Sucrose:sucrose fructosyltransferase and fructan:fructan fructosyltransferase from allium cepa

Sucrose: sucrose fructosyltransferase and fructan:fructan fructosyltransferase were isolated from the inner leaf bases of bulbing onion plants (Allium cepa) and separated by gel filtration on Bio-Gel P-150. Sucrose:sucrose fructosyltransferase produced only one trisaccharide, 1^F-fructosylsucrose, from sucrose. Fructan:fructan fructosyltransferase produced tetrasaccharide and higher polymers from trisaccharide. The trisaccharide found in the greatest concentration in onion, 6^G-fructosylsucrose, was produced from 1^F-fructosylsucrose by fructan:fructan fructosyltransferase and was not a product of sucrose:sucrose fructosyltransferase.     

Exopolysaccharide and Kestose Production by Lactobacillus sanfranciscensis LTH2590

The effect was investigated of sucrose concentration on sucrose metabolism and on the formation of exopolysaccharide (EPS) by Lactobacillus sanfranciscensis LTH2590 in pH-controlled fermentations with sucrose concentrations ranging from 20 to 160 g liter⁻¹. The EPS production increased and the relative sucrose hydrolysis activity decreased by increasing the sucrose concentration in the medium. The carbon recovery decreased from 95% at a sucrose concentration of 30 g liter⁻¹ to 58% at a sucrose concentration of 160 g liter⁻¹ because of the production of an unknown metabolite by L. sanfranciscensis. This metabolite was characterized as a fructo-oligosaccharide. The oligosaccharide produced by L. sanfranciscensis was purified and characterized as a trisaccharide with a glucose/fructose ratio of 1:2. The comparison of the retention time of this oligosaccharide and that of pure oligosaccharide standards using two different chromatography methods revealed that the oligosaccharide produced by L. sanfranciscensis LTH2590 is 1-kestose. Kestose production increased concomitantly with the initial sucrose concentration in the medium.     

P1 Trisaccharide (Galα1,4Galβ1,4GlcNAc) Synthesis by Enzyme Glycosylation Reactions Using Recombinant Escherichia coli

The frequency of Escherichia coli infection has lead to concerns over pathogenic bacteria in our food supply and a demand for therapeutics. Glycolipids on gut cells serve as receptors for the Shiga-like toxin produced by E. coli. Oligosaccharide moiety analogues of these glycolipids can compete with receptors for the toxin, thus acting as antibacterials. An enzymatic synthesis of the P1 trisaccharide (Galα1,4Galβ1,4GlcNAc), one of the oligosaccharide analogues, was assessed in this study. In the proposed synthetic pathway, UDP-glucose was generated from sucrose with an Anabaena sp. sucrose synthase and then converted with an E. coli UDP-glucose 4-epimerase to UDP-galactose. Two molecules of galactose were linked to N-acetylglucosamine subsequently with a Helicobacter pylori β-l,4-galactosyltransferase and a Neisseria meningitidis α-1,4-galactosyltransferase to produce one molecule of P1 trisaccharide. The four enzymes were coexpressed in a single genetically engineered E. coli strain that was then permeabilized and used to catalyze the enzymatic reaction. P1 trisaccharide was accumulated up to 50 mM (5.4 g in a 200-ml reaction volume), with a 67% yield based on the consumption of N-acetylglucosamine. This study provides an efficient approach for the preparative-scale synthesis of P1 trisaccharide with recombinant bacteria.     

Current studies on physiological functions and biological production of lactosucrose

Lactosucrose (O-β-d-galactopyranosyl-(1,4)-O-α-d-glucopyranosyl-(1,2)-β-d-fructofuranoside) is a trisaccharide formed from lactose and sucrose by enzymatic transglycosylation. This rare trisaccharide is a kind of indigestible carbohydrate, has good prebiotic effect, and promotes intestinal mineral absorption. It has been used as a functional ingredient in a range of food products which are approved as foods for specified health uses in Japan. Using lactose and sucrose as substrates, lactosucrose can be produced through transfructosylation by β-fructofuranosidase from Arthrobacter sp. K-1 or a range of levansucrases, or through transgalactosylation by β-galactosidase from Bacillus circulans. This article presented a review of recent studies on the physiological functions of lactosucrose and the biological production from lactose and sucrose by different enzymes.     

Biosynthesis of Umbelliferose in Aegopodium podagraria

The following reaction leading to the synthesis of the trisaccharide umbelliferose was demonstrated in an enzyme preparation from leaves of Aegopodium podagraria L.: sucrose + UDP-gal-¹⁴C → umbelliferose-¹⁴C + UDP. Neither galactinol nor galactose 1-phosphate could replace UDP-gal. Among 10 different sugars tested only sucrose was a suitable galatosyl acceptor.     

Oligosaccharide intermediates of fructan synthesis in Lolium temulentum

Evidence is presented that fructan accumulation in leaves of Lolium temulentum plants grown at 5° proceeded via the synthesis of trisaccharide intermediates. Studies on the oligosaccharide components of this tissue indicated that the major intermediate was probably 1^F-fructosyl sucrose (isokestose) but that two distinct series of oligofructosides could be isolated. One of these had chromatographic properties identical to the 1,2-linked inulin series from Helianthus tuberosus. The relationship of this synthetic pattern to the structure of grass fructans and their accumulation in other species is discussed.     

Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel l-FucNAc and d-FucNAc synthons and immunochemical evaluation

Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysaccharide is: →4-β-d-ManNAcA-(1→4)-α-l-FucNAc(3OAc)-(1→3)-β-d-FucNAc-(1→. The stereocontrolled insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the preparation of two novel versatile l- and d-fucosamine synthons from commercially available starting materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to CRM₁₉₇. The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to significantly compete with the native type 5 polymer in the binding with sera from mice immunized with S. aureus type 5 polysaccharide–CRM₁₉₇ conjugate.     

Evidence for Plasmid Linkage of Raffinose Utilization and Associated α-Galactosidase and Sucrose Hydrolase Activity in Pediococcus pentosaceus

The ability to ferment the trisaccharide raffinose was linked with the presence of plasmid DNA in three strains of Pediococcus pentosaceus. Parental strains showed associated inducible α-galactosidase and sucrose hydrolase activities when grown in α-galactosides and sucrose, respectively. Derivative strains of PPE1.0, PPE2.0, and PPE5.0, which had lost 30-, 28-, and 23-megadalton plasmids, respectively, had no α-galactosidase or sucrose hydrolase activity.     

Production of 6-kestose by the filamentous fungus Gliocladium virens as affected by sucrose concentration

The filamentous fungus Gliocladium virens is able to produce fructooligosaccharides (FOS), fructose-containing sugars, used as functional ingredients to improve nutritional and technological properties of foods. In this work we evaluated FOS production by G. virens when grown in a wide range of sucrose concentrations (10–400 g l^?1). High sucrose concentrations increased both biomass and FOS production, including 6-kestose, a trisaccharide comprising β (2 → 6) linked fructosyl units, with enhanced stability and prebiotic activity when compared to the typical FOS β (2 → 1) linked. The highest 6-kestose yield (3 g l^?1) was achieved in media containing 150 g l^?1 sucrose after 4–5 days of culture, production being 90% greater than in media containing 10, 30, or 50 g l^?1 sucrose. After 5 days, FOS production declined markedly, following complete sucrose depletion in the medium. Although most of the β-fructofuranosidases preferentially catalyze sucrose hydrolysis, FOS production in G. virens grown in high sucrose concentration, might be attributed to a reverse hydrolysis by these enzymes. In conclusion, high sucrose concentrations increase growth of G. virens whilst 6-kestose accumulation in the medium seems to be controlled both by specific properties of β-fructofuranosidases and on the sucrose concentration.     

Engineering Escherichia coli to produce Bordetella pertussis oligosaccharide with multiple trisaccharide units

The immunogenicity of the pertussis vaccine can be significantly improved by adding Bordetella pertussis oligosaccharide with multiple trisaccharide units. The more trisaccharide units there are, the better the efficiency of the immune response induction. However, natural B. pertussis oligosaccharides usually contain only a single terminal trisaccharide unit. In addition, B. pertussis is pathogenic, and there are potential safety hazards when preparing oligosaccharides from B. pertussis. In this study, Escherichia coli MG1655 was engineered to produce B. pertussis oligosaccharides containing multiple trisaccharide units. Fifty-nine genes relevant to the biosynthesis of the O-antigen and core oligosaccharide of lipopolysaccharide, enterobacterial common antigen, and colanic acid were deleted in MG1655, resulting in strain MDCO020. Then, 25 genes relevant to the biosynthesis of the oligosaccharide antigen in B. pertussis and 3 genes relevant to the repeating trisaccharide unit in Pseudomonas aeruginosa PAO1 were overexpressed in MDCO020, resulting in the recombinant E. coli MDCO020/pWpBpD5. The production of B. pertussis oligosaccharide with multiple trisaccharide units by MDCO020/pWpBpD5 was confirmed by SDS-PAGE and ¹H NMR analyses, and its immune response-stimulating activity was confirmed by using rabbit anti-pertussis serum.     

Detection of Kestoses and Kestose-Related Oligosaccharides in Extracts of Festuca arundinacea, Dactylis glomerata L., and Asparagus officinalis L. Root Cultures and Invertase by C and H Nuclear Magnetic Resonance Spectroscopy

A previous study (KL Forsythe, MS Feather [1989] Carbohydr Res 185: 315-319) showed that ¹³C nuclear magnetic resonance spectroscopy can be used to detect and identify mixtures of 1-kestose and neokestose after conversion to the acetate derivatives. In this study, unequivocal assignments are made for the anomeric carbon and proton signals for the above two trisaccharide acetates as well as for 6-kestose hendecaacetate and for nystose tetradecaacetate (a 1-kestose-derived tetrasaccharide). A number of oligosaccharide fractions were isolated from several plant species, converted to the acetates, and nuclear magnetic resonance spectra obtained. Using the above reference data, the following information was obtained. The trisaccharide fraction from Dactylis glomerata L. stem tissue and Asparagus officinalis L. roots contain both 1-kestose and neokestose, and the tetrasaccharide fractions contain three components, one of which is nystose. Penta- and hexasaccharide acetates were also isolated from A. officinalis L. roots and were found to contain, respectively, four and at least five components. All components of both of the above species appear to contain a kestose residue and to be produced by the sequential addition of fructofuranosyl units to these. The trisaccharide fraction from Festuca arundinacea is complex, and contains at least five different components, two of which appear to be 1-kestose and neokestose.     

A dolichol-linked trisaccharide from central nervous tissue: structure and biosynthesis.

Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[¹⁴C]glucosamine from UDP-N-acetyl[¹⁴C]glucosamine into N-acetyl[¹⁴C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[¹⁴C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [¹⁴C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys.181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[¹⁴C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [¹⁴C]trisaccharide lipid. The intact [¹⁴C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [¹⁴C]trisaccharide is converted to N,N′-diacetyl[¹⁴C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl-N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [¹⁴C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP-N-acetylglucosamine to exogenous N-acetyl[¹⁴C] glucosaminylpyrophosphoryldolichol to form [¹⁴C]disaccharide lipid. The biosynthesis of [¹⁴C]disaccharide lipid is stimulated by unlabeled UDP-N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [¹⁴C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [¹⁴C] trisaccharide lipid. The formation of the [¹⁴C]trisaccharide lipid from exogenous [¹⁴C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg²⁺, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor.     

Synthesis of glycosyl esters of oleanolic acid

The trisaccharide, O-(2,3,4-tri-O-benzoyl-β-L-rhamnopyranosyl)-(1→4)-O-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→6)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranose has been prepared by two different routes. Condensation of this trisaccharide with oleanolic acid afforded the corresponding 1,2-trans glycosyl ester. Some other glycosyl esters of oleanolic acid were also prepared by the same method.     

Sink Metabolism in Tomato Fruit : III. Analysis of Carbohydrate Assimilation in a Wild Species

Carbohydrate composition and key enzymes involved in carbohydrate metabolism were assayed throughout development of Lycopersicon esculentum and L. chmielewskii fruit. Translocation and assimilation of asymmetric sucrose and total soluble solids content was also determined in both species. The data showed that L. chmielewskii accumulated less starch than L. esculentum, and this was related to a lower level of ADPglucose pyrophosphorylase and a higher level of phosphorylase in L. chmielewskii. L. chmielewskii accumulated sucrose throughout fruit development rather than glucose and fructose which were accumulated by L. esculentum. A low level of invertase and nondetectable levels of sucrose synthase were associated with the high level of sucrose in L. chmielewskii. Translocation and assimilation of asymmetrically labeled sucrose indicated that sucrose accumulated in L. chmielewskii fruit was imported and stored directly in the fruit without intervening metabolism along the translocation path. In contrast, the relatively low level of radioactive sucrose found in L. esculentum fruit appeared to arise from hydrolysis and resynthesis of sucrose. The possible relationship between the level of soluble solids and differences in carbohydrate metabolism in sink tissue of the two species is discussed.     

Food-grade sugar can promote differentiation in melon (Cucumis melo L.) tissue culture

The objective of the present study was to investigate the origin of discrepancy between experimental results in in vitro culture of Turkish melon (Cucumis melo L.) cultivars, conducted by the same individual using the same protocol and same seed batches in two different laboratories. The difference in the sucrose source was found to be the major reason for the deviation in results between the two laboratories. The percentage of regenerating explants and the number of bud-like protuberances and/or shoots were significantly greater when a food-grade Turkish sucrose was used in the medium compared with analytical-grade sucrose. Media formulated with the food-grade sucrose regenerated 37 and 67 % more explants and bud-like protuberances and/or shoots per explant, respectively, than media containing analytical-grade sucrose. No meaningful differences were found in added elements or anions between the sucrose sources or by liquid chromatography/mass spectroscopy. The only significant chemical difference observed between the sucrose samples was the presence of melanoidins (Maillard reaction products) in the food-grade sucrose. The melanoidins were of high molecular weight (>3,000 Da determined by ultrafiltration), with characteristic ultraviolet?Cvisible spectra and in vitro antioxidant activity. Melanoidin-containing sucrose can be differentiated by color and spectroscopy.     

Transport-limited sucrose utilization and neokestose production by Phaffia rhodozyma

Summary Growth of an astaxanthin hyper-producing strain of Phaffia rhodozyma on sucrose is accompanied by the accumulation of glucose and fructose in the medium due to the limited capacity of the corresponding monosaccharide transport system or systems. This is accompanied by the production of the trisaccharide neokestose by transglycosylation reactions.     

Comparative Sucrose Responsiveness in Apis mellifera and A. cerana Foragers

In the European honey bee, Apis mellifera, pollen foragers have a higher sucrose responsiveness than nectar foragers when tested using a proboscis extension response (PER) assay. In addition, Africanized honey bees have a higher sucrose responsiveness than European honey bees. Based on the biology of the Eastern honey bee, A. cerana, we hypothesized that A. cerana should also have a higher responsiveness to sucrose than A. mellifera. To test this hypothesis, we compared the sucrose thresholds of pollen foragers and nectar foragers in both A. cerana and A. mellifera in Fujian Province, China. Pollen foragers were more responsive to sucrose than nectar foragers in both species, consistent with previous studies. However, contrary to our hypothesis, A. mellifera was more responsive than A. cerana. We also demonstrated that this higher sucrose responsiveness in A. mellifera was not due to differences in the colony environment by co-fostering two species of bees in the same mixed-species colonies. Because A. mellifera foragers were more responsive to sucrose, we predicted that their nectar foragers should bring in less concentrated nectar compared to that of A. cerana. However, we found no differences between the two species. We conclude that A. cerana shows a different pattern in sucrose responsiveness from that of Africanized bees. There may be other mechanisms that enable A. cerana to perform well in areas with sparse nectar resources.     

Proton-Coupled Sucrose Transport in Plasmalemma Vesicles Isolated from Sugar Beet (Beta vulgaris L. cv Great Western) Leaves

Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton-coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. ΔpH-dependent sucrose transport exhibited saturation kinetics with an apparent K_m of 1.20 ± 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. ΔpH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because ΔpH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K⁺, Mg²⁺-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue.     

Sugar transport in isolated corn root protoplasts

Isolated corn (Zea mays L.) root protoplasts were used to study sucrose and hexose uptake. It is found that glucose was preferentially taken up by the protoplasts over sucrose and other hexoses. Glucose uptake showed a biphasic dependence on external glucose concentration with saturable (K_m of 7 millimolar) and linear components. In contrast, sucrose uptake only showed a linear kinetic curve. Sucrose and glucose uptake were linear over a minimum of 1 hour at pH 6.0 and 1 millimolar exogenous sugar concentration. Glucose uptake showed a sharp 42°C temperature optimum, while sucrose uptake showed a lower temperature sensitivity which did not reach a maximum below 50°C. Uptake of both sugars was sensitive to several metabolic inhibitors and external pH. Differences between sucrose and glucose uptake in two different sink tissue (i.e. protoplasts from corn roots and soybean cotyledons) are discussed.     

Identification of Prebiotic Fructooligosaccharide Metabolism in Lactobacillus plantarum WCFS1 through Microarrays

Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-color microarrays were used to screen for differentially expressed genes when grown on scFOS compared to glucose (control). A significant up-regulation (8- to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a β-fructofuranosidase, a fructokinase, an α-glucosidase, and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTSs. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a β-fructofuranosidase that could be used for scFOS degradation.