfer products to CTS. The structure of main transgalactosylation product was 3-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-D-Galp-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These results showed that beta-galactosidase transferred galactose directly to the ring glucose residue of CTS.     

Transglycosylation of glycosyl residues to cyclic tetrasaccharide by Bacillus stearothermophilus cyclomaltodextrin glucanotransferase using cyclomaltodextrin as the glycosyl donor

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as 4-mono-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], and sachharide D was 4,4'-di-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.     

Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp. NRRL B-21195

A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE. The pH optimum was 7.0, and the enzyme was stable from pH 6.0 to 9.2. The temperature optimum was 35 degrees C and the enzyme exhibited stability up to 50 degrees C. The enzyme hydrolyzed cycloalternan [CA; cyclo(-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(-->3)-alpha-d-Glcp-(1-->)] as the best substrate, to produce only isomaltose via an intermediate, alpha-isomaltosyl-(1-->3)-isomaltose. This enzyme also hydrolyzed isomaltosyl substrates, such as panose, alpha-isomaltosyl-(1-->4)-maltooligosaccharides, alpha-isomaltosyl-(1-->3)-glucose, and alpha-isomaltosyl-(1-->3)-isomaltose to liberate isomaltose. Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires alpha-isomaltosyl residues connected with (1-->4)- or (1-->3)-linkages. The K(m) value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The k(cat) value on panose (14.4s(-1)) was not significantly different from that of cycloalternan (10.8 s(-1)). Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase. This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp. NRRL B-21195, its role being to hydrolyze cycloalternan inside the cells.     

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside. With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue. alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4). Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue. Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product. The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol.     

Identification of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-d-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->)

A structural characterization of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was carried out by NMR spectroscopy. H-1', 2'-OH, H-3', and 4'-OH of the 3-O-glycosylated residue and H-1 of the 6-O-glycosylated residue were found to cross-relax with protons of bound waters using the double-pulsed field-gradient spin-echo ROESY experiment. In the crystal structure, one water molecule is located in the center of the plate, and its temperature factor is very low, indicating that this water molecule is an intrinsic component.     

Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.     

An enzymatically produced novel cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->} (cyclic maltosyl-(1-->6)-maltose), from starch

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1-->4)- and alpha-(1-->6)-linkages. The cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages.     

A novel glucanotransferase from a Bacillus circulans strain that produces a cyclomaltopentaose cyclized by an alpha-1,6-linkage

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 degrees C, and stable from pH 4.5 to 9.0 at up to 35 degrees C. The addition of 1 mM Ca(2+) enhanced the thermal stability of the enzyme up to 40 degrees C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular alpha-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular alpha-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.     

An enzymatically produced novel cyclomaltopentaose cyclized from amylose by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}

A bacterial strain AM7, isolated from soil and identified as Bacillus circulans, produced two kinds of novel cyclic oligosaccharides. The cyclic oligosaccharides were produced from amylose using a culture supernatant of the strain as the enzyme preparation. The major product was a cyclomaltopentaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. The other minor product was cyclomaltohexaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one alpha-(1-->6)-linkage. ICG5 was digested by alpha-amylase derived from Aspergillus oryzae, cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus, and maltogenic alpha-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans, and maltogenic alpha-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an alpha-(1-->6)-linkage in their molecules.     

Aglycone specificity of Escherichia coli alpha-xylosidase investigated by transxylosylation

The specificity of the aglycone-binding site of Escherichia coli alpha-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and alpha-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl-enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, alpha-D-Xylp-(1-->6)-D-Manp, alpha-D-Xylp-(1-->6)-D-Fruf, and alpha-d-Xylp-(1-->3)-D-Frup, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by alpha-1,6-xylosidic linkages. Of the transxylosylation products, alpha-d-Xylp-(1-->6)-D-Manp and alpha-d-Xylp-(1-->6)-D-Fruf inhibited intestinal alpha-glucosidases.     

Steroidal saponins from the fruits of Asparagus racemosus

Three steroidal saponins, racemosides A (1), B (2) and C (3), were isolated from the methanolic extract of the fruits of Asparagus racemosus, and characterized as (25S)-5beta-spirostan-3beta-ol-3-O-{beta-D- glucopyranosyl (1-->6)-[alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->4)]-beta-D-glucopyranoside}, (25S)-5beta-spirostan-3beta-ol-3-O-alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranoside and (25S)-5beta-spirostan-3beta-ol-3-O-{alpha-L-rhamnopyranosyl-(1-->6)-[alpha-L-rhamnopyranosyl (1-->4)]-beta-D-glucopyranoside}, respectively, by spectrometric analysis and some chemical strategies.     

Synthesis of 3-O-beta-N-acetylglucosaminyl cyclic tetrasaccharide through a lysozyme-catalyzed transfer reaction

Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS). Structural analysis showed that the transfer product was 3-O-beta-N-acetylglucosaminyl CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-GlcNAc-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.     

Oligosaccharide synthesis by dextransucrase: new unconventional acceptors

The acceptor reactions of dextransucrase offer the potential for a targeted synthesis of a wide range of di-, tri- and higher oligosaccharides by the transfer of a glucosyl group from sucrose to the acceptor. We here report on results which show that the synthetic potential of this enzyme is not restricted to 'normal' saccharides. Additionally functionalized saccharides, such as alditols, aldosuloses, sugar acids, alkyl saccharides, and glycals, and rather unconventional saccharides, such as fructose dianhydride, may also act as acceptors. Some of these acceptors even turned out to be relatively efficient: alpha-D-glucopyranosyl-(1-->5)-D-arabinonic acid, alpha-D-glucopyranosyl-(1-->4)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-mannitol, alpha-D-fructofuranosyl-beta-D-fructofuranosyl-(1,2':2,3')-dianhydride, 1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol ('D-glucal'), and may therefore be of interest for future applications of the dextransucrase acceptor reaction.     

Enzymatic synthesis of beta-xylanase substrates: transglycosylation reactions of the beta-xylosidase from Aspergillus sp

A beta-D-xylosidase with molecular mass of 250+/-5 kDa consisting of two identical subunits was purified to homogeneity from a cultural filtrate of Aspergillus sp. The enzyme manifested high transglycosylation activity in transxylosylation with p-nitrophenyl beta-D-xylopyranoside (PNP-X) as substrate, resulting in regio- and stereoselective synthesis of p-nitrophenyl (PNP) beta-(1-->4)-D-xylooligosaccharides with dp 2-7. All transfer products were isolated from the reaction mixtures by HPLC and their structures established by electrospray mass spectrometry and 1H and 13C NMR spectroscopy. The glycosides synthesised, beta-Xyl-1-->(4-beta-Xyl-1-->)(n)4-beta-Xyl-OC6H4NO2-p (n=1-5), were tested as chromogenic substrates for family 10 beta-xylanase from Aspergillus orizae (XynA) and family 11 beta-xylanase I from Trichoderma reesei (XynT) by reversed-phase HPLC and UV-spectroscopy techniques. The action pattern of XynA against the foregoing PNP beta-(1-->4)-D-xylooligosaccharides differed from that of XynT in that the latter released PNP mainly from short PNP xylosides (dp 2-3) while the former liberated PNP from the entire set of substrates synthesised.     

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003.

Structural analysis of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae strain 1003 has been achieved by the application of high-field NMR techniques, ESI-MS, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PP Etn-->4]-alpha-Kdop-(2-->6)-Lipid A, in which the beta-D-Glcp residue is substituted by phosphocholine at O-6 and an acetyl group at O-4. A second acetyl group is located at O-3 of the distal heptose residue (HepIII). HepIII is chain elongated at O-2 by either a beta-D-Glcp residue (major), lactose or sialyllactose (minor, i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp), where a third minor acetylation site was identified at the glucose residue. Disialylated species were also detected. In addition, a minor substitution of ester-linked glycine at HepIII and Kdo was observed.     

Synthesis of cellobiose-containing oligosaccharides by intermolecular transglucosylation of cyclodextrin glycosyltransferase from Paenibacillus sp. A11

Intermolecular transglucosylation of cyclodextrin glycosyltransferase (CGTase) was investigated for its use in oligosaccharide synthesis. From the kinetic parameters of the CGTase-catalyzed transglucosylation reaction, using β-cyclodextrin (β-CD) as the glucosyl donor and various saccharides or derivatives as acceptors, the efficient acceptors of the Paenibacillus sp. A11 enzyme were glucose, sorbose, lactose and cellobiose. Amongst these acceptors, cellobiose showed the highest k_cat/K_m value. The transglucosylation yields of the reactions for cellobiose, sorbose and glucose acceptors were 78, 57 and 54%, respectively, making cellobiose the most efficient acceptor of the tested saccharides in coupling with β-CD. The optimal condition for the coupling reaction was determined as: 2% (w/v) β-CD and 0.5% (w/v) cellobiose, incubated with 64 U/mL of CGTase at 30 °C for 2 h. Two main transfer products detected by HPLC, PC1 and PC2, with retention times of 3.81 and 4.42 min, respectively, and a product ratio of 3:1, had a molecular mass of 504 and 666 Da, respectively, as analyzed by mass spectrometry. The structures suggested by NMR were a trisaccharide and a novel tetrasaccharide-containing cellobiose of the structures glc (α1 → 4) glc (β1 → 4) glc and glc (α1 → 4) glc (α1 → 4) glc (β1 → 4) glc, respectively. The products were found to be resistant to hydrolysis by α-amylase.     

Novel carbohydrate-induced modification of peptides: crystal structure and NMR analysis of ester-bridged bicyclic galactose-enkephalin adduct containing imidazolidinone moiety.

Comparative studies based on x-ray crystallography and NMR spectroscopy were used for structural characterization of the novel minor, imidazolidinone moiety containing, product 2b of the Maillard reaction obtained in vitro by using the galactose-modified endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) 1. The x-ray analysis uniquely defined the molecular structure as cyclo-(N-(12-[-4)-D-galacto-pentitol-1-yl]-4-(4-hydroxybenzyl)-5-oxoimidazolidin-1-yl-(1 --> O]acetyl]glycyl-L-phenylalanyl-L-leucyl-] (3), having an 18-membered ring with an ester bond between the secondary (C4') hydroxyl group of a D-galacto-pentitolyl residue and the C-terminal carboxy group of leucine-enkephalin. The absolute configuration of the new chiral centre at the imidazolidinone moiety was established as C2(S), indicating a cis arrangement of C2 and C4 substituents at the 5-membered heterocyclic ring. The NMR analysis of compound 2b carried out in CH3CN-d3 and DMSO-d6, indicated the existence of two isomers in solution, differing only in the position of the ester group in the molecule. NMR data for the minor isomer (13%-16%) are in agreement with structure 3. The migratory tendency of the peptidyl group from the primary (2b) to the secondary hydroxyl group (3) of a D-galacto-pentitolyl residue in methanol/water solution was confirmed by RP HPLC analysis.     

Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation

The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEtn at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> or beta-D-Glcp-(1-->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MSn in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS.     

Transglycosylation of Glycosyl Residues to Cyclic Tetrasaccharide by Bacillus stearothermophilus Cyclomaltodextrin Glucanotransferase Using Cyclomaltodextrin as the Glycosyl Donor

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}, CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as4-mono-O-α-glucosyl-CTS, {→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}, and sachharide D was 4,4′-di-O-α-glucosyl-CTS, {→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→}. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.     

Structural characterization of a novel branching pattern in the lipopolysaccharide from nontypeable Haemophilus influenzae.

Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as by ESI-MSn on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcxp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-d-Glcp residue was either substituted by PCho or the disaccharide branch beta-d-Galp-(1-->4)-d-alpha-d-Hepp, while the O-4 position was substituted by the globotetraose unit, beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp, or sequentially truncated versions thereof. This is the first time a branching sugar residue has been reported in the outer-core region of H. influenzae LPS. Additionally, a PEtn group was identified at O-3 of the distal heptose residue in the inner-core.