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Cyclophellitols are potent inhibitors of exo- and endoglycosidases. Efficient synthetic methodologies are needed to fully capitalize on this intriguing class of mechanism-based enzyme deactivators. We report the synthesis of an orthogonally protected cyclitol from d-glucal (19% yield over 12 steps) and its use in the synthesis of α-(1,3)-linked di- and trisaccharide dextran mimetics. These new glycomimetics may find use as Dextranase inhibitors, and the developed chemistries in widening the palette of glycoprocessing enzyme-targeting glycomimetics.

The design and structural optimisation of a novel polysaccharide-based nanomaterial for the controlled and sustained release of doxorubicin are here reported. A cross-linked polymer was obtained by reacting a tetraglucose, named cyclic nigerosyl-1-6-nigerose (CNN), with pyromellitic dianhydride. The cross-linking reaction formed solid nanoparticles, named nanosponges, able to swell as a function of the pH. Nanoparticle sizes were reduced using High Pressure Homogenization, to obtain uniform nanosuspensions. Doxorubicin was incorporated into the CNN-nanosponges in a good extent. DSC and solid state NMR analyses proved the drug interaction with the polymer matrix. In vitro studies demonstrated pH-dependent slow and prolonged release kinetics of the drug from the nanoformulation. Doxorubicin-loaded CNN-nanosponges were easily internalized in A2780 cell line. They might considered an intracellular doxorubicin reservoir, able to slowly release the drug over time. CNN-nanosponges may be promising biocompatible nanocarriers for the sustained delivery of doxorubicin with potential localised application in cancer treatments.

The susceptibility to glycation of all d-glucose-containing reducing disaccharides (kojibiose, sophorose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, and gentiobiose) was evaluated by Maillard browning and the percentages of their acyclic forms estimated using a novel method to evaluate reactivity toward oxime formation were compared for the first time. This new method is facile and applicable to non-labeled carbohydrates, and it is extremely sensitive, more so than any other previously reported methods. The disaccharides linked by 1-6 bonds displayed both high browning intensity and oxime formation reactivity, and they had the greatest amount of the acyclic form. On the other hand, the proportion of acyclic form was generally very low when glucoses were linked by 1-2, 1-3 and 1-4 bonds. The stability of the 1-3 linkage was drastically reduced when basicity was increased due to β-elimination and the production of a highly reactive dehydrated hexose. The 1-4-linked structures, involved in the formation of amylose and cellulose, respectively, were found to be advantageous due to their relatively low susceptibility to glycation.

The knowledge of carbohydrate composition is greatly important to determine the properties of natural matrices such as foodstuff and food ingredients. However, because of the structural similarity and the multiple isomeric forms of carbohydrates in solution, their analysis is often a complex task. Here we propose an NMR analytical procedure based on highly selective chemical shift filters followed by TOCSY, which allows us to acquire specific background-free signals for each sugar. The method was tested on raw honey samples dissolved in water with no other pretreatment. In total, 22 sugars typically found in honey were quantified: 4 monosaccharides (glucose, fructose, mannose, rhamnose), 11 disaccharides (sucrose, trehalose, turanose, maltose, maltulose, palatinose, melibiose and melezitose, isomaltose, gentiobiose nigerose, and kojibiose), and 7 trisaccharides (raffinose, isomaltotriose, erlose, melezitose, maltotriose, panose, and 1-kestose). Satisfactory results in terms of limit of quantification (0.03-0.4 g/100g honey), precision (% RSD: 0.99-4.03), trueness (bias % 0.4-4.2), and recovery (97-104%) were obtained. An accurate control of the instrumental temperature and of the sample pH endows an optimal chemical shift reproducibility, making the procedure amenable to automation and suitable to routine analysis. While validated on honey, which is one of the most complex natural matrices in terms of saccharides composition, this innovative approach can be easily transferred to other natural matrices.

Functionalized rare sugars were synthesized with 2-, 3-, and 6-tosylated glucose derivatives as acceptor substrates by transglucosylation with sucrose and the glucansucrase GTFR from Streptococcus oralis. The 2- and 3-tosylated glucose derivatives yielded the corresponding 1,6-linked disaccharides (isomaltose analogues), whereas the 6-tosylated glucose derivatives resulted in 1,3-linked disaccharides (nigerose analogue) with high regioselectivity in up to 95 % yield. Docking studies provided insight into the binding mode of the acceptors and suggested two different orientations that were responsible for the change in regioselectivity.

3-O-Substituted reducing aldoses are commonly unstable under heat treatment at neutral and alkaline pH. In this study, to evaluate the decomposition products, nigerose (3-O-α- d -glucopyranosyl- d -glucose) and 3-O-methyl glucose were heated at 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Decomposition via β-elimination was observed that formed a mixture of 3-deoxy-arabino-hexonic acid and 3-deoxy-ribo-hexonic acid; upon further acid treatment, it was converted to their γ-lactones. Similarly, turanose (3-O-α- d -glucopyranosyl- d -fructose), a ketose isomer of nigerose, decomposed more rapidly than nigerose under the same conditions, forming the same products. These findings indicate that 3-O-substituted reducing glucose and fructose decompose via the same 1,2-enediol intermediate. The alkoxycarbonyl elimination of 3-O-substituted reducing glucose and fructose occurs readily if an O-glycosidic bond is located on the carbon adjacent to the 1,2-enediol intermediate. Following these experiments, we proposed a kinetic model for the3- decomposition of nigerose and turanose by heat treatment under neutral pH conditions. The proposed model showed a good fit with the experimental data collected in this study. The rate constant of the decomposition for nigerose was (1.2 ± 0.1) × 10−4 s−1, whereas that for turanose [(2.6 ± 0.2) × 10−4 s−1] was about 2.2 times higher.

The detailed reaction behavior of cellulose in an ionic liquid, 1-ethyl-3-methylimidazolium chloride ([C2mim][Cl]), was investigated. Glucose is a component of cellulose; its oligomerization was studied during treatment with [C2mim][Cl] at around 100 °C. Glucose was polymerized to produce oligosaccharides such as disaccharides and trisaccharides. These oligosaccharides, however, disappeared upon prolonging the treatment time. Maltose, nigerose, kojibiose, laminaribiose, isomaltose, and gentiobiose were identified by gas chromatograph–mass spectrometer in the disaccharides produced. The yield of these disaccharides increased as the initial concentration of glucose in [C2mim][Cl] increased. [C2mim][Cl], therefore, is a reagent capable of producing various oligosaccharides from glucose in the absence of a catalyst.

For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.

Glycoside hydrolase family (GH) 31 enzymes exhibit various substrate specificities, although the majority of members are α-glucosidases. Here, we constructed a heterologous expression system of a GH31 enzyme, Fjoh_4430, from Flavobacterium johnsoniae NBRC 14942, using Escherichia coli, and characterized its enzymatic properties. The enzyme hydrolyzed dextran and pullulan to produce isomaltooligosaccharides and isopanose, respectively. When isomaltose was used as a substrate, the enzyme catalyzed disproportionation to form isomaltooligosaccharides. The enzyme also acted, albeit inefficiently, on p-nitrophenyl α-D-glucopyranoside, and p-nitrophenyl α-isomaltoside was the main product of the reaction. In contrast, Fjoh_4430 did not act on trehalose, kojibiose, nigerose, maltose, maltotriose, or soluble starch. The optimal pH and temperature were pH 6.0 and 60 °C, respectively. Our results indicate that Fjoh_4430 is a novel GH31 dextranase with high transglucosylation activity.

We found an unreported activity of phosphorylase catalyzed by a protein (Cphy1019) belonging to glycoside hydrolase family 65 (GH65) from Clostridium phytofermentans. The recombinant Cphy1019 produced in Escherichia coli did not phosphorolyze α-linked glucobioses, such as trehalose (α1-α1), kojibiose (α1-2), nigerose (α1-3), and maltose (α1-4), which are typical substrates for GH65 enzymes. In reverse phosphorolysis, Cphy1019 utilized only l-rhamnose as the acceptor among various sugars examined with β-d-glucose 1-phosphate as the donor. The reaction product was determined to be 3-O-α-d-glucopyranosyl-l-rhamnose, indicating strict α1-3 regioselectivity. We propose 3-O-α-d-glucopyranosyl-l-rhamnose: phosphate β-d-glucosyltransferase as the systematic name and 3-O-α-d-glucopyranosyl-l-rhamnose phosphorylase as the short name for this novel GH65 phosphorylase.

The transglycosylation reaction of maltodextrin glucosidase (MalZ) cloned and purified from Escherichia coli K12 was characterized and applied to the synthesis of branched oligosaccharides. Purified MalZ preferentially catalyzed the hydrolysis of maltodextrin, gamma-cyclodextrin (CD), and cycloamylose (CA). In addition, when the enzyme was incubated with 5% maltotriose (G3), a series of transfer products were produced. The resulting major transfer products, annotated as T1, T2, and T3, were purified and their structures were determined by TLC, MALDI-TOF/MS, (13)C NMR, and enzymatic analysis. T1 was identified as a novel compound, maltosyl alpha-1,3-maltose, whereas T2 and T3 were determined to be isopanose and maltosyl-alpha-1,6-maltose, respectively. These results indicated that MalZ transferred sugar moiety mainly to C-3 or C-6-OH of glucose of the acceptor molecule. To obtain highly concentrated transfer products, the enzyme was reacted with 10% liquefied cornstarch, and then glucose and maltose were removed by immobilized yeast. The T1 content of the resulting reaction mixture reached 9.0%. The mixture of T1 containing a nigerose moiety can have an immunopotentiating effect on the human body and may be a potential functional sugar stuff.

The infrared spectral characteristics of ten different types of disaccharides (trehalose, kojibiose, nigerose, maltose, isomaltose, trehalulose, sucrose, turanose, maltulose, and palatinose) and five different types of monosaccharides (glucose, mannose, galactose, talose, and fructose) in aqueous solutions (H2O and D2O) were determined. The infrared spectra were collected using the Fourier transform infrared attenuated total reflectance (FT-IR/ATR) method and comparisons between the degrees of absorption band-shift of the saccharide spectra in the H2O solution with those in the D2O solution with respect to the saccharide concentrations were done. The study revealed that the wavenumber shifts in the bands of mono- and disaccharides in the H2O and D2O solutions could be used as an indicator of the level of interaction between the saccharides and water. The study also focused on the glycosidic linkage position and the constituent monosaccharides and found that they have a significant influence on the infrared spectroscopic characterization of disaccharides in an aqueous solution.

A fungus producing an alpha-glucosidase that synthesizes alpha-1,3- and alpha-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65 degres C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitro-phenyl alpha-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing alpha-1,3- and alpha-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-alpha-nigerosyl-D-glucose, and 4-O-alpha-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.

Δ H f values were calculated with the bond- and group-enthalpy method for analogs of 13 disaccharides to learn the relative stabilities for various configurations about the carbon atoms connected to the linkage oxygen atom. The analogs were based on tetrahydropyran and, in the case of the sucrose analogs, tetrahydrofuran. The molecular mechanics program MM3 calculates these values as an option. The method was also used with RHF/6-31G ∗ and B3LYP/6-31G ∗ quantum mechanics (QM) theory. Δ H f values had ranges of 15–17 kcal (all energies herein are molar) by the different methods, with the analogs of the three trehaloses and sucrose having the lowest values. Δ H f values for the isomeric analogs of non-reducing sugars with two anomeric centers are about −150 kcal, about 8 kcal lower than for the analogs of the reducing dimers (nigerose, maltose, laminarabiose, cellobiose and galabiose (α- d -galactosyl- d -galactose)) by all three types of calculation. The analogs of the di-axial, axial–equatorial and di-equatorial, non-glycosidic pseudo-disaccharides had Δ H f values within 1.0 kcal by each of the three methods. They were about 8 kcal higher than for the molecules that contain one glycosidic sequence. The relative Δ H f values by QM were within 1.0 kcal of their corresponding relative electronic energies, except for the methylated sucrose analog, for which the discrepancy was about 1.85 kcal at both QM levels. Compared to our B3LYP results, the RHF-based calculations overestimated the stability of all molecules by about 2.85 kcal. The MM3 values were close to the B3LYP numbers, with the largest discrepancy for the cellobiose analog, 1.76 kcal. The stabilization embodied in the group enthalpy increment for anomeric centers is another manifestation of the anomeric effect.

The immunopotentiating activity of nigerooligosaccharides (NOS), a mixture of nigerose, nigerosyl glucose and nigerosyl maltose, was studied in vitro and in vivo in mice. Mitogen-induced proliferation of splenocytes from normal mice was augmented in a dose-dependent manner by nigerose of NOS. NOS enhanced interleukin 12 (IL-12) and interferon-gamma (IFN-gamma) production by normal splenocytes in the presence of the potent IL-12 inducer, heat-killed Lactobacillus plantarum L-137, in vitro. Consistent with the in vitro finding, L. plantarum L-137-induced IL-12 production and IL-2-induced IFN-gamma production were augmented in mice fed with a 14.6% NOS diet for 2 weeks compared with mice fed with a control diet. Notably, mice fed with the NOS diet showed significantly longer survival time than the control mice after the induction of an endogenous infection by administering 5-fluorouracil in a lethal dose. Taken together, these results suggest that NOS may exert immunopotentiating activity through the activation of an IL-12-dependent T helper 1-like immune response.

From a complex mixture of mono- and di-2-anthracenecarboxylic acid (AC) esters of cyclic nigerosylnigerose (CNN), two monoesters (2(B) and 6(A)) and four diesters in which AC was introduced on the transannular B/D (2(B)2(D)), adjacent A/B and A/D (6(A)2(B) and 6(A)2(D)), and same B/B (2(B)3(B)) nigerose rings were isolated. Possessing two ACs at distant positions, 2(B)2(D) and 6(A)2(D) showed negative Cotton effects for the (1)Bb band, the intensities of which were stronger than that of 6(A). 2(B)2(D) and 6(A)2(D) slowly photocyclodimerized to give HH dimers 3* and 4 with 57% and 81% HH selectivity, respectively, which were appreciably higher than that for 6(A) (34%), while the enantiomeric excesses (ee's) of anti-HH dimer 3* were 2% and -18%, respectively. In contrast, 6(A)2(B) and 2(B)3(B) carrying two ACs on adjacent A and B rings or at vicinal positions on the B ring, respectively, exhibited strong positive CD couplets, the amplitudes of which amounted to 97 and 409 M(-1) cm(-1), respectively. Upon irradiation, 6(A)2(B) afforded 3* with -62% ee and 4 in 96% combined yield, whereas 2(B)3(B) gave almost exclusively 3* with -99% ee in 96% yield, likely as a result of the introduction of two ACs at the vicinal positions of the rigid CNN scaffold.

Substrate specificity and subsite affinities of high pl α-glucosidase from germinated barley ( Hordeum vulgare L.) seeds were investigated by kinetics. The enzyme has only one maltose binding site per molecule and shows high activity on small maltooligosaccharides and nigerose. Hydrolysis of isomaltose and p -nitrophenyl α-glucoside is moderate. Trehalose is not hydrolyzed at detectable rates. The ratios of the maximum velocities for maltose, nigerose, isomaltose, p -nitrophenyl α-glucoside and malto-triose, -tetraose, -pentaose, -hexaose, -heptaose are 100:95:21:9:111:116:119:104:111. The K m values for these substrates are 1.91, 1.29, 5.32, 1.04, 1.11, 2.37, 2.92, 5.44 and 7.89 mM, respectively. Based on the rate parameters for maltooligosaccharides, the subsite affinities ( A i s) in the active site of the enzyme were evaluated according to subsite theory. Subsites 1, 2 and 3, having positive A i values ( A 1 , A 2 and A 3 ; 1.34, 5.37 and 0.27 kcal/mol, respectively), were considered to be effective for the binding of substrate to the active site. The different arrangement of subsite affinities among α-glucosidases, glucoamylases and amylases was used to explain their substrate specificities.

Five α-linked diglucosyl disaccharides (isomaltose, nigerose, kojibiose, maltose, and α,β-trehalose) and two trisaccharides (isomaltotriose and panose) were produced in 90% aqueous solutions of 2-methoxyethyl ether, 2-ethoxyethyl ether, or triethylene glycol dimethyl ether by glucoamylase-catalyzed glucose condensation. Precipitation of the products and phase separation caused higher net production of disaccharides than expected by comparison with the same reactions occurring in solvent-free solutions. Glucoamylase was equally stable in water and in aqueous 90% 2-methoxyethyl ether, but was stabilized by further decreases of water concentration. Aqueous ethers neither modified the product specificity of glucoamylase nor changed the relative order of initial rates of disaccharide formation. Because of its broad specificity (five disaccharides and two trisaccharides), glucoamylase would not be a suitable biocatalyst for oligosaccharide synthesis.

The substrate specificity of honeybee α-glucosidase II, a monomeric protein, was investigated in detail. The enzyme hydrolyzed phenyl α-glucoside and p-nitrophenyl α-glucoside more rapidly than maltooligosaccharides. Unusual kinetics were observed in hydrolysis of sucrose, turanose, kojibiose, and soluble starch. The s versus v plots showed sigmoidal curves, and so the Lineweaver-Burk plots became concave, indicating positive kinetic cooperativity. The Hill coefficients for sucrose, turanose, kojibiose, and soluble starch were calculated to be 1.46, 1.34, 1.15, and 1.28, respectively. The Km values for these substrates were calculated as the concentration at one half of the maximum velocity. The ratios of the maximum velocities for maltose, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose, maltodextrin ( = 13), kojibiose, nigerose, isomaltose, sucrose, turanose, phenyl α-glucoside, p-nitrophenyl α-glucoside, phenyl α-maltoside, and soluble starch were estimated to be 100:163:159:156:149:144:113:96....

An enzyme hydrolyzing nigeran (alternating alpha-1,3- and alpha-1,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by percipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50 degrees C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50 degrees C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the alpha-1,4-glucosidic linkages in nigeran.

An extracellular α-glucosidase was purified from the culture broth of Paecilomyces varioti AHU 9417 by a combination of precipitation with ethanol, chromatography on DEAE-Sepharose CL-6B and Bio-Gel P-150, and preparative gel electrophoresis. The enzyme migrated as a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 100,000 by SDS-disc electrophoresis, and the optimum pH of activity was 4.5. The enzyme had a wide substrate specificity, as it rapidly hydrolysed maltooligosaccharides and isomaltooligosaccharides. The ratios of Vmax for maltose, isomaltose, kojibiose, nigerose, phenyl α-glucoside, maltotriose, isomaltotriose, panose, phenyl α-maltoside, and soluble starch were estimated to be 100, 72, 31, 105, 9.6, 103, 73, 47, 115, and 66, and the Km (mM of nonreducing terminal) for these substrates were 0.40, 2.4, 1.6, 2.5, 0.23, 0.33, 4.6, 0.67, 0.24, and 4.7 mM, respectively. The enzyme is characterized by a high activity toward isomaltose.

Eight monosaccharides ( l -arabinose, l -fucose, d -galactose, d -glucose, d -lyxose, d -mannose, l -rhamnose, and d -xylose), eight disaccharides (cellobiose, gentiobiose, isomaltose, lactose, maltose, nigerose, sophorose, and xylobiose), and three trisaccharides (isomaltotriose, maltotriose, and xylotriose) were derivatized with N-methylbis-(trifluoroacetamide) in pyridine solution to form trifluoroacetylated derivatives. These were analyzed by 1H-n.m.r. spectroscopy to determine the characteristics of the spectra and distributions of the reaction products. Peaks corresponding to reducing-residue anomeric protons were located significantly downfield of all others, and were in general 0.4 p.p.m. or more downfield of equivalent signals from the same carbohydrates when they were free or derivatized with other groups. Neither the location of anomeric proton peaks relative to each other nor the degree of spin-spin coupling between H-1 and H-2 varied greatly with type of derivatization. Spin-spin coupling, however, decreased for some β-pyranose forms of xylobiose and the three trisaccharides. In all examples except some where H-2 was oriented equatorially to a pyranose ring, the proportion of the α-pyranose was either enhanced or not changed in concentration by trifluoroacetylation.

The structural features of carbohydrates are a combination of 1) sequence and types of mono-sugars, 2) stereochemistry of their glycosidic linkages, and 3) their glycosidic linkage sites. We performed the first systematic VCD study on glycoside linking site discrimination. VCD spectra, in the CH stretching region from 2000 to 4000 cm(-1), of eleven glucobioses (trehalose (alpha1-alpha1), neotrehalose (alpha1-beta1), isotrehalose (beta1-beta1), kojibiose (alpha1-2), nigerose (alpha1-3), maltose (alpha1-4), isomaltose (alpha1-6), sophorose (beta1-2), laminaribiose (beta1-3), cellobiose (beta1-4), gentiobiose (beta1-6)) suggested a possible new discrimination method for glyco analysis, while VCD spectra in the mid-IR region distinguished the stereochemistry (alpha or beta) of the glycosidic linkage. Both reducing and nonreducing glucobioses showed different VCD spectral features compared to their constituent D-glucose and the anomer-fixed model compounds. Interresidue interaction such as hydrogen bonding was suggested to cause these spectral differences. Interplay between residues is a common phenomenon and thus VCD analysis could be applicable to other di-, oligo- or poly-saccharides. Several isotropic labeled compounds were also measured to support spectral assignment and interpretation.

In a previous study (Oku, K.; Watanabe, H.; Kubota, M.; Fukuda, S.; Kurimoto, M.; Tujisaka, Y.; Komori, M.; Inoue, Y.; Sakurai, M. J. Am. Chem. Soc. 2003, 125, 12739), we investigated the mechanism of the antioxidant function of trehalose against unsaturated fatty acids (UFAs) and revealed that the key factor relevant to the function is the formation of OH...pi and CH...O hydrogen bonds between trehalose and the cis double bonds of the UFA. Here, we investigate whether such intriguing interactions also occur between this sugar and cis double bonds in other unsaturated compounds. For this purpose, we selected various diene compounds (1,3-butadiene, 1,3-pentadiene, 1,4-pentadiene, and 2,5-heptadiene) as interaction partners. All NMR experiments performed, including 1H-1H NOESY measurements, indicated that trehalose selectively interacts with the cis-olefin proton pair in the above diene with a 1:1 stoichiometry, and the C-3 (C-3') and C-6' (C-6) sites of the sugar are responsible for the interaction. Similar interactions were not observed for the mixtures of the diene and other saccharides (neotrehalose, kojibiose, nigerose, maltose, isomaltose, sucrose, maltitol, and sorbitol). Quantum chemical calculations revealed that the OH-3 and OH-6 groups bind to the olefin double bonds of the diene through OH...pi and CH...O types of hydrogen bonds, respectively, and the stabilization energy of the resulting complex is 5-6 kcal mol(-1). These results strongly support the above NMR results. Finally, the activation energies were calculated for the hydrogen abstraction reactions from the activated methylene group of heptadiene. In particular, when the reaction was initiated by a methyl radical, the activation energy was only 10 kcal mol(-1) for the free heptadiene, but on complexation with trehalose it drastically increased to ca. 40 kcal mol(-1). This indicates that trehalose has a significant depression effect on the oxidation of the diene compounds. These results strongly support the antioxidant mechanism deduced in the previous study and indicate that the formation of unique multiple hydrogen bonds between trehalose and cis-olefin bonds is rather a general event not confined to the case of UFA.

Chimeric phosphorylases were constructed of the kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.

Nigerose and nigerooligosaccharides served as acceptors for a glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus to give a series of homologous acceptor products. The soluble oligosaccharides (dp 5-9) strongly activated the acceptor reaction, resulting in the accumulation of water-insoluble (1-->3)-alpha-D-glucan. The enzyme transferred the labeled glucosyl residue from D-[U-13C]sucrose to the 3-hydroxyl group at the non-reducing end of the (1-->3)-alpha-D-oligosaccharides, as unequivocally shown by NMR 13C-13C coupling patterns. The values of the 13C-13C one-bond coupling constant (1J) are also presented for the C-1-C-6 of the 13C-labeled alpha-(1-->3)-linked glucosyl residue and of the non-reducing-end residue.

A strain of Acremonium sp. S4G13, which produced an enzyme producing nigerose, nigerosyl glucose, and nigerosyl maltose from maltooligosaccharides in high yield, was isolated from soil. From these observations, the enzyme was strongly supposed to be a novel enzyme that hydrolyzed maltooligosaccharides and released glucose, but also transferred the glucosyl moiety into the C-3 or C-4 position of the acceptor molecule by alpha-form in significant amounts.

When Mucor javanicus was cultured with castanospermine, two alpha-glucosidases (CS-I, II) were produced. The two enzymes were purified by two CM-cellulofine column chromatographies, and compared with the alpha-glucosidase (C) from a culture without castanospermine. The molecular weights of the three enzymes were calculated to be 97,000 (CS-I, II) and 94,000 (C) from SDS-PAGE. CS-II was similar in hydrolysis of alpha-1,4-linkage to C, but hydrolyzed alpha-1,2-, alpha-1,3-, and alpha-1,6-linkages at a lower rate than C. The Michaelis constant of CS-I for malto-oligosaccharides was several times as high as the constants of CS-II and C, but CS-I hydrolyzed nigerose at a faster rate than maltose.

The Glcalpha(1--3)Glcalpha(1--3)Manalpha(1--2)Man tetrasaccharide (Glc(2)Man(2)-fragment), a substructure of the natural N-glycan precursor, was synthesized. The interaction of this fragment with the protein malectin, a carbohydrate binding protein localized in the endoplasmatic reticulum, was investigated by (1)H(15)N HSQC experiments and isothermal calorimetry. The chemical shift perturbations of nuclei in the protein's backbone caused by the binding of the Glc(2)Man(2)-fragment to malectin suggest a binding mode like the known ligand nigerose.

Energy surfaces were computed for relative orientations of the relaxed pyranosyl rings of the two anomeric forms of kojibiose, nigerose, and maltose, the (1----2)-alpha, (1----3)-alpha, and (1----4)-alpha-linked D-glucosyl disaccharides, respectively. Twenty-four combinations of starting conformations of the rotatable side-groups were considered for each disaccharide. Optimized structures were calculated using MM3 on a 20 degree grid spacing of the torsional angles about the glycosidic bonds. The energy surfaces of the six disaccharides were similar in many respects but differed in detail within the low-energy regions. The maps also illustrate the importance of the exo-anomeric effect and linkage type in determining the conformational flexibility of disaccharides. Torsional conformations of known crystal structures of maltosyl-containing molecules lie in a lower MM3 energy range than previously reported.

Nigerose [alpha-D-Glcp-(1----3)-D-Glcp], nigerotriose, nigerotetraose, and nigeropentaose have been synthesized by chain elongation starting at the reducing end, from the corresponding octa-, undeca-, tetradeca-, and heptadeca-beta-D-acetates, respectively, via thioglycoside-mediated 1,2-cis coupling, using 1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose as the glucosyl acceptor and methyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside, methyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside, and methyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1----3)-2,4,6-tri-O- benzyl-1-thio-beta-D-glucopyranoside as the donors.

A new α- D -glucan, designated elsinan, has been isolated from the culture filtrate of Elsinoe leucospila grown in potato extract-sucrose medium. Acid hydrolysis of the methylated polysaccharide gave 2,3,6- and 2,4,6-tri- O -methyl- D -glucose, in the ratio of 2.5:1.0, together with small proportions of 2,3,4,6-tetra- (0.7%) and 2,4-di- O -methyl- D -glucose (0.5%), indicating that the glucan is an essentially linear polymer containing (1→4)- and (1→3)-α- D -glucosidic linkages. Periodate oxidation, followed by borohydride reduction and mild hydrolysis with acid (mild Smith degradation) yielded 2- O -α- D -glucosyl- D -erythritol and erythritol, in the molar ratio of 1.0:1.4, and a trace of glycerol. Partial acid hydrolysis, and also acetolysis, of elsinan gave nigerose, maltose, O -α- D -glucopyranosyl-(1→3)- O -α- D -glucopyranosyl (1→4)- D -glucopyranose, O -α- D -glucopyranosyl-(1→4)- O -α- D -glucopyranosyl-(1→3)- D -glucopyranose, maltotriose, and a small proportion of maltotetraose. It is concluded that elsinan is composed mainly of maltotriose residues joined by α-(1→3)-linkages, in the sequence →3)-α- D -Gl cp -(1→4)-α- D -Gl cp -(1→.The unique structural features of elsinan are discussed in comparison with other glucans.

Piricularia oryzae, the pathogenic fungus of rice blast disease, produced a glucoamylase as a sole amylolytic enzyme with little concomitant activity of other related enzymes. The purified enzyme preparation showed a single band on electrophoresis. The enzyme had optimum pH at 6.5 and optimum temperature between 50 and 55°C. It was completely inactivated by heating for 15 min at 60°C. The molecular weight of the enzyme was estimated as about 94,000. The enzyme hydrolyzed various kinds of amylaceous substrates and some malto-oligosaccharides into glucose. Glucobioses such as isomaltose, nigerose, kojibiose, α,α-trehalose, β,β-trehalose as well as phenyl-α-glucoside were also hydrolyzed.

A p -nitrophenyl α- d -glucopyranoside-hydrolyzing α-glucosidase of an obligate thermophile, Bacillus stearothermophilus ATCC 12016, was purified to an electrophoretically and immunologically homogeneous state. The specific activity was 113 μmol p -nitrophenyl α- d -glucopyranoside hydrolyzed/min per mg protein at 60°C and pH 6.8. The molecular weight, Stokes radius, sedimentation coefficient (s 20,w ), extinction coefficient at 280 nm and pH 6.8, and isoelectric point were estimated as 47 000, 3.06 nm, 3.98 S, 2.88 cm 2 · mg −1 and 5.2, respectively. The NH 2 -terminal amino acid of the enzyme was menthionine. Neither carbohydrate nor cysteine residue was present in the enzyme. The pH and temperature optima for activity were 6.3 and 70°C, respectively. The K m for the nitrophenyl glucoside was 0.63 mM. The enzyme produced α-glucose by the cleavage of single glucose units from the non-reducing terminal α-1,4-bonds of maltosaccharides (chain length = 2–6 glucose units), α-limit dextrins, phenyl α- d -maltoside, dextrin, amylose, amylopectin and soluble starch. The α-1,6-bonds in these saccharides, were not attacked. Sucrose, turanose and melezitose were weakly hydrolyzed, while there was no activity towards the α-glucosidic linkages in methyl α- d -glucoside, nigerose, kojibiose, panose, β-limit dextrins, isomaltosaccharides (2–6 glucose units), α- and β-cyclodextrins, pullulan and dextran. The greatly restricted substrate specificity proposed the assignment of the enzyme to an exo-α-1,4-glucosidase. Other catalytic properties, along with amino acid composition, are described in this report.

Near-homogeneous forms of glucoamylases I and II, previously purified from an industrial Aspergillus niger preparation, were incubated with D-glucose at a number of temperatures and pH values. Kinetics and equilibria of the formation of alpha,beta-trehalose, kojibiose, nigerose, maltose, isomaltose, panose, and isomaltotriose, which with isomaltotetraose were the only products formed, were determined. There was no difference in the abilities of GA I and GA II to form these products. Activation energies for the formation of maltose and panose were lower than those of the other Oligosaccharides. Relative rates of oligosaccharide production based on glucoamylase hydrolytic activity did not vary significantly between pH 3.5 and 4.5 but were lower at pH 5.5. Maltose was formed much faster than any other product. Equilibrium concentrations at higher dissolved solids concentrations decreased in the order isomaltose, isomaltotriose, kojibiose, nigerose, maltose, alpha, beta-Mrehalose, panose, and isomaltotetraose. They were not appreciably affected by changes in temperature or pH. A kinetic model based on adsorption of D-glucose and the seven di- and trisaccharides by the first three glucoamylase subsites was formulated. Oligosaccharide formation was simulated with the model, using equilibrium data gathered for this article and subsite binding energies and kinetic parameters for oligosaccharide hydrolysis measured earlier. Agreement of simulated and actual oligosaccharide formation data through the course of the reaction was excellent except at very high solid concentrations.

A transglucosidase, an α- d -glucosidase with a high transferase activity, has been purified to homogeneity from culture broths of A. niger. The enzyme, which gave a single protein band on SDS-gel electrophoresis (mol. wt. 116,000) and two protein bands on isoelectric focusing (pI values 5.1 and 5.0), is a glycoprotein, containing 27.6% of carbohydrate, most of which is mannose, has an optimal pH of 4.0–4.5, and is stable in the pH range 4.0–6.0 and at m ) for the following substrates were obtained: maltose (73.5, 1.7), isomaltose (31.9, 7.7), nigerose (63.6, 20.0), kojibiose (22.1, 3.6), phenyl α- d -glucopyranoside (2.5, 2.4), p-nitrophenyl α- d -glucopyranoside (2.1, 0.7), and methyl α- d -glucopyranoside (1.0, 26.3). The effect of the enzyme on the conversion, by amyloglucosidase, of wort saccharides into glucose has been quantified. A procedure, based on the use of phenyl α- d -glucopyranoside as substrate, is recommended for the assay of this enzyme in crude culture filtrates.

The acceptor reaction of dextransucrase consists of the transfer of d -glucosyl groups from sucrose to other carbohydrates, and occurs at the expense of dextran synthesis. In the present study, solutions of [ 14 C]sucrose and of each of seventeen acceptor sugars were digested with highly purified Leuconostoc mesenteroides B-512F dextransucrase. The products were separated by paper chromatography, and quantitated by liquid scintillation counting. Maltose was the most effective acceptor; its products, members of an isomaltodextrinyl-maltose series (d.p. 3 to 6), accounted for >75% of the d -glucosyl groups of sucrose. Other acceptors giving rise to a similar series of oligosaccharide products were (in order of decreasing effectiveness): isomaltose, nigerose, methyl α- d -glucoside, 1,5-anhydro- d -glucitol, d -glucose, turanose, methyl β- d -glucoside, cellobiose, and l -sorbose. Lactose, raffinose, melibiose, d -galactose, and d -xylose each gave a single, mono- d -glucosylated product; d -fructose and d -mannose each gave a pair of mono- d -glucosylated (disaccharide) products. Another series of digests contained sucrose and various proportions of maltose. As the level of maltose increased, the size of the largest oligosaccharide acceptor-product decreased, and less dextran was produced. The virtual absence of high-d.p. (8 to 13) oligosaccharide products in all acceptor digests is interpreted as evidence against a role for acceptors as primers of dextran synthesis.

Fragmentation by controlled acetolysis of an alkali-soluble d -glucan, isolated from the fruit body of Laetiporus sulphureus , followed by fractionation of the products on a column of silica gel, provide a simple, preparative approach to the peracetates of 3- O -α- d -glucopyranosyl- d -glucose (nigerose) and of O -α- d -glucopyranosyl-(1→3)- O -α- d -glucopyranosyl-(1→3)- d -glucose (nigerotriose). The synthesis of methyl β-, benzyl β-, phenyl α-, and phenyl β-nigeroside, and of methyl β-nigerotrioside is described.

a-Glucosidases which are essentially hydrolase have been found to catalyze also the trans-glucosylation which means the transfer action on the acceptors other than water. The action patterns of some a-glucosidases were compared with one another. Sacch. logos a-glucosidase, an a-1, 6-transglucosylase, showed the transfer activity of a glucosyl moiety to glucose and maltose to form isomaltose and panose mainly. Honey bee a-glucosidase, an a-1, 4-transglucosylase, produced maltotriose and phenyl-a-maltoside as the major products from maltose and phenyl-a-glucoside, respectively. However, buckwheat a-glucosidase, an a-1, 2-, a-1, 3- and a-1, 4-transglucosylase, catalyzed the predominant formation of kojibiose, nigerose and maltose from not only maltose but also soluble starch. Isomaltose was the minor product by buckwheat a-glucosidase. Such a multiplicity The anomalous reaction of honey bee a-glucosidase I on phenyl-a-glucoside was demonstrated, that is, the appreciable activation of only the transfer action depending on the concentration of substrate. A probable mechanism of the reaction of a-glucosidase was proposed, by which the hydrolysis and the transglucosylation were explained.

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [ 14 C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [ 14 C]sucrose. After extensive washings with buffer, the 14 C label remained attached to BGD. This labeled material was previously shown to be [ 14 C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [ 14 C]dextran whereas essentially no [ 14 C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [ 14 C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [ 14 C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C 3 -OH on an acceptor dextran acts as a nucleophile on C 1 of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.

A gas-liquid chromatographic method was examined in order to determine kojibiose and maltose in their mixture. The two disaccharides proved to be determined by this method, in which they were estimated from the ratio between the heights of the peaks on the gas-liquid chromatogram of α-maltose and α-kojibiose. The amounts of disaccharides produced from soluble starch through the transglucosylation of buckwheat α-glucosidase were quantitatively determined for the first time by a combination of a gas-liquid and a paper chromatographic methods. In the initial stage of the transglucosylation of the α-glucosidase, kojibiose, nigerose and maltose were remarkably formed, and isomaltose was the minor product. However, isomaltose was gradually accumulated, and other disaccharides disappeared rapidly with the elapse of reaction time.

The formation of 1,6-anhydro-β- d -glucopyranose and several d -glucosyl oligosaccharides has been observed during the action of a purified, fungal glucosyltransferase (EC 2.4.1.24) on maltose. Such products are synthesized by a transglucosylation mechanism involving the formation of a d -glucosyl-enzyme complex and the displacement of the d -glucosyl group by appropriate acceptor-substrates. The formation of the 1,6-anhydro bond is a novel type of transfer reaction and occurs by displacement of the enzyme from the d -glucosyl-enzyme complex by the proton of the primary hydroxyl group of the same glucosyl group. This reaction is characterized by inversion of configuration at the position of glucosidic bond-cleavage of the substrate. Synthesis of the d -glucosyl oligosaccharides occurs by displacement of the d -glucosyl groups from the enzyme by suitable acceptor-substrates. In these cases, the reactions are characterized by retention of configuration of the d -glucosidic bonds of the substrate. The list of oligosaccharides produced from maltose includes nigerose, kojibiose, isomaltose, maltotriose, panose, isomaltotriose, and 6- O - d -glucosyl-panose. The identity of these compounds has been established by methylation analysis and enzymic hydrolysis. d -Glucose is also a product of the reaction and arises from both the reducing and the non-reducing groups of maltose.

Extracellular glucansucrases from various bacterial sources, including Leuconostoc mesenteroides, have been shown to catalyze the transfer of glucosyl groups from sucrose to low-molecular-weight acceptor sugars, forming a series of oligosaccharides. An extracellular glucansucrase recently isolated from Leuconostoc mesenteroides NRRL B-1355 synthesizes a polysaccharide consisting of alternating α-(1→6)- and α-(1→3)-linked d-glucose residues. We have found that this enzyme preparation is capable of forming both α-(1→6)- and α-(1→3)-linked acceptor-products in the presence of a number of low-molecular-weight acceptor sugars. d-Glucose gave isomaltose only, no nigerose being formed. Similarly, methyl α-d-glucoside, methyl β-d-glucoside, maltose, and nigerose gave methyl α-isomaltoside, methyl β-isomaltoside, panose, and 62-O-α-d-glucosylnigerose, respectively. However, isomaltose gave both isomaltotriose and 32-O-α-d-glucosylisomaltose. These initial acceptor-products may also act as acceptors, and the structures of the products of higher d.p. show that a (1→3)-α-d-glucosidic bond is formed only when the nonreducing glucose acceptor group is linked through an α-(1→6) bond to another glucose residue. Experiments using [14C]sucrose and unlabeled acceptors showed that acceptor reactions occur by transfer of a glucosyl group from sucrose to O-3 or O-6 of the glucosyl group at the nonreducing end of the acceptor saccharide. If a “good” acceptor is defined as one that gives rise to a greater amount of oligosaccharide and less polysaccharide, then the acceptors tested may be listed as follows: maltose ≥ nigerose > methyl α-d-glucoside > isomaltose >d-glucose > methyl β-d-glucoside.

The relative retention times of seventeen trimethylsilyl disaccharides (cellobiose, gentiobiose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose, maltulose, melibiose, nigerose, palatinose, sophorose, sucrose, αα-trehalose, turanose and xylobiose) on a fused-silica capillary column coated with SE-54 are reported. Equilibrium anomeric compositions of fourteen of these disaccharides in pyridine are also given. Isothermal operation at 240°C allowed complete quantitation of the mixture.

Cladosporium resinae (1→3)-α-D-glucanase has been characterized as an endoglucanase capable of completely hydrolysing insoluble (1→3)-α-D-glucans isolated from fungal cell-walls. D-Glucose was the major product, but a small amount of nigerose was also produced. The enzyme was specific for the hydrolysis of (1→3) bonds that occur in sequence, and nigerotetraose was the smallest substrate that was rapidly attacked. Isolated (1→3)-α-D-glucosidic linkages that occur in mycodextran, isolichenin, dextrans, and oligosaccharides derived from dextran were not hydrolysed. Insoluble glucans synthesised from sucrose by culture filtrates of Streptococcus spp. were all hydrolysed to various limits; the range was 11-61%. A soluble glucan, synthesised by an extracellular D-glucosyltransferase of S. mutans OMZ176, was not a substrate, whereas insoluble glucans synthesised by a different D-glucosyltransferase, isolated from S. mutans strains OMZ176 and K1-R, were extensively hydrolysed (84 and 92%, respectively). It is suggested that dextranase-CB, a bacterial endo(1→6)-α-D-glucanase that does not release D-glucose from any substrate, could be used together with C. resinae (1→3)-α-D-glucanase to determine the relative proportions of(1→6)-linked to (1→3)-linked sequences of D-glucose residues in the insoluble glucans produced by oral streptococci. The simultaneous action of the two D-glucanases was highly effective in solubilizing the glucans.

Cell walls of the Basidiomycete fungus Polyporus tumulosus (Cooke) were fractionated, and the polysaccharide content of the fractions investigated. The major constituents of the cell wall include four polysaccharides, chitin, a β-1, 3-glucan and the alkali soluble α-glucan and xylomannan. The glucan is highly dextrotatory with an [α] D 2 1 of + 221° and gave on partial acid hydrolysis and acetolysis an homologous series of oligosaccharides. The disaccharide was shown to be nigerose 3-0-α-D-glucopyranosyl-D-glucose. Periodate oxidation and methylation studies provided supporting evidence that the polysaccharide is an essentially unbranched polymer of 1,3-linked glucose residues. The other alkali-soluble polysaccharide, a xylomannan, is a polymer of mannose and xylose in the approximate molar proportions of 1.2:1. It has an [ α ] D = + 56° and on partial acid hydrolysis and acetolysis gave an homologous series of 1,3-linked mannodextrins but no oligosaccharides containing xylose were obtained. An α-1,3-linked mannan was prepared from the xylomannan by degradation with mild acid or by degradation of the periodate-oxidased and reduced xylomannan. The structure therefore is visualised as having a backbone of 1,3-linked mannan, to which xylose residues are attached. Methylation studies showed that branching occurs at C-4 of the mannopyranose units; the presence of 2,3-di- o -methyl- d -xylose in the hydrolysate of the methylated polysaccharide indicated that some of the xylose residues are 1,4-linked. The possible structure of the fungal cell wall is discussed in the light of the results obtained.