Solution effects on the thermotropic phase transition of unilamellar liposomes

We have investigated the effect of two monosaccharides, glucose and fructose, and two disaccharides, sucrose and trehalose, on the thermotropic phase transition of unilamellar extruded vesicles of DPPC. All the sugars investigated raise the main transition temperature (Tm) of some fraction of the lipid, but there are differences between the effect of glucose and the other three sugars. At low concentrations of glucose, Tm is lowered. At high concentrations of glucose there are two transitions, one with a low Tm and one with a high Tm. The data suggest that at low concentrations, all of the glucose present may bind to the bilayer and increase headgroup spacing by physical intercalation or increased hydration. The appearance of a Tm above that of pure hydrated DPPC suggests the possibility of the dehydration of some other population of phospholipid molecules. The other three sugars increase Tm, but at high concentrations of trehalose, sucrose, and fructose a second peak occurs at a low Tm. The other sugars appear to dehydrate the bilayer at low concentrations, but may show some binding or increased hydration of some portion of the lipid at very high concentrations. The sugar effects on unilamellar vesicles are strikingly different from the effects of these sugars on multilamellar vesicles.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Improved biovar test for Ralstonia solanacearum

Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test uses bromothymol blue as a pH indicator in 15 ml culture tubes containing 3 to 5 ml of test media, and takes weeks to complete at 24 or 28 °C. We improved the biovar test by using phenol red as a pH indicator that changes color at a higher pH when a carbohydrate is utilized. We also conducted the test at 32 °C in 0.2 ml of 8-tube strips that reduced the medium needed by at least 20 fold. Using the improved test, biovars of R. solanacearum strains can be determined in 4 days when a panel of seven carbohydrates is used including glucose, trehalose, mannitol, sorbitol, dulcitol, maltose and cellobiose. To differentiate biovars 1, 2, 3 and 4, the test can be further simplified and completed in 3 days using a panel of four carbohydrates containing glucose, trehalose, maltose and dulcitol, significantly saving money, space and time.     

Improved resistance against oxidative stress of engineered cellobiose-fermenting <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> revealed by metabolite profiling

Cellobiose has garnered attention as an alternative carbon source for numerous biotechnological processes because it is produced when lignocellulosic biomass is treated with endo and exo-glucanases. An engineered Saccharomyces cerevisiae (CEL), expressing cellobiose transporter and intracellular beta-glucosidase utilized cellobiose efficiently. As compared to the culture using glucose, the CEL strain grown on cellobiose produced a similar yield of ethanol with slightly reduced growth rate. In this study, concentrations of central metabolites were monitored at mid-log phase with GC/MS to compare cellobiose- and glucose-grown CEL strain. When the CEL strain was grown on cellobiose, intracellular trehalose concentration increased 6-fold as compared with the glucosegrown cells. Interestingly, the higher level of trehalose in cells grown on cellobiose resulted in physiological changes which might be beneficial for biotechnological processes. We observed higher resistance against oxidative stress when cellobiose was used. Oxidative stress is commonly occurred by the byproducts of pretreatment process of lignocellulosic biomass, such as 2-furaldehyde (furfural) and 5-hydroxymethylfurfural (HMF). Our study demonstrated that intracellular metabolite profiling of yeast strains can be employed for linking intracellular concentrations of metabolite with physiological changes of cells upon genetic and environmental perturbations.     

Effects of carbohydrates on membrane stability at low water activities

The relative effectiveness of a variety of carbohydrates in preserving the structural and functional integrity of membranes at low water activities was studied, using Ca-transporting microsomes from muscle as a model membrane. The order of effectiveness (greatest to lowest) was: trehalose, lactose, maltose, cellobiose, sucrose, glucose, fructose, sorbitol, raffinose, myo-inositol, glycerol. At the highest concentrations of the most effective sugars tested, microsomes were obtained upon rehydration that were similar structurally and functionally to fresh membranes. The least effective carbohydrates, alcohol sugars, all appear to be fusogenic. A structural explanation for relative effectiveness of the sugars was sought, but no clear relationship was found, except that effectiveness does not appear to be related to the number of position of hydroxyl groups available for hydrogen bonding.     

The effects of carbohydrates upon the survival and reproduction of adult female Pimpla turionellae L. (Hym., Ichneumonidae)

In this study the effects of 23 carbohydrates belonging to various groups upon the survival egg production and egg of female adult Pimpla turionellae L. were investigated. The best results among the carbohydrates tested was obtained with sucrose which was also employed as control. Glucose, maltose, trehalose and melezitose on the other hand showed no significant effect. The egg production was observed to be unaffected by glucose and maltose although it showed significant increase with trehalose and significant decrease with melezitose. Fructose and sorbitol caused a significant decrease in the survival of the insect. Fructose and sorbitol did not have any significant effect upon egg production whereas galactose caused a significant decrease. With the exception of galactose, no carbohydrate caused any significant effect upon egg hatching. Although they did not produce any eggs the female insects survived for 13.17, 15.13, 11.58 and 15.83 days in mannose, melibiose, raffinose and mannitol, respectively. The shortest life span was observed in arabinose followed by α-methyl- D -glucoside, dulcitol, rhamnose, cellobiose, xylose, starch, lactose, sarbose, ribose and glycogen.     

Transport and hydrolysis of disaccharides by Trichosporon cutaneum.

Trichosporon cutaneum is shown to utilize six disaccharides, cellobiose, maltose, lactose, sucrose, melibiose, and trehalose. T. cutaneum can thus be counted with the rather restricted group of yeasts (11 to 12% of all investigated) which can utilize lactose and melibiose. The half-saturation constants for uptake were 10 +/- 3 mM sucrose or lactose and 5 +/- 1 mM maltose, which is of the same order of magnitude as those reported for Saccharomyces cerevisiae. Our results indicate that maltose shares a common transport system with sucrose and that there may be some interaction between the uptake systems for lactose, cellobiose, and glucose. Lactose, cellobiose, and melibiose are hydrolyzed by cell wall-bound glycosidase(s), suggesting hydrolysis before or in connection with uptake. In contrast, maltose, sucrose, and trehalose seem to be taken up as such. The uptake of sucrose and lactose is dependent on a proton gradient across the cell membrane. In contrast, there were no indications of the involvement of gradients of H+, K+, or Na+ in the uptake of maltose. The uptake of lactose is to a large extent inducible, as is the corresponding glycosidase. Also the glycosidases for cellobiose, trehalose, and melibiose are inducible. In contrast, the uptake of sucrose and maltose and the corresponding glycosidases is constitutive.     

Products of enzymatic hydrolysis of cellulose and its derivatives

Cellobiose and glucose were determined in a mixture of the two carbohydrates by methods involving the use of glucose oxidase and of β-glucosidase.Paper-partition chromatography is used as a confirmatory method in the identification of the hydrolysis products and in the detection of the various constituents.The cellulolytic organisms studied produce large amounts of the enzyme Cx, which diffuses into the medium. Only small amounts of β-glucosidase are found outside the cell. Cellobiose resulting from Cx activity can enter the cells as rapidly as can glucose.The role of cellobiose as a principal product in the hydrolysis of cellulose is confirmed. It is hypothesized that the principal final product of Cx activity is cellobiose, and that the presence of cellobiase in the medium is not a prerequisite to utilization of cellobiose by the organism. This is a correction of the hypothesis previously published stating that glucose appeared to be the final product of Cx activity.     

The freeze-dried preservation of liposome encapsulated hemoglobin: a potential blood substitute

In this report, the ability of carbohydrates (trehalose, sucrose, and glucose) to preserve the blood substitute liposome-encapsulated hemoglobin (LEH) in the freeze-dried state is examined. The water-free stabilization of individual components of this blood substitute and LEH is reported. Lyophilization of hemoglobin solutions in the absence of carbohydrates results in significant oxidative degradation of Hb as measured by a large increase (approximately 60%) in methemoglobin. Hb samples lyophilized in increasing carbohydrate concentrations show reduced levels of methemoglobin, and at 0.5 M trehalose, sucrose, or glucose, these levels are reduced to nearly the same levels as unlyophilized controls. Storage of lyophilized Hb samples following rehydration at 4 degrees C shows the same rate of methemoglobin formation regardless of whether carbohydrates are present. This suggests that carbohydrates prevent Hb oxidation in the dry state but are less effective at retarding oxidative damage to Hb in solution. The addition of 0.25 M trehalose or sucrose to LEH results in the maintenance of liposomal size following lyophilization. In these experiments, glucose was least effective at inhibiting dehydration-induced LEH fusion. Lyophilization of LEH in 0.25 M trehalose or sucrose also results in significantly greater retention of the encapsulated hemoglobin following lyophilization and rehydration. These results suggest that the long-term stabilization of LEH in the dry state is a realizable goal.     

Relationship between sporulation medium and germination ability of Mucor racemosus sporangiospores

Asexual sporangiospores of Mucor racemosus produced on a minimal sporulation medium (M spores) germinated only if glucose, mannose or a complex substrate such as peptone, yeast extract or Casamino acids was present. Once germinated, growth was supported by a wide range of substrates including amino acids, carbohydrates or organic acids. Sporangiospores produced on a nutritionally complex sporulation medium (C-spores) germinated on a wide range of carbon sources. C-spore phenotype was pleiotropic in that sporangiospores capable of germinating on cellobiose could always germinate on glutamate or xylose; but C-spores capable of germinating on xylose or glutamate did not always germinate on cellobiose. There was a hierarchy of substrates capable of initiating germination with glucose = mannose greater than xylose greater than glutamate greater than cellobiose. C-spores also differed from M-spores by initiating germination in the presence of the non-metabolizable glucose analogue 3-O-methylglucose. These results suggest that at least two sporangiospore phenotypes are produced depending upon the concentration and type of ingredients present in the sporulation medium.     

Carbohydrate Transport by the Anaerobic Thermophile Clostridium thermocellum LQRI

Clostridium thermocellum is an anaerobic thermophilic bacterium which degrades cellulose and ferments the resulting glucose, cellobiose, and cellodextrins predominantly to ethanol. However, relatively little information was available on carbohydrate uptake by this bacterium. Washed cells internalized intact oligomers as large as cellopentaose. Since cellobiose and cellodextrin phosphorylase activities were detected in the cytosol and were not associated with cell membranes, phosphorylation of carbohydrates occurred intracellularly. Kinetic studies indicated that cellobiose and larger cellodextrins were taken up by a common uptake system while glucose entered via a separate mechanism. When cells were treated with metabolic inhibitors including iodoacetate and arsenate, the uptake of radiolabeled glucose or cellobiose was reduced by as much as 90%, and this reduction was associated with a 95% decline in intracellular ATP content. A combination of the ionophores nigericin and valinomycin abolished the proton-motive force but only slightly decreased transport and ATP. These results suggested that the two modes of carbohydrate transport in C. thermocellum were ATP dependent. This work is the first demonstration of cellodextrin transport by a cellulolytic bacterium.     

Examining the interaction of light,nutrients and carbohydrates on seed germination and early seedling development of <Emphasis Type="Italic">Bletia purpurea</Emphasis> (Orchidaceae)

The effects of carbohydrate availability, carbohydrate source, nutrient availability and illumination on germination and early development of Bletia purpurea (Orchidaceae) seeds were investigated using asymbiotic seed germination. Of special interest was determining the minimum nutritional and light requirements for the completion of germination. Germination and development was limited when seeds were cultured in darkness without sucrose. Seeds were able to germinate under illuminated conditions even in the absence of sucrose and this effect was enhanced when mineral nutrients were incorporated into media. Sucrose, fructose, glucose and trehalose enhanced germination and seedling development while mannitol and sorbitol did not. These data suggest that carbohydrates, either as products of photosynthesis, from symbiotic fungi in situ or as exogenously supplied sugars in vitro, play an important role in regulating seed germination by fulfilling an energy requirement. This hypothesis has been often expressed but rarely satisfactorily tested. Mineral nutrients appear to be less important for germination than carbohydrates. The differential effect of sucrose, fructose, glucose and trehalose at two different concentrations on rhizoid production indicates carbohydrates may play a role in regulating rhizoid production.     

Hyperinduction of enzymes of the phosphorylative pathway of glucose dissimilation inPseudomonas cepacia

Glucose-dehydrogenase-deficient (Gcd^–) strains ofPseudomonas cepacia 249 compensated for loss of operation of the direct oxidative pathway by expanding the phosphorylative pathway. When grown on glucose, they had between two- and fourfold higher than normal levels of glucokinase and NAD-linked glucose-6-phosphate dehydrogenase activity and a comparable increase in capacity to transport glucose. Similar expansion of the phosphorylative pathway was noted when the wild type was grown on cellobiose or trehalose. Gcd^– strains grew normally on cellobiose and trehalose, but not if also deficient in glucokinase; this indicates that the disaccharides were converted to glucose and metabolized via the phosphorylative pathway. The expansion of the phosphorylative pathway during growth of the wild type on disaccharides or of Gcd^– mutants on glucose was a consequence of hyperinduction of pathway enzymes. Other compounds that promoted such hyperinduction included aromatic conjugates of glucose such as arbutin and salicin, and mannose. Under conditions leading to expansion of the phosphorylative pathway, enzymes related to the direct oxidative pathway, such as gluconate dehydrogenase and the 6-phosphogluconate dehydrogenase active with NAD, were not formed. The results indicate that intracellular glucose and extracellular glucose are metabolized to 6-phosphogluconate via different routes.     

Location and Formation of Cellulases in Trichoderma viride

The location and formation of cellulases and β-glucosidase in the fungus Trichoderma viride were studied on different substrates. The cellulases were found to be cell-bound during active growth on cellulose, CMC, and cellobiose. On CMC, much CM-cellulase was found cell-free but sonication released cellulase from the washed mycelium. Analysis of the carbohydrates of the mycelial cell wall after hydrolysis revealed glucose, mannose, and galactose—the same carbohydrates as reported to be present in purified cellulase from the same organism. Glucose repressed the formation of both CM-cellulase and Avicelase and cellobiose apparently the formation of Avicelase. Relatively little CM-cellulase was formed on cellobiose but a straight line was obtained when a differential plot of CM-cellulase versus protein was made.     

Redundancy in periplasmic binding protein-dependent transport systems for trehalose,sucrose, and maltose in Sinorhizobium meliloti

We have identified a cluster of six genes involved in trehalose transport and utilization (thu) in Sinorhizobium meliloti. Four of these genes, thuE, -F, -G, and -K, were found to encode components of a binding protein-dependent trehalose/maltose/sucrose ABC transporter. Their deduced gene products comprise a trehalose/maltose-binding protein (ThuE), two integral membrane proteins (ThuF and ThuG), and an ATP-binding protein (ThuK). In addition, a putative regulatory protein (ThuR) was found divergently transcribed from the thuEFGK operon. When the thuE locus was inactivated by gene replacement, the resulting S. meliloti strain was impaired in its ability to grow on trehalose, and a significant retardation in growth was seen on maltose as well. The wild type and the thuE mutant were indistinguishable for growth on glucose and sucrose. This suggested a possible overlap in function of the thuEFGK operon with the aglEFGAK operon, which was identified as a binding protein-dependent ATP-binding transport system for sucrose, maltose, and trehalose. The K(m)s for trehalose transport were 8 +/- 1 nM and 55 +/- 5 nM in the uninduced and induced cultures, respectively. Transport and growth experiments using mutants impaired in either or both of these transport systems show that these systems form the major transport systems for trehalose, maltose, and sucrose. By using a thuE'-lacZ fusion, we show that thuE is induced only by trehalose and not by cellobiose, glucose, maltopentaose, maltose, mannitol, or sucrose, suggesting that the thuEFGK system is primarily targeted toward trehalose. The aglEFGAK operon, on the other hand, is induced primarily by sucrose and to a lesser extent by trehalose. Tests for root colonization, nodulation, and nitrogen fixation suggest that uptake of disaccharides can be critical for colonization of alfalfa roots but is not important for nodulation and nitrogen fixation per se.     

Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation.

The amounts of glycogen and trehalose have been measured in cells of a prototrophic diploid yeast strain subjected to a variety of nutrient limitations. Both glycogen and trehalose were accumulated in cells deprived specifically of nirogen, sulfur, or phosphorus, suggesting that reserve carbohydrate accumulation is a general response to nutrient limitation. The patterns of accumulation and utilization of glycogen and trehalose were not identical under these conditions, suggesting that the two carbohydrates may play distinct physiological roles. Glycogen and trehalose were also accumulated by cells undergoing carbon and energy limitation, both during diauxic growth in a relatively poor medium and during the approach to stationary phase in a rich medium. Growth in the rich medium was shown to be carbon or energy limited or both, although the interaction between carbon source limitation and oxygen limitation was complex. In both media, the pattern of glycogen accumulation and utilization was compatible with its serving as a source of energy both during respiratory adaptation and during a subsequent starvation. In contrast, the pattern of trehalose accumulation and utilization seemed compatible only with the latter role. In cultures that were depleting their supplies of exogenous glucose, the accumulation of glycogen began at glucose concentrations well above those sufficient to suppress glycogen accumulation in cultures growing with a constant concentration of exogenous glucose. The mechanism of this effect is not clear, but may involve a response to the rapid rate of change in the glucose concentration.