Influence of alkaline concentration on molecular association structure and viscoelastic properties of curdlan aqueous systems

Curdlan is an extracellular polysaccharide produced from soil microorganism Alcaligens faecalis var. 10C3K, and the linear structure consists of β-1,3-glycoside linkages. Curdlan is not soluble in water but it is soluble in alkaline aqueous solution, and we can obtain the gel when curdlan alkaline solution is heated above 60°C or neutralized by acids. In the present study, the gelation mechanism and dispersing structure of curdlan in the alkaline solutions are studied in terms of correlation between the molecular association structure and viscoelastic properties, using static light scattering and rheological measurements. The degree of association for the curdlan molecules in dilute solution increases with decreasing alkaline concentration. The viscoelastic properties also depend strongly on the alkaline concentration. The concentrated curdlan solution shows almost Newtonian flow at high alkaline concentrations and shows a gel-like behavior at low alkaline concentrations. It was elucidated that the molecular association in the dilute solution reflects on the viscoelastic properties of the concentrated solution and that the gelation mechanism is related to the association structure of curdlan molecules. In the case of lower NaOH concentration systems, the molecular association is likely to consist of a hydrophobic core and hydrophilic surface. The gelation mechanism above 60°C is considered to include the dissociation process of the molecular association and reformation of the network structure. © 1997 John Wiley & Sons, Inc. Biopoly 42: 479–487, 1997     

Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment

Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3⁴) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120 °C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20 PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40 °C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9 × 10¹¹ CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS.     

Purified curdlan and its hydroxyalkyl derivatives: preparation, properties and applications

Curdlan, a β-1,3-glucan produced by fermentation of Alkaligenes faecalis, is a non-ionic gel-forming polysaccharide that is, along with its hydroxalkyl derivatives, a potentially important matrix for life science applications. The commercially available material contains residual nucleic acids, cellular debris and other contaminants that can interfere with electrophoretic separations and visualization procedures. Simple procedures have been developed for purification of the curdlan and coherent gel formation. Curdlan gels can be formed in a variety of chaotrope-containing, or chaotropic, solvents, including 40% formamide, 7 M urea. The chaotropes can be retained or subsequently removed by leaching. Heat treatment before or after leaching enables thermostable gel formation. Hydroxyethyl and glyceryl derivatives of curdlan have been prepared. Depending on the degree of substitution (DS), a whole spectrum of derivatives with a range of unique properties can be obtained. Lower DS hydroxyethyl derivatives form clear, elastic gels in 6 M urea, while the higher DS hydroxyethyl derivatives are soluble in hot water and gel on cooling to form clear, elastic, thermoreversible gels. Partial depolymerization of curdlan by γ-irradiation reduces the viscosity of subsequent preparations, enabling the preparation of higher concentration, more sieving gels. Use of selected preparations to form unique matrices for electrophoretic separations has been demonstrated.     

The Use of Protein Silver for Staining Protozoa

When commercially prepared silver products suitable for staining protozoa by the Bodian silver technic apparently became unavailable, a substitute for Protargol was prepared as follows: 0.9 g. of gelatin is dissolved by heat in 100 ml. of distilled water; to this 0.1 g. of silver nitrate is added at 60°C; this solution is poured into Columbia staining dishes (10 ml.) in which one or two drops of M/10 sodium hydroxide have been added. Copper is not used in the impregnating bath. Smears fixed in Hollande's or Schaudinn's fixatives are bleached and impregnated for 36 hours or more at 35°C. Impregnated smears are reduced with a mixture of hydroquinone and sodium sulfite, and toned with gold chloride as recommended by Kirby (1945).     

Composition of cell wall preparations of rice bran and germ

Cell walls of petrol-defatted non-waxy IR32 rice bran and germ were prepared by protein removal with 0.5% SDS—0.6% β-mercaptoethanol, heating the residue to 80°, and destarching with Bacillus licheniformis α-amylase. A waxy rice, IR29, had a similar cell wall composition as IR32. Principal wall sugars were arabinose, xylose, and glucose. The 0.5 M sodium or potassium hydroxide and 8 M urea preferentially extracted arabinose-, xylose- and uronic acid-rich polysaccharides but 6 M sodium hydroxide—0.81 M boric acid extracted mannose-rich polysaccharides. DEAE-cellulose BO₃^3? chromatography of the 0.5 M sodium hydroxide extracts gave fractions of similar arabinose— xylose ratios. Proteins in the cell wall preparations had only 0.4–1.6% hydroxyproline, and were bound mainly to polysaccharides, based on disc gel electrophoresis. The preparations were autofluorescent in UV and rich in phenols, mainly ferulic acid. The cell wall preparations and their 8 M urea fractions had a softening effect on defatted waxy starch aqueous gel at 0.2–2% of the starch.     

Relationship between DNA acid-solubility and frequency of single-strand breaks near apurinic sites

Using [32P]DNA alkylated with [3H]methyl methanesulfonate, depurinated by heating at 50 degrees C for various periods, then treated with sodium hydroxide, a table was constructed giving the DNA fraction soluble in 5% perchloric acid at 0 degree C as a function of the frequency of strand breaks. The alkaline treatment placed a break near each apurinic site; the apurinic sites were counted in two ways which gave consonant results: by the loss of [3H]methyl groups and by reaction with [14C]methoxyamine. The 32P label of DNA was used to measure the acid-solubility.     

Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.     

Stabilization of heat-induced changes in plant peroxidase preparations by ClpX, a bacterial heat shock protein

Peroxidases (PODs) are known to be quite stable at elevated temperatures. Moreover, partially denatured peroxidases are able to regain their catalytic activity during incubation at room temperature. In this paper, we describe the effects of some heat shock proteins on the self-reactivation of plant peroxidase preparations. Horseradish and artichoke peroxidases (HRP and ARP, respectively) were first heated (at 60 °C or 90 °C), then incubated at a slightly elevated temperature (30 °C). The heat-treatment resulted in a considerable loss of activity of both enzymes but the subsequent incubation allowed their reactivation. However, no reactivation could be detected when incubation was carried out in the presence of the molecular chaperone ClpX. Other chaperones that were tested (DnaK, DnaJ and GrpE) did not show the inhibitory effect. Electrophoretic analyses further indicated that the heat-treated horseradish peroxidase, but not the native enzyme, binds to ClpX eliminating the possibility of undesirable protein refolding that would result in aggregation.     

Chiral recognition ability of curdlan triacetate: solvent and temperature effects

The chiral recognition ability of the chiral stationary phase (CSP) consisting of curdlan (beta-1,3-glucan) triacetate coated on silica gel was clearly changed by the contacting solvents and heat treatment. The chiral recognition ability significantly decreased, particularly at temperatures above 45 degrees C, depending on the racemates. The molecular weight of the curdlan triacetate slightly influenced its ability. The recognition abilities of curdlan tricetate that was lost by heat treatment were partially recovered by contact with methanol. However, when it was contacted with ethanol a different selectivity was observed. The labile chiral recognition ability of curdlan triacetate is in striking contrast to the very stable chiral recognition of cellulose (beta-1,4-glucan) triacetate (Chiralcel OA). This difference may be ascribed to the conformational stability of the acetates consisting of curdlan (beta-1,3-glucan) and cellulose (beta-1,4-glucan) with different sugar linkages.     

Rapid purification of commercial gellan gum to highly soluble and gellable monovalent cation salts

Commercial gellan gum contains divalent cation contaminants (mainly Ca²⁺ and Mg²⁺) in levels sufficient to neutralize over one-third of its car☐yl groups. Consequently, in order to dissolve gellan gum in water, the mixtures must be heated to more than 90°C. This has prevented applications of gellan gum to such uses as the immobilization of viable enzymes and cells in beads. A rapid two-step method is described here to purify commercial gellan gum to the monovalent cation salts in an overall yield of 85%, through the intermediate free acid form. The gellan monovalent cation salts were highly soluble at temperatures as low as 5°C, and readily gelled upon exposure to solutions of divalent cations. Laboratory-scale preparations of 100 g of gellan monovalent cation salts were readily achieved in a day.     

Induction of heat sensitivity of wheat roots and its effects on mitochondria, adenosine triphosphate, triglyceride, and total lipid content

Wheat seedlings were subjected to heat shock for 2 min at 45 °C. After heat treatment, the wheat seedlings were incubated at 25 or 35 °C. At 25 °C, but not at 35 °C, the root tips survived the heat shock. Immediately after the heat treatment the free triglyceride content in the treated root tips was higher than in the untreated roots, but the total lipid content was not changed. The ATP content immediately after the heat treatment was variable, but after about 1 h it stabilized at the same level as in the control or at a higher level. After 45 min at 25 °C after heat shock, the endoplasmic reticulum cisternae had expanded, giving rise to small irregular vacuoles. Golgi vesicles were also irregular. Four hours after heat treatment the endoplasmic reticulum and Golgi vesicles again were normal, but mitochondria were irregular with fewer tubules and with adhering membrane curls containing lipids. These membrane curls were not observed 24 h after heat treatment. When incubated at 35 °C after heat shock wheat root meristems died. Some cells in the meristem were still alive 4 h after treatment. They had large vacuoles with membrane whorls and plasmalemmasomes, and in some cases the cells were partly lysed.     

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.     

Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from the stem of the pineapple plant (Ananas comosus) and is unique for it contains a single histidine residue (His-158) in the polypeptide. Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of bromelain was selected. Bromelain was successfully immobilized on iminodiacetic acid carrier Sepharose 6B. Cu²⁺ complexed with iminodiacetate (IDA) was used as the chelating ligand to bind the lone histidine on bromelain. Simultaneously, preparation of a high affinity immobilized preparation was attempted using a soluble cross-linked preparation of bromelain on Cu-IDA-Sepharose. However this second method proved unsuccessful, possibly due to poor histidine accessibility in the cross-linked preparation. The immobilized preparation obtained using uncrosslinked bromelain was more resistant to thermal inactivation, as evidenced by retention of over enzyme 50% activity after incubation at 60 °C, as compared to 20% retained by the native enzyme. The immobilized preparation also exhibited a broader pH-activity profile in acidic range. The native, immobilized and soluble cross-linked bromelain showed apparent Michaelis constant (K_m) values of 1.08, 0.42, 1.56 mg/ml, respectively, using casein as the substrate. While the maximum velocity (V_max) values of the soluble and immobilized preparations were comparable, cross-linked preparation showed a 20% decrease, suggesting inactivation. The mild conditions used for predominantly oriented immobilization exploiting the unique property of single histidine, the high recovery of immobilized preparations, the stability, reusability and the regenerability of the matrix are the main features of the method reported here.     

A 13C-nuclear magnetic resonance study of polysaccharide gels. Molecular architecture in the gels consisting of fungal,branched (1 → 3)-β-d-glucans (lentinan and schizophyllan) as manifested by conformational changes induced by sodium hydroxide

To gain further insight into the architecture of the gel network of some branched (1 → 3)-β-d-glucans, a ¹³C-n.m.r. study of sodium hydroxide-induced, conformational change was performed. The branched d-glucans examined were lentinan from Lentinusedodes, a lower-molecular-weight fraction thereof, and schizophyllan from Scilizophyllum commune; these (1 → 3)-β-d-glucans have two branches for every five d-glucopyranosyl residues (lentinan), or one for every three or four (schizophyllan) at 0-6. In contrast to the gel oflinear (1 → 3)-β-d-glucan (curdlan), all of the ¹³C signals due to the β-d-(1 → 3)-linked d-glucosyl residues were completely suppressed in the gel state. As the peak intensity and line width of the ¹³C-resonance peaks for the gel state are strongly influenced by the degree of cross-linking, such a complete loss of the peak areas can be explained in terms of a higher degree of cross-linking than that of the linear d-glucans. As demonstrated previously, the cross-links involve physical association of the helical segments, such as the double- or triple-stranded helices. These helix forms were found to be converted, at 0.2M sodium hydroxide, into the random-coil form (gel-to-sol transition), which gives rise to full peak-areas, because of complete breaking of the physical cross-links. Also, in contrast to the linear d-glucan, such helix-coil transition of the branched d-glucans proceeded in a noncooperative way: the peak intensity and line width gradually changed with the concentration of sodium hydroxide. This behavior is best interpreted in terms of distribution of the various degrees of cross-linking. Some loose cross-links are readily broken in the lower range of concentration of alkali (0.09M), and others are resistant until complete conversion into the random coil occurs (0.2M). This result is consistent with the view that the primary structure of the branched (1 → 3)-β-d-glucans is hi-highly branched, as in a tree-like structure.     

Acclimation to temperature and death at changing lethal temperatures in the freshwater fish Chalcalburnues chalcoides

1. 1.|In the freshwater fish Chalcalburnus chalcoides, an increase in the body (standard) size caused decreases in the upper LT-50 from 36.6° to 36.0°C and lower LT-50 from 6.3° to 5.3°C

2. 2.|The fish acclimated to constant temperatures between 10°C and 30°C showed reasonable heat acclimation and also reasonable cold acclimation. Thus, an increase in the acclimation temperature from 10°C to 30°C caused increases in the upper LT-50 from 34° to 36.2°C and the lower LT-50 from 1.25 to 6.5°C.

3. 3|The mean survival time — temperature curves of 10°, 20° and 30°C acclimated fish at various constant temperatures showed decreased in the survival tim ewith increasing lethal temperatures. Furthermore, an increase in the acclimation temperature causes a shift in the survival duration-temperature curve to the right, i.e., the fish become more heat resistant. Thus, the mean survival duration of 10°, 20° and 30°C acclimated fish at 35°C were 7.5, 79.6 and 530 minutes, respectively.

4. 4.|The effect of the thermal experience to changing lethal temperatures depends on the first lethal temperature to which the fish were exposed as well as the sequence of temperature changes. In the experiments in which the first lethal temperatures were between 32° and 34°C and the temperature was varied in an ascending order, their thermal resistance was increased and the fish required 114 to 174% of the expected lethal doses to die while in the experiments in which the starting temperature were between 38° and 40°C and the temperature varied in descending order, the fish become more sensitive to the upper lethal temperature and they died after receiving only 62 to 81% of the expected lethal doses. Thus, with a gradual increase in the lethal temperature, the fish show additional acclimation in the zone of resistance which in turn causes an increase in the thermal resistance. This may have ecological significance in nature.

13C NMR structural investigation of scleroglucan

This paper concerns the ¹³C NMR signal assignment in the DMSO of a neutral polysaccharide, scleroglucan. The previously proposed chemical structure is confirmed. The ¹³C NMR spectrum shows that scleroglucan is a regular poly (A, B, C, D) type glucan. The relaxation times of the different series of carbon atoms demonstrate that a single, pendant glucose group is attached to each third monomer along the main chain of what is a β(1 → 3)-glucan. Partial acid hydrolysis gives a spectrum analogous to that of the β(1 → 3)-d-glucan, curdlan, and confirms the structure of the polymer backbone.In aqueous solution, no signal has been obtained due to the existence of a rigid, ordered conformation as demonstrated by optical rotation; in the presence of sodium hydroxide, a conformational transition is produced just as with curdlan. The conclusion is that the behaviour of scleroglucan in solution is similar to that of other β(1 → 3)-d-glucans even though it is more soluble.     

Loss of Symbiodinium from bleached soft corals Sarcophyton ehrenbergi, Sinularia sp. and Xenia sp.

The deleterious effects of temperature-induced coral bleaching, a process by which corals lose their endosymbiotic algae (zooxanthellae; genus Symbiodinium) primarily at temperatures above mean yearly maximums, has not been well described for alcyonacean soft corals (Coelenterata, Octocorallia). The study of Symbiodinium cells lost from Sarcophyton ehrenbergi, Sinularia sp., and Xenia sp., which have not been compared in bleaching studies, indicate that the soft coral S. ehrenbergi released the greatest number of symbiont cells, however, it was less susceptible to heat stress surviving temperatures of 34 °C for >39 h. Sinularia sp. showed intermediate levels of bleaching tolerance to elevated temperatures, surviving prolonged exposures at 32 °C, but dying within 24 h at 34 °C. Xenia sp., however, was the most vulnerable to high heat stress maximally releasing Symbiodinium at temperatures ≤30 °C. This evidence indicates that Xenia sp. is even more susceptible to elevated temperatures than Acropora spp., previously reported to be the most vulnerable coral species to elevated temperature-induced bleaching.

Molecular analysis showed that the more resistant soft coral species (S. ehrenbergi) had the same type of Symbiodinium (clade C) as less resistant soft corals (Xenia sp.). In comparison to scleractinian corals collected from the same region that show similar bleaching resistance to high temperatures (e.g. Porities solida—more robust; Favites complanata—moderate resistance; Acropora hyacinthus—less robust), all scleractinian corals were symbiotic with Symbiodinium from clade C. A. hyacinthus, however, was found to possess multiple symbionts (clades B and C), and this represents a first report of Clade B in any Acropora species.     

Impact of microwave heating on the physico-chemical properties of a starch–water model system

The objective of this work was to understand the physico-chemical changes induced in a wheat starch model system as a result of microwave heating. Wheat starch dispersions in water, with final solids content of 33%, 40% or 50%, were heated in a microwave oven. Following heating the samples were stored at 25 °C for up to 120 h and analyzed periodically. Microwave heated gels were significantly different from conduction heated gels in all parameters measured. The differences in properties are a reflection of the differences in the heat and mass transfer of the different modes of heating. The lack of granule swelling and the resulting soft gel are two key observations. The results of this study suggest a different mechanism of starch gelatinization compared to conduction heating. The vibrational motion and the rapid increase in temperature also result in granule rupture and formation of film polymers coating the granule surface.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Exercise in the heat: Effects of dinitrophenol administration

1. 1.|Dinitrophenol (DNP) was administered to rats in two equal dosages (20 mg/kg, 30 min interval); the second injection was followed immediately by exercise (9.14 m/min) in the heat (30°C) or at room temperature (21°C).

2. 2.|At 21°C control (saline-treated) rats manifested a mean endurance of 94 min which was reduced to 32 min among DNP-treated animals.

3. 3.|At 30°C, control rats ran for 65 min (δT_re/min = 0.05°C) while DNP-treated animals had a mean endurance of only 12 min (δT_re/min = 0.22°C).

4. 4.|DNP-treated rats (30°C) manifested no decrements in tail-skin heat loss (δT_sk/min = 0.17°C vs 0.10°C) or saliva secretion (0.78 g/min, DNP vs. 0.19 g/min, control) for their brief treadmill duration.

5. 5.|The increased metabolic heat production of DNP severely reduced performance.