Effets des sucres sur le comportement alimentaire de Lambdina fiscellaria

Quatorze sucres (D-arabinose, L-arabinose, fructose, galactose, glucose, maltose, manose, mélézitose, mélibiose, raffinose, rhammose, sucrose, tréhalose, xylose) ont été étudiés en fonction de leur consommation, à différentes concentrations, par les stades IV et V de Lambdina fiscellaria fiscellaria Guén. Les indices de consommation mesurés varient avec la concentration de la solution sucrée, à l'exception du D-arabinose qui induit une consommation comparable (t_0.05) à 0.5 M, 0.1 M et 0.02 M et ce, pour les 2 stades. Une diminution de la consommation est parallèle à une diminution de la concentration des solutions de galactose et de xylose pour le stade IV, et de sucrose et xylose pour le stade V. La prise alimentaire la plus élevée pour le stade IV est mesurée avec le sucrose aux 3 molarités, tandis que celle du stade V est notée avec le sucrose et le glucose. L'écart entre les indices de comsommation des sucres diminue avec une baisse de la concentration de la solution sucrée pour les 2 stades, créant ainsi de nombreuses interrelations entre les sucres.

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Summary Fourteen sugars (D-arabionose, L-arabinose, fructose, galactose, glucose, maltose, mannose, melezitose, melibiose, raffinose, rhamnose, sucrose, trehalose, xylose) at different concentrations were studied with regard to consumption, by the fourth and fifth larval instars of Lambdina fiscellaria fiscellaria Guén. The consumption indices vary with the concentration of sugars, except for D-arabinose which induces comparable consumptions (t_0.05) at 0.5 M, 0.1 M and 0.02 M in both larval instars. A reduction in the consumption of sugars parallels decreases of galactose and xylose concentrations for the fourth larval instar, and sucrose and xylose concentrations for the fifth larval instar. According to food intake data, the fourth larval instar prefers sucrose while the fifth larval instar shows increased glucose responses, and even prefers glucose at 0.02 M. The differences among sugar consumption indices decrease when the sugar concentration is reduced.

     

Isolation of an extremely thermophilic chemoorganotrophic anaerobe similar to Dictyoglomus thermophilum from new zealand hot springs

A strain similar to Dictyoglomus thermophilum, isolated from a New Zealand hot spring, is described. This strictly anaerobic, Gram-negative, non-motile and nonsporulating bacterium usually exists as long thin filaments of 5 to 25 m by 0.35 to 0.45 m. Rotund bodies are commonly observed. Thin sections of the cells revealed a two-layered cell wall. The optimum temperature and pH for growth was 70°C and 7.0 and 7.5 respectively. No growth was observed at 40°C and 85°C or at pH 4.5 to pH 9.0. The organism fermented glucose, maltose, mannose, xylose, lactose, cellobiose, galactose and sucrose and produced acetate as the major end-product with significant amounts of lactate, H₂ and CO₂ and only traces of ethanol. The doubling time on glucose was 10 h. The DNA base composition was 29.5% guanine plus cytosine as determined by the thermal denaturation method. Growth was inhibited by penicillin, tetracycline and chloramphenicol indicating that the organism was a eubacterium. These features are in common with the newly described species Dictyoglomus thermophilum to which the New Zealand isolate belongs.     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

The effect of sugars on niger embryogenesis and plant regeneration in anther culture

The influence of different sugars (sucrose, maltose, glucose and fructose, 0.05–0.5 M) on embryogenesis and plant regeneration from cultured anthers of niger [Guizotia abyssinica (L. f.) Cass.] have been studied. Among the different sugars tested, 0.2 M sucrose was the best for embryo induction and plant regeneration. Maximum of 57 embryos per 60 anthers were induced on embryo induction medium [Gamborg’s B5 medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2 μM kinetin (KIN)] containing 0.2 M sucrose. Embryo differentiation was achieved on B5 medium supplemented with 0.5 μM benzyladenine (BA) and 0.09 M sucrose. Embryo maturation was on B5 medium containing 10 μM abscisic acid (ABA) and 0.09 M sucrose. Embryo germination was achieved on B5 medium with 0.09 M sucrose. Embryos that were developed on B5 medium supplemented with 0.2 M sucrose showed highest frequency (68 %) of plant regeneration.     

Growth characteristics of an obligately psychrophilic Vibrio sp.

The growth characteristics of an obligately psychrophilic Vibrio sp. have been studied in a chemostat with glucose or lactose as the limiting substrate over a temperature range 0–23°C. Vibrio AF-1 has an optimum growth temperature of 15°C and maximum growth temperature which is dependent upon the carbon source. On glucose growth ceases at 20°C whereas on lactose growth continues to 23°C. Growth rate is also a function of the carbon source provided. When grown on glucose, fructose, sucrose, maltose and galactose _max values of 0.046 h^-1 at 15°C were recorded whereas on lactose, mannose, ribose and xylose _max values of 0.020 h^-1 were obtained. Substrate affinities (K _s ) for the 9 sugars also fall into 2 divisions as for _max and are temperature dependent. Those sugars which support a high growth rate have highest K _s values at 0°C whereas these which give a low growth rate show maximum affinities at 15°C. Vibrio AF-1 produces the maximum cell yield (0.6 g/g sugar consumed) at temperature <8°C irrespective of the carbon source utilised and correlated with maximum rates of sugar uptake and minimum O₂ consumption. Maintenance energy determination on glucose grown cells show that at 2° C 2% of the carbon input is used for maintenance whereas at 20°C the requirement increases to 10% of the carbon input.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

Production of a New Acidic Polysaccharide,Succinoglucan by Alcaligenes faecalis var. myxogenes

A slimy non-spore-forming bacterium strain 10C3 isolated from soil was motile with peritrichous flagella and named Alcaligenes faecalis var. myxogenes. Studies were made on the conditions necessary for maximal production of a new acidic succinoglucan polysaccharide by this strain in shaken cultures. Much production was observed with sucrose, glucose, xylose, galactose, cellobiose, maltose, fructose, mannose and rhamnose. The yield was greatest with sucrose and decreased in order with the above sugars from about 36 to 23 per cent. The most suitable medium contained 4 per cent sugar, 0.5 per cent yeast extract and one per cent calcium carbonate in tap water. The optimum temperature was 28°C.     

Role of polyols in thermal inactivation of shark ornithine transcarbamoylase

The ability of activity modulators of ornithine transcarbamoylase (OCT) from the liver of the thresher shark Alopias vulpinus to stabilize the enzyme against thermal denaturation was investigated in the tri-buffer at pH 7.8, at temperatures ranging from 60 to 70 (o)C, in the presence of polyhydroxylic molecules such as glycerol and sugars. The study indicated that in the presence of 0.5 M sugars and 1.6 M glycerol in the preincubation medium the OCT activity increases. When trehalose is introduced directly in the reaction mixture in a range of concentration of 0.25-0.5 M, the activity is lower than that with maltose, glycerol and buffer alone. Kinetic data for carbamoyl phosphate and ornithine with and without maltose and glycerol are similar, whereas trehalose increases the kinetic values. Arrhenius plots show an increase of activation energy due to trehalose, whereas values obtained with maltose and glycerol are similar to the control.     

Variation in the Nature of Organic Acid Secretion and Mineral Phosphate Solubilization by Citrobacter sp. DHRSS in the Presence of Different Sugars

A novel phosphate solubilizing bacterium (PSB) was isolated from the rhizosphere of sugarcane and is capable of utilizing sucrose and rock phosphate as the sole carbon and phosphate source, respectively. This PSB exhibited mineral phosphate solubilizing (MPS) phenotype on sugars such as sucrose and fructose, which are not substrates for enzyme glucose dehydrogenase (GDH), along with GDH substrates, viz., glucose, xylose, and maltose, as carbon sources. PCR amplification of the rRNA gene and sequence analysis identified this bacterium as Citrobacter sp. DHRSS. On sucrose and fructose Citrobacter sp. DHRSS liberated 170 and 100 μM free phosphate from rock phosphate and secreted 49 mM (2.94 g/L) and 35 mM (2.1 g/L) acetic acid, respectively. Growth of Citrobacter sp. DHRSS on sucrose is mediated by an intracellular inducible neutral invertase. Interestingly, in the presence of GDH substrates like glucose and maltose, Citrobacter sp. DHRSS produced approximately 20 mM (4.36 g/L) gluconic acid and phosphate released was 520 and 570 μM, respectively. Citrobacter sp. DHRSS GDH activity was found when grown on GDH and non-GDH substrates, indicating that it is constitutive and could act on a wide range of aldose sugars. This study demonstrates the role of different organic acids in mineral phosphate solubilization by rhizobacteria depending on the nature of the available carbon source.     

碳源对早花百子莲愈伤组织诱导及其增殖的生理特性影响

为揭示碳源对早花百子莲愈伤组织诱导与增殖的影响机理,该研究以早花百子莲的小花梗为外植体,比较分析30.0 g/L蔗糖、葡萄糖、麦芽糖在愈伤组织诱导、增殖中的效果,测定不同碳源种类处理下愈伤组织增殖相关生理特性,并根据细胞增殖效果、生理指标相关性进行优化验证。结果表明:(1)蔗糖、葡萄糖和麦芽糖碳源处理下,愈伤组织诱导率分别为86.00%、72.00%和59.67%,蔗糖碳源的愈伤组织诱导率比葡萄糖和麦芽糖分别显著提高19.44%和44.13%(P<0.05),蔗糖碳源较葡萄糖和麦芽糖碳源的愈伤组织大小分别显著增加22.44%和90.09%(P<0.05);愈伤组织增殖阶段,蔗糖碳源能够同时维持良好的细胞增殖效率及活性,而葡萄糖碳源的愈伤组织增殖快、状态差,麦芽糖处理增殖慢、状态佳;蔗糖转换葡萄糖碳源后愈伤组织细胞团大小、细胞活性明显下降;蔗糖转换蔗糖、蔗糖转换麦芽糖的效果较好。(2)培养基碳源显著调节愈伤组织增殖阶段的糖代谢、内源激素代谢和氧化胁迫平衡。(3)愈伤组织的主要糖组分为淀粉、葡萄糖;淀粉、麦芽糖含量与细胞团大小相关性高,以蔗糖为碳源的培养基中添加麦芽糖,愈伤组织...     

Variability of the thermal behavior of honeybees on a feeding place

Summary The thermoregulation of honeybees (Apis mellifera carnica) was investigated under field conditions, on a feeding place 335 m away from the hive, where 0.25M or 0.5M sucrose solution was offered. By means of real-time tele-thermography, contactless body surface temperature measurements of undisturbed animals were made.The foraging bees showed highly developed individual thermoregulatory abilities. Complex behavioral patterns such as food uptake, active body temperature regulation, and preparation for flight were performed simultaneously. However, body temperature was more variable than expected. When bees drank 0.5M sucrose solution, they generally had a higher thoracic surface temperature (T _Ths) and showed smaller temperature fluctuations (e.g., cooling down after landing) than with 0.25M solution. Given 0.5M sucrose they stayed for shorter periods at the feeding place. The highest (maximum)T _Ths during the stop was positively (linearly) correlated with the ambient temperature (T _a=18–30°C) for both 0.25M and 0.5M sucrose feeding. At aT _a of 19°C the mean (interpolated) maximum values forT _Ths were 37.2°C (0.25M) and 38.5°C (0.5M); at aT _a of 27°C they were 39.2°C (0.25M) and 40.9°C (0.5M). The minimumT _Ths was correlated withT _a only with 0.5M feeding, whereas with 0.25M feeding a great variability was observed. Similarly as the maximumT _Ths,T _Ths upon landing and taking off were positively (linearly) correlated withT _a and were higher during 0.5M feeding.The quality (concentration) of the food offered to the bees obviously influenced their thermal behavior at the feeding place.Abbreviations T _a ambient temperature - T _Ths thoracic surface temperature - IR infra-red     

Interacting effects of time,temperature, pH and simple sugars on biomass and toxic metabolite production by three Alternaria spp.

Biomass and toxic metabolite production by Altemaria hemicota, A. solani and A. tenuis in modified Czapek Dox broth (mCDB) as affected by incubation time (up to 4 wk) temperature (6, 15 and 25 °C), pH (3.0, 5.0 and 8.0) and sugars (glucose, fructose and sucrose) was studied. Biomass production was greatest at 25 ° C and pH 8.0 but toxin production was enhanced at 15 °C. In general, biomass production was promoted by up to 9% glucose and sucrose and 6% fructose; however, toxin production was simultaneously reduced by elevated sugars in mCDB over a 4-wk incubation period.     

Evaluating potential sugar food sources from the olive grove agroecosystems for Prays oleae parasitoid Chelonus elaeaphilus

Chelonus elaeaphilus Silvestri (Hymenoptera: Braconidae) is a host-specific parasitoid of the olive moth, Prays oleae (Bernard), that can cause parasitism rates of up to 80% in Mediterranean olive groves. A laboratory study was carried out to assess the potential of sugars provided by wild plant species in olive grove agroecosystem to enhance the fitness of C. elaeaphilus. Insects were reared in a climate-controlled chamber at 25?±?2°C, 60?±?5% relative humidity (RH) with a photoperiod of 16:8 (L:D) h. Five naturally occurring wild plant nectar sugars (sucrose, fructose, glucose, maltose and mannose) were tested for their effect on insect longevity. The nectar sugar content of sucrose, fructose and glucose in 12 selected olive grove agroecosystem plant species was analysed and categorised on the basis of sugar ratios. Female insect longevity was increased when they were fed on both sucrose and glucose compared to either maltose or fructose, suggesting that sucrose-dominant nectars would bene?t this parasitoid. Sucrose was predominant in the nectar of five of the studied plant species (Silene gallica, Borago officinalis, Echium plantagineum, Lavandula stoechas and Lonicera hispânica). The results are discussed in terms of potential enhancement of the biological control of P. oleae.     

The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora

The response of five Coffea canephora Pierre genotypes with regard to somatic embryogenesis was tested on media containing silver nitrate (AgNO₃) and different carbohydrates (sucrose, fructose, maltose and glucose). The presence of AgNO₃ caused only small modifications to the ionic equilibrium of the media. At concentrations between 30–60 M, AgNO₃ improved embryo yield for the genotypes evaluated, while higher doses negatively affected the regenerative capacity. The substitution of maltose, glucose or fructose for sucrose produced different responses depending on the genotype. Fructose significantly increased somatic embryo production in genotypes N91 and N128, while maltose was highly effective for N75. In addition, more synchronous embryo development was observed in genotype N91 when glucose was used instead of sucrose.     

Thermostable amylolytic enzymes from a cellulolytic fungusMyceliophthora thermophila D14 (ATCC 48 104)

Summary The production of amylolytic enzymes by a thermophilic cellulolytic fungus,Myceliophthora thermophila D14 was investigated by batch cultivation in Czapek-Dox medium at 45° C. Among various nitrogenous compounds used, NaNO₃ and KNO₃ were found to be the best for amylase production. Starch, cellobiose and maltose induced the synthesis of amylase while glucose, fructose, galactose, lactose, arabinose, xylose, sorbitol, mesoinositol and sucrose did not. Calcium ions had the most stimulating effect on enzyme formation amongst many ions investigated. The synthesis of amylolytic enzymes was dependent on growth and occurred predominantly in the mid-stationary phase. The enzyme was active in a broad temperature range (50° C–60° C) and displayed activity optima at 60° C and pH 5.6.     

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW fresh weight - MS Murashige and Skoog (1962) - 1/2MS medium MS medium containing half strength mineral salts - 1/2MS-0 1/2MS medium containing no growth regulators - NAA 1-naphthaleneacetic acid - p-calli protoplast-derived calli - PE plating efficiency - picloram 4-amino-3,5,6-trichloro-picolinic acid     

Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects on nonenzymatic glycosylation

A variety of neutral and acidic sugars and related compounds were evaluated in terms of their effect on the midpoint, T_d, of the thermal denaturation curve of antithrombin III. The objectives were to determine which structural features of these molecules are responsible for their stabilizing properties and to identify more efficient stabilizers which combine the effects of lyotropic anions such as citrate with those of the polyols in a single molecule. The presence of one or more carboxylate groups in a sugar molecule invariably increased its stabilizing potency, whereas the number and position of hydroxyl groups appeared to have no influence on the molecules' stabilizing ability. Several compounds were shown to be effective in preserving antithrombin III activity during pasteurization for 10 h at 60°C. However, the presence of reducing sugars invariably resulted in a decrease in activity following pasteurization, in spite of their ablity to increase T_d. In fact, when antithrombin III was pasteurized in the presence of 2 M glucose and 0.5 M citrate, it steadily losts its ability to inhibit thrombin even though T_d under the conditions was 10°C higher than in citrate alone where activity was preserved. This effect was shown to be coincident with the covalent incorporation of glucose into the protein molecule.     

Glucose fermentation to acetate,CO2 and H2 in the anaerobic hyperthermophilic eubacterium Thermotoga maritima: involvement of the Embden-Meyerhof pathway

The hyperthermophilic anaerobic eubacterium Thermotoga maritima was grown on glucose as carbon and energy source. During growth 1 mol glucose was fermented to 2 mol acetate, 2 mol CO₂ and 4 mol H₂. The molar growth yicld on glucose (Y_glucose) was about 45 g cell dry mass/mol glucose. In the presence of elemental sulfur growing cultures of T. maritima converted 1 mol glucose to 2 mol acetate, 2 mol CO₂ about 0.5 mol H₂ and about 3.5 mol H₂S. Y_glucose was about 45 g/mol. Cell extracts contained all enzymes of the Embden-Meyerhof pathway: hexokinase (0.29 U/mg, 50°C), glucose-6-phosphate isomerase (0.56 U/mg, 50°C), phosphofructokinase (0.19 U/mg, 50° C), fructose-1,6-bisphosphate aldolase (0.033 U/mg, 50°C), triosephosphate isomerase (6.3 U/mg, 50°C), glyceraldehyde-3-phosphate dehydrogenase (NAD⁺ reducing: 0.63 U/mg, 50°C), phosphoglycerate kinase (3.7 U/mg, 50°C), phosphoglycerate mutase (0.4 U/mg, 50°C); enolase (4 U/mg, 80°C), pyruvate kinase (0.05 U/mg, 50°C). Furthermore, cell extracts contained pyruvate: ferredoxin oxidoreductasee (0.43 U/mg, 60°C); NADH: ferredoxin oxidoreductase (benzylviologen reduction: 0.46 U/mg, 80°C); hydrogenase (benzylviologen reduction: 15 U/mg, 80°C), phosphate acetyltransferase (0.13 U/mg, 80°C), acetate kinase (1.2 U/mg, 55°C), lactate dehydrogenase (0.16 U/mg, 80°C) and pyruvate carboxylase (0.02 U/mg, 50°C). The findings indicate that the hyperthermophilic eubacterium T. maritima ferments sugars (glucose) to acetate, CO₂ and H₂ involving the Embden-Meyerhof pathway, phosphate acetyltransferase and acetate kinase. Thus, the organism differs from the hyperthermophilic archaeon Pyrococcus furiosus which ferments sugars to acetate, CO₂ and H₂ involving a modified non-phosphorylated Entner-Doudoroff pathway and acetyl-CoA synthetase (ADP forming).     

Heat-stable pullulanase from Bacillus acidopullulyticus: characterization and refolding after guanidinium chloride-induced unfolding

Heat-stable pullulanase from Bacillus acidopullulyticus was characterized with respect to its stability against thermal and chemical denaturation and its reactivation after complete chemical unfolding. The enzyme was quite thermostable and retained 55% of activity after heating at 60°C for 30 min at pH 5.5. At pH 6.0, only 9% residual activity was observed. The addition of sucrose, polyols, and Na₂SO₄ strongly stabilized the enzyme against thermal inactivation. The processes of chemical unfolding by guanidinium chloride (GdmCl) and refolding were studied by enzymological and spectroscopic criteria. B. acidopullulyticus pullulanase was very sensitive to GdmCl denaturation and had a transition midpoint at 1.2 M GdmCl. Reactivation after complete unfolding in 5 M GdmCl was initiated by dilution of the unfolding mixture; 67% reactivation was observed under standard conditions. The influence of some chemical and physical parameters (pH, chemical agents, temperature, and unfolding and refolding time) on refolding was investigated. Of the additives tested to assist reactivation, only bovine serum albumin (BSA) increased the yield of activity to 80%. The full regain of structure and activity was proven by comparing the enzymological, physicochemical, and spectroscopic properties of the native and refolded pullulanase. Received: June 22, 1998 / Accepted: December 11, 1998