Non-reducing Trisaccharide Research Articles

Identification of two kinds of Colchicum corms by DNA barcoding, physicochemical, chromatographic and chemometric analyses along with pharmacognostic parameters

Corms of “Suranjan” are widely used in traditional medicine for the management of rheumatism and various ailments. In traditional medicinal systems of India, Suranjan is divided into two categories, i.e. “Suranjan Talkh”, which is known as “Bitter Suranjan” and “Suranjan Shirin”, which is known as “Sweet Suranjan”. There is confusion between both species even though both have the same genus, i.e. Colchicum. In the present study, DNA barcoding, pharmacognostic parameters, chromatographic analysis (including HPTLC, HPLC, LC-MS and GC–MS) and a safety parameter and multivariant analysis was performed to identify the corms of both species. DNA barcoding-based results indicated that the bitter species is Colchicum luteum Baker whereas the sweet species is Colchicum autumnale L. Chemical profiling revealed that colchicine is a basic biomarker of both varieties. Moreover, based upon HPTLC and HPLC analyses, the major difference is that Colchicum autumnale L. contained caffeic acid along with colchicine. The phytochemicals observed in HPTLC and HPLC analyses was confirmed by LC-MS analysis. More interestingly, Colchicum autumnale L. had 27.34 % more colchicine as compared to Colchicum luteum Baker. As per organoleptic evaluation, Colchicum autumnale L. has a sweeter taste as compared to Colchicum luteum Baker. GC–MS analysis revealed that Colchicum autumnale L. had 90.87 % more melezitose as compared to Colchicum luteum Baker. Melezitose is a non-reducing trisaccharide sugar molecule and considered as a masking agent in the food industry and a replacement for sugar. Melezitose is the molecule that suppresses an unpleasant taste or sensation, i.e. organoleptic parameters. For the HPLC method-based sugar analysis, the observed sucrose content was 130.66 mg/mL in Colchicum autumnale L, whereas no sugar content was observed in Colchicum luteum Baker. Hence, Colchicum autumnaleL.is the sweet variety even in the presence of a high content of colchicine having low bitterness or sweetness due to the presence of Melezitose in a large content. Antioxidant studies (like TPC, TFC and DPPH assays) demonstrated that Colchicum autumnale L. has far better antioxidant activities than Colchicum luteum Baker. In conclusion, the present study may be a breakthrough for the authentication and identification of market samples of Colchicum autumnale L. and Colchicum luteum Baker. Newly established (as per ICH guidelines) HPTLC and HPLC based methods may help analysts to quantify caffeic acid along with colchicine content.

Differentiating planteose and raffinose using negative ion mode mass spectrometry

Planteose, a non-reducing trisaccharide, is present in seeds of many higher plants. It is a structural isomer of raffinose comprising α-d-galactopyranosyl linked to C (6') of fructose residue. Planteose-centric studies are at a disadvantage due to the lack of relatively more straightforward methods to identify it. In the present study, the fragmentation pattern associated with planteose was demonstrated using collision-induced dissociation in negative ion mode. MS2 of planteose revealed the presence of a unique peak at m/z 233 that is three times more intense than that at m/z 221. The m/z 221 peak is commonly seen in the MS2 of raffinose. Furthermore, we evaluated the effect of the presence of a reducing end sugar building block in a similar trisaccharide (levantriose) that was revealed to affect the generation of ion at m/z 233. Overall our analysis indicated that planteose can be clearly distinguished from raffinose based on fragment patterns in the negative ion mode.

Raffinose positively regulates maize drought tolerance by reducing leaf transpiration.

Drought stress is one of the major constraints of global crop production. Raffinose, a non-reducing trisaccharide, has been considered to regulate positively the plant drought stress tolerance; however, evidence that augmenting raffinose production in leaves results in enhanced plant drought stress tolerance is lacking. The biochemical mechanism through which raffinose might act to mitigate plant drought stress remains unidentified. ZmRAFS encodes Zea mays RAFFINOSE SYNTHASE, a key enzyme that transfers galactose from the galactoside galactinol to sucrose for raffinose production. Overexpression of ZmRAFS in maize increased the RAFS protein and the raffinose content and decreased the water loss of leaves and enhanced plant drought stress tolerance. The biomass of the ZmRAFS overexpressing plants was similar to that of non-transgenic control plants when grown under optimal conditions, but was significantly greater than that of non-transgenic plants when grown under drought stress conditions. In contrast, the percentage of water loss of the detached leaves from two independent zmrafs mutant lines, incapable of synthesizing raffinose, was greater than that from null segregant controls and this phenomenon was partially rescued by supplementation of raffinose to detached zmrafs leaves. In addition, while there were differences in water loss among different maize lines, there was no difference in stomata density or aperture. Taken together, our work demonstrated that overexpression of the ZmRAFS gene in maize, in contrast to Arabidopsis, increased the raffinose content in leaves, assisted the leaf to retain water, and enhanced the plant drought stress tolerance without causing a detectable growth penalty.

Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66T.

β-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 β-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied β-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc β-1,4 and β-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM being 341 and 344 mM−1·s−1, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide β-Gal-1,4-β-Glc-1,1-β-GlcNAc, and in low amounts the β-1,2- or β-1,3-linked trisaccharide β-Gal-1,4(β-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 β-N-acetylhexosaminidases.

Production of isomelezitose from sucrose by engineered glucansucrases

AbstractCertain lactic acid bacteria produce glycosyltransferases known as glucansucrases, which synthesize α-D-glucans via glucosyl transfer from sucrose. We recently reported on the formation of the unusual trisaccharide isomelezitose in low yields by a variety of glucansucrases. Isomelezitose is a rare non-reducing trisaccharide, with the structure α-d-glucopyranosyl- (1→6)-β-d-fructofuranosyl-(2↔1)-α-d-glucopyranoside. In this work, we describe the synthesis of isomelezitose in high yields by variants of glucansucrases engineered to contain a single point mutation at a key leucine residue involved in acceptor substrate binding. Some variants produce isomelezitose in yields up to 57%. This method is amenable to large-scale production of isomelezitose for food, industrial and biomedical applications.

Hemolytic activity and solubilizing capacity of raffinose and melezitose fatty acid monoesters prepared by enzymatic synthesis

The hemolytic activity and solubilizing capacity of two families of non-reducing trisaccharide fatty acid monoesters have been studied to assess their usefulness as surfactants for pharmaceutical applications. The carbohydrate-based surfactants investigated included homologous series of raffinose and melezitose monoesters bearing C10 to C18 acyl chains prepared by lipase-catalyzed synthesis in organic media. The hemolytic activity was determined in vitro using a static method based on the addition of the surfactants to an erythrocyte suspension and subsequent spectrophotometric determination of the released hemoglobin. The effect of the carbohydrate head group, the acyl chain length and the regioisomeric purity was investigated. In all cases, the carbohydrate monoester surfactants decreased their hemolytic activity (with respect to their critical micelle concentration) when increasing the length of the acyl chain. A very similar behaviour was observed either the carbohydrate head-group (raffinose and melezitose) or regardless of the regioisomeric purity. Interestingly, decanoyl (C10) and lauroyl (C12) monoesters were just marginally hemolytic at their critical micelle concentrations while the longer palmitoyl (C16) and (C18) stearoyl monoesters become hemolytic at concentrations much higher than their respective cmc. The palmitoyl and stearoyl monoesters also displayed higher solubilization capacity than the shorter acyl chain monoesters in a solubilization assay of a hydrophobic dye as a model drug mimic. These results suggest that raffinose and melezitose monoesters with long-chain fatty acids (C16 to C18) are promising surfactants for pharmaceutical applications and could be an alternative to the use of current commercial nonionic polyoxyethylene-based surfactants in parenteral formulations.

Non-reducing trisaccharide fatty acid monoesters: Novel detergents in membrane biochemistry

Three families of non-reducing trisaccharide fatty acid monoesters bearing C10 to C18 acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to μM. The new compounds are capable of dissolving phospolipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed β-dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies.

ChemInform Abstract: A Novel Trisaccharide Glycolipid Biosurfactant Containing Trehalose Bears Ester-Linked Hexanoate, Succinate, and Acyloxyacyl Moieties: NMR and MS Characterization of the Underivatized Structure.

A Gram-positive actinomycete growing on n-hexadecane secreted a family of anionic glycolipid surfactant homologs. The major homolog, with a molecular weight of 1210.6347, had the formula C58H98O26. Following mild alkaline saponification, 1H and 13C NMR spectroscopy were used to characterize the non-reducing trisaccharide backbone: β-Glcp-(1→3)-α-Glcp-(1↔1)-α-Glcp (‘laminaratrehalose’). Hexanoate, succinate, 3-hydroxyoctanoate, and 3-hydroxydecanoate were found in 3:1:1:1 molar ratio using GC–EIMS analysis of fatty acid methyl esters (FAME) prepared by transesterification. We found that the β-hydroxy acids bore secondary hexanoate chains in 3-O-ester linkage, giving acyloxyacyl anions of appropriate m/z in FABMS and FABMS/MS spectra. COSY, HETCOR, HMBC, and HMQC NMR experiments established the acylation pattern: succinate at C-2 of the terminal α-glucopyranose ring; hexanoate at C-3′′ of the β-glucopyranose ring; 3-hexanoyloxyoctanoate and 3-hexanoyloxydecanoate at the 2′- and 4-positions. In FABMS spectra, the homologs flanked the molecular ion by ±14 and ±28 amu, suggesting heterogeneity in acyl chain length.

Positive‐ion ESI mass spectrometry of regioisomeric nonreducing oligosaccharide fatty acid monoesters: In‐source fragmentation of sodium adducts

Structural characterization and differentiation of a novel group of regioisomeric monolaurate esters of the nonreducing trisaccharides raffinose and melezitose, and the nonreducing tetrasaccharide stachyose has been obtained using positive electrospray ionization (ESI) mass spectrometry with in-source fragmentation. The surfactant nature and high polarity of these compounds make them appropriate analytes for being studied by conventional ESI-MS. The position of the acyl chain in each particular regioisomer has been used as a reporter group that allows unambiguous rationalization of the fragmentation routes of the corresponding natriated molecular ions [M + Na](+). In all cases, the main fragment ions were produced through cleavage of the glycosidic bond involving two anomeric carbons, the C-1' and C-2'' of the alpha-D-Glcp-(1-2)-beta-D-Fruf bond, and it was observed that sodium cation retention occurred on the heavier mass fragment of the two formed fragments, (e.g. di- or trisaccharide type vs monosaccharide type). Our results may help to better understand the fragmentation behavior of nonreducing oligosaccharides (as sodium adducts) in positive ESI mass spectrometry.

One- and two-step enzymatic synthesis of polymerizable vinyladipoyl mono- and diesters of non-reducing trisaccharides

The enzymatic transesterification of non-reducing trisaccharides raffinose and melezitose with divinyl adipate using two lipases (Novozym 435, Lipozyme TL IM) and the protease subtilisin Carlsberg is described. Polymerizable vinyladipoyl sugar esters were obtained with good to excellent selectivity and high yields. Monoesters prepared with lipase catalysis have also been used as substrates for a second acylation step catalyzed by the protease. Diesters were obtained with high selectivity although the isolated yields were slightly lower than those obtained for the corresponding monoester derivatives. It is important to note that the preferential acylation positions for subtilisin are the same in the free sugar and in the corresponding used monoesters for both trisaccharides. This two-step enzymatic approach allows regioselective control in the incorporation of the sugar inside the polymer structure.

Acceptor specificity of trehalose phosphorylase from Thermoanaerobacter brockii: Production of novel nonreducing trisaccharide, 6-O-α-D-galactopyranosyl trehalose

We investigated the acceptor specificity of a thermostable trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047 (TbTP) was examined using beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor and oligosaccharides as the acceptor. Oligosaccharides with a reducing-end glucose residue as the C-6 substituent (e.g., isomaltose, gentiobiose, melibiose, isomaltotriose, and isopanose) were found to be successful acceptors. The transfer products of isomaltose, gentiobiose, and melibiose were isolated and characterized as 6-O-alpha-D-glucopyranosyl trehalose (alpha-GlcTre), 6-O-beta-D-glucopyranosyl trehalose (beta-GlcTre), and 6-O-alpha-D-galactopyranosyl trehalose (alpha-GalTre), respectively. To produce alpha-GalTre, a novel nonreducing trisaccharide, the reaction conditions of alpha-GalTre were examined using trehalose as a glucosyl donor. As a result, the yield of alpha-GalTre reached 40.5%.

Hyper expression of kojibiose phosphorylase gene and trehalose phosphorylase gene from Thermoanaerobacter brockii ATCC35047 in Bacillus subtilis and selaginose synthesis utilizing two phosphorylases

The kojibiose phosphorylase (KP) gene and trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii ATCC35047 were intracellularly hyper-expressed under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be 2.1 g of KP and 4.9 g of TP per liter of medium. Selaginose, non-reducing trisaccharide, was synthesized from trehalose utilizing the recombinant KP and TP from B. subtilis. Selaginose was not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.

Biosynthetic origin and longevity in vivo of alpha-d-mannopyranosyl-(1 --> 4)-alpha-d-glucuronopyranosyl-(1 --> 2)-myo-inositol, an unusual extracellular oligosaccharide produced by cultured rose cells

A non-reducing trisaccharide, alpha-D-mannopyranosyl-(1 --> 4)-alpha-D-glucuronopyranosyl-(1 --> 2)-myo-inositol (MGI) accumulated in the spent medium of cell-suspension cultures of 'Paul's Scarlet' rose (Rosa sp.) predominantly during the period of rapid cell growth. This trisaccharide was also produced by cultures of sycamore (Acer pseudoplatanus L.) but not by those of the graminaceous monocots maize (Zea mays L.) and tall fescue grass (Festuca arundinacea Schreb.). When added to cultured Rosa cells, [(14)C]MGI was neither taken up by the cells nor bound to the cell surface and was not metabolised extracellularly. When D-[6-(14)C]glucuronic acid was fed to cultured Rosa cells, extracellular [(14)C]MGI started to appear only after a 5-h lag period, compared with a 0.5-h lag period for labelling of extracelluar polysaccharides. Furthermore, [(14)C]MGI continued to accumulate in the medium for at least 20 h after the accumulation of (14)C-polymers had ceased. These observations indicate that extracellular MGI was produced from a slowly turning-over pool of a pre-formed intermediate. Structural considerations indicate that the intermediate could be a glucuronomannan or a phytoglycolipid (glycophosphosphingolipid). No Rosa polysaccharides could be found that generated MGI in the presence of living Rosa cells. We therefore favour phytoglycolipids as the probable biosynthetic origin of MGI.

α-d-Mannopyranosyl-(1→4)-α-d-glucuronopyranosyl-(1→2)-myo-inositol, a new and unusual oligosaccharide from cultured rose cells

Abstract Cell-suspension cultures of rose (Rosa sp.) accumulated approximately micromolar concentrations of a novel, extracellular, non-reducing trisaccharide, characterised here as α- d -mannopyranosyl-(1→4)-α- d -glucuronopyranosyl-(1→2)-myo-inositol (MGI). Acid hydrolysis of [glucuronic acid-14C]MGI, isolated after incubation of the cells with d -[6-14C]glucuronic acid, readily yielded [14C]glucuronic acid-inositol, which was relatively resistant to further hydrolysis, as revealed by chromatography and electrophoresis. The complete structure and stereochemistry of MGI was elucidated using one and two dimensional NMR spectroscopic techniques. The structure of MGI suggests a biosynthetic origin from a membrane-bound glycophosphosphingolipid (GPS). We propose that GPSs represent a hitherto unrecognised source of oligosaccharin signalling molecules in plants.

A novel trisaccharide glycolipid biosurfactant containing trehalose bears ester-linked hexanoate, succinate, and acyloxyacyl moieties: NMR and MS characterization of the underivatized structure

A Gram-positive actinomycete growing on n-hexadecane secreted a family of anionic glycolipid surfactant homologs. The major homolog, with a molecular weight of 1210.6347, had the formula C 58H 98O 26. Following mild alkaline saponification, 1H and 13C NMR spectroscopy were used to characterize the non-reducing trisaccharide backbone: β-Glc p-(1→3)-α-Glc p-(1↔1)-α-Glc p (‘laminaratrehalose’). Hexanoate, succinate, 3-hydroxyoctanoate, and 3-hydroxydecanoate were found in 3:1:1:1 molar ratio using GC–EIMS analysis of fatty acid methyl esters (FAME) prepared by transesterification. We found that the β-hydroxy acids bore secondary hexanoate chains in 3- O-ester linkage, giving acyloxyacyl anions of appropriate m/ z in FABMS and FABMS/MS spectra. COSY, HETCOR, HMBC, and HMQC NMR experiments established the acylation pattern: succinate at C-2 of the terminal α-glucopyranose ring; hexanoate at C-3′′ of the β-glucopyranose ring; 3-hexanoyloxyoctanoate and 3-hexanoyloxydecanoate at the 2′- and 4-positions. In FABMS spectra, the homologs flanked the molecular ion by ±14 and ±28 amu, suggesting heterogeneity in acyl chain length.

Formation of a Nonreducing Trisaccharide, Selaginose, from Trehalose by a Cell-free System of Thermoanaerobium brockii.

A nonreducing trisaccharide was formed in a reaction mixture containing trehalose and the cell-free extract of a thermophilic anaerobe, Thermoanaerobium brockii ATCC 35047. This saccharide was isolated from the mixture by trehalase digestion, alkaline treatment and preparative HPLC. From the results of partial acid hydrolysis, methylation analysis, and 13C-NMR analysis, the chemical structure of the saccharide was determined to be 2-O-α-D-glucopyranosyl α-D-glucopyranosyl α-D-glucopyrano- side (selaginose). Two enzymes related to Selaginose synthesis were partially purified by DEAEToyopearl column chromatography from the cell-free extract of T brockii. One was a trehalose phosphorylase (EC 2.4.1.64), which catalyzes the reversible phosphorolysis of trehalose into β-glucose-1-phosphate and glucose. The other synthesized kojibiose from β-glucose 1-phosphate and glucose. The latter enzyme was considered to be a novel phosphorylase. The hypothetical mechanism of selaginose synthesis was proposed as follows: trehalose+Pi→glucose+β-glucose 1-phosphate, β-glucose 1-phosphate +trehalose→selaginose+Pi.

Production of Maltosylfructose (Erlose) with Levansucrase from Bacillus Subtilis

Levansucrase (E.C.2.4.1.10) from B. subtilis was used in a synthesis reaction for the production of maltosylfructose using maltose as acceptor and sucrose as the fructosyl donor. The effect of various parameters was studied in order to minimize the hydrolysis of sucrose and the production of levan. A high total solute concentration (240 g/L of sucrose and maltose) reduced hydrolysis to 20%. Further reductions could only be obtained by decreasing the sucrose/maltose concentration ratio. Optimum fructoside production was obtained at around 70% sucrose conversion, as afterwards, the product was hydrolyzed by the enzyme. Under these conditions the yield could be as high as 70-80% with respect to converted sugar or 46% with respect to total sucrose in the reaction. The product was purified and identified as O-α-D-glucopyranosyl-(1-4)-0-α-D-glucopyranosyl-(l-2)-β-fructofuranoside, a non-reducing trisaccharide known as Erlose which occurs naturally in Aphis pomi honeydew.

The tumor association of a trisaccharide epitope: specificity of antiserum developed to galactose beta1->3 N-acetyl glucosamine beta1-->3 galactose.

A pentasaccharide carbohydrate epitope described by Nozawa et at (1) is expressed by 35% of the neoplastic tissue samples from patients with endometrial cancer but not by normal endometrium. This epitope was detected using a human monoclonal antibody (HMST-1) produced by fusion of lymphocytes from an endometrial cancer patient. We chemically linked a synthetically produced nonreducing terminal trisaccharide portion of this pentasaccharide to bovine serum albumin to create an effective immunogen, Galbeta1->3GlcNAcbeta1->3Gal-BSA. A rabbit polyclonal antibody was produced and tested against panels of tumor and normal tissues. In contrast to the results obtained with HMST-1, 100% of the endometrial adenocarcinomas we studied stained with this polyclonal antiserum while normal endometrium was non-reactive. The reactivity with other tyes of adenocarcinomas was approximately 80%, whereas most normal tissues were not reactive with the antiserum. Immunological specificity analysis was performed with structurally related carbohydrates and this shows the fine specificity reaction of the antiserum. This antigen may be clinically useful for immunolocalization and for immunotargeting.

Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin.

Three discrete tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate). The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III. Isolation of the pentasulfated component has never been reported. Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these tetrasaccharides to the enzymes. The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units. The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site.

Enzymatic Synthesis of a Novel Trisaccharide, Glucosyl Lactoside

Cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus produced a series of oligosaccharides by the transglycosylation reaction with cyclomaltohexaose as a glycosyl donor and D-lactose as its acceptor. The content of oligosaccharide A as the main transfer product was 22.1% of the total sugar. The content was increased to 34.1% by hydrolysis with glucoamylase. Oligosaccharide A was isolated by column chromatography and crystallized. By chemical and enzymatic analyses, oligosaccharide A was found to be a novel non-reducing trisaccharide; O-beta-D-galactopyranosyl-(1-->4)-O- beta-D-glucopyranosyl alpha-D-glucopyranoside.