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Background/Aims: The use of novel cryo-additive agents to increase cell viability post-cryopreservation is paramount to improve future cell based-therapy treatments. We aimed to establish the Human Leukemia (HL-60) cells lipidomic and biological patterns when cryo-preserved in DMSO alone and with 300 µM Nigerose (Nig), 200 µM Salidroside (Sal) or a combination of Nig (150 µM) and Sal (100 µM). Methods: HL-60 cells were pre-incubated with Nig/Sal prior, during and post cryopreservation, and subjected to global lipidomic analysis. Malondialdeyhde (MDA), released lactate dehydrogenase (LDH) and reactive oxygen scavenger (ROS) measurements were also carried out to evaluate levels of lipid peroxidation and cytotoxicity. Results: Cryopreserving HL-60 cells in DMSO with Nig and Sal provided optimal protection against unsaturated fatty acid oxidation. Post-thaw, cellular phospholipids and mitochondrial cardiolipins were increased by Nig/Sal as the ratio of unsaturated to saturated fatty acids 2.08 +/- 0.03 and 0.95 +/- 0.09 folds respectively in comparison to cells cryopreserved in DMSO alone (0.49 +/- 0.05 and 0.86 +/- 0.10 folds). HL-60 lipid peroxidation levels in the presence of DMSO + Nig and Sal combined were significantly reduced relative to pre-cryopreservation levels (10.91 +/- 2.13 nmole) compared to DMSO (17.1 +/- 3.96 nmole). DMSO + Nig/Sal combined also significantly reduced cell cytotoxicity post-thaw (0.0128 +/- 0.00182 mU/mL) in comparison to DMSO (0.0164 +/- 0.00126 mU/mL). The combination of Nig/Sal also reduced significantly ROS levels to the levels of prior cryopreservation of HL-60. Conclusion: Overall, the establishment of the cryopreserved HL-60 cells lipidomic and the corresponding biological profiles showed an improved cryo-formulation in the presence of DMSO with the Nig/Sal combination by protecting the, mitochondrial inner membrane, unsaturated fatty acid components (i. e. Cardiolipins) and total phospholipids.

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 α-1,3-glucanase from Paenibacillus glycanilyticus FH11 (Agl-FH1), a member of the glycoside hydrolase family 87 (GH87), hydrolyzes α-1,3-glucan with an endo-action. GH87 enzymes are known to degrade dental plaque produced by oral pathogenic Streptococcus species. In this study, the kinetic analyses revealed that this enzyme hydrolyzed α-1,3-tetraglucan into glucose and α-1,3-triglucan with β-configuration at the reducing end by an inverting mechanism. The crystal structures of the catalytic domain (CatAgl-FH1) complexed with or without oligosaccharides at 1.4-2.5 or 1.6 Å resolutions, respectively, are also presented. The initial crystal structure of CatAgl-FH1 was determined by native single-wavelength anomalous diffraction. The catalytic domain was composed of two modules, a β-sandwich fold module, and a right-handed β-helix fold module. The structure of the β-sandwich was similar to those of the carbohydrate-binding module family 35 members. The glycerol or nigerose enzyme complex structures demonstrated that this β-sandwich fold module is a novel carbohydrate-binding module with the capabilities to bind saccharides and to promote the degradation of polysaccharides. The structures of the inactive mutant in complexes with oligosaccharide showed that at least eight subsites for glucose binding were located in the active cleft of the β-helix fold and the architecture of the active cleft was suitable for the recognition and hydrolysis of α-1,3-glucan by the inverting mechanism. The structural similarity to GH28 and GH49 enzymes and the results of site-directed mutagenesis indicated that three Asp residues, Asp1045, Asp1068, and Asp1069, are the most likely candidates for the catalytic residues of Agl-FH1. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers 6K0M (CatAgl-FH1), 6K0N (WT/nigerose), 6K0P (D1045A/nigerose), 6K0Q (D1068A/nigerose), 6K0S (D1069A/ nigerose), 6K0U (D1068A/oligo), and 6K0V (D1069A/oligo). ENZYMES: Agl-FH1, α-1,3-glucanase (EC3.2.1.59) from Paenibacillus glycanilyticus FH11.

Koji amazake, prepared from rice koji, is a traditional Japanese sweet beverage. The main source of sweetness is glucose derived from rice starch following digestion by enzymes of Aspergillus oryzae during saccharification. The temperature of this process was empirically determined as 45°C-60°C, but no studies have systematically investigated the effect of temperature on saccharification efficiency. We addressed this in the present study by evaluating saccharification efficiency at various temperatures. We found that glucose content was the highest at 50°C (100%) and was reduced at temperatures of 40°C (66.4%), 60°C (91.9%), and 70°C (76.6%). We previously reported that 12 types of oligosaccharides are present in koji amazake; the levels of eight of these, namely nigerose, kojibiose, trehalose, isomaltose, gentiobiose, raffinose, panose, and isomaltotriose, were the highest at 50°C-60°C, whereas sophorose production was maximal at 70°C. Based on these findings, we initially performed saccharification at 50°C and then switched the temperature to 70°C. The maximum amount of each saccharide including sophorose that was produced was close to the values obtained at these two temperatures. Thus, oligosaccharide composition of koji amazake is dependent on saccharification temperature. These findings provide useful information for improving the consumer appeal of koji amazake by enhancing oligosaccharide content.

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The k

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 sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) can be used as a transglucosylase for the production of rare sugars. We designed variants of BaSP for the efficient synthesis of nigerose from sucrose and glucose, thereby adding to the inventory of rare sugars that can conveniently be produced from bulk sugars.

Pyrobaculum arsenaticum is a hyperthermophilic archaeon that thrives at 95°C. This strain encodes a putative GH31 intracellular α-glucosidase (Pars_2044, PyAG) in its genome. The recombinant PyAG (rPyAG) was optimally expressed in Escherichia coli at 37°C for 4 h after IPTG induction. The purified rPyAG is a homotetrameric α-glucosidase that exhibited highly thermostable properties. Maximum p-nitrophenyl-α-D-glucopyranoside (pNPG) hydrolysis activity was observed at 90°C and pH 5.0. The enzyme mainly recognized the non-reducing end of the substrate, releasing the glucose unit. rPyAG also had broad substrate specificity, cleaving maltose (α-1,4-linkage), kojibiose (α-1,2-linkage), and nigerose (α-1,3-linkage) with similar efficiency. Based on these results, rPyAG can be used to modify health-relevant sugar conjugates linked by α-1,2- or α-1,3-bonds.

Mutanase, α-(1→3)-glucanase, belonging to the family glucanohydrolase, catalyses mutan [α-(1→3)-glucan] synthesized by cariogenic streptococci and hence has potential in caries prophylaxis. A novel bacterial strain with potential to produce higher mutanase (glucanohydrolase) activity was isolated from soils contaminated with cellulosic waste. One of the isolated strains, RSP-02, was subjected to biochemical and 16S rRNA molecular analysis, and we noticed that it belongs to the genus Paracoccus. The mutanase production (800- 1200 U l-1) in this strain was growth associated and substrate induced, and the activity was comparable with the strains reported earlier. The enzyme displayed a molecular mass of 138 kDa by native PAGE studies, showed endolytic activity and produced nigerose as end product. In vitro studies revealed production of 140±2.82 µg of glucose equivalents in 30 min from the biofilm formed on glass surface indicating its potentiality in dentistry. To the best of our knowledge, this is the first report on the production of mutanase by Paracoccus sp.; hence, this isolated bacterial strain is designated as Paracoccus mutanolyticus RSP-02.

The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1 -> 6)-alpha-nigerosyl (CNN), a cyclic glucotetraose with alternate alpha-(1 -> 6)- and alpha-(1 -> 3)-glucosidic linkages, from starch in the culture medium. We identified gene clusters associated with the production and intracellular catabolism of CNN in the K. flavida genome. One cluster encodes 6-alpha-glucosyl-transferase and 3-alpha-isomaltosyltransferase, which are known to coproduce CNN from starch. The other cluster contains four genes annotated as a transcriptional regulator, sugar transporter, glycoside hydrolase family (GH) 31 protein (Kfla1895), and GH15 protein (Kfla1896). Kfla1895 hydrolyzed the alpha-(1 -> 3)-glucosidic linkages of CNN and produced isomaltose via a possible linear tetrasaccharide. The initial rate of hydrolysis of CNN (11.6 s(-1)) was much higher than that of panose (0.242 s(-1)), and hydrolysis of isomaltotriose and nigerose was extremely low. Because Kfla1895 has a strong preference for the alpha-(1 -> 3)-isomaltosyl moiety and effectively hydrolyzes the alpha-(1 -> 3)-glucosidic linkage, it should be termed 1,3-alpha-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of beta-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The k(cat)/K-m for isomaltose (4.81 +/- 0.18 s(-1) mM(-1)) was 6.9- and 19-fold higher than those for panose and isomaltotriose, respectively. These results indicate that Kfla1896 is a new GH15 enzyme with high substrate specificity for isomaltose, suggesting the enzyme should be designated an isomaltose glucohydrolase. This is the first report to identify a starch-utilization pathway that proceeds via CNN.

Isomalto-oligosaccharides (IMO) significantly contribute to the global oligosaccharide market. IMO are linear α-(1 → 6) linked oligosaccharides with isomaltotriose as the representative trisaccharide. Commercial IMO preparations ypically also contain panose-series oligosaccharides as a major component. In humans, IMO are partially digestible but the digestibility of specific components of commerical IMO preparations remains unknown. This study aimed to compare the in vitro digestibility of reference compounds, experimental α-gluco-oligosaccharides and commercial IMO. Experimental α-gluco-oligosaccharides were synthesized with the recombinant dextransucrase DsrM. Two in vitro digestion methods were used, a reference method matching the AOAC method for dietary fibre, and a protocol that uses brush border glycosyl hydrolases from the rat intestine. The α-gluco-oligosaccharides patterns after hydrolysis remain were analyzed by high performance anion exchange chromatography coupled to pulsed amperometric detection. Panose-series oligosaccharides were hydrolysed more rapidly by amylase and amyloglucosidase when compared to hydrolysis by rat intestinal enzymes. The rate of hydrolysis by rat intestinal enzymes decreased in the order panose > isomaltose, kojibiose or nigerose. Hydrolysis of panose-series oligosaccharides but not the hydrolysis of isomalto-oligosaccharides was dependent on the degree of polymerization. Qualitative analysis of oligosaccharides remaining after hydrolysis indicated that rat small intestinal enzymes hydrolyse their substrates from the non-reducing end. Taken together, results inform on the modification or optimization of current production processes for IMO to obtain tailored oligosaccharide preparations with reduced digestibility and an increased content of dietary fibre.

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.

α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of N-glycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)- and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4-glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short- and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (

Background Cryopreservation is a routinely used methodology for prolonged storage of viable cells. The use of cryo-protective agents (CPAs) such as dimethylsulfoxide (DMSO), glycerol, or trehalose is paramount to reducing cellular cryo-injury, but their effectiveness is still limited. The current study focuses on establishing and modulating the proteomic and the corresponding biological profiles associated with the cryo-injury of human leukemia (HL-60) cells cryopreserved in DMSO alone or DMSO +/- novel CPAs (e.g., nigerose [Nig] or salidroside [Sal]). Findings To reduce cryo-damage, HL-60 cells were cultured prior and post cryopreservation in malondialdehyde Roswell Park Memorial Institute medium-1640 media +/- Nig or Sal. Shotgun proteomic analysis showed significant alterations in the levels of proteins in cells cryopreserved in Nig or Sal compared to DMSO. Nig mostly affected cellular metabolism and energy pathways, whereas Sal increased the levels of proteins associated with DNA repair/duplication, RNA transcription, and cell proliferation. Validation testing showed that the proteome profile associated with Sal was correlated with a 2.8-fold increase in cell proliferative rate. At the functional level, both Nig and Sal increased glutathione reductase (0.0012±6.19E-05 and 0.0016±3.04E-05 mU/mL, respectively) compared to DMSO controls (0.0003±3.7E-05 mU/mL) and reduced cytotoxicity by decreasing lactate dehydrogenase activities (from -2.5 to -4.75 fold) and lipid oxidation (-1.6 fold). In contrast, only Nig attenuated protein carbonylation or oxidation. Conclusions We have identified key molecules and corresponding functional pathways underpinning the effect of cryopreservation (+/- CPAs) of HL-60 cells. We also validated the proteomic findings by identifying the corresponding biological profiles associated with promoting an anti-oxidative environment post cryopreservation. Nig or Sal in comparison to DMSO showed differential or additive effects in regard to reducing cryo-injury and enhancing cell survival/proliferation post thaw. These results can provide useful insight to cryo-damage and the design of enhanced cryomedia formulation.

European Food Research and Technology, 225 (3-4), ISSN:1438-2377, ISSN:1438-2385

Sugar profiles of 45 Korean honey samples (15 acacia, 15 multi-floral, 10 chestnut, and 5 artificial honey samples), which are commercially available in the Korean markets, were analyzed using gas chromatography/mass spectrometry (GC/MS) through TMS-oxime and TMS-methoxime derivatization. The average invert sugar contents in acacia, multi-floral, chestnut, and artificial honey samples were 71.2±1.05, 68.7±3.26, 63.2±1.85, and 68.0±2.10%, respectively. Fourteen disaccharides were detected from the samples, and the average content of major disaccharides was higher in order of turanose, maltulose, maltose, trehalulose, kojibiose, isomaltose, and nigerose. The average content of total disaccharides was highest in chestnut and lowest in acacia. Seven trisaccharides were detected from the samples, and the average content of trisaccharides was the highest in artificial honeys, which had high erlose content. The total content of disaccharides and trisaccharides was highest (16.0±2.03%) in chestnut honey and lowest (9.70±1.75%) in acacia honey.

An efficient NMR approach is described for determining the chemical structures of the monosaccharide glucose and four disaccharides, namely, nigerose, gentiobiose, leucrose and isomaltulose. This approach uses the 1H resonances of the -OH groups, which are observable in the NMR spectrum of a supercooled aqueous solution, as the starting point for further analysis. The 2D-NMR technique, HSQC-TOCSY, is then applied to fully define the covalent structure (i.e., the topological relationship between C-C, C-H, and O-H bonds) that must be established for a novel carbohydrate before proceeding to further conformational studies. This process also leads to complete assignment of all 1H and 13C resonances. The approach is exemplified by analyzing the monosaccharide glucose, which is treated as if it were an "unknown", and also by fully assigning all the NMR resonances for the four disaccharides that contain glucose. It is proposed that this technique should be equally applicable to the determination of chemical structures for larger carbohydrates of unknown composition, including those that are only available in limited quantities from biological studies. The advantages of commencing the structure elucidation of a carbohydrate at the -OH groups are discussed with reference to the now well-established 2D-/3D-NMR strategy for investigation of peptides/proteins, which employs the -NH resonances as the starting point.

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.

In the absence of the natural acceptor inorganic phosphate wild-type sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) produces maltose (4-O-α-d-glucopyranosyl-d-glucose) and kojibiose (2-O-α-d-glucopyranosyl-d-glucose) as sole transfer products. A Q345F exchange switches the enzyme's regioselectivity from 2 to 3 exclusively, yielding the rare sugar nigerose (3-O-α-d-glucopyranosyl-d-glucose, sakebiose).

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.

Cyclic nigerosyl nigerose (CNN) is a cyclic tetrasaccharide that exhibits properties distinct from other conventional cyclodextrins. Herein, we demonstrate that treatment of B16 melanoma with CNN results in a dose-dependent decrease in melanin synthesis, even under conditions that stimulate melanin synthesis, without significant cytotoxity. The effects of CNN were prolonged for more than 27 days, and were gradually reversed following removal of CNN. Undigested CNN was found to accumulate within B16 cells at relatively high levels. Further, CNN showed a weak but significant direct inhibitory effect on the enzymatic activity of tyrosinase, suggesting one possible mechanism of hypopigmentation. While a slight reduction in tyrosinase expression was observed, tyrosinase expression was maintained at significant levels, processed into a mature form, and transported to late-stage melanosomes. Immunocytochemical analysis demonstrated that CNN treatment induced drastic morphological changes of Pmel17-positive and LAMP-1-positive organelles within B16 cells, suggesting that CNN is a potent organelle modulator. Colocalization of both tyrosinase-positive and LAMP-1-positive regions in CNN-treated cells indicated possible degradation of tyrosinase in LAMP-1-positive organelles; however, that possibility was ruled out by subsequent inhibition experiments. Taken together, this study opens a new paradigm of functional oligosaccharides, and offers CNN as a novel hypopigmenting molecule and organelle modulator.

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.

The most common type of carbohydrate-transport system in Streptococcus mutans is the phosphoenolpyruvate (PEP)-sugar phosphotransferase system (PTS). We previously showed that fourteen PTSs exist in S. mutans UA159 (Ajdic et al., 2002). Several studies have shown that microorganisms growing in biofilms express different genes as compared to their planktonic counterparts. In this study, we showed that one PTS of S. mutans was expressed in sucrose-grown biofilms. Furthermore, the same PTS was also responsible for the transport and metabolism of disaccharide nigerose (3-O-α-D-glucopyranosyl-D-glucose). Additionally, the results indicate that the studied PTS might be involved in the transport and metabolism of carbohydrates synthesized by glucosyltransferase B (GtfB) and glucosyltransferase C (GtfC) of S. mutans. To our knowledge, this is the first report that shows PTS transport of a disaccharide (and possibly extracellular oligosaccharides) with α-1,3 linkage.

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.

Recently, we found that Arthrobacter crystallopoietes N-08 isolated from soil directly produces trehalose from maltose by a resting cell reaction. In this study, the optimal set of conditions and substrate specificity for the trehalose production using resting cells was investigated. Optimum temperature and pH of the resting cell reaction were 55℃ and pH 5.5, respectively, and the reaction was stable for two hours at 37～55℃ and for one hour at the wide pH ranges of 3～9. Various disaccharide substrates with different glycosidic linkages, such as maltose, isomaltose, cellobiose, nigerose, sophorose, and laminaribiose, were converted into trehalose-like spots in thin layer chromatography (TLC). These results indicated broad substrate specificity of this reaction and the possibility that cellobiose could be converted into other trehalose anomers such as α,β- and -trehalose. Therefore, the product after the resting cell reaction with cellobiose was purified by -glucosidase treatment and Dowex-1 (OH?) column chromatography and its structure was analyzed. Component sugar and methylation analyses indicated that this cellobiose-conversion product was composed of only non-reducing terminal glucopyranoside. MALDI-TOF and ESI-MS/MS analyses suggested that this oligosaccharide contained a non-reducing disaccharide unit with a 1,1-glucosidic linkage. When this disaccharide was analyzed by ¹H-NMR and 13 C-NMR, it gave the same signals with α-D-glucopyranosyl-(1,1)-α-D-glucopyranoside. These results suggest that cellobiose can be converted to α,α-trehalose by the resting cells of A. crystallopoietes N-08.

The gene (Ta0298) encoding a putative α-glucosidase from hyperthermophilic archaeon Thermoplasma acidophilum (AglA) was cloned and expressed in Escherichia coli. Gel filtration chromatography of the purified enzyme indicated that the native form was a pentamer with strong maltose (α-1,4 linkage)-hydrolyzing activity. AglA was optimally active at pH 5–6 and 80 °C and had a half-life of 16.8 h and 1.4 h at 80 °C and 85 °C, respectively. The enzyme also hydrolyzes kojibiose (α-1,2), nigerose (α-1,3), and isomaltose (α-1,6) to a lesser extent. Analysis of the reaction with maltooligosaccharides and panose as substrates show that AglA specifically liberates glucose from the non-reducing end indicating that it is typical of a glycoside hydrolase family 31 (GH31) α-glucosidase. Kinetic analyses revealed that the hydrolytic activity of AglA was greatly affected by the chain length of the substrate and the regiospecificity of the glucosidic linkages. The enzyme showed highest specificity for maltose and decreasing values of catalytic efficiency (kcat/Km) toward higher maltooligosaccahrides, although these still serve as substrates. The inhibition profile of AglA toward aesculin was revealed to be a mixed type of noncompetitive inhibition with a Ki value of 4.30 μM and K ′ i of 12.5 μM, whereas that toward acarbose showed a competitive inhibition pattern with a Ki of 2.99 μM. Structural analyses of two arbutin transglucosylation products using NMR indicated that the glucose unit of maltose was transferred to the C-3 and C-6 position in the glucose moiety of arbutin.

The early steps of glycoprotein biosynthesis involve processing of the N-glycan core by endoplasmic reticulum α-glucosidases I and II which sequentially trim the outermost α1,2-linked and the two more internal α1,3-linked glucose units, respectively. We have demonstrated the presence of some components of the enzymic machinery required for glycoprotein synthesis in Sporothrix schenckii, the etiological agent of human and animal sporotrichosis. However, information on this process is still very limited. Here, a distribution analysis of α-glucosidase revealed that 38 and 50% of total enzyme activity were present in a soluble and in a mixed membrane fraction, respectively. From the latter, the enzyme was solubilized, purified to apparent homogeneity and biochemically characterized. Analysis of the enzyme by denaturing electrophoresis and size exclusion chromatography revealed molecular masses of 75.4 and 152.7 kDa, respectively, suggesting a homodimeric structure. Purified α-glucosidase cleaved the fluorogenic substrate 4-methylumbelliferyl-α-D: -glucopyranoside with high affinity as judged from K(m) and V(max) values of 0.3 μM and 250 nmol of MU/min/mg protein, respectively. Analysis of linkage specificity using a number of glucose α-disaccharides as substrates demonstrated a clear preference of the enzyme for nigerose, an α1,3-linked disaccharide, over other substrates such as kojibiose (α1,2), trehalose (α1,1) and isomaltose (α1,6). Use of selective inhibitors of processing α-glucosidases such as 1-deoxynojirimycin, castanospermine and australine provided further evidence of the possible type of α-glucosidase. Accordingly, 1-deoxynojirimycin, a more specific inhibitor of α-glucosidase II than I, was a stronger inhibitor of hydrolysis of 4-methylumbelliferyl-α-D: -glucopyranoside and nigerose than castanospermine, a preferential inhibitor of α-glucosidase I. Inhibition of hydrolysis of kojibiose and maltose by 1-deoxynojirimycin and castanoespermine was significantly lower than that of nigerose. Taken together, these properties are consistent with a type II-like α-glucosidase probably involved in N-glycan processing. To our knowledge, this is the first report of such an activity in a truly dimorphic fungus.

The endoplasmic reticulum malectin is a highly conserved protein in the animal kingdom that has no counterpart so far in lower organisms. We recently determined the structure of its conserved domain and found a highly selective binding to Glc(2)Man(9)GlcNAc(2), an intermediate of N-glycosylation. In our quest for putative ligands during the initial characterization of the protein, we noticed that the malectin domain is highly specific for diglucosides but quite tolerant towards the linkage of the glucosidic bond. To understand the molecular requirements for the observed promiscuity of the malectin domain, here we analyze the binding to a range of diglucosides through comparison of the protein chemical shift perturbation patterns and the saturation transfer difference spectra of the ligands including two maltose-mimicking drugs. A comparison of the maltose-bound structure of the malectin domain with the complex of the native ligand nigerose reveals why malectin is able to tolerate such a diversity of ligands.

We present an extension of the Martini coarse-grained force field to carbohydrates. The parametrization follows the same philosophy as was used previously for lipids and proteins, focusing on the reproduction of partitioning free energies of small compounds between polar and nonpolar phases. The carbohydrate building blocks considered are the monosaccharides glucose and fructose and the disaccharides sucrose, trehalose, maltose, cellobiose, nigerose, laminarabiose, kojibiose, and sophorose. Bonded parameters for these saccharides are optimized by comparison to conformations sampled with an atomistic force field, in particular with respect to the representation of the most populated rotameric state for the glycosidic bond. Application of the new coarse-grained carbohydrate model to the oligosaccharides amylose and Curdlan shows a preservation of the main structural properties with 3 orders of magnitude more efficient sampling than the atomistic counterpart. Finally, we investigate the cryo- and anhydro-protective effect of glucose and trehalose on a lipid bilayer and find a strong decrease of the melting temperature, in good agreement with both experimental findings and atomistic simulation studies.

Explicit-solvent molecular dynamics simulations (50 ns, 300 K) of the eight reducing glucose disaccharides (kojibiose, sophorose, nigerose, laminarabiose, maltose, cellobiose, isomaltose, and gentiobiose) have been carried out using the GROMOS 45A4 force field (including a recently reoptimized carbohydrate parameter set), to investigate and compare their conformational preferences, intramolecular hydrogen-bonding patterns, torsional dynamics, and configurational entropies. The calculated average values of the glycosidic torsional angles agree well with available experimental data, providing validation for the force field and simulation methodology employed in this study. These simulations show in particular that: 1) (1--6)-linked disaccharides are characterized by an increased flexibility, the absence of any persistent intramolecular hydrogen bond and a significantly higher configurational entropy (compared to the other disaccharides); 2) cellobiose presents a highly persistent interresidue hydrogen bond and a significantly lower configurational entropy (compared to the other disaccharides); 3) persistent hydrogen bonds are observed for all disaccharides (except (1--6)-linked) and typically involve a hydrogen donor in the reducing residue and an acceptor in the nonreducing one; 4) the probability distributions associated with the glycosidic dihedral angles and psi are essentially unimodal for all disaccharides, and full rotation around these angles occurs at most once or twice for (never for psi) on the 50-ns timescale; and 5) the timescales associated with torsional transitions (except around and psi) range from approximately 30 ps (rotation of hydroxyl groups) to the nanosecond range (rotation of the lactol and hydroxymethyl groups, and around the omega-glycosidic dihedral angle in (1--6)-linked disaccharides).

An improved method for the quantitative analysis of isomaltooligosaccharide (IMO) products by HPLC with a polymer-based amino column was developed. The column was much higher in durability than a silica-based amino column used for the conventional method. The column durability enabled us to determine each IMO using the calibration curve of RI-detector response against a concentration of standard IMO and maltosaccharide reagents. The linear relationship between peak height of RI response and concentration of saccharide was found for glucose, maltose, kojibiose, nigerose, isomaltose, maltotriose, panose, isomatotriose, maltotetraose and isomaltotetraose. The linearity was obtained at concentrations of up to 17 mg/mL, and correlation coefficients were ≥0.999. The slope of peak height versus concentration differed from saccharides, of which glucose was the highest while isomaltotetraose was the lowest. The relative slope of each saccharide to glucose, (slope for saccharide)/(slope for glucose), referred as a conversion factor, was calculated, and the concentration of each saccharide in commercial IMO products was determined from peak height on a HPLC chromatogram by the following equation: (concentration of saccharide A, mg/mL)=(concentration of standard glucose, mg/mL)×(peak height of A)/(conversion factor of A)/(peak height of standard glucose). A commercial IMO product was analyzed and the result obtained was as follows: isomaltose (19.2 g), isomaltotriose (10.3 g), panose (4.9 g), nigerose (2.0 g), kojibiose (3.5 g) and isomaltotetraose (2.8 g), respectively. The total amount of the sugars identified by the improved method from IMO was higher than those determined by the conventional method, which may have resulted from the higher resolution of each saccharide. The method showed clearly the presence of nigerose and kojibiose together with four unknown components. A major unknown component was identified to be isomaltotriosylglucose by 1H- and 13C-NMR analyses.

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.

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.

The proteins encoded in the yicI and yihQ gene of Escherichia coli have similarities in the amino acid sequences to glycoside hydrolase family 31 enzymes, but they have not been detected as the active enzymes. The functions of the two proteins have been first clarified in this study. Recombinant YicI and YihQ produced in E. coli were purified and characterized. YicI has the activity of alpha-xylosidase. YicI existing as a hexamer shows optimal pH at 7.0 and is stable in the pH range of 4.7-10.1 with incubation for 24h at 4 degrees C and also is stable up to 47 degrees C with incubation for 15 min. The enzyme shows higher activity against alpha-xylosyl fluoride, isoprimeverose (6-O-alpha-xylopyranosyl-glucopyranose), and alpha-xyloside in xyloglucan oligosaccharides. The alpha-xylosidase catalyzes the transfer of alpha-xylosyl residue from alpha-xyloside to xylose, glucose, mannose, fructose, maltose, isomaltose, nigerose, kojibiose, sucrose, and trehalose. YihQ exhibits the hydrolysis activity against alpha-glucosyl fluoride, and so is an alpha-glucosidase, although the natural substrates, such as alpha-glucobioses, are scarcely hydrolyzed. alpha-Glucosidase has been found for the first time in E. coli.

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.

A soluble α-glucosidase was partially purified from Candida albicans cells by a three-step procedure consisting of size-exclusion, ion-exchange and adsorption chromatographies. After the last step, enzyme was enriched about 8.7-fold with a yield of 13% over the starting material and analysis of the purified preparation revealed two major polypeptides of 36 and 47 kDa. The latter was responsible for enzyme activity as visualized with a fluorescent substrate. Nigerose, an α-1,3-linked glucose disaccharide, was preferentially hydrolyzed by the purified enzyme over other glucosedisaccharides bearing distinct α-linkages. The purified α-glucosidase also converted the GlcMan9GlcNAc2 oligosaccharide into the Man9GlcNAc2 product in a time-dependent manner. These and other determined properties are consistent with a type GII α-glucosidase probably involved in N-glycan processing.