Tuning the catalytic performances of a sucrose isomerase for production of isomaltulose with high concentration.

Obtaining a sucrose isomerase (SIase) with high catalytic performance is of great importance in industrial production of isomaltulose (a reducing sugar). In order to obtain such SIase mutant, a high-throughput screening system in microtiter plate format was developed based on a widely used 2,4-dinitrosalicylic acid (DNS) method for determination of reducing sugar. An SIase from Erwinia sp. Ejp617 (ErSIase) was selected to improve its catalytic efficiency. After screening of ~ 8000 mutants from a random mutagenesis library, Q209 and R456 were identified as beneficial positions. Saturation mutagenesis of the two positions resulted in a double-site mutant ErSIase_Q209S-R456H that showed the highest catalytic efficiency, and its specific activity reached 684 U/mg that is 17.5-fold higher than that of the wild-type ErSIase. By employing the lyophilized Escherichia coli (E. coli) cells harboring ErSIase_Q209S-R456H, a high space-time yield (STY = 3.9 kg/(L·d)) was achieved toward 600 g/L sucrose. Furthermore, the in silico analysis suggested that the hydrogen bond network was improved and steric hindrance was reduced due to the beneficial substitutions.Key points• A sucrose isomerase mutant with high catalytic efficiency was obtained.• The highest space-time yield was achieved toward high-concentration sucrose.• The optimized H-bond network contributed to the enhanced catalytic efficiency.

Simple and efficient preparation of high-purity trehalulose from the waste syrup of isomaltulose production using solid-phase extraction followed by hydrophilic interaction chromatography.

A simple and efficient method was developed for the preparation of high-purity trehalulose from the waste syrup of isomaltulose production. The waste syrup was pre-treated with C18 solid-phase extraction, where 98% decolorization and 97% reducing sugar recovery were obtained, followed by hydrophilic interaction liquid chromatography separation on a cysteine-bonded zwitterionic column. Under optimized conditions, trehalulose was separated from isomaltulose isomer and prepared on a semi-preparative scale with >99% purity. The structure of the prepared trehalulose was subsequently confirmed by nuclear magnetic resonance, and three tautomers of trehalulose (α-D-glucosylpyranosyl-1,1-ß-D-fructopyranose, α-D-glucosylpyranosyl-1,1-ß-D-fructofuranose, and α-D-glucosylpyranosyl-1,1-α-D-fructofuranose) were detected and completely characterized by 13 C NMR spectroscopy for the first time in this study. The tautomerization of α-D and ß-D type transition was observed by hydrophilic interaction liquid chromatography on an AdvanceBio Glycan Mapping column, with smaller particle size (2.7 µm). Furthermore, the prepared trehalulose was applied as a standard for trehalulose quantification during the sucrose conversion by Klebsiella sp. LX3. The combination of solid-phase extraction and hydrophilic interaction liquid chromatography offers a new avenue for the preparation of sugar isomers from complex natural or fermentation products.

Characterization of a recombinant sucrose isomerase and its application to enzymatic production of isomaltulose.

OBJECTIVE: To characterize a recombinant isomerase that can catalyze the isomerization of sucrose into isomaltulose and investigate its application for the enzymatic production of isomaltulose. RESULTS: A sucrose isomerase gene from Erwinia sp. Ejp617 was synthesized and expressed in Escherichia coli BL21(DE3). The enzymatic characterization revealed that the optimal pH and temperature of the purified sucrose isomerase were 6.0 and 40 °C, respectively. The enzyme activity was slightly activated by Mn2+and Mg2+, but partially inhibited by Ca2+, Ba2+, Cu2+, Zn2+ and EDTA. The kinetic parameters of Km and Vmax for sucrose were 69.28 mM and 118.87 U/mg, respectively. The time course showed that 240.9 g/L of isomaltulose was produced from 300 g/L of sucrose, and the yield reached 80.3% after bioreaction for 180 min. CONCLUSIONS: This recombinant enzyme showed excellent capability for biotransforming sucrose to isomaltulose at the substrate concentration of 300 g/L. Further investigations should be carried out focusing on selection of suitable heterologous expression system with the aim to improve its expression level.

Economical production of isomaltulose from agricultural residues in a system with sucrose isomerase displayed on Bacillus subtilis spores.

A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background.

Sucrose isomers as alternative sweeteners: properties, production, and applications.

In the daily diet, sweeteners play an indispensable role. Among them, sucrose, a widely occurring disaccharide in nature, is a commonly used sweetener. However, the intake of sucrose can cause a rapid increase in blood glucose, which leads to a number of health problems. Therefore, there is an urgent need for possible alternatives to sucrose. Currently, four naturally occurring sucrose isomers, trehalulose, turanose, leucrose, and isomaltulose are considered to be possible alternatives to sucrose due to their suitable sweetness, potential physiological benefits, and feasible production processes. This review covers the properties of these alternative sweeteners, including their structure, sweetness, hydrolysis rate, toxicology, and cariogenicity, and exhibits their potential applications in chronic diseases management, anti-inflammatory supplement, prebiotic dietary supplement, and stabilizing agent. The biosynthesis of these sucrose isomers using carbohydrate-active enzymes and their industrial production processes are also systematically summarized.

Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose⁻Oligosaccharide Synthesis.

Isomaltose-oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 °C, and the half-life was 84 h at 60 °C, whereas the enzyme was active over the range of pH 6.0-10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl ß-D-glucopyranoside (pNPG) as substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 °C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.

Production of keto-disaccharides from aldo-disaccharides in subcritical aqueous ethanol.

Isomerization of disaccharides (maltose, isomaltose, cellobiose, lactose, melibiose, palatinose, sucrose, and trehalose) was investigated in subcritical aqueous ethanol. A marked increase in the isomerization of aldo-disaccharides to keto-disaccharides was noted and their hydrolytic reactions were suppressed with increasing ethanol concentration. Under any study condition, the maximum yield of keto-disaccharides produced from aldo-disaccharides linked by ß-glycosidic bond was higher than that produced from aldo-disaccharides linked by α-glycosidic bond. Palatinose, a keto-disaccharide, mainly underwent decomposition rather than isomerization in subcritical water and subcritical aqueous ethanol. No isomerization was noted for the non-reducing disaccharides trehalose and sucrose. The rate constant of maltose to maltulose isomerization almost doubled by changing solvent from subcritical water to 80 wt% aqueous ethanol at 220 °C. Increased maltose monohydrate concentration in feed decreased the conversion of maltose and the maximum yield of maltulose, but increased the productivity of maltulose. The maximum productivity of maltulose was ca. 41 g/(h kg-solution).

A glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae.

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.

Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase.

Isomaltulose is a natural isomer of sucrose. It is widely used as a functional sweetener with promising properties, including slower digestion, lower glycemic index, prolonged energy release, lower insulin reaction, and less cariogenicity. It has been approved as a safe sucrose substitute by the Food and Drug Administration of the US; Ministry of Health, Labor and Welfare of Japan; and the Commission of the European Communities. This article presents a review of recent studies on the properties, physiological effects, and food application of isomaltulose. In addition, the biochemical properties of sucrose isomerases producing isomaltulose are compared; the heterologous expression, fermentation optimization, structural determination, and catalysis mechanism of sucrose isomerase are reviewed; and the biotechnological production of isomaltulose from sucrose is summarized.

Characterization of a unique pH-dependent amylosucrase from Deinococcus cellulosilyticus.

The amylosucrase (ASase, EC 2.4.1.4) utilizes sucrose as the sole substrate to catalyze multifunctional reactions. It can naturally synthesize α-1,4-linked glucans such as amylose as well as sucrose isomers with more favorable properties than sucrose with a lower intestinal digestibility and non-cariogenic properties. The amino acid sequence of the asase gene from Deinococcus cellulosilyticus (DceAS) exhibits low homology with those of other ASases from other Deinococcus species. In this study, we cloned and expressed DceAS and demonstrated its high activity at pH 6 and pH 8 and maintained stability. It showed higher polymerization activity at pH 6 than at pH 8, but similar isomerization activity and produced more turanose and trehalulose at pH 6 than at pH 8 and produced more isomaltulose at pH 8. Furthermore, the molecular weight of DceAS was 226.6 kDa at pH 6 and 145.5 kDa at pH 8, indicating that it existed as a trimer and dimer, respectively under those conditions. Additionally, circular dichroism spectra showed that the DceAS secondary structure was different at pH 6 and pH 8. These differences in reaction products at different pHs can be harnessed to naturally produce sucrose alternatives that are more beneficial to human health.

Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

The effect of lactose-isomaltulose-containing growing-up milks on cognitive performance of Indonesian children: a cross-over study.

Glycaemic response to dietary carbohydrates might have an impact on cognitive performance. The present study investigated the effects of growing-up milks (GUM) with isomaltulose and extra minerals and vitamins or lower protein content on cognitive parameters in children aged 5­6 years. In a blinded, partly randomised, controlled, cross-over study, four GUM were provided, each taken over 14 d (2 × 200 ml/d): standard (Std) GUM; Std GUM+5 g isomaltulose (Iso-5 GUM); Iso-5 GUM with 26 % less protein (Iso-5 LP GUM); Std GUM with 2·5 g isomaltulose and extra Mg, Zn, Se, D3, B1, B2, B12, folic acid and choline (Iso-2·5 GUM). At test days, when GUM replaced breakfast, repeated (0, 60, 120 and 180 min post-dose) cognitive tasks were performed (picture presentation, simple reaction time, digit vigilance, choice reaction time, spatial and numeric working memory and picture recognition). Task performance of all subjects (n 50) worsened over the morning. Best performance was seen on isomaltulose GUM, most notably at 180 min. Iso-2·5 GUM showed best performance on several parameters of attention and memory, Iso-5 GUM performed best on parameters of memory and Iso-5 LP GUM was positively associated with parameters of attention but less with memory. Std GUM showed only a benefit on one attention and one memory task. Thus, isomaltulose-enriched GUM positively affected parameters of attention and memory at 180 min post-dose when compared with Std GUM. Extra minerals and vitamins seem beneficial, whereas lowering protein content might improve attention in particular.

sp2-Iminosugar O-, S-, and N-glycosides as conformational mimics of α-linked disaccharides; implications for glycosidase inhibition.

The synthesis of mimics of the α(1→6)- and α(1→4)-linked disaccharides isomaltose and maltose featuring a bicyclic sp(2)-iminosugar nonreducing moiety O-, S-, or N-linked to a glucopyranoside residue is reported. The strong generalized anomeric effect operating in sp(2)-iminosugars determines the α-stereochemical outcome of the glycosylation reactions, independent of the presence or not of participating protecting groups and of the nature of the heteroatom. It also imparts chemical stability to the resulting aminoacetal, aminothioacetal, or gem-diamine functionalities. All the three isomaltose mimics behave as potent and very selective inhibitors of isomaltase and maltase, two α-glucosidases that bind the parent disaccharides either as substrate or inhibitor. In contrast, large differences in the inhibitory properties were observed among the maltose mimics, with the O-linked derivative being a more potent inhibitor than the N-linked analogue; the S-linked pseudodisaccharide did not inhibit either of the two target enzymes. A comparative conformational analysis based on NMR and molecular modelling revealed remarkable differences in the flexibility about the glycosidic linkage as a function of the nature of the linking atom in this series. Thus, the N-pseudodisaccharide is more rigid than the O-linked derivative, which exhibits conformational properties very similar to those of the natural maltose. The analogous pseudothiomaltoside is much more flexible than the N- or O-linked derivatives, and can access a broader area of the conformational space, which probably implies a strong entropic penalty upon binding to the enzymes. Together, the present results illustrate the importance of taking conformational aspects into consideration in the design of functional oligosaccharide mimetics.

Unique substrate specificity of a thermostable glycosyl hydrolase from an uncultured Anaerolinea, derived from bacterial mat on a subsurface geothermal water stream.

To investigate novel extremozymes encoded by sequenced metagenes from a microbial community in an extreme environment, we have characterized a recombinant glycosyl hydrolase (rGH) from an uncultured bacterium within the order Chloroflexi. rGH formed insoluble bodies in an Escherichia coli protein expression system. The protein was partially dissolved by a surfactant and was enzymatically characterized. The MW of the monomeric peptide was ~62 kDa, and it formed a homodimers in buffer. It was optimally active at 65 °C and from pH 4 to 8. rGH showed hydrolytic activity for α-1,1, α-1,2 and α-1,6 linkages, including isomaltose, but not α-1,4 and ß-linkages.

Isomerization of 6-O-substituted glucose and fructose under neutral pH conditions and subsequent ß-elimination reactions.

Isomaltose (6-O-α-d-glucopyranosyl-d-glucose) and isomaltulose (palatinose; 6-O-ß-d-glucopyranosyl-d-fructose) were heated to 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Aldose-ketose isomerization between isomaltose, isomaltulose, and epi-isomaltulose was observed in the early stage of the reaction, alongside the release of a small amount of glucose. The total concentration of these disaccharides gradually decreased as the heating time increased. However, this decrease did not correlate with the amount of glucose or fructose released, suggesting that the releases of these monosaccharides were not caused by the hydrolysis of glycosidic linkages. A slight decrease in the pH of the reaction solution was attributed to the formation of two organic acids, 6-O-ß-d-glucopyranosyl-3-deoxy-d-arabino-hexonic acid (1) and 6-O-ß-d-glucopyranosyl-3-deoxy-d-ribo-hexonic acid (2). These compounds were formed from the ß-elimination of the hydroxyl group at the C-3 of fructose, leaving a substituted glucose residue at the C-6 position, followed by keto-enol tautomerization and benzilic acid rearrangement. Although approximately 30% of 1 and 2 were degraded after 360 min of heating at 90 °C in 100 mM sodium phosphate, a little release of glucose was observed, indicating no hydrolysis of the glucoside bond at C-6. Besides 1 and 2, time-dependent changes in the NMR spectra of the reaction mixture in water indicated the formation of formic acid and the presence of species possibly resulting from the ß-elimination of the hydroxyl group from 3- and 4-ulose. The glucose released by heating isomaltose and isomaltulose may be generated via tautomerizations of keto-enols between the C-4 and C-5 positions and cleavage of 6-O-glycosidic linkage via ß-elimination.

Structural insights reveal the second base catalyst of isomaltose glucohydrolase.

Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-kcat and pH-kcat /Km profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a ß-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.

Effect of sugars on the molecular motion of freeze-dried protein formulations reflected by NMR relaxation times.

PURPOSE: To relate NMR relaxation times to instability-related molecular motions of freeze-dried protein formulations and to examine the effect of sugars on these motions. METHODS: Rotating-frame spin-lattice relaxation time (T(1ρ)) was determined for both protein and sugar carbons in freeze-dried lysozyme-sugar (trehalose, sucrose and isomaltose) formulations using solid-state (13)C NMR. RESULTS: The temperature dependence of T(1ρ) for the lysozyme carbonyl carbons in lysozyme with and without sugars was describable with a model that includes two different types of molecular motion with different correlation times (τ(c)) for the carbon with each τ(c) showing Arrhenius temperature dependence. Both relaxation modes have much smaller relaxation time constant (τ(c)) and temperature coefficient (Ea) than structural relaxation and may be classified as ß-relaxation and γ-relaxation. The τ(c) and Ea for γ-relaxation were not affected by sugars, but those for ß-relaxation were increased by sucrose, changed little by trehalose, and decreased by isomaltose, suggesting that the ß-mobility of the lysozyme carbonyl carbons is decreased by sucrose and increased by isomaltose. CONCLUSION: T(1ρ) determined for the lysozyme carbonyl carbons can reflect the effect of sugars on molecular mobility in lysozyme. However, interpretation of relaxation time data is complex and may demand data over an extended temperature range.

NMR analysis and molecular dynamics conformation of α-1,6-linear and α-1,3-branched isomaltose oligomers as mimetics of α-1,6-linked dextran.

The conformational preferences of several α-1,6-linear and α-1,3-branched isomalto-oligosaccharides were investigated by NMR and MD-simulations. Right-handed helical structure contributed to the solution geometry in isomaltotriose and isomaltotetraose with one nearly complete helix turn and stabilizing intramolecular hydrogen bonds in the latter by MD-simulation. Decreased helix contribution was observed in α-1,3-glucopyranosyl- and α-1,3-isomaltosyl-branched saccharide chains. Especially the latter modification was predicted to cause a more compact structure consistent with literature rheology measurements as well as with published dextranase-resistant α-1,3-branched oligosaccharides. The findings presented here are significant because they shed further light on the conformational preference of isomalto-oligosaccharides and provide possible help for the design of dextran-based drug delivery systems or for the targeted degradation of capsular polysaccharides by dextranases in multi-drug resistant bacteria.

Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia pastoris and its efficient recovery for synthesis of isomaltooligosaccharides.

A gene conferring α-glucosidase (AG) with high transglycosylation activity from Aspergillus neoniger (a non-niger strain belonging to section Nigri) was cloned and expressed in Pichia pastoris. As the cDNA construction retained intronic portions due to alternative splicing, the exonic portions of the gene were stitched using restriction digestion and overlap extension PCR. Pre-determined open-loop exponential feeding strategies were evaluated for methanol dosage to improve the recombinant enzyme synthesis during high-cell density cultivation in 5 L bioreactor. Specific growth rate of 0.1 h-1 resulted in the highest enzyme activity of 182.3 mU/mL in the supernatant, whereas the activity of 3.8 U/g dry cell weight was obtained in the cell pellet. There was negligible enzyme activity in the cell lysate, indicating that approximately 80 % accumulation of total enzyme is in the periplasm. Later, this unreleased fraction was extracted to 90 % yield using 25 mM cysteine. The enzyme was purified and validated using western blot analysis and MS/MS profile. The SDS PAGE analysis revealed three bands corresponding to 80, 38, and 33 kDa indicating the multimeric nature of the enzyme. Thus, obtained enzyme was utilized in synthesis of a potential prebiotic molecule, isomaltooligosaccharides (IMOs), which can be used as a sweetener and bulk filler in the food industry. This is the first report to demonstrate challenges in cloning and expression of transglycosylating α-glucosidase from Aspergillus neoniger.

NMR solution geometry of saccharides containing the 6-O-(α-D-glucopyranosyl)-α/ß-D-glucopyranose (isomaltose) or 6-O-(α-D-galactopyranosyl)-α/ß-D-glucopyranose (melibiose) core.

The solution geometries of D-Glcp, Me-D-Glcp, 6-O-Me-D-Glcp, Me-6-O-Me-D-Glcp, D-Glcp-(α-1,6)-D-Glcp (isomaltose), D-Glcp-(α-1,6)-D-Glcp-(α-1,6)-D-Glcp (isomaltotriose), D-Galp-(α-1,6)-D-Glcp (melibiose), D-Galp-(α-1,6)-D-Glcp-(α-1,2)-D-Fruf (raffinose), and D-Galp-(α-1,6)-D-Galp-(α-1,6)-D-Glcp-(α-1,2)-D-Fruf (stachyose) in water are described by NMR spectroscopy, molecular dynamic simulations and quantum mechanical calculations. Overall, a change in anomeric configuration at the reducing end and/or anomeric substitution (methylation) changed the conformational space of the terminal CH2OH group significantly. Conformational analysis of the free monosaccharides matched literature results very well. Dihedral angle histograms weighted against published Karplus equations yielded excellent matches of experimental J-values in some cases but significant deviations in other. The anomeric hemiacetal configuration appeared to have a significant remote influence on the conformational space of the α-1,6-glycosidic linkage. Rigid glycosidic φ-conformations (g+) combined with mostly st-conformations for glycosidic ψ-angles from computations matched experimental nuclear Overhauser enhancements in all cases. While the investigated Glcp-α-1,6-Glcp linkages were nearly identical in φ/ψ-conformation, differences were apparent in the Galp-α-1,6-Galp linkage of stachyose. Of twenty-one crystal structures, a total of fourteen had ligand conformations corresponding to the most abundant or second-most abundant solution geometry determined in this study.