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At least 181 records · Page 10Linked to original sources
PubMed1998

Evidence from temperature studies that the human erythrocyte hexose transporter has a transient memory of its dissociated ligands.

The inhibition constant of L-sorbose efflux (Ki(sorbose)) from human erythrocytes for inhibition by D-glucose increases from 5.15 +/- 0.89 to 12.24 +/- 1.9 mM on cooling from 50 degrees C to 30 degrees C; the Ki(sorbose) of D-mannose increases similarly on cooling. The activation energy Ea(sorbose) of net L-sorbose exit from human erythrocytes is 62.9 +/- 3.1 kJ mol-1; but in the co-presence of 5 mM D-glucose Ea(sorbose) is reduced to 41.7 +/- 1.6 kJ mol-1 (P < 0.005). These data are consistent with the view that when D-glucose binds to the hexose transporter it leads to an activated transporter state which remains transiently activated after glucose dissociates; if L-sorbose binds to the excited state it is more mobile than otherwise and consequently the apparent Ki(sorbose) of D-glucose is raised. Cooling prolongs the decay time of the activated state; hence the Ki(sorbose) of D-glucose rises as temperature is reduced.

Biological Transport↗
PubMed1995

Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans.

Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob+) in Escherichia coli S17-1 (Tra+). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose.

Acetobacter↗
PubMed1962

Mechanism of glucose transport across the yeast cell membrane.

Cirillo, Vincent P. (Seton Hall College of Medicine and Dentistry, Jersey City, N.J.). Mechanism of glucose transport across the yeast cell membrane. J. Bacteriol. 84:485-491. 1962.-The kinetics of d-glucose and l-sorbose transport was studied in Saccharomyces cerevisiae inhibited with iodoacetic acid under nitrogen to prevent glucose metabolism. d-Glucose was found to compete with l-sorbose for a common membrane transport system with an apparent affinity greater than 25 times that of sorbose. A comparison of the net rate of glucose and sorbose transport at 50 and 500 mm external concentration showed that glucose transport is greater than that of sorbose from the lower concentration, but sorbose transport is greater than glucose at the higher concentration. This reversal of transport rate of two sugars with markedly different affinities is predicted by the membrane carrier theory. A further prediction of carrier theory was confirmed by the demonstration that the rate of glucose transport into fructose-loaded cells is greater than into unloaded cells.

Biological Transport↗
PubMed2000

Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures.

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10 degrees C to 37 degrees C and had average optimum growth temperature between 30-33 degrees C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37 degrees C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37 degrees C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264T was unable to do such fermentation at 37 degrees C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30 degrees C. Even oxidative fermentation of D-fructose done at 37 degrees C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37 degrees C was superior to that observed at 30 degrees C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.

Catalysis↗
PubMed2003

Water mobility in multicomponent model media as studied by 2H and 17O NMR.

Molecular mobility of water was studied in a microbiological media containing complex and heterogeneous mixtures of cellulose, l-sorbose, and orange serum broth (OSB) using (2)H and (17)O high-resolution NMR. All mixtures showed Lorentian (17)O NMR spectra but complex (2)H NMR line shapes. Sorbose, when solubilized, caused line-narrowing where as cellulose-OSB mixtures showed wide peaks with flat plateaus. Presence of liquid or solvent water had a profound effect on a marked increase in T(2) relaxation time observed in sorbose-containing samples. (17)O NMR data were not composition dependent, while (2)H NMR data were highly sorbose dependent.

Cellulose↗
PubMed1999

Role of water mobility on mold spore germination.

A sugar transport defected strain of Aspergillus nidulans (biA-1 sorA-2) was tested for spore germination in nutrient media containing various water activity (a(w)) values and varying amounts of non-nutritive, nontoxic carbohydrates (L-sorbose and cellulose). Freeze-dried media [containing the same nutrient level but different in sorbose/cellulose (s:c) ratio] were adjusted to 0.75-0.97a(w) at 25 degrees C before inoculation. Minimum a(w) for germination varied with s:c ratio. Because both sorbose and cellulose were not metabolizable and unable to be transported into the cells, the results reflected the molecular mobility of water. (2)H NMR T(2) relaxation time correlated well with spore germination time, and it distinguished the difference between water sorbed to cellulose and water in a solution associated with dissolved sorbose. On the other hand, mold germination time correlated poorly with a(w). It was highly dependent on the s:c ratio. Water mobility was found to correlate better with biological activity than a(w) because it differentiated the availability between water in dissolved sorbose and adsorbed water in cellulose.

Aspergillus↗
PubMed1987

Biotyping as a method of screening for enterotoxigenic Escherichia coli.

A scheme of biotyping described originally for E. coli isolated from urine was used to investigate enterotoxigenic (ETEC) and non-enterotoxigenic (non-ETEC) fecal E. coli. Primary biotype was determined by fermentation of raffinose, sorbose and dulcitol and decarboxylation of ornithine. Failure to ferment sorbose correlated best with enterotoxicity; 95.5% of ETEC and less than 40% of non-ETEC did not ferment sorbose. The sensitivity compares favourably with reported results of the use of polyvalent antisera for recognition of serotypes associated with ETEC, although specificity was lower using biotyping. Strains included in our study were mainly from Australia, New Zealand or Indonesia and we do not know if our observations apply to E. coli isolated elsewhere. If failure to ferment sorbose proved to be a characteristic of most ETEC, this reaction offers the possibility of developing selective media to increase the yield of ETEC from primary plates for laboratories in which gene probe techniques for recognition of ETEC are not available.

Bacteriological Techniques↗
PubMed1975

New mechanisms for the biosynthesis and metabolism of 2-keto-L-gulonic acid in bacteria.

L-Sorbose is oxidized to 2-keto-L-gulonic acid (KGA) via the following sequence of reactions which we call the "sorbosone pathway": L-sorbose in equilibrium L-sorbosone leads to KGA. The first step is reversible and is mediated by enzymes found in a soluble fraction obtained from Pseudomonas putida ATCC 21812. Although no cofactor requirements were found for the forward reaction, the reverse reaction clearly required NADH. Enzymes for this NADH-dependent synthesis of L-sorbose could be differentiated on the basis of molecular weights. The second step in the sorbosone pathway is catalyzed by a particulate enzyme found in extracts from P. putida and Gluconobacter melanogenus IFO 3293. The rate limiting reaction in the sorbosone pathway is the synthesis of L-sorbosone. In addition to P. putida, Klebsiella pneumoniae (ATCC 27858) and Serratia marcescens (ATCC 27857) also contain the enzymes which catalyze the reactions of the sorbosone pathway. Two of the bacteria studied, P. putida and G. melanogenus, also contain an enzyme involved in the further metabolism of KGA to L-idonic acid. This enzyme, referred to as KGA-reductase, is found in the soluble fraction of cell-free extracts and is dependent on NADH or NADPH.

Bacteria↗
PubMed1975

Reversible permeability changes in the membrane of a yeast cell sugar compartment.

Sorbose uptake by Saccharomyces cerevisiae was increased 40 to 60% by glucose and other metabolizable sugars. Neither growth nor binding accounted for the increased uptake. However, accessibility of a restrictive intracellular compartment was increased as shown by counterflow and efflux measurements. Efflux from the compartment was more than doubled by glucose. This effect was reversed by washing and was prevented by iodoacetic acid and other inhibitors, but not by cycloheximide. No evidence was found for a facilitated transport system in the compartment membrane such as exists in the external cell membrane. It was concluded that sorbose crosses the compartment membrane by simple diffusion and that a reaction requiring the metabolism of sugars increases the permeability of the membrane. Arabinose and fucose entered and were lost from the compartment like sorbose, whereas dimethylsulfoxide was unaffected by the compartment. All three of these later compounds were bound by the cells when glucose was available in uptake suspensions. Binding was prevented by iodoacetic acid, but not by cycloheximide.

Arabinose↗
PubMed2005

Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency.

To improve the stability and catalytic efficiency of pyranose 2-oxidase (P2Ox) by molecular enzyme evolution, we cloned P2Ox cDNA by RACE-PCR from a cDNA library derived from the basidiomycete Peniophora gigantea. The P2Ox gene was expressed in Escherichia coli BL21(DE3), yielding an intracellular and enzymatically active P2OxB with a volumetric yield of 500 units/l. Site-directed mutagenesis was employed to construct the P2Ox variant E540K (termed P2OxB1), which exhibited increased thermo- and pH-stability compared with the wild type, concomitantly with increased catalytic efficiencies (k(cat)/K(m)) for D-xylose and L-sorbose. P2OxB1 was provided with a C-terminal His(6)-tag (termed P2OxB1H) and subjected to directed evolution using error-prone PCR. Screening based on a chromogenic assay yielded the new P2Ox variant K312E (termed P2OxB2H) that showed significant improvements with respect to k(cat)/K(m) for D-glucose (5.3-fold), methyl-beta-D-glucoside (2.0-fold), D-galactose (4.8-fold), D-xylose (59.9-fold), and L-sorbose (69.0-fold), compared with wild-type P2Ox. The improved catalytic performance of P2OxB2H was demonstrated by bioconversions of L-sorbose that initially was a poor substrate for wild-type P2Ox. This is the first report on the improvement of a pyranose 2-oxidase by a dual approach of site-directed mutagenesis and directed evolution, and the application of the engineered P2Ox in bioconversions.

Amino Acid Substitution↗
PubMed1993

Purification and characterization of a pyranose oxidase from the basidiomycete Peniophora gigantea and chemical analyses of its reaction products.

A pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea. This enzyme was purified 104-fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxidase has a relative molecular mass (M(r)) of 322,800 +/- 18,300 as determined on the basis of its Stokes' radius (rs = 6.2 nm) and sedimentation coefficient (S20,w = 10.6), dynamic light-scattering experiments, gradient-gel electrophoresis and cross-linking studies. SDS/PAGE resulted in one single polypeptide band of M(r) 76,000 indicating that the enzyme consists of four subunits of identical size. The pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates D-glucose, D-xylose, L-sorbose, D-galactose, methyl beta-D-glucoside, maltose and D-fucose. Regioselective oxidation of D-glucose, L-sorbose and D-xylose to 2-keto-D-glucose, 5-keto-D-fructose and 2-keto-D-xylose, was demonstrated by identifying the reaction products by mass spectroscopy 13C-NMR spectroscopy and 1H-NMR spectroscopy after purification and derivatization. The pH optimum of the pyranose oxidase was in the range pH 6.0-6.5 in 0.1 M potassium phosphate, and its activation energy (delta H degree) for the conversion of D-glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis-Menten kinetics, and the Km values determined for D-glucose, L-sorbose, D-xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reagents and bivalent cations each at 1 mM.

Basidiomycota↗
PubMed2002

L-glucitol catabolism in Stenotrophomonas maltophilia Ac.

The carbohydrate catabolism of the bacterium Stenotrophomonas maltophilia Ac (previously named Pseudomonas sp. strain Ac), which is known to convert the unnatural polyol L-glucitol to D-sorbose during growth on the former as the sole source of carbon and energy, was studied in detail. All enzymes operating in a pathway that channels L-glucitol via D-sorbose into compounds of the intermediary metabolism were demonstrated, and for some prominent reactions the products of conversion were identified. D-Sorbose was converted by C-3 epimerization to D-tagatose, which, in turn, was isomerized to D-galactose. D-Galactose was the initial substrate of the De Ley-Doudoroff pathway, involving reactions of NAD-dependent oxidation of D-galactose to D-galactonate, its dehydration to 2-keto-3-deoxy-D-galactonate, and its phosphorylation to 2-keto-3-deoxy-D-galactonate 6-phosphate. Finally, aldol cleavage yielded pyruvate and D-glycerate 3-phosphate as the central metabolic intermediates.

Chromatography, High Pressure Liquid↗
PubMed1977

Monosaccharide transport in protein-depleted vesicles from erythrocyte membranes.

Treatment of human erythrocyte membranes with dilute alkali (pH 11.5) generates sealed, protein-depleted vesicles that can be isolated by density gradient centrifugation. The vesicles are 0.5 to 2.0 micrometers in diameter, and their membranes are predominantly oriented inside-out. The vesicles lack protein bands 1, 2, 5, and 6 (nomenclature of Steck, T.L. (1974) J. Cell Biol. 62, 1-19) of the erythrocyte membrane. L-Sorbose, a substrate of the monosaccharide transport system in erythrocytes, is transported by the vesicles. Based on comparisons between erythrocytes and vesicles with regard to specificity, temparture dependence, and effects of inhibitors, we conclude that sorbose uptake into the vesicles occurs by way of the monosaccharide transport system. The specific activity of the transport system in vesicles, as determined by initial rate measurements of sorbose uptake, averaged 58% of that in erythrocytes. This finding indicates that the major polypeptides of Bands 1, 2, 5, and 6 do not play an obligatory role in monosaccharide transport.

Biological Transport↗