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Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M(r) 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M(r) 37000, pI 3.6 and a 60-fold preference for NADPH over NADH. In this study, the influence of aeration and the response to the addition of electron acceptors on xylose fermentation by F. oxysporum were also studied. The batch cultivation of F. oxysporum on xylose was performed under aerobic, anaerobic and oxygen-limited conditions in stirred tank reactors. Oxygen limitation had considerable influence on xylose metabolism. Under anaerobic conditions (0 vvm), xylitol was the main product with a maximum yield of 0.34 mole of xylitol/mole of xylose while the maximum ethanol yield (1.02 moles of ethanol/mole of xylose) was obtained under aerobic conditions (0.3 vvm). When the artificial electron acceptor acetoin was added to an anaerobic batch fermentation of xylose by F. oxysporum, the ethanol yield increased while xylitol excretion was also decreased.
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PMID:NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum. 1623 33

Radiolabeled d-[1-(3)H]glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the (3)H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained (3)H, showing that de novo synthesis of myo-inositol from [1-(3)H]glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The (3)H percentage in trifluoroacetic acid-soluble arabinose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, (3)H percentages in hexose residues were higher than their mole percentages. The results suggest that myo-inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo-inositol oxidation pathway.
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PMID:myo-Inositol Synthesis from [1-H]Glucose in Phaseolus vulgaris L. during Early Stages of Germination. 1666 44

Dissimilatory nitrate reduction metabolism, of the natural xylose-fermenting fungus Fusarium oxysporum, was used as a strategy to achieve anaerobic growth and ethanol production from xylose. Beneficial alterations of the redox fluxes and thereby of the xylose metabolism were obtained by taking advantage of the regeneration of the cofactor NAD(+) during the denitrification process. In batch cultivations, nitrate sustained growth under anaerobic conditions (1.21 g L(-1) biomass) and simultaneously a maximum yield of 0.55 moles of ethanol per mole of xylose was achieved, whereas substitution of nitrate with ammonium limited the growth significantly (0.15 g L(-1) biomass). Using nitrate, the maximum acetate yield was 0.21 moles per mole of xylose and no xylitol excretion was observed. Furthermore, the network structure in the central carbon metabolism of F. oxysporum was characterized in steady state. F. oxysporum grew anaerobically on [1-(13)C] labelled glucose and unlabelled xylose in chemostat cultivation with nitrate as nitrogen source. The use of labelled substrate allowed the precise determination of the glucose and xylose contribution to the carbon fluxes in the central metabolism of this poorly described microorganism. It was demonstrated that dissimilatory nitrate reduction allows F. oxysporum to exhibit typical respiratory metabolic behaviour with a highly active TCA cycle and a large demand for NADPH.
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PMID:Engineering of the redox imbalance of Fusarium oxysporum enables anaerobic growth on xylose. 1679 96

The range of value-added chemicals produced by Escherichia coli from simple sugars has been expanded to include xylitol. This was accomplished by screening the in vivo activity of a number of heterologous xylitol-producing enzymes. Xylose reductases from Candida boidinii (CbXR), Candida tenuis (CtXR), Pichia stipitis (PsXR), and Saccharmoyces cerivisiae (ScXR), and xylitol dehydrogenases from Gluconobacter oxydans (GoXDH) and Pichia stipitis (PsXDH) were all functional in E. coli to varying extents. Replacement of E. coli's native cyclic AMP receptor protein (CRP) with a cyclic AMP-independent mutant (CRP*) facilitated xylose uptake and xylitol production from mixtures of glucose and xylose, with glucose serving as the growth substrate and source of reducing equivalents. Of the enzymes tested, overexpression of NADPH-dependent CbXR produced the highest concentrations of xylitol in shake-flask cultures (approximately 275 mM in LB cultures, approximately 180 mM using minimal medium). Expression of CbXR in strain PC09 (crp*, DeltaxylB) in a 10-L controlled fermentation containing minimal medium resulted in production of approximately 250 mM xylitol (38 g/L), with concomitant utilization of approximately 150 mM glucose. The ratio of moles xylitol produced (from xylose) per mole glucose consumed was improved to > 3.7:1 using metabolically active "resting" cells.
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PMID:Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. 1683 79

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.
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PMID:Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans. 1721 40

Saturating wood particles with HCl gas under pressure was found to be an effective pretreatment prior to subjecting wood to dilute acid hydrolysis. Pretreament is necessary to release sugars from wood because of the tight lattice structure of cellulose. The HCl gas makes the cellulose more susceptible to subsequent acid hydrolysis and the glucose yield is doubled when dilute acid hydrolysis is preceded by HCl saturation at high pressure. The saturation was most effectively performed in a fluidized bed reactor, with pure HCl gas fluidizing an equal volume of ground wood plus inert particles. The fluidized bed effectively dissipated the large amount of heat released upon HCl absorption into the wood. Batch reaction times of 1 h at 315 psia gave glucose yields of 80 degrees and xylose yields of 95 degrees after dilute acid hydrolysis. A model was developed which proposed gas diffusing through the solid as limiting the reaction rate and this was found to effectively describe the HCl-wood reaction in the fluidized bed. The HCl was found to form a stable adduct with the lignin residue in the wood, in a ratio of 3.33 mole of lignin monomer. The adduct was broken upon the addition of water.
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PMID:Production of sugars from wood using high-pressure hydrogen chloride. 1854 4

A new experimental technique, called oxygen programmed fermentation (OPF), was used to study microbial cultures of the years Pichia stipitis and Candida utilis growing on xylose as carbon and energy source. In the oxygen programmed fermentation, the inlet oxygen mole fraction was continuously changed to scan through a wide range of oxygen uptake rates in a continuous culture. The largest ethanol yields and productivities of P. stipitis were found at oxygen transfer rates below 1.5 mmol L(-1) h(-1). It was found that the ratio between the culture fluorescence and near-IR absorbance increased at oxygen transfer rates lower than 1.5 mmol L(-1) h(-1). Small amounts of ethanol were produced also by C. utilis when the oxygen transfer rate was between 0 and 3 mmol L(-1) h(-1). It is suggested that OPF will form a nice complement to ordinary, microaerobic chemostat experiments, by making the identification of interesting regions of oxygen transfer rates possible in an efficient and time-saving initial experiment. (c) 1994 John Wiley & Sons, Inc.
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PMID:A new method for studying microaerobic fermentations. II. An experimental investigation of xylose fermentation. 1861 76

The titrimetric determination of glucose, fructose, mannose, galactose, arabinose, xylose and sucrose with potassium ditelluratocuprate(III) is described. On heating, pentoses and hexoses consume 20 and 24 equivalents of copper(III) per mole respectively, and sucrose consumes 48 equivalents.
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PMID:Oxidation of some sugars with copper(III). 1896 Mar 4

The [Pd(II){(R,R)-chxn}(OH)(2)] reagent (chxn=1,2-diaminocyclohexane) is introduced as a metal probe for the detection of the bidentate chelating sites of a glycose. Two moles of hydroxide per mole palladium support double deprotonation of potentially chelating diol functions at a glycose's backbone. The individual chelating sites are detected using one- and two-dimensional NMR techniques. At equimolar amounts of palladium(II) and aldose, the metal-binding sites include mostly the hydroxy function at the anomeric carbon atom. Chelators are derived from both the pyranose and the furanose isomers. Most pyranose-based chelators form five-membered chelate rings by using their 1,2-diol function. Though 1,2-diolate bonding is also common to the furanoses, the formation of six-membered chelate rings by 1,3-bonding is more significant for them. Metal-excess conditions provoke mostly bis-bidentate 1,2;3,4-chelation but unusual isomers form also: thus d-xylose is dimetallated in its all-axial beta-pyranose form, and erythrose's dimetallation results in the formation of two isomers of a metal derivative of the open-chain hydrate. The spectroscopic results are supported by crystal-structure determinations on [Pd{(R,R)-chxn}(alpha-D-Xylp1,2H(-2)-kappaO(1,2))].H(2)O (Xyl=xylose), [Pd{(R,R)-chxn}(alpha-D-Ribp1,2H(-2)-kappaO(1,2))].2.25H(2)O (Rib=ribose), [Pd{(R,R)-chxn}(alpha-L-Thrf1,3H(-2)-kappaO(1,3))].2H(2)O (Thr=threose) and [Pd{(R,R)-chxn}(alpha-D-Eryf1,3H(-2)-kappaO(1,3))].3H(2)O (Ery=erythrose).
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PMID:Bidentate palladium(II) chelation by the common aldoses. 1950 13

Psidium guajava L. is a valuable farm fruit plant having many medicinal uses. Previously its budding leaves (PE) were shown to contain huge amounts of soluble polyphenolics (SP) including (in mg/g) gallic acid (348), catechin (102), epicatechin (60), rutin (100), quercetin (102), and rutin (100) and to exhibit potent anticancer activity. However, reconstitution of these polyphenolics recovered only 40% of the original bioactivity, and the soluble carbohydrate (SC) portion in PE was suspected to contribute the remaining. PE contained a novel rhamnoallosan, which had a carbohydrate/protein (w/w) ratio = 29.06%/10.27% (=2.83, average molecular mass of 5029 kDa), characteristically evidencing a peptidoglycan, consisting of a composition (mole % ratio) of rhamnose/allose/arabinose/tallose/xylose/fucose/glucose/mannose/galactose = 36.05:24.24:8.76:7.95:7.37:5.90:3.69:3.19:2.85 and of amino acid (in wt %) glycine/leucine/proline/alanine/methionine/isoleucine/valine/histidine/tyrosine/phenylalanine/cysteine/aspartic acid/lysine/glutamic acid = 37.12:12.68:10.05:8.97:5.99:4.89:4.83:4.25:4.05:2.78:1.86:1.10:0.73:0.70. Kinetic analysis showed comparable apparent cell-killing rate coefficients (k(app)) to be 4.03 x 10(3) and 2.92 x 10(3) cells mg(-1) h(-1), respectively, by SP and SC, evidencing the complementary anti-DU-145 bioactivity in nature.
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PMID:Anticancer activity of rhamnoallosan against DU-145 cells is kinetically complementary to coexisting Polyphenolics in Psidium guajava budding leaves. 1955 30


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