Abstract  With anticipation of the improvement in biological aspects in our SGLT2 program, novel pyridazinyl and thiazolyl analogs were designed and efficiently synthesized. The installation of the pyridazine ring at the anomeric carbon of d-glucopyranose was carried out in a stereoselective fashion. On the other hand, a series of thiazolyl analogs was also synthesized through a coupling reaction between perbenzyl gluconolactone 9 and 2-lithiothiazole. Biological activities of the compounds thus prepared were evaluated by the in vitro SGLT2 inhibition assay. Considering assay results, the novel benzylpyridazinyl and benzylthiazolyl analogs, disclosed in this article, could be a quick reference to prospective SGLT2 inhibitors useful for pharmacotherapy.

Abstract  There is a wide interest in developing management and feeding strategies to stimulate gut development and health in newly-weaned pigs, in order to improve growth performance while minimizing the use of antibiotics and rather expensive feed ingredients, such as milk products. A better understanding of the mechanisms whereby antibiotics influence animal physiology, as well as appropriate use of disease models and in vitro techniques, will lead to the development of alternatives to in-feed antibiotic. Given the considerable advances made in the understanding of intestinal nutrient utilization and metabolism, a complimentary goal in nutrition might be to formulate young pig diets with the specific task of optimizing the growth, function and health of the gut. Important aspects of gut health-promoting pig diets are: reduced content of protein that is fermented in the pigs' gut, minimal buffering capacity, minimal content of anti-nutritional factors, and supply of beneficial compounds such as immunoglobulins. The optimum dietary level and type of fibre will vary with the nature of enteric disease challenges and production objectives. These diet characteristics are influenced by feed ingredient composition and feed processing, including feed fermentation and application of enzymes. A large number of feed additives have been evaluated that are aimed at (1) enhancing the pig's immune response (e.g. immunoglobulin; omega-3 fatty acids, yeast derived beta-glucans), (2) reducing pathogen load in the pig's gut (e.g. organic and inorganic acids, high levels of zinc oxide, essential oils, herbs and spices, some types of prebiotics, bacteriophages, and anti-microbial peptides), (3) stimulate establishment of beneficial gut microbes (probiotics and some types of prebiotics), and (4) stimulate digestive function (e.g. butyric acid, gluconic acid, lactic acid, glutamine, threonine, cysteine, and nucleotides). When manipulating gut microbiota the positive effects of gut health-promoting microbes should be weighed against the increased energy and nutrient costs to support these microbes. In some instances feed additives have been proven effective in vitro but not effective in vivo. The latter applies in particular to essential oils that have strong anti-microbial activity but appear not to be effective in controlling bacterial pathogens when fed to pigs. A combination of different approaches may provide the most effective alternative to in-feed antibiotics. (C) 2010 Elsevier B.V. All rights reserved.

Abstract  The synthesis and uses of different kinds of carbohydrate-based lactones are described. This group of compounds includes aldonolactones, other related monocyclic lactones and bicyclic systems. The latter can arise from uronic acids, carboxymethyl ethers or glycosides, or from C-branched sugars.

Abstract  The recently described species Acetobacter diazotrophicus isolated from sugar cane roots and stems was found capable of growth at pH 3.0 and showed high nitrogenase activity even at pH 2.5. No growth occurred at pH 7.5. Extracellular oxidation of glucose followed by gluconic acid formation was necessary for initiation of logarithmic growth, which proceeded with N2 as the sole nitrogen source. N2-dependent growth did not occur in N-free liquid medium under air, but starter doses of 0.6 to 1 mM (NH4)2SO4 led to active N2-fixing cultures after 34 h. Nitrogenase activity was only partially inhibited by 20 mM (NH4+)2SO4 and several amino acids showed similar effects. However, NO3- did not inhibit or repress nitrogenase activity.

Abstract  For gluconic acid fermentation with Aspergillus niger oxygen-enriched air was used to increase dissolved oxygen concentration during the production phase from c. 30% to 100% saturation related to air at 1 bar. At the highest dissolved oxygen concentration the activity of glucose oxidase increased more than three-fold. At the same time, the gluconic acid production rate increased by a factor of two. During the fermentations, glucose oxidase was present endocellularly as well as exocellularly; the presence of this enzyme in the broth was due to a controlled secretion. It was found that the two fractions of the enzyme showed different activities in their contribution to overall acid production.

Abstract  Based on the immobilization enzyme technology and the fluorescence capillary analysis method, the authors have developed a highly sensitive fluorescence reaction system and a novel immobilization multienzyme glucose fluorescence capillary biosensor for determining glucose contents. Reaction principle of the system is that under the catalysis of glucose oxidase (GOD) and horseradish peroxidase (HRP) immobilized on inner surface of a medical capillary, beta-D-glucose reacts with dissolved oxygen to form gluconic acid-delta-lactone and hydrogen peroxide, and then the latter reacts with l-tyrosine to produce a tyrosine dimer, which has maximal excitation and emission wavelengths at 320 nm and 410 nm, respectively. Fluorescence of the dimer is proportional to the concentration of the beta-D-glucose. Optimization conditions suitable for the reaction system and the biosensor were as follows. Concentration of the L-tyrosine used as fluorescence reagent was 0.15 mol L(-1), the active concentrations of the GOD and the HRP for the immobilization were 15 kU L(-1) and 8 kU L(-1), respectively. Consumptions of the sample and reagents in one determination were 5.0 microL and 15 microL, respectively. Quantitative range of the biosensor for the glucose was in the range 1-10 micromol L(-1), its relative standard deviation was less than 4.9%, and its detection limit was 0.62 micromol L(-1). The biosensor's recovery was in the range 96-105%. Results of some serum determined with the biosensor and with a commercial glucose-kit were well coincident to each other. Accordingly, the biosensor can be applied to the determination of serum glucose contents in the diagnosis of diabetes.

Abstract  Sixty-eight isolates of the filamentous fungus Aspergillus niger were examined for calcium gluconate production under submerged culture conditions in 500-ml Erlenmeyer flasks. The isolate Chem-15 was selected for improvement through ultraviolet (UV) light-induced mutagenesis. Among viable mutants, strain 32 exhibited the best gluconate productivity, and it was subjected to N-methyl N-nitro N-nitroso guanidine (NG) treatment. Mutant strain NG-7 gave the highest gluconate production (86.48g/L) which varied significantly (p0.05) from that of the wild type. The mutant was cultured overnight and plated on 5-fluorocytosine-PDA medium. Gluconate productivity was increased by 35% when the process parameters, incubation period (72h), initial pH (6.5), glucose as carbon source (15%), inoculum size (1.875x10(6)CFU/ml) and corn steep liquor (CSL) as nitrogen source (0.5%) were optimized using a 2-factorial Plackett-Burman design. Maximal glucose oxidase activity (28U/ml/min) was achieved at the optimal fermentation conditions with 26.5g/L DCM. The model terms were highly significant thus suggesting the potential commercial utility of the mutant (HS, df=3 approximately 0.0182).

Abstract  The acceptor reactions of dextransucrase offer the potential for a targeted synthesis of a wide range of di-, tri- and higher oligosaccharides by the transfer of a glucosyl group from sucrose to the acceptor. We here report on results which show that the synthetic potential of this enzyme is not restricted to 'normal' saccharides. Additionally functionalized saccharides, such as alditols, aldosuloses, sugar acids, alkyl saccharides, and glycals, and rather unconventional saccharides, such as fructose dianhydride, may also act as acceptors. Some of these acceptors even turned out to be relatively efficient: OC-D-glucopyranosyl-(1 --> 5)-D-arabinonic acid, alpha-D-glucopyranosyl-(1, 4)-D-glucpyranosol, alpha-D-glucopyranosyl-(1 --> 6)-D-glucitol, alpha-D-glucopyranosyl-(1 --> 6)-D-mannitol, alpha-D-fructofuranosyl-beta-D-fructofuranosyl-(1,2':2,3')-dianhydride, 1, 5-anhydro-2-deoxy-D-arabino-hex-1-enitol ('D-glucal'), and may therefore be of interest for future applications of the dextransucrase acceptor reaction. (C) 2002 Elsevier Science Ltd. All rights reserved.

Abstract  Two classes of polymers that are currently receiving widespread attention in biosensor development are hydrogels and conducting electroactive polymers. The present study reports on the integration of these two materials to produce electroactive hydrogel composites that physically entrap enzymes within their matrices for biosensor construction and chemically stimulated controlled release. Enhanced biosensing capabilities of these membranes have been demonstrated in the fabrication of glucose, cholesterol and galactose amperometric biosensors. All biosensors displayed extended linear response ranges (10(-5)-10(-2) M), rapid response times (< 60 s), retained storage stabilities of up to I year, and excellent screening of the physiological interferents ascorbic acid, uric acid, and acetaminophen. When the cross-linked hydrogel components of these composite membranes were prepared with the amine containing dimethylaminoethyl methacrylate monomer the result was polymeric devices that swelled in response to pH changes (neutral to acidic). Entrapment of glucose oxidase within these materials made them glucose-responsive through the formation of gluconic acid. When insulin was co-loaded with glucose oxidase into these 'bio-smart' devices, there was a twofold increase in insulin release rate when the devices were immersed in glucose solutions. This demonstrates the potential of such systems to function as a chemically-synthesized artificial pancreas. (C) 2002 Elsevier Science B.V. All rights reserved.

Abstract  The side products formed in the TEMPO-mediated oxidation of glucose to glucaric acid were determined by GC. Next to glucaric acid, gluconic acid, the intermediate in the oxidation, the degradation products, oxalic acid, tartronic acid, meso(erythraric) and DL-threaric (tartaric) acid were detected. Chiral GC determined the DL-tartaric acid to be non-racemic mixtures of L- and D-tartaric acids, with inverse D/L-ratios depending on the oxidation of D- or L-glucose. The origin of all degradation products is rationalized. This study details a fast screening method to optimize the reaction conditions toward minimal degradation. (C) 2002 Published by Elsevier Science Ltd.

Abstract  The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of thioesterase II, are discussed.

Abstract  A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. (1)H nuclear magnetic resonance ((1)H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.

Abstract  BACKGROUND: In recent years inulin-type prebiotics have attracted much attention due to consumers' awareness of the health benefits of functional foods. Currently no information is available about the possible texture-modifying effect of these non-ionizable polar carbohydrates in different soy-based food systems. In this study, the effect of inulin/oligofructose on the cold aggregation and gelation of preheated soy protein isolate (SPI) and its fractions (7S, 1 IS, and their mixture), induced by glucono-delta-lactone (GDL), were evaluated by turbidity (A(600)) and dynamic rheological measurements.

RESULTS: Oligofructose significantly delayed the aggregation of all protein samples and decreased the end-point optical density of 11 S fraction and SPI. Inulin, a long-chain fructan, only delayed the aggregation of 7S globulin and reduced the capacity of aggregation (A600) of SPI. While oligofructose showed no significant effect, the addition of 5% (w/v) inulin enhanced the gelation of SPI and the 7S/11S mixture, which was demonstrated by the increase in gel storage modulus up to 13.6% and 10.1% (P < 0.05), respectively.

CONCLUSION: Inulin was found to enhance the viscoelastic properties of GDL-induced cold-set soy protein gels. It is expected that 'functional' cold-set gel products with improved texture can be prepared from preheated soy proteins and inulin. (C) 2009 Society of Chemical Industry

Abstract  To better regulate the biodistribution of the vinblastine-folate conjugate, EC145, a new folate-spacer that incorporates 1-amino-1-deoxy-D-glucitol-gamma-glutamate subunits into a peptidic backbone, was synthesized. Synthesis of Fmoc-3,4;5,6-di-O-isopropylidene-1-amino-1-deoxy-D-glucitol-gamma-glutamate 20, suitable for Fmoc-strategy solid-phase peptide synthesis (SPPS), was achieved in four steps from delta-gluconolactone. Addition of alternating glutamic acid and 20 moieties onto a cysteine-loaded resin, followed by the addition of folate, deprotection, and cleavage, resulted in the isolation of the new folate-spacer: Pte-gammaGlu-(Glu(1-amino-1-deoxy-D-glucitol)-Glu)(2)-Glu(1-amino-1-deoxy-D-glucitol)-Cys-OH (21). The addition of 21 to an appropriately modified desacetylvinblastine hydrazide (DAVLBH) resulted in a conjugate (25) with an improved therapeutic index. Treatment of 25 with DTT in neutral buffer at room temperature demonstrated that free DAVLBH would be released under the reductive environment of the internalized endosome.

Abstract  The effect of pure gluconic acid and of gluconic-acid-producing bacteria on the activity of three protozoan species, Colpoda steinii (a ciliate), Vahlkampfia sp. (an amoeba) and Neobodo designis (a flagellate), was determined in vitro and in soil microcosms. Pure gluconic acid was shown to mediate disappearance of active cells, due to encystment and/or death of protozoa, at 0.15 mM in saline medium. Similarly, the presence of gluconic acid inhibited excystment of the three protozoa tested. Enterobacter intermedium 60-2G (Wt), a gluconic acid-producing rhizobacterium, elicited the same effects on protozoa when co-cultured in the presence of 5 g L(-1) glucose. However, the effect was not observed when glucose was omitted from the medium. Similarly, a pqqA isogenic mutant strain, unable to produce gluconic acid from glucose, exhibited a reduced effect on protozoan activity. Rhizosphere-microcosm studies performed with wheat (Triticum aestivum L) confirmed the reduced ability of the pqqA mutant to limit protozoa reproduction compared to the Wt strain. Since the sodium salt of gluconic acid did not cause any significant stress to protozoa and considering that addition of 50 mM Tris-CI (pH 7.2) abolished the deleterious effect of gluconic acid, acidification of the medium appeared as the key factor that induced encystment/death of protozoa. We propose that production and excretion of gluconic acid should be considered an efficient mechanism evolved by bacteria to escape, tolerate or defend themselves against protozoan grazing in rhizosphere environments. (C) 2010 Elsevier Ltd. All rights reserved.

Abstract  Lithium iron phosphate, LiFePO4 (LFP), is considered to be a potential cathode material for lithium-ion batteries but its rate performance is significantly restricted by sluggish kinetics of electrons and lithium ions. A simple solvothermal method has been described in this article to synthesize carbon coated LFP (LFP/C) nanoplates with varying thickness from 20 to 500 nm by using different iron precursors. The influence of solvents on the morphology of the LFP in the solvothermal synthesis is also investigated. A uniform carbon coverage at the surfaces has been achieved by a selective chelating carbonising source, D-gluconic acid lactone. The smallest dimension of the nanoplates has been found to be the b-axis where the Li+ ion diffuses quickly. The overall capacity and rate performance have, in general, been found to increase with the decrease of thickness of the nanoplates. Hierarchical LFP/C with similar to 30 nm thickness shows the best electrochemical performance of 167 mA h g(-1), followed by spindle (<20 nm thickness but aggregated, 121 mA h g(-1)), plates (200-300 nm thickness, 110 mA h g(-1)) and diamond shaped LFP/C (300-500 nm thickness, 82 mA h g(-1)) at a current rate of 17mA g(-1) (0.1C rate). The spindle shaped LFP/C shows unexpected electrochemical performance since the nanoplates are heavily agglomerated in the bulk which prevents access for the liquid electrolyte, as well as additive Super P carbon, between neighbouring nanoplates during the fabrication of the composite electrodes. Hence, only the peripheral plates of the spindle are actively involved in the insertion/extraction of Li+, while the core of the spindle shaped LFP/C is almost inactive, resulting in moderate storage behaviour.

Abstract  The capacity to produce an aldonic acid from aldose sugars is widespread among acinetobacters. The enzyme responsible for this reaction is a membrane-bound, pyrrolo-quinoline quinone (PQQ)-dependent aldose dehydrogenase (E.C. 1.1.99.17). Fermentor studies with Acinetobacter calcoaceticus strain LMD 79.41 showed that the aldose dehydrogenase is synthesized constitutively. Preliminary results obtained with carbon-limited chemostat cultures of this organism revealed that the cell yield on mixtures of acetate and glucose was significantly higher than on acetate alone (molar growth yield (g multiplied by mole super(-1)) for acetate 14.6 versus 21.3 for acetate plus glucose). Since glucose is almost quantitatively oxidized to gluconic acid it follows that the aldose dehydrogenase may play a role in energy metabolism of acinetobacters.