D-glucosyl Residues Research Articles

Effects of maltogenic α-amylase treatment on the proportion of slowly digestible starch and the structural properties of pea starch

In this study, pea starch was modified with maltogenic α-amylase and then its physicochemical properties, chain length distribution, and in vitro digestibility were examined. An analysis of the starch products resulting from the maltogenic α-amylase treatment revealed a decrease in the molecular weight, an increase in the proportion of short chains [degree of polymerization (DP) <13], a decrease in the proportion of long chains (DP > 13), and a decrease in viscosity, resulting in slower digestion. The results indicated that maltogenic α-amylase can selectively hydrolyze α-1,4- and α-1,6-glycosides to some extent. Cleaving the α-1,4 linkage of starch facilitated the transfer of the non-reducing D-glucosyl residues of maltose, thereby generating an α-1,6 branch linkage. The reaction between maltogenic α-amylase and pea starch was affected by the reaction time and enzyme concentration. The maximum slowly digestible starch content (38.5%) was obtained by digesting samples with 60.0 ppm maltogenic α-amylase for 4.0 h. These findings demonstrate that modifying pea starch using maltogenic α-amylase can generate starch products with new branching structures conducive to slow digestion.

Structure-Reactivity Relationships of Conformationally Armed Disaccharide Donors and Their Use in the Synthesis of a Hexasaccharide Related to the Capsular Polysaccharide from Streptococcus pneumoniae Type 37.

To advance the field of glycobiology, efficient synthesis methods of oligosaccharides and glycoconjugates are a requisite. In glycosylation reactions using superarmed donors, both selectivity and reactivity issues must be considered, and we herein investigate these aspects for differently protected β-linked 2-O-glycosylated glucosyl donors carrying bulky tert-butyldimethylsilyl groups to different extents. The acceptors in reactions being secondary alcohols presents a challenging situation with respect to steric crowding. Conformational pyranose ring equilibria of the superarmed disaccharide donors with axial-rich substituents contained skew and boat conformations, and three-state models were generally assumed. With NIS/TfOH as the promotor, 2,6-di-tert-butyl-4-methylpyridine as the base, and a dichloromethane/toluene solvent mixture, ethyl 1-thio-β-d-glucosyl disaccharide donors having 6-O-benzyl group(s) besides tert-butyldimethylsilyl groups were efficiently coupled at -40 °C to the hydroxyl group at position 3 of glucopyranosyl acceptors to form β-(1 → 2),β-(1 → 3)-linked trisaccharides, isolated in excellent 95% yield. The more axial-rich donors in skew and boat conformations are thus preorganized closer to the assumed transition state in these glycosylation reactions. The developed methodology was subsequently applied in the synthesis of a multibranched hexasaccharide related to the capsular polysaccharide from Streptococcus pneumoniae type 37, which consists of a β-(1 → 3)-linked backbone and a β-(1 → 2)-linked side chain of d-glucosyl residues in disaccharide repeating units.

Colourimetric and fluorometric substrates for measurement of pullulanase activity

Specific and highly sensitive colourimetric and fluorometric substrate mixtures have been prepared for the measurement of pullulanase and limit-dextrinase activity and assays employing these substrates have been developed. These mixtures comprise thermostable α- and β-glucosidases and either 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl (1-6) α-maltotrioside (BzCNPG3G3, 1) as a colourimetric substrate or 4,6-O-benzylidene-4-methylumbelliferyl-β-maltotriosyl (1-6) α-maltotrioside (BzMUG3G3, 2) as a fluorometric substrate. Hydrolysis of substrates 1 and 2 by exo-acting enzymes such as amyloglucosidase, β-amylase and α-glucosidase is prevented by the presence of the 4,6-O-benzylidene group on the non-reducing end d-glucosyl residue. The substrates are not hydrolysed by any α-amylases studied, (including those from Aspergillus niger and porcine pancreas) and are resistant to hydrolysis by Pseudomonas sp. isoamylase. On hydrolysis by pullulanase, the 2-chloro-4-nitrophenyl-β-maltotrioside (3) or 4-methylumbelliferyl-β-maltotrioside (4) liberated is immediately hydrolysed to d-glucose and 2-chloro-4-nitrophenol or 4-methylumbelliferone. The reaction is terminated by the addition of a weak alkaline solution leading to the formation of phenolate ions in solution whose concentration can be determined using either spectrophotometric or fluorometric analysis. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation.

Dual-enzymatic modification of maize starch for increasing slow digestion property

Maize starch was modified using β-amylase and transglucosidase and their molecular fine structure and in vitro digestibility were investigated. By dual-enzymes treatment, the molecule weight decreased, the amount of short chains and α-1, 6 linkages increased. This indicated that α-1,4 linkage of starch was cleaved and non-reducing D-glucosyl residues of maltose was transferred to forming α-1,6 branch linkage. A maximum SDS content (33.5%) was obtained using double enzymes hydrolysis for 6 h compared to native starch. Both the increase in amount of shortened chain length and α-1,6 linkage were likely attributed to slow digestion property of starch. The results suggested that starches using combined β-amylase and transglucosidase treatment produced new branched structures with slowly digestible character.

Novel substrates for the measurement of endo-1,4-β-glucanase (endo-cellulase)

A specific and sensitive substrate for the assay of endo-1,4-β-glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-β-cellotrioside (BzCNPG3) in the presence of thermostable β-glucosidase. Hydrolysis by exo-acting enzymes such as β-glucosidase and exo-β-glucanase is prevented by the presence of the benzylidene group on the non-reducing end d-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-β-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free d-glucose by the β-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-β-glucanase in general.

Spotlight on Starch: An Update from the Carbohydrate Division

Amylopectin is the major component in starch. It is elongated through α-(1→4)-linked D-glucosyl residues with α-(1→6)linkages as branches. In the granules, the branches form the internal parts of amylopectin, are found inside amorphous lamellae, and from these external chains are clustered in double helices and build up the crystalline lamellae. The internal structure of amylopectin (i.e., the composition and arrangement of branches) and their relationships to the functionality of starch are still largely unclear.

Xanthan Gum and Its Derivatives as a Potential Bio-polymeric Carrier for Drug Delivery System

Xanthan gum is a high molecular weight natural polysaccharide produced by fermentation process. It consists of 1, 4-linked β-D-glucose residues, having a trisaccharide side chain attached to alternate D-glucosyl residues. Although the gum has many properties desirable for drug delivery, its practical use is mainly confined to the unmodified forms due to slow dissolution and substantial swelling in biological fluids. Xanthan gum has been chemically modified by conventional chemical methods like carboxymethylation, and grafting such as free radical, microwave-assisted, chemoenzymatic and plasma assisted chemical grafting to alter physicochemical properties for a wide spectrum of biological applications. This article reviews various techniques utilized for modification of xanthan gum and its applications in a range of drug delivery systems.

Identification of Bacillus selenitireducens MLS10 maltose phosphorylase possessing synthetic ability for branched α-d-glucosyl trisaccharides

We discovered an inverting maltose phosphorylase (Bsel2056) belonging to glycoside hydrolase family 65 from Bacillus selenitireducens MLS10, which possesses synthetic ability for α-d-glucosyl disaccharides and trisaccharides through the reverse phosphorolysis with β-d-glucose 1-phosphate as the donor. Bsel2056 showed the flexibility for monosaccharide acceptors with alternative C2 substituent (2-amino-2-deoxy-d-glucose, 2-deoxy-d-arabino-hexose, 2-acetamido-2-deoxy-d-glucose, d-mannose), resulting in production of 1,4-α-d-glucosyl disaccharides with strict regioselectivity. In addition, Bsel2056 synthesized two maltose derivatives possessing additional d-glucosyl residue bound to C2 position of the d-glucose residue at the reducing end, 1,4-α-d-glucopyranosyl-[1,2-α-d-glucopyranosyl]-d-glucose and 1,4-α-d-glucopyranosyl-[1,2-β-d-glucopyranosyl]-d-glucose, from 1,2-α-d-glucopyranosyl-d-glucose (kojibiose) and 1,2-β-d-glucopyranosyl-d-glucose (sophorose), respectively, as the acceptors. These results suggested that Bsel2056 possessed a binding space to accommodate the bulky C2 substituent of d-glucose.

Branching Structure and Chain Conformation of Water-Soluble Glucan Extracted from Auricularia auricula-judae

A water-soluble neutral polysaccharide (AF1) was extracted from Auricularia (A.) auricula-judae with 0.15 M aqueous NaCl at 80-100 °C. Its chemical components and structure were analyzed by GC, GC-MS, and NMR. AF1 was identified as a β-(1→3)-D-glucan with two β-(1→6)-D-glucosyl residues for every three main chain glucose residues, showing a comb-branched structure. The M(w) values of AF1 in both aqueous solution and DMSO determined by LLS and SEC-LLS were in the narrow range of 2.07-2.15 × 10(6), indicating AF1 existed as single chains in the two solvents. The high intrinsic viscosity [η] of 1753 mL/g and the structure-sensitive parameter ρ (≡R(g)/R(h)) value of 2.3 in water revealed that AF1 existed as stiff chain conformation. Moreover, we directly observed the extended stiff chain conformation by AFM. The branching structure led to the water solubility of AF1, and the intramolecular hydrogen bonds sustained the stiff chain conformation. The rheological results showed that this polysaccharide aqueous solution had higher viscosity than even xanthan, a pronounced thickening agent. This work provided important information for developing new thickeners in food fields, and how neutral polysaccharides can be used as good candidates.

Active-site Mapping of a Populus Xyloglucan endo-Transglycosylase with a Library of Xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.

PREPARATION OF GLUCOSYL-SUBSTITUTED POLYPHOSPHAZENE HYDROGELS

Two kinds of polyphosphazenes,that bear α-D-glucosyl side groups together with methoxyethoxy or ethyl glycinato as cosubstituent groups,have been synthesized and characterized by IR and ~1H-NMR analyses.They were examined in order to investigate their possible use as matrix for glucose-sensitive insulin delivery basing on the specific interaction of glucose with lection. Briefly, the polymers were prepared via nucleophilic displacement of the reactive chlorines of polydichlorophosphazene with diisopropylidene D-glucose at first, and subsequently with methoxyethanol (polymer a) or glycine ethyl ester (polymer b) to substitute the residual P-Cl. After filtration and dialysis, the purified polymers were then treated with 90% trifluoroacetic acid to remove isopropylidene groups to get glucosyl groups. The formation of aggregates of glucosyl-substituted polyphosphazenes physically crosslinked by Concanavalin A (Con A) was studied by monitoring the transmittance at 360 nm of dilute polymeric aqueous solutions. Hydrogels were made by mixing concentrated glucosyl-substituted polyphosphazene aqueous solution with Con A aqueous solution. Different polyphosphazenes with various compositions of α-D-glucosyl side group and methoxyethoxy or ethyl glycinato side group combinations were obtained by adjusting the feeding dose of the nucleophiles. And their hydrophilicity differed from each other for the methoxyethoxy group is hydrophilic, and the ethyl glycinato group is hydrophobic, which would affect the formation of hydrogel and the drug release thereof in different way. In the deprotection reaction, the treating time needed to be prolonged to complete the hydrolysis of isopropylidene groups, however, it would cause significant cleavage of polyphosphazene backbones and degradation of cosubstituted ethyl glycinato groups (while almost no effect to methoxyethoxy) in the strong acidic condition. As a result, the deprotection ratio of glucoses was maintained at about 60% in this study to avoid remarkable decrease in molecular weight, and at the same time, the remained diisopropylidene D-glucosyl groups were considered biocompatible and might play a role in balancing polymeric hydrophilicity/hydrophobicity. Con A is one of the most widely used lectins in studying the interaction of specific saccharide with protein, particularly, for nonreducing D-mannosyl and D-glucosyl residues. For both polymers a and b, their dilute solutions rapidly turned turbid when Con A was mixed into, and turbidity increased with increasing the concentration of polymeric solutions and the content of glucosyl side groups in the polymers due to the formation of more and larger aggregates. The aggregates could be dissociated by addition of free glucose, and transmittance of the solution increased. On the other hand, it has been found that the transmittance data of solutions of polymer a mixed with Con A were much larger that those of polymer b, even if they had the similar amount of glucosyl side groups and the same polymeric concentration. One possible reason for this phenomenon was that polymer b could not dissolve well in water due to the presence of hydrophobic ethyl glycinato side groups, and the interaction between Con A and glucosyl groups was hindered. Thus, hydrogels were made from polymer a and Con A by increasing the polymeric concentration above 10 wt%. Higher concentrations of glucosyl-substituted polyphosphazene, more hydrophilic co-substituted side groups and higher amounts of glucosyl side groups would be in favor of hydrogel formation. This hydrogel can be a potential matrix for glusoce-sensitive insulin delivery.

Studies on isolation and structural features of a polysaccharide from the mycelium of an Chinese edible fungus (Cordyceps sinensis)

The mycelium of an Chinese edible fungus ( Cordyceps sinensis) was found to contain a d-glucan. Methylation, Smith degradation, acetolysis, NMR spectroscopy ( 1H, 13C, 13C– 1H 2D-COSY) and acid hydrolysis studies were conducted to elucidate its structure. The results indicated that the d-glucan consisted of a backbone composed of (1→4)- d-glucosyl residues and carried a single (1→6)-linked d-glucosyl residue. α- d-glucosidic linkages were present in the polysaccharide according to i.r. and NMR spectra. The d-glucan gave with iodine a faint blue color that had λ max 564 nm, indicating the polysaccharide of α-(1→4)-linkages with short, exterior chains.

Structural Analysis of a Neutral (1→3),(1→4)-β-d-Glucan from the Mycelia of Cordyceps sinensis

The extracellular polysaccharide (1) extracted from the mycelia of Cordyceps sinensis with hot water was fractionated and purified by ion-exchange and gel-filtration chromatography. The structure was investigated using methylation and hydrolysis analysis, periodate oxidation, NMR spectroscopy, and reaction with beta-D-glucanase, and the results indicated that this D-glucan consisted of a backbone composed of (1-->3)-beta-D-glucosyl residues and carried a single (1-->4)-beta-linked D-glucosyl residue. NMR and IR spectroscopic measurements showed that the sugar residues were beta-glycosidically linked.

Production of Cyclic Tetrasaccharide with 6-.ALPHA.-Glucosyltransferase and .ALPHA.-Isomaltosyltransferase

Cyclic tetrasaccharide (CTS; cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}) is a cyclic oligosaccharide four D-glucosyl residues linked in an alternating α-1,3- and α-1,6-fashion. CTS has been reported for enzymatic synthesis from alternan, an α-(1→3)-α-(1→6)-D-glucan synthesized from sucrose by Leuconostoc mesenteroides NRRL B-1355 alternansucrase, by endo-glucanase (alternanase) from Bacillus sp. NRRL B-21195. While searching for nonreducing oligosaccharides that can be produced from α-1,4-glucan as the substrate, we screened bacteria isolated from soil in our own way, and obtained Bacillus globisporus C11, which produces CTS from starch. Two kinds of glycosyltransferase, 6-α-glucosyltransferase (6GT) and α-isomaltosyltransferase (IMT), were purified from the culture supernatant of this strain. It was found that CTS is produced by the sequential action of both enzymes. The genes for IMT (CtsY) and 6GT (CtsZ) were linked together on the chromosome, forming ctsYZ. Both of the gene products showed similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserved two aspartic acids corresponding to the putative catalytic residues of these enzymes. CtsYZ and four open reading frames upstream of ctsYZ form a gene cluster, ctsUVWXYZ. The reaction conditions for CTS synthesis were examined using 6GT and IMT from B. globisporus C11. The optimum reaction conditions to obtain CTS from Pinedex #100 (partial hydrolyzate of starch, 1.3%-hydrolysis) were the following: substrate concentration, 3%; pH, 6-7; temperature, 30°C; enzyme dosages, 1 U/g-dry solid 6GT, 10 U/g-dry solid IMT. In these optimum conditions the CTS yields reached 62% at the reaction time of 48 h. A mass-production method of highly purified CTS crystals at a reasonable cost was established, and the functions and characteristics of CTS were studied.

Changes in the Structure of Xyloglucan of Apple Fruit during Development

Cell-wall materials (80% methanol-insoluble fraction) of 'Starking Delicious' apple fruit obtained at different developmental stages were separated into water-soluble and water-insoluble polysaccharide fractions. Crude xyloglucans, prepared from both polysaccharide fractions, were hydrolyzed with Geotrichm sp. M128 xyloglucanase. The xyloglucanase- hydrolyzates were subjected to high-performance anion-exchange chromatography with pulsed amperometric detection before and after digestion with Eupenicillium sp. M9 isoprimeverose-producing oligoxyloglucan hydrolase. Both xyloglucan preparations were composed mainly of XXG, XXXG, XXLG, XLXG, XXFG, XLLG and XLFG [where each (1→4)-β-linked D-glucosyl residue in the backbone is given a one-letter code according to its substituents: G=β-D-Glc; X=α-D-Xyl-(1→6)-β-D-Glc; L=β-D-Gal-(1→2)-α-D-Xyl-(1→6)-β-D-Glc; F=α-L-Fuc-(1→2)-β-D-Gal-(1→2)-α-D-Xyl-(1→6)-β-D-Glc]. The ratios of fucose-containing oligosaccharide units (XXFG and XLFG) to fucose-noncontaining oligosaccharide units (XXG, XXXG, XXLG, XLXG and XLLG) in the water-insoluble xyloglucan molecules decreased with fruit development. The molar ratio of a pentasaccharide, XXG, in the water-soluble and -insoluble xyloglucan molecules increased with fruit development.

Synthesis with Glycosynthases: Cello-Oligomers of Isofagomine and a Tetrahydrooxazine as Cellulase Inhibitors

Isofagomine and a carbohydrate-like tetrahydrooxazine, as their N-benzyloxycarbonyl derivatives, have been subjected to a glycosynthase in the presence of α-D-glucopyranosyl fluoride as a glucosyl donor. In each case, after protecting group removal, a mixture of 1,4-β-linked di-, tri-, and tetra-'saccharides' was obtained. These novel oligosaccharide derivatives were tested as inhibitors of the endo-glycanase Cex from Cellulomonasfimi. Affinities increased progressively as additional D-glucosyl residues were incorporated, which is consistent with the known substrate specificity of this enzyme.

Molecular origin for the thermal stability of Koshihikari rice amylopectin

The non-Newtonian behavior and dynamic viscoelasticity of Koshihikari rice amylopectin (waxy type) in aqueous solution were investigated. The flow curves, at 25°C, of Koshihikari amylopectin showed plastic behavior at various concentrations. The viscosity increased slightly with increase in temperature up to 25°C, then it decreased a little with further increase in the temperature at a concentration of 6%. The dynamic modulus showed very large values and stayed constant during increase in temperature at 2.0 and 4.0%, but a little increase of dynamic modulus was observed at 6.0% solution. The dynamic modulus decreased slightly upon addition of urea (4.0 M) or 85% DMSO and decreased gradually with increase in the temperature. The dynamic modulus, however, was lower in 0.10 M NaOH solution than in aqueous solution and decreased rapidly when the temperature reached 45°C, which was estimated to be a transition temperature. The results obtained support the mode of intramolecular associations within rice amylopectin molecules previously proposed. An anomeric hydrogen atom may take part in an intramolecular van der Waals forces of attraction with an adjacent OH-6 or methylene group of d-glucosyl residue.

The Crystal Structure of a Family 5 Endoglucanase Mutant in Complexed and Uncomplexed Forms Reveals an Induced Fit Activation Mechanism

The structures of the Glu140→Gln mutant of Clostridium thermocellumendoglucanase CelC in unliganded form (CelC E140Q) and in complex with cellohexaose (CelC E140Q– Glc 6) have been refined to crystallographic R-factors of 19.4% at 1.9 Å and 17.8% at 2.3 Å resolution, respectively. The structure of CelC E140Q– Glc 6complex shows two D-glucosyl residues bound to the non-reducing end of the substrate-binding cleft. Comparison of the unliganded and complexes structures reveals conformational changes due to substrate binding, including a significant reorientation of the loop 138 – 141 which carries the general acid/base catalyst Glu140 in wild-type CelC. Endoglucanase CelC, a family 5 glycohydrolase, exhibits a (β/α) 8-fold with an additional subdomain of 54 amino acids inserted between β-strand 6 and α-helix 6. Seven amino acid residues (Arg46, His90, Asn139, Glu140, His198, Tyr200, and Glu280) located close to the catalytic reaction center are strictly conserved in family 5 cellulases. Only three of these residues (His90, Gln140 and Glu280) make direct contacts with the substrate, but all participate in a network of hydrogen bonds which contribute to the stability of the active site architecture and may influence the protonation state of the two catalytic residues. Residue Trp313, which interacts with the nucleophile Glu280 and is within hydrogen bonding distance of the substrate, is involved in a non-proline cis-peptide bond. An aromatic residue occurs at an equivalent position in many other (β/α) 8-barrel glycosidases; the presence of a cis-peptide bond at this position in the structures of family 1 β-glucosidases, family 2 β-galactosidases, family 5 cellulases, family 17 β-glucanases, and family 18 chitinases provides further evidence of an evolutionary relationship between glycosyl hydrolases with a (β/α) 8- architecture.

Transition state structures for the hydrolysis of alpha-D-glucopyranosyl fluoride by retaining and inverting reactions of glycosylases.

Secondary tritium and primary 14C kinetic isotope effects were measured for the hydrolysis of alpha-D-glucopyranosyl fluoride catalyzed by sugar beet seed alpha-D-glucosidase, forming alpha-D-glucose, and by Rhizopus niveus glucoamylase forming beta-D-glucose. The data provided a novel opportunity to model and directly compare the transition state structures for the hydrolysis of a substrate promoted with retention or inversion of configuration according to the enzyme catalyst. The isotope effects for the reaction catalyzed by each enzyme are most consistent with an SN1 rather than an SN2 mechanism. The modeled transition state structures for the hydrolysis promoted by the alpha-glucosidase and the glucoamylase both bear significant oxocarbonium ion character, with the D-glucosyl residue having a flattened 4C1 conformation and a C-1-O-5 bond order of 1.92, even though opposite D-glucose anomers were produced from the substrate. The transition states show some modest differences, but their general similarity strongly suggests that the stereochemical outcome of glycosylase reactions does not predict the transition state structure, nor does the transition state structure of such reactions predict the stereochemical outcome. The results support previously reported evidence for the separate topological control of product configuration by protein structures in these and other glycosylases.

Enzymically produced cyclic alpha-1,3-linked and alpha-1,6-linked oligosaccharides of D-glucose.

A new type of bacterial enzyme hydrolyzed alternan (Leuconostoc mesenteroides NRRL B-1355 fraction S dextran, an alternating alpha-1,3-alpha-1,6-D-glucan) to give rise to a series of oligosaccharides. The oligosaccharide formed in the greatest proportion was a cyclic tetrasaccharide of D-glucosyl residues linked in an alternating alpha-1,3-alpha-1,6 fashion. Other saccharide products included isomaltose and alpha-D-glucopyranosyl-1,3-alpha-D-glucopyranosyl-1,6-D-glucose. Oligosaccharides of higher degrees of polymerization were also formed, and included alpha-D-glucosylated derivatives of the cyclic tetrasaccharide. This is the first report of a naturally produced cyclic tetrasaccharide.