Effect of light and media upon growth and melanin formation in aureobasidium pullulans (de By.)Arn. (=pullularia pullulans)

Summary Polysaccharides like dextrine and starch are shown to be the best carbon sources for the growth ofAureobasidium pullulans although growth is good upon a variety of other carbon sources. Light increases growth markedly when polysaccharides are the carbon source but not when other sugars are used. Variation in cell morphology is described in response to sugars and light. Extracellular granules, whose properties resemble those of melanin, are produced when dextrine is the carbon source in a defined medium containing asparagine as the source of nitrogen. The dark pigment was extracted from the walls of thick-walled brown cells ofA. pullulans and characterized as a melanin on the basis of several tests, including solubility and absorption spectrum.A. pullulans was grown on several defined and undefined media and the response of the fungus to light is shown to be determined by the medium, and the temperature at which the cultures are grown.     

Chemical Analysis of the Lamella Walls of Agaricus bisporus Fruit Bodies

Purified lamella wall fragments of Agaricus bisporus fruit bodies were analyzed and shown to consist of neutral sugars (46.5%), hexosamines (31.7%), proteins (9.5%), some lipid material (10.0%), and ash (1.4%). The cell walls were fractionated on the basis of their polysaccharide solubility in water and alkaline solutions. The isolated fractions, using methylation analysis, exhibited striking chemical structural differences compared with the same fractions obtained from the corresponding vegetative cells and fruit bodies (stipe and pileus) walls. The structural differences detected in the wall seem to correspond to the ultimate differentiation of the mycelium inside the fruit body of A. bisporus. Received: 30 November 1998 / Accepted: 29 January 1999     

Influence of Sugars on in vitro Rooting and Acclimatization of Carob Tree

Carob tree (Cerafoma siliqua L.) micropropagated shoots were rooted on half-strength Murashige and Skoog medium, supplemented with different types and concentrations of sugars, in order to determine the effects of sugar composition and concentration on in vitro rooting and in vivo establishment of the plantlets. Among the various sugars tested, the best rooting response was obtained with 145 mM sucrose, both in terms of rooting frequency and index of rooting. The use of filter-sterilized rather that autoclaved fructose increased root number and root length. Sugar treatment during rooting slightly influenced plantlet survival and growth during acclimatization. A reduction in the glucose concentration during rooting was beneficial for plantlet acclimatization.     

UDP‐sugar pyrophosphorylase is essential for arabinose and xylose recycling,and is required during vegetative and reproductive growth in Arabidopsis

Numerous nucleotide sugars are needed in plants to synthesize cell wall polymers and glycoproteins. The de novo synthesis of nucleotide sugars is of major importance. During growth, however, some polymers are broken down to monosaccharides. Reactivation of these sugars into nucleotide sugars occurs in two steps: first, by a substrate‐specific sugar‐1‐kinase and, second, by UDP‐sugar‐pyrophosphorylase (USP), which has broad substrate specificity. A knock‐out of the USP gene results in non‐fertile pollen. By using various genetic complementation approaches we obtained a strong (>95%) knock‐down line in USP that allowed us to investigate the physiological role of the enzyme during the life cycle. Mutant plants show an arabinose reduction in the cell wall, and accumulate mainly two sugars, arabinose and xylose, in the cytoplasm. The arabinogalactanproteins in usp mutants show no significant reduction in size. USP is also part of the myo‐inositol oxygenation pathway to UDP‐glucuronic acid; however, free glucuronic acid does not accumulate in cells, suggesting alternative conversion pathways of this monosaccharide. The knock‐down plants are mostly sterile because of the improper formation of anthers and pollen sacks.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

On accuracy of predicting densities and solubility parameters of polymers using atomistic simulations

Abstract

Molecular dynamics simulation is performed to compute densities and solubility parameters of polymers. TEAM force field is validated on monomers and then employed for predictions for polymers built with different chain lengths. The calculated densities converge when the polymer chain lengths are >10 repeat units. With the consideration of polymer crystallinity, the predictions agree well with experimental data. The solubility parameters calculated from the cohesive energy densities exhibit systematical and progressive underestimates as the chain lengths increase, indicating the energy contribution from polymer chain conformation changes from bulk state to solution must be included in order to calculate the solubility parameters correctly. A model using the radiuses of gyration to represent the conformation changes is proposed to correct the systematical error.     

VARIATIONS IN THE SUBSTITUTED 3‐LINKED MANNANS CLOSELY ASSOCIATED WITH THE SILICIFIED WALLS OF DIATOMS1

The polysaccharides from cleaned frustules of the diatoms Pinnularia viridis (Nitzsch) Ehrenberg, Craspedostauros australis Cox, Thalassiosira pseudonana Hasle et Heimdal, and Nitzschia navis‐varingica Lundholm et Moestrup were extracted with hot alkali that dissolved the silica and were characterized by constituent sugar and linkage analyses. The polysaccharides from P. viridis were investigated further by permethylation, partitioning according to solubility, desulfation, and CD₃I‐methylation. Yields of carbohydrate in the hot alkali extracts ranged from 0.9% to 1.8% w/w based on the dry weight of the silica. Mannose was the dominant sugar in the polysaccharides from all four species (54–69 mol% of constituent sugars), although 14 other monosaccharides, including neutral sugars (glucose, galactose, xylose, arabinose, rhamnose, fucose), acidic sugars (glucuronic acid, galacturonic acid, 2‐O‐methylglucuronic acid), and O‐methylated neutral sugars (2‐O‐methylrhamnose, 3‐O‐methylrhamnose, 2,3‐di‐O‐methylrhamnose, 3‐O‐methylxylose, 4‐O‐methylxylose) were also detected in varying proportions among the four samples. The polysaccharides were predominantly composed of a 3‐linked mannopyranose backbone with a prevalence of linkage and/or substitution at O‐2 of the 3‐linked mannopyranosyl residues, and they were polyanionic, bearing uronic acid residues and/or sulfate esters. There were, however, species‐specific differences in the degree and position of substitution on the mannan backbone, the type and substitution patterns of the anionic substituents, and the type and linkage patterns of sugars other than mannose. Although definitive functions for these polysaccharides in diatom biology remain uncertain, a possible role in biosilicification is discussed.     

Influence ďune alimentation continue avec une solution glucosée sur ľévolution des glucides et des protéines dans les divers organes de la rose coupée (Rosa hybrida cv. Carina)

Influence of continuous supply with a solution of glucose on the changes in the content of soluble sugars and proteins in various organs of the cut rose (Rosa hybrida cv. Carina). Exogenous glucose continuously supplied to the cut rose is immediately converted into saccharose in the stem tissues. This saccharose migrates to the flower, where it is immediately hyd-rolysed, and to the leaves where its hydrolysis occurs more slowly. The reducing sugars resulting from the hydrolysis of saccharose in the flower and, therefore, possibly from the hydrolysis of saccharose in leaves, induce a large accumulation of hexoses (glucose and fructose) in the flower. The protein content does not depend on the level of reducing sugars in the flower.     

Isolation of a New Intermediary Substance Consisting of Colistin Fatty Acid and l-α,δ-Diaminobutyric Acid from Colistin-producing Cells of Bacillus colistinus Koyama

Several saccharides were found to be significantly effective in providing protection against hydrostatic pressure and high temperature damage in the yeast Saccharomyces cerevisiae. The extent of barotolerance and thermotolerance with seven different sugars showed a linear relationship to their mean number of equatorial OH groups. The same linear relatioship is seen when sugars protect protein molecules against elevated temperatures in vitro. Some sugars were more effective in providing protection against hydrostatic pressure nearly a hundred times than high temperature. Pre-heat shock treatment on yeast cells induce various stress tolerances. In this report, pre-heat shocked cells showed potent protection against elevated temperature, but these cells showed faint protection against elevated pressure.

These results suggest that sugars may protect cells against hydrostatic pressure and high temperature in a similar manner, probably by stabilizing the macromolecule(s), and such type of protection may be suited for pressure stress.     

Mobilization of cadmium and other metals from two soils by root exudates of Zea mays L., Nicotiana tabacum L. and Nicotiana rustica L.

Soluble root exudates were collected from three plants (Nicotiana tabacum L., Nicotiana rustica L. and Zea mays L.), grown under axenic and hydroponic conditions, in order to study their metal-solubilizing ability for Cd and other cations (Cu, Fe, Mn, Ni, Zn). Nicotiana spp. and Zea mays L. root exudates differed markedly in C/N ratio, sugars vs. amino acids ratio and organic acids content. Metals from two soils were extracted with either root exudate solutions, containing equal amounts of organic carbon, or distilled water as control. In the presence or absence of root exudates, the solubility of Fe and Mn was much higher than of the four other metals tested. Root exudates increased the solubilities of Mn and Cu, whereas those of Ni and Zn were not affected. Root exudates of Nicotiana spp. enhanced the solubility of Cd. The extent of Cd extraction by root exudates (N. tabacum L. N. rustica L. Zea mays L.) was similar to the order of Cd bioavailability to these three plants when grown on soil. An increase in Cd solubility in the rhizosphere of apical root zones due to root exudates is likely to be an important cause of the relatively high Cd accumulation in Nicotiana spp.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins

Summary A new general method has been developed for the specific histochemical identification ofO-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose andN-acetylhexosamine with an ester substituenent(s) located on a potentialvicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-Bh*-Bh-PAS) method] the initial PA-Bh treatment rendersvicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from bothO-acyl sugars andO-acyl sialic acids by saponification (KOH), sialic acidvicinal diols are selectively removed by the PA^*-Bh sequence andO-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections.Using the PA-Bh-KOH-PA^*-Bh-PAS method we have demonstrated, for the first time, thatO-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence ofO-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA^*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars andO-acyl sialic acids are selected.     

Evaluation of the biocompatibile ionic liquid 1-methyl-3-methylimidazolium dimethylphosphite pretreatment of corn cob for improved saccharification

Ionic liquid (IL) pretreatment of lignocellulose materials is a promising process in biomass conversion to renewable biofuel. More in-depth research involving environment-friendly IL is much needed to explore pretreatment green route. In our case, IL 1-methyl-3-methylimidazolium dimethylphosphite ([Mmim]DMP) was chosen as an environment-friendly solvent to pretreat corn cob in view of its biocompatibility with both lignocellulose solubility and cellulase activity. The pretreatment/saccharification process and in situ saccharification process involving [Mmim]DMP were efficiently performed in bioconversion of corn cob to sugars, and more than 70% saccharification rates were obtained. Furthermore, the fermentability of reducing sugars obtained from the hydrolyzates was evaluated using Rhodococcus opacus strain ACCC41043 (R. opacus). High lipid production 41–43% of cell dry matter was obtained after 30 h of cultivation. GC/MS analysis indicated that lipids from R. opacus contained mainly long-chain fatty acids with four major constituent/oleic acid, stearic acid, palmitic acid, palmitoleic acid which are good candidates for biodiesel. These elucidated that corn cob pretreated by IL [Mmim]DMP did not bring negative effects on saccharification, cell growth, and accumulation of lipid of R. opacus. In conclusion, the IL [Mmim]DMP shows promise as green pretreatment solvent for cellulosic materials.     

The influence of type and concentration of the carbon source on production of citric acid by Aspergillus niger

Summary The influence of various carbon sources and their concentration on the production of citrate by Aspergillus niger has been investigated. The sugars maltose, sucrose, glucose, mannose and fructose (in the given order) were carbon sources giving high yields of citric acid. Optimal yields were observed at sugar concentrations of 10% (w/v), with the exception of glucose (7.5%). No citric acid was produced on media containing less than 2.5% sugar. Precultivation of A. niger on 1% sucrose and transference to a 14% concentration of various other sugars induced citrate accumulation. This could be blocked by the addition of cycloheximide, an inhibitor of de novo protein synthesis. This induction was achieved using maltose, sucrose, glucose, mannose and fructose, and also by some other carbon sources (e.g. glycerol) that gave no citric acid accumulation in direct fermentation. Precultivation of A. niger at high (14%) sucrose concentrations and subsequent transfer to the same concentrations of various other carbohydrates, normally not leading to citric acid production, led to formation of citrate. Endogenous carbon sources were also converted to citrate under these conditions. A 14%-sucrose precultivated mycelium continued producing some citrate upon transfer to 1% sugar. These results indicate that high concentrations of certain carbon sources are required for high citrate yields, because they induce the appropriate metabolic imbalance required for acidogenesis.     

Sensitivity of antennal gustatory receptor neurones to aphid honeydew sugars in the carabid Anchomenus dorsalis

Using electrophysiology, the stimulating effect of 13 sugars and three sugar alcohols (each at a concentration of 100 mm ) to antennal gustatory receptor neurones (GRNs) is tested in the carabid beetle Anchomenus dorsalis (Pontoppidan, 1763) (Coleoptera, Carabidae). Maltose, sucrose, glucose and raffinose are the most stimulating sugars for the sugar‐sensitive neurone (SuN), evoking 6.7–18.6 spikes s^?1 in fed insects, whereas the others had little or no effect. The firing rate of the antennal GRNs is not affected by any of the tested sugar alcohols, dulcitol, inositol and sorbitol. Additionally, concentration/response curves for sucrose and maltose are obtained in the range 0.01–100 mm . The responses of beetles starved for 96 h to this range of sucrose are two‐ to three‐fold higher compared with those of fed beetles. The presence of a terminal α‐glucose unit is an important feature of the molecular structure determining the stimulating properties of the two disaccharides, maltose and sucrose, as well as glucose. The other monosaccharide unit of the molecule is also of great importance in determining the stimulating properties of various disaccharides. The sensitivity of the SuN to the four most prevalent aphid honeydew sugars suggests that A. dorsalis uses these chemicals as sensory cues when searching for aphids as prey.     

Experimental and theoretical discrepancies in growth yields of Acinetobacter calcoaceticus: a correction of published data

Acinetobacter calcoaceticus can incompletely oxidize aldose sugars to the corresponding aldonic acids. This reaction can serve as an auxiliary energy source for the organism. An increase in biomass yields is observed in acetate-limited chemostat cultures grown in the presence of, for example, xylose. However, experimental and theoretical discrepancies exist with respect to the magnitude of the yield enhancement as a result of xylose addition. We previously observed increases in cell yields that were unexpectedly high. In contrast, other data were in agreement with the theoretical predictions. In this paper, evidence is presented indicating that this discrepancy is likely to be due to errors in the methodology used for our previous investigation, in particular with respect to the determination of biomass concentrations. Correspondence to: J. P. van Dijken     

Metabolite Profiling of Cheonggukjang,a Fermented Soybean Paste,Inoculated with Various Bacillus Strains during Fermentation

Metabolite profiling of Cheonggukjang inoculated with different Bacillus strains including Bacillus amyloliqueciens CH86-1, Bacillus licheniformis 58, and Bacillus licheniformis 67 during fermentation, was performed using gas chromatography-time of flight-mass spectrometry after derivatization, combined with multivariate statistical analysis. A total of 20 amino acids, 10 sugars, five sugar alcohols, and seven organic acids were identified in three Cheonggukjang samples. With fermentation time, most of the amino acids showed increasing amounts. On the other hand, most of the sugars including sucrose, fructose, and glucose decreasing patterns, and the amounts of organic acids varied. In order to observe differences in metabolites with fermentation time and inoculated Bacillus strains, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were carried out, respectively. On PCA plots, some sugars and organic acids including sucrose, fructose, glucose, mannose, succinic acid, and malonic acid, as well as most of the amino acids, contributed mainly to differentiation of the Cheonggukjang samples fermentation time. On the other hand, on PLS-DA, mannose, xylose, glutamic acid, and proline were mainly responsible for differentiating the Cheonggukjang among into various inoculated strains.     

Fed‐batch synthesis of galacto‐oligosaccharides with Aspergillus oryzae β‐galactosidase using optimal control strategy

Fed‐batch synthesis of galacto‐oligosaccharides (GOS) from lactose with β‐galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed‐batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L^?1 as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40°C). Simulations predicted a GOS yield of 32.5 g g^?1 in fed‐batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g^?1). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS‐5 and GOS‐6) was observed in the fed‐batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:59–67, 2014     

Carbon isotopic tracing of sugars throughout whole‐trees exposed to climate warming

Trees allocate C from sources to sinks by way of a series of processes involving carbohydrate transport and utilization. Yet these dynamics are not well characterized in trees, and it is unclear how these dynamics will respond to a warmer world. Here, we conducted a warming and pulse‐chase experiment on Eucalyptus parramattensis growing in a whole‐tree chamber system to test whether warming impacts carbon allocation by increasing the speed of carbohydrate dynamics. We pulse‐labelled large (6‐m tall) trees with ¹³C‐CO₂ to follow recently fixed C through different organs by using compound‐specific isotope analysis of sugars. We then compared concentrations and mean residence times of individual sugars between ambient and warmed (+3°C) treatments. Trees dynamically allocated ¹³C‐labelled sugars throughout the aboveground‐belowground continuum. We did not, however, find a significant treatment effect on C dynamics, as sugar concentrations and mean residence times were not altered by warming. From the canopy to the root system, ¹³C enrichment of sugars decreased, and mean residence times increased, reflecting dilution and mixing of recent photoassimilates with older reserves along the transport pathway. Our results suggest that a locally endemic eucalypt was seemingly able to adjust its physiology to warming representative of future temperature predictions for Australia.