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In a competitive microbial environment, nutrient acquisition is a major contributor to the survival of any individual bacterial species, and the ability to access uncommon energy sources can provide a fitness advantage. One set of soluble carbohydrates that have attracted increased attention for use in biotechnology and biomedicine is the α-diglucosides. Maltose is the most well-studied member of this class; however, the remaining four less common α-diglucosides (trehalose, kojibiose, nigerose, and isomaltose) are increasingly used in processed food and fermented beverages. The consumption of trehalose has recently been shown to be a contributing factor in gut microbiome disease as certain pathogens are using α-diglucosides to outcompete native gut flora. Kojibiose and nigerose have also been examined as potential prebiotics and alternative sweeteners for a variety of foods. Compared to the study of maltose metabolism, our understanding of the synthesis and degradation of uncommon α-diglucosides is lacking, and several fundamental questions remain unanswered, particularly with regard to the regulation of bacterial metabolism for α-diglucosides. Therefore, this minireview attempts to provide a focused analysis of uncommon α-diglucoside metabolism in bacteria and suggests some future directions for this research area that could potentially accelerate biotechnology and biomedicine developments. Key points: • α-diglucosides are increasingly important but understudied bacterial metabolites. •Kinetically superior α-diglucoside enzymes require few amino acid substitutions. •In vivo studies are required to realize the biotechnology potential of α-diglucosides. [ABSTRACT FROM AUTHOR]

Background/Aims: The use of novel cryo-additive agents to increase cell viability postcryopreservation is paramount to improve future cell based-therapy treatments. We aimed to establish the Human Leukemia (HL-60) cells lipidomic and biological patterns when cryopreserved 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. [ABSTRACT FROM AUTHOR]

Abstract: 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, γ-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, 13C NMR, and enzymatic analysis. T1 was identified as a novel compound, maltosyl α-1,3-maltose, whereas T2 and T3 were determined to be isopanose and maltosyl-α-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. [Copyright &y& Elsevier]

Abstract: Proteins belonging to the glycoside hydrolase family 63 (GH63) are found in bacteria, archaea, and eukaryotes. Eukaryotic GH63 proteins are processing α-glucosidase I enzymes that hydrolyze an oligosaccharide precursor of eukaryotic N-linked glycoproteins. In contrast, the functions of the bacterial and archaeal GH63 proteins are unclear. Here we determined the crystal structure of a bacterial GH63 enzyme, Escherichia coli K12 YgjK, at 1.78 Å resolution and investigated some properties of the enzyme. YgjK consists of the N-domain and the A-domain, joined by a linker region. The N-domain is composed of 18 antiparallel β-strands and is classified as a super-β-sandwich. The A-domain contains 16 α-helices, 12 of which form an (α/α)6-barrel; the remaining 4 α-helices are found in an extra structural unit that we designated as the A′-region. YgjK, a member of the glycoside hydrolase clan GH-G, shares structural similarity with glucoamylase (GH15) and chitobiose phosphorylase (GH65), both of which belong to clan GH-L. In crystal structures of YgjK in complex with glucose, mannose, and galactose, all of the glucose, mannose, and galactose units were located in the catalytic cleft. YgjK showed the highest activity for the α-1,3-glucosidic linkage of nigerose, but also hydrolyzed trehalose, kojibiose, and maltooligosaccharides from maltose to maltoheptaose, although the activities were low. These findings suggest that YgjK is a glucosidase with relaxed specificity for sugars. [Copyright &y& Elsevier]

Two neutral disaccharides, gentiobiose [β-D-Glcp-(1→6)-D-Glc] and nigerose [α-D-Glcp-(1→3)-D-Glc], were detected in tomato (Lycopersicon esculentum Mill.) pericarp and locule. Gentiobiose was present in the locule of green fruit and ripe fruit at 0.88 and 5.8 µmol (kg fresh weight)–1, respectively. When vacuum-infiltrated into green tomato fruit, exogenous gentiobiose (50 or 200 µg per fruit) hastened the initiation of ripening (as judged by colour change) by 1–3 days relative to fruit that were infiltrated with glucose or isomaltose. Nigerose plus gentiobiose was particularly effective, but nigerose alone had no significant effect. The endogenous disaccharides were found to be present in the apoplastic fluid of the fruit, compatible with a proposed intercellular signalling role. The origin and metabolic fate of the disaccharides were investigated. Phenolic esters of these disaccharides were not detectable in tomato fruit and it is therefore unlikely that the free disaccharides were formed from a pool of such esters. An alternative possible biosynthetic origin, via transglycosylation, is discussed. When [14C]gentiobiose was vacuum-infiltrated into unripe or ripe fruit, the disaccharide remained intact for at least 1 h but was largely degraded within 24 h. The results suggest that gentiobiose is a new, naturally occurring oligosaccharin with a rapid turnover rate. [ABSTRACT FROM AUTHOR]

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 cat /K m values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members., Competing Interests: Conflict of interest The authors have no conflicts of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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. Image 1 • Nigerose and turanose are unstable against heat treatment under neutral pH. • The decomposition by heating results in formation of 3-deoxy-hexonic acids. • γ-Lactones are generated by acid treatment of 3-deoxy-hexonic acids. • The elimination occurs on the carbon adjacent to the 1,2-enediol intermediate. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (<15 min) synthesized 3 II - O-α-glucosyl-maltose and maltotriose, indicating α-1,3-/α-1,4-transferring specificity. A long-term reaction (<24 h) accumulated kojibiose and nigerose using formed glucose as an acceptor substrate. AgdA and AgdB are distinct α-glucosidases. At a high concentration of glucose added exogenously, AgdB largely generated the rare sugars kojibiose and nigerose (exhibiting beneficial physiological functions) with 19% and 24% yields from maltose, respectively.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)