Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates.     

Specificity of sugar-phospholipid interactions

Previous studies by Lefevre et al. (6) have shown that phospholipids stimulate uptake of glucose and other sugars by lipid solvents and enhance transfer of glucose through solvent layers into water. In this paper the specificity of the process for different sugars is investigated by following uptake from thin films of sugars or from glass fiber strips coated with radioactive sugars. Hexoses were taken up slowly to molar ratios of sugar to lipid phosphorus of about 1:1. Pentoses and deoxy sugars were taken up 5–10 times more rapidly to molar ratios of between 1.5 and 2.5:1. Relative rates of formation of the complexes at 25 °C were (d-glucose = 1.0):l-fucose, 9.1; d-ribose, 6.1; d-arabinose, 5.5; l-rhamnose, 3.8; l-arabinose, 3.7; d-xylose, 3.6; d-lyxose, 3.1; 3-O-methyl-d-glucose, 1.52; d-mannose, 1.36; d-galactose, 1.13; sucrose, 0.03; and lactose, 0.015. Radioactive sugars bound to phospholipids exchanged readily with unlabeled sugar in the anhydrous state and the sugars passed slowly into the aqueous phase when the complexes were shaken with water. The relative rates of dissociation (d-glucose = 1.0): l-arabinose, 2.82; d-arabinose, 2.49; l-rhamnose, 2.26; l-fucose, 1.96; d-xylose, 1.65; 3-O-methyl-d-glucose, 0.37; d-galactose, 0.28 were in the same general order as formation, suggesting that a common intermediate may be involved in both processes. In general, sugars with high mutarotation rates reacted most rapidly indicating a possible relationship between the structural features which favor interaction with phospholipids and those which enhance mutarotation.     

Catabolite Repression of induction of aldose reductase activity and utilization of mixed hemicellulosic surgars in Candida guilliermondii

NADPH-dependent aldose reductase activity induced by d-xylose or l-arabinose was detected in cell-free extracts of Candida guilliermondii, but only negligible activities were observed if d-glucose served as carbon source. The induction of aldose reductase activity on mixed sugars was investigated under resting cell conditions. d-Glucose repressed enzyme induction by d-xylose or l-arabinose to varying degrees, and l-arabinose inhibited enzyme induction by d-xylose. During incubation in a mixture of d-xylose-d-glucose, glucose consumption by cells was fast and simultaneous with d-xylose utilization. l-arabinose consumption was poor when it was present as the only sugar and in a mixture with d-glucose; this pentose depletion occurred only when all hexose was consumed. When d-xylose and l-arabinose were present in a mixture, the consumption of both pentoses was reduced by the presence of the second sugar, although both sugars were consumed simultaneously by cells. The results show that induction of aldose reductase activity and d-xylose utilization by cells of Candida guilliermondii are under control of glucose repression.     

Cloning of Xanthomonas DNA that expresses d-xylose catabolic enzymes

A gene bank of the d-xylose utilizing, cellulolytic Xanthomonas (XA1-1) DNA, inserted into the Hind III site of pKT230, was screened for clones which encoded d-xylose isomerase. One clone (pND70) was identified which complemented d-xylose isomerase negative mutants of Escherichia coli and this clone contained an insert of XA1-1 DNA of approximately 15 kb. Enzyme assays showed that pND70 appeared to encode d-xylose permease, and xylulose kinase in addition to d-xylose isomerase. Specific activities of all 3 enzymes from E. coli JA200 (pND70) grown in d-xylose were double those detected in XA1-1 when also grown on d-xylose.     

Dynamic characteristics of sugar accumulation and related enzyme activities in sweet and non-sweet watermelon fruits

Sugar content largely determines watermelon fruit quality. We compared changes in sugar accumulation and activities of carbohydrate enzymes in the flesh (central portion) and mesocarp of elite sweet watermelon line 97103 (Citrullus lanatus subsp. vulgaris) and exotic non-sweet line PI296341-FR (C. lanatus subsp. lanatus) to elucidate the physiological and biochemical mechanisms of sugar accumulation in watermelon fruit. The major translocated sugars, raffinose and stachyose, were more unloaded into sweet watermelon fruit than non-sweet fruit. During the fruit development, acid α-galactosidase activity was much higher in flesh of 97103 than in mesocarp of 97103, in flesh and mesocarp of PI296341-FR fruit. Insoluble acid invertase activity was higher in 97103 flesh than in 97103 mesocarp, PI296341-FR flesh or mesocarp from 18 days after pollination (DAP) to 34 DAP. Changes in soluble acid invertase activity in 97103 flesh were similar to those in PI296341-FR flesh and mesocarp from 18 DAP to full ripening. Sucrose synthase and sucrose phosphate synthase activities in 97103 flesh were significantly higher than those in 97103 mesocarp and PI296341-FR fruits from 18 to 34 DAP. Only insoluble acid invertase and sucrose phosphate synthase activities were significantly positively correlated with sucrose content in 97103 flesh. Therefore, phloem loading, distribution and metabolism of major translocated sugars, which are controlled by key sugar metabolism enzymes, determine fruit sugar accumulation in sweet and non-sweet watermelon and reflect the distribution diversity of translocated sugars between subspecies.     

Xylitol production by a methanol yeast,Candida boidinii (Kloeckera sp.) No. 2201

A methanol yeast, Candida boidinii no. 2201, could produce xylitol and also d-xylulose during cultivation on d-xylose medium. These fermentative products were identified by high performance liquid chromatography. A large amount of xylitol was obtained from a d-xylose medium containing ammonium acetate and yeast extract at the initial pH of 7.0. Maximum productivities of xylitol and enzymes concerned with the production were observed after 4–5 d of cultivation. A d-xylose (100 g/l) medium supplemented with 2% (v/v) methanol gave higher amounts of xylitol (48.5 g/l) and d-xylulose (3.3 g/l). Enzyme activities for d-xylose reduction, d-xylulose reduction, d-xylose isomerization, and d-xylulose phosphorylation, which could be involved in the xylitol production, were measured in cell-free extracts during cultivation and a possible pathway of xylitol production was discussed.     

Partitioning of Sugar between Growth and Nitrate Reduction in Cotton Roots

The level of endogenous sugars was inversely related to nitrate availability in young cotton (Gossypium hirsutum L.) plants, with high nitrate causing a greater decline in sugar content of roots than of shoots. High nitrate (low sugar) plants also displayed relatively more shoot growth and less root growth than low nitrate (high sugar) plants. These data are consistent with the theory that roots are poor competitors for sugar, and that sugar supply is a major factor limiting root growth in vivo.

The effects of endogenous sugar level on root growth and on nitrate reductase activity in the root were different. When root sugar level was experimentally controlled by varying nitrate concentration in the nutrient solution, root growth was less sensitive than nitrate reductase activity to sugar deficiency. Also, in sterile root tips cultured on media containing a wide range of sucrose concentrations, growth rate was considerably less sensitive to endogenous sugar deficiency than was nitrate assimilation rate. Similarly, in plants which were detopped or girdled, nitrate reductase activity in the roots declined more rapidly than did root sugars, especially glucose and fructose. These results suggest that when sugar is deficient, cotton roots preferentially use it for growth at the expense of nitrate reduction.

     

Utilization of degraded peach gum polysaccharide by Aspergillus flavus

d-Galactose, d-mannose, d-xylose, l-arabinose, and d-glucuronic acid and its γ-lactone were examined as carbon sources for the culture of Aspergillus flavus. d-Mannose was taken up the most rapidly and d-glucuronic acid and its γ-lactone the least rapidly. A partially degraded polysaccharide from peach tree gum (Prunus persica [L.] Batsch containing the above sugars together with d-glucuronic acid and its 4-O-methyl ether was used as substrate for another A. flavus culture. It was found that d-galactose was the major sugar passing into the culture medium with lower proportions of d-xylose, l-arabinose, 2-O-β-d-glucopyranuronosyl-d-mannose, and 6-O-β-d-glucopyranuronosyl-d-galactose. This indicates that the fungus produces extracellular exo- and endo-glycanohydrolases which may be useful in structural studies on polysaccharides.     

Effects of chilling stress on the accumulation of soluble sugars and their key enzymes in <Emphasis Type="Italic">Jatropha curcas</Emphasis> seedlings

As osmolytes and signaling molecules, soluble sugars participate in the response and adaptation of plants to environmental stresses. In the present study, we measured the effect of chilling (12 °C) stress on the contents of eight soluble sugars in the leaves, cotyledons, stems, and roots of Jatropha curcas seedlings, as well as on the activities of eight rate-limiting enzymes that are critical to the metabolism of those soluble sugars. Chilling stress promoted both starch hydrolysis and soluble sugar accumulation. The soluble sugar contents of the leaves and cotyledons were affected more than that of the stems and roots. Meanwhile, the activities of the corresponding metabolic enzymes (e.g., β-amylase, uridine diphosphate glucose phosphorylase, and sucrose phosphate synthase) also increased in some organs. The gradual increase of soluble neutral alkaline invertase activity in the four studied organs suggested that sucrose catabolic production, such as glucose and fructose, was especially important in determining resistance to chilling stress and hexose signal transduction pathway. In addition, the substantial accumulation of raffinose family oligosaccharides and increase in corresponding metabolic enzyme activity suggested that galactinol and raffinose play an important role in determining the chilling resistance of J. curcas. Together, these findings establish a foundation for determining the relationship between the chilling resistance and soluble sugar accumulation of J. curcas and for investigating the mechanisms underlying sugar signaling transduction and stress responses.     

Identification and inactivation of pleiotropic regulator CcpA to eliminate glucose repression of xylose utilization in Clostridium acetobutylicum

d-xylose utilization is a key issue for lignocellulosic biomass fermentation, and a major problem in this process is carbon catabolite repression (CCR). In this investigation, solvent-producing bacterium Clostridium acetobutylicum ATCC 824 was metabolically engineered to eliminate d-glucose repression of d-xylose utilization. The ccpA gene, encoding the pleiotropic regulator CcpA, was experimentally characterized and then disrupted. Under pH-controlled conditions, the ccpA-disrupted mutant (824ccpA) can use a mixture of d-xylose and d-glucose simultaneously without CCR. Moreover, this engineered strain produced acetone, butanol and ethanol (ABE) at a maximal titer of 4.94, 12.05 and 1.04 g/L, respectively, which was close to the solvent level of maize- or molasses-based fermentation by wild type C. acetobutylicum. Molar balance analysis for improved process of mixed sugars utilization also revealed less acid accumulation and more butanol yield by the engineered strain as compared to the wild type. This study offers a genetic modification strategy for improving simultaneous utilization of mixed sugars by Clostridium, which is essential for commercial exploitation of lignocellulose for the production of solvents and biofuels.     

Sugars as complementary alternative food for the establishment of Nesidiocoris tenuis in greenhouse tomato

Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is an omnivorous generalist predator which is augmentatively released and conserved for control of whiteflies (Hemiptera: Aleyrodidae) and Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) in tomato crops. Eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) are often provided as factitious prey to improve the establishment of N. tenuis after its release. We first tested different amounts of E. kuehniella eggs per plant to optimize N. tenuis establishment and then investigated whether the amount of eggs that optimized N. tenuis establishment might be reduced by adding sugars (hydrocapsules filled with 0.5 m sucrose) under walk‐in cage and commercial greenhouse conditions. These experiments demonstrated that the addition of sugar to the diet of N. tenuis could half the amount of E. kuehniella eggs required to establish N. tenuis. Under greenhouse conditions, the progeny of N. tenuis per plant did not differ significantly between E. kuehniella alone or the half amount of E. kuehniella plus hydrocapsules. These results demonstrated that the sugar could partially substitute for E. kuehniella eggs improve establishment of N. tenuis and suggest that natural sugars such as nectar and honeydew might also beneficial.     

A kinetic analysis of d-xylose transport in rhodotorula glutinis

The kinetics of D-xylose transport were studied in Rhodotorula glutinis. Analysis of the saturation isotherm revealed the presence of at least two carriers for d-xylose in the Rhodotorula plasma membrane. These two carriers exhibited Km values differing by more than an order of magnitude. The low K_m carrier was repressed in rapidly growing cells and depressed by starvation of the cells.Several hexoses were observed to inhibit d-xylose transport. In the studies reported here, the inhibitions produced by d-galactose and 2-deoxy-d-glucose were examined in some detail in order to define the interactions of these sugars with the d-xylose carriers. 2-Deoxy-d-glucose competitively inhibited both of the d-xylose carriers. In contrast, only the low-K_m carrier was competitively inhibited by d-galactose.     

The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised pisum sativum roots

Nitrate reductase (NR) induction is enhanced by exogenously supplied sucrose in excised pea roots exposed to both exogenous nitrate and exogenous nitrite. NR synthesis is preferentially supported by sugars transported to the cells at the moment, however NR induction can take place for some time without exogenous sugar influx if roots are saturated with sugars during precultivation. Steady high NR levels are dependent on steady sugar and nitrate influxes. NR induction is low in roots precultivated for 20 h without sucrose although sugar content is still high in them. This suggests that compartmentation of sugars in the cells is of major importance during NR induction. Total nitrate content in roots exposed to nitrate is not influenced by sucrose supplied together with nitrate. Some nitrite is oxidized to nitrate in roots exposed to exogenous nitrite ; we assume that this nitrate is responsible for NR induction. Our results indicate that sugars, besides many indirect effects on NR induction, may also directly influence NR synthesis either as coinducers or as derepressors of NR synthesis. Our results further show that NR is not a product-inducible enzyme.     

Sugar as nutritional supplement for the zoophytophagous predator Nesidiocoris tenuis

Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is a zoophytophagous predator widely used in integrated pest management programs in both greenhouse and open-field tomato crops. Mass rearing of N. tenuis is greatly dependent on Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs as food source. Moreover, the addition of this factitious prey after the inoculative releases of N. tenuis under field conditions is recommended to facilitate establishment of this mirid. However, E. kuehniella eggs are expensive and availability is limited. One possible strategy to reduce the amount of E. kuehniella eggs needed is the provision of sugar. In this work, the effect of sucrose as nutritional supplement on selected life-history traits of N. tenuis was studied. The addition of sucrose (0.5 M) ad libitum to a diet of E. kuehniella eggs significantly increased the progeny of N. tenuis and did not affect survival of nymphs nor developmental time. Moreover, addition of sucrose significantly reduced the number of E. kuehniella eggs consumed. These results may have practical implications of interest in mass rearing systems of N. tenuis and its management in fields and greenhouses as a part of biological control programs.     

Genetic control of serine dehydratase and phosphoenolpyruvate carboxykinase in mice

Inbred strains of mice were found to differ with regard to their endogenous activities of the liver enzymes serine dehydratase (SD) and phosphoenolpyruvate carboxykinase (PEPCK). The strain distribution patterns for the activity of each enzyme were identical. On feeding of high-protein diets or on fasting, the activities of both enzymes were induced in a concordant fashion which suggested the control of both enzymes by a single gene. Genetic analysis established that the induction of both enzymes on feeding of high-protein diets was controlled by a single gene (Sdr-1), whereas the induction of SD, but not of PEPCK, on fasting was controlled by different single gene (Sdr-2). The lack of segregation of the backcross generations with respect to PEPCK activities obtained on fasting precluded the establishment of any association of the response of PEPCK to fasting with either the Sdr-1 or Sdr-2 locus. The strain of mice (BALB/cJ) that had the ability to maximally induce both gluconeogenic enzymes under both dietary treatments failed to survive a fast as long as those strains with less ability to induce. This suggests that the ability to induce key enzymes in gluconeogenesis when food is unavailable is of little consequence with regard to their ability to produce essential nutrients necessary for survival.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

Studies on sugar isomerases of Lactobacillus sp.: Whole cell immobilization of d-glucose isomerase

A new soil isolate of Lactobacillus sp. grown in Yamanaka medium under submerged conditions showed the presence of d-glucose, d-xylose and d-ribose isomerases in washed cell suspension and cell free extracts. d-Xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) and d-ribose isomerase (d-ribose ketol-isomerase, EC 5.3.1.20) activities reached a maximum in 48 h of growth and then declined. d-Glucose isomerase (d-glucose 6-phosphate isomerase, d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) activity was maximum after 72 h and remained constant for ～120 h of growth. d-Glucose isomerase activity increased with the increase in number of generations of culture and reached a maximum in 5–6 generations, whereas d-xylose and d-ribose isomerase activities decreased. The washed and starved whole cells could be heat treated and immobilized on the rough surface of glass rods or glass slides using acetone treatment. The heat treated immobilized cells showed only the presence of d-glucose isomerase activity and showed no d-xylose and d-ribose isomerase activities. d-Glucose isomerase activity of heat treated immobilized cells was inhibited less by sorbitol, mannitol, sodium arsenate, cysteine and calcium ions than the free d-glucose isomerase activity in fresh untreated washed whole cells and cell free extracts. EDTA inhibition had the same effect for both forms. Ca²⁺inhibition could be reversed by adding Mg²⁺ions.     

Excess nickel modulates activities of carbohydrate metabolizing enzymes and induces accumulation of sugars by upregulating acid invertase and sucrose synthase in rice seedlings

The effects of increasing concentrations of nickel sulfate, NiSO₄ (200 and 400 μM) in the growth medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism were examined in seedlings of the two Indica rice cvs. Malviya-36 and Pant-12. During a 5–20 day growth period of seedlings in sand cultures, with Ni treatment, no definite pattern of alteration in starch level could be observed in the seedlings. In both roots and shoots of the seedlings Ni treatment led to a significant decrease in activities of starch degrading enzymes α-amylase, β-amylase, whereas starch phosphorylase activity increased. The contents of reducing, non-reducing, and total sugars increased in Ni-treated rice seedlings with a concomitant increase in the activities of sucrose degrading enzymes acid invertase and sucrose synthase. However, the activity of sucrose synthesizing enzyme sucrose phosphate synthase declined. These results suggest that Ni toxicity in rice seedlings causes marked perturbation in metabolism of carbohydrates leading to increased accumulation of soluble sugars. Such perturbation could serve as a limiting factor for growth of rice seedlings in Ni polluted environments and accumulating soluble sugars could serve as compatible solutes in the cells under Ni toxicity conditions.     

Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula

The coordinate appearance of the bispecific NAD(P)H-nitrate reductase (NR; EC 1.6.6.2) and nitrite reductase (NiR; EC 1.7.7.1) was investigated in leaves and roots from European white birch seedlings (Betula pendula Roth). Induction by nitrate and light of both enzymes was analyzed by in vitro assays and by measuring NR- and NiR-encoding mRNA pools with homologous cDNAs as probes. When birch seedlings were grown on a medium containing ammonium as the sole nitrogen source, low constitutive expression of NR and NiR was observed in leaves, whereas only NiR was significantly expressed in roots. Upon transfer of the seedlings to a nitrate-containing medium, mRNA pools and activities of NR and NiR dramatically increased in leaves and roots, with a more rapid induction in leaves. Peak accumulations of mRNA pools preceded the maximum activities of NR and NiR, suggesting that the appearance of both activities can be mainly attributed to an increased expression of NR and NiR genes. Expression of NR was strictly light-dependent in leaves and roots and was repressed by ammonium in roots but not in leaves. In contrast with NR, constitutive expression of NiR was not affected by light, and even a slight induction following the addition of nitrate was found in the dark in roots but not in leaves. No effect of ammonium on NiR expression was detectable in both organs. In leaves as well as in roots, NiR was induced more rapidly than NR, which appears to be a safety measure to prevent nitrite accumulation.