Presence Of Sorbitol Research Articles

Redox switching of carbohydrate binding to ferrocene boronic acid

The redox properties of ferrocene boronic acid in aqueous buffers from pH 4 to pH 12 have been investigated using cyclic voltammetry. It is shown that the proton transfer equilibrium is under thermodynamic control over the range of scan rates employed (20-5000 mV/s), leading to pKa values of 10.8 and 5.8 for the ferrocene and ferrocenium forms, respectively. In the presence of sorbitol, fructose, or glucose, the voltammetric behaviour of ferrocene boronic acid at pH 7 is consistent with reversible formation of a carbohydrate - boronic acid complex. However, in all cases, the complexation-dissociation is under kinetic control. Analysis of the voltammetric response as a function of scan rate and carbohydrate concentration, together with an independent spectroscopic determination of the binding constant to the ferrocene form, allows all of the kinetic and thermodynamic constants for each system to be determined. In general, it is found that the binding constants ferrocenium form are about two orders of magnitude greater than those for the ferrocene form. It is possible that this redox dependent switching of the carbohydrate binding can be exploited in amperometric or potentiometric sensing devices.

Influence of site-directed mutagenesis on protein assembly and solubility of tadpole H-chain ferritin

In order to understand the influence of ferroxidase center on the protein assembly and solubility of tadpole ferritin, three mutant plasmids, pTH58K, pTH61G, and pTHKG were constructed with the aid of site-directed mutagenesis and mutant proteins were produced inEscherichia coli. Mutant ferritin H-subunits produced by the cells carrying plasmids pTH58K and pTHKG were active soluble proteins, whereas the mutant obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence of sorbitol and betaine. Especially, the cells carrying pTH61G together with the plasmid pGroESL harboring the molecular chaperone genes produced soluble ferritin. The mutant ferritin H-subunits were all assembled into ferritin-like holoproteins. These mutant ferritins were capable of forming stable iron cores, which means the mutants are able to accumulate iron with such modified ferroxidase sites. Further functional analysis was also made on the individual amino acid residues of ferroxidase center.

Thermal stabilization by polyols of β-xylanase from Bacillus amyloliquefaciens

Purified endo-β-1,4-xylanase of Bacillus amyloliquefaciens MIR 32 retained 100% of its activity after 4 days of incubation at 50°C. Sorbitol (400 mg cm -3 ) produced a 63-fold increase in the half-life of the enzyme at 65°C, which was only 29 min at this temperature in the absence of the polyol. This thermal stabilizing activity increased exponentially in respect to sorbitol concentration in the range 250-400 mg cm -3 and was dependent on the pH, showing a maximum at pH values between 5.25 and 8.0. The circular dichroism (CD) thermal scanning profile (50°C h -1 ) at 224 nm showed that changes in the secondary structure of xylanase started at 65°C, while in the presence of sorbitol (400 mg cm -3 ) these modifications started at 80°C. This study indicated that sorbitol might be a valuable stabilizer for the use of β-xylanase from B. amyloliquefaciens at high temperatures.

Thermal stabilization by polyols ofβ-xylanase fromBacillus amyloliquefaciens

Purified endo-β-1,4-xylanase of Bacillus amyloliquefaciens MIR 32 retained 100% of its activity after 4 days of incubation at 50°C. Sorbitol (400 mg cm−3) produced a 63-fold increase in the half-life of the enzyme at 65°C, which was only 29 min at this temperature in the absence of the polyol. This thermal stabilizing activity increased exponentially in respect to sorbitol concentration in the range 250–400 mg cm−3 and was dependent on the pH, showing a maximum at pH values between 5·25 and 8·0. The circular dichroism (CD) thermal scanning profile (50°C h−1) at 224 nm showed that changes in the secondary structure of xylanase started at 65°C, while in the presence of sorbitol (400 mg cm−3) these modifications started at 80°C. This study indicated that sorbitol might be a valuable stabilizer for the use of β-xylanase from B. amyloliquefaciens at high temperatures. © 1998 SCI

Biochemical characterization of a Δ12 acyl-lipid desaturase after overexpression of the enzyme in Escherichia coli

The Δ12 acyl-lipid desaturase of Synechocystis sp. PCC 6803 was overexpressed in Escherichia coli as an active enzyme. The overexpressed protein was associated with cell membranes; it represented about 10% of the total cellular protein and 25% of the total membrane protein. The enzyme in the membrane fraction exhibited strong fatty-acid desaturase activity. The desaturase in salt-washed membranes was stabilized by the presence of sorbitol. Storage of salt-washed membranes in 2 M sorbitol at 4°C and at pH 7–8 for six days resulted in the loss of less than 10% of the desaturase activity. The desaturase activity had a positive temperature coefficient, a result that suggests that the increase in the desaturation of fatty acids at low temperature might not be caused by the activation of desaturases at low temperature but, rather, by the increased synthesis of desaturases de novo.

Towards a molecular level understanding of protein stabilization: the interaction between lysozyme and sorbitol

The paper is investigating the mechanism of stabilization of proteins by polyols at the molecular level. It is addressing the interactions of sorbitol, a polyol commonly used as a protein stabilizing agent, with hen egg white lysozyme, a well studied protein. Differential scanning calorimetry shows an increase in denaturation temperature of lysozyme upon addition of sorbitol at a concentration of 250 mM and above. Increasing sorbitol concentration also caused an increase in signal intensity of the CD spectrum of lysozyme in the wavelength region of 280–300 nm. Two-dimensional nuclear magnetic resonance spectroscopy was used to examine interactions between lysozyme and sorbitol. Most significant changes are manifest in the anomalous relaxation properties of Ala and Thr methyl groups indicating modifications of local motions and possibly compression of the entire structure. This is further corroborated by new intra-protein nuclear Overhauser effects in the presence of sorbitol. There is also evidence that water is displaced from the enzyme surface close to Ile-88 upon addition of sorbitol. In combination these results reveal a complex interplay of different interactions. Comparison to NMR-spectra of lysozyme with a bound inhibitor (tri- N-acetyl-glucosamine) shows that the interaction with sorbitol affects spatially disparate regions of the protein.

Evidence for a Role of Glucose-induced Translocation of Glucokinase in the Control of Hepatic Glycogen Synthesis

Glucokinase reversibly partitions between a bound and a free state in the hepatocyte in response to the metabolic status of the cell. Maximum binding occurs at low [glucose] (<5 mM) and minimum binding at high [glucose] or in the presence of sorbitol or fructose. In this study we determined the binding characteristics of glucokinase in the hepatocyte in situ, by adenovirus-mediated glucokinase overexpression combined with the digitonin-permeabilization technique. We also determined the sensitivity of glycogen synthesis to changes in either total glucokinase overexpression or in free glucokinase activity. Glucokinase overexpression is associated with an increase in both free and bound activity, with an overall decrease in the proportion of bound activity. In hepatocytes incubated at low [glucose] (0-5 mM), glucokinase binding involves a high-affinity binding site with a Kd of approximately 0.1 microM and a binding capacity of approximately 3 pmol/mg total cell protein and low-affinity binding with a Kd of approximately 1.6 microM. Increasing glucose concentration to 20 mM causes a dose-dependent increase in the Kd of the high- affinity site to approximately 0.6 microM, and this effect was mimicked by 50 microM sorbitol, a precursor of fructose 1-P, confirming that this site is the regulatory protein of glucokinase. Glycogen synthesis determined from the incorporation of [2-3H,U-14C]glucose into glycogen at 5 mM or 10 mM glucose was very sensitive to small increases in total glucokinase activity and correlated more closely with the increase in free glucokinase activity. The relation between glycogenic flux and glucokinase activity is sigmoidal. Expression of the sensitivity of glycogen synthesis to glucokinase activity as the control coefficient reveals that the coefficient is greater for the incorporation of 2-tritium (which occurs exclusively by the direct pathway) than for incorporation of 14C label (which involves direct and indirect pathways) and is greater at 5 mM glucose (when glucokinase is maximally sequestered at its high-affinity site) than at 10 mM glucose. The results support the hypothesis that compartmentation of glucokinase in the hepatocyte increases the sensitivity of glycogen synthesis to small changes in total glucokinase activity and that glucose-induced translocation of glucokinase has a major role in the acute control of glycogen synthesis.

Studies on fructose metabolism in cultured astroglial cells and control hepatocytes: lack of fructokinase activity and immunoreactivity in astrocytes.

Astroglia-rich primary cultures derived from the brains of newborn rats can be grown in the presence of sorbitol or fructose. In the present study, evidence was obtained by enzymatic analysis and immunocytochemistry that fructose is further metabolized to fructose-6-phosphate and that fructokinase is lacking in the astrocytes. In contrast, fructose-1-phosphate as well as fructokinase immunoreactivity could be detected in cultured hepatocytes. Considerable amounts of astroglial glycogen were synthesized from fructose. Lactate release in fructose-fed cultures was still 30% that of glucose-fed cells and was abolished in the presence of 2-deoxyglucose. No glycogen was synthesized when sorbitol, which is converted intracellularly to fructose, replaced glucose in the incubation medium. However, lactate release from sorbitol-fed cultures was still significant and was not abolished by 2-deoxyglucose. The results are compatible with the idea of astroglial glycogen being a store of lactate rather than glucose. Furthermore, the results demonstrate that sorbitol is an adequate substrate for astroglial glycolysis but, in contrast to fructose, cannot be utilized for the buildup of glycogen stores.

Sorbitol Increases Shelf Life of Fresh Mushrooms Stored in Conventional Packages

ABSTRACTTrays containing 100g of mushrooms overwrapped with PVC film were held at 12°C. Two 3 mm holes were made on top of the ovetwrap for ventilation and a TyvekR pouch containing sorbitol was placed at the bottom of the tray before storage. Surface moisture content of mushrooms decreased in the presence of sorbitol. Mushrooms packaged with 10g sorbitol had a constant surface moisture content and those packaged with 15g sorbitol had the best overall color. Principal component analysis of Vis‐NIR spectra revealed that surface moisture content affected the scattering of incident light and mushroom color. Lowering of the in‐package relative humidity did not affect the maturation rate of mushrooms, but reduced bacterial growth, suggesting that improvement in color was probably due to reduced bacterial activity.

Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media

The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (a(w) = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol-containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N-acetyl-L-phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol-containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin-sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89 degrees C). The transition temperatures of the substrate-containing chymotrypsin and of the control were identical (72 degrees C).

Purification and characterization of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro.

Plant ferritin subunits are synthesized as precursor molecules; the transit peptide (TP) in their NH2 extremity, responsible for plastid targeting, is cleaved during translocation to this compartment. In addition, the N-terminus of the mature subunit contains a plant-specific sequence named extension peptide (EP) [Ragland, Briat, Gagnon, Laulhère, Massenet, and Theil, E.C. (1990) J. Biol. Chem. 265, 18339-18344], the function of which is unknown. A novel pea-seed ferritin cDNA, with a consensus ferroxidase centre conserved within H-type animal ferritins has been characterized. This pea-seed ferritin cDNA has been engineered using oligonucleotide-directed mutagenesis to produce DNA fragments (1) corresponding to the wild-type (WT) ferritin precursor, (2) with the TP deleted, (3) with both the TP and the plant specific EP sequences deleted and (4) containing the TP but with the EP deleted. These four DNA fragments have been cloned in an Escherichia coli expression vector to produce the corresponding recombinant pea-seed ferritins. Expression at 37 degrees C led to the accumulation of recombinant pea-seed ferritins in inclusion bodies, whatever the construct introduced in E. coli. Expression at 25 degrees C in the presence of sorbitol and betaine allowed soluble proteins to accumulate when constructs with the TP deleted were used; under this condition, E. coli cells transformed with constructs containing the TP were unable to accumulate recombinant protein. Recombinant ferritins purified from inclusion bodies were found to be assembled only when the TP was deleted; however assembled ferritin under this condition had a ferroxidase activity undetectable at acid pH. On the other hand, soluble recombinant ferritins with the TP deleted and expressed at 25 degrees C were purified as 24-mers containing an average of 40-50 iron atoms per molecule. Despite the conservation in the plant ferritin subunit of a consensus ferroxidase centre, the iron uptake activity in vitro at pH 6.8 was found to be lower than that of the recombinant human H-ferritin, though it was much more active than the recombinant human L-ferritin. The recombinant ferritin with both the TP and the EP deleted (r delta TP/EP) assembled correctly as a 24-mer; it has slightly higher ferroxidase activity and decreased solubility compared with the wild-type protein with the TP deleted (r delta TP). In addition, on denaturation by urea followed by renaturation by dialysis the r delta TP/EP protein showed a 25% increase in core-formation in vitro compared with the r delta TP protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Visualization of capsule formation by Erwinia amylovora and assays to determine amylovoran synthesis

Exopolysaccharide (EPS) synthesis by Erwinia amylovora depends on environmental and genetic predispositions. To measure the amount of the acidic EPS amylovoran synthesized by E. amylovora cell cultures, a turbidity assay using cetylpyridinium salt was developed. The EPS produced by bacteria grown on solid media was additionally characterized by its water content. The amylovoran capsules were visualized in situ by staining with fluorescein isothiocyanate (FITC)-labelled lectin from Abrus precatorius, which reacts with the galactose residue of the EPS side chain. The staining and the turbidity assays were applied to suspension cell cultures or to cells from colonies and did not require any purification steps. Lectin staining was superior to electron microscopic (EM) techniques for visualization of capsules. For EM, the capsule was stabilized with polycationic ferritin. In contrast to lectin staining, only a small fraction of the cells was found to be EPS-coated in the EM assay. An increase in capsulation and in amylovoran production was found in conjunction with mutations in a ribosomal protein conferring resistance to streptomycin. Furthermore, the presence of sorbitol in the growth environment resulted in high synthesis of amylovoran. Cells in the stationary growth phase continued to produce amylovoran. Apparently, the strong dependence of the fireblight pathogen on capsules requires the capacity for EPS synthesis in all growth stages in order to escape plant defence reactions.

Effects of xylitol/sorbitol combinations on bacterial growth and metabolism in Streptococcus sobrinus OMZ 176.

Combinations of the non-cariogenic sugar alcohols xylitol and sorbitol are widely used as sucrose substitutes in lozenges and chewing-gums. In S. sobrinus OMZ 176 xylitol inhibits the metabolism of sorbitol, and the xylitol-induced bacterial growth inhibition of S. sobrinus OMZ 176 has been shown to be enhanced by sorbitol. In combination with xylitol the sorbitol transport changes from a pts- to a non-pts transport, while the transport of xylitol is unaffected by the presence of sorbitol. It would be of interest to know how much xylitol has to be added to a sorbitol-containing medium to inhibit the metabolism of this sugar alcohol. In the present study no optimal inhibitory concentrations of xylitol/sorbitol combinations were found. Growth and acid production in S. sobrinus OMZ 176 are essentially the same for xylitol: sorbitol ratios in the range from 1:7 to 7:1. The presence of intracellular xylitol metabolites seems to be essential.

Sorbitol Content of Selected Oral Liquids

Excipients in pharmaceuticals usually are considered inert, and may be overlooked in the differential diagnosis of diarrhea. Sorbitol-containing medicinal liquids are capable of inducing osmotic diarrhea. We reviewed the oral liquids in our formulary to determine their sorbitol content and to evaluate the availability of this information. The oral liquids stocked by our hospital were determined through a computer search and manual inspection of the pharmacy storeroom. Three common sources of drug information were consulted to determine each product's sorbitol content: manufacturers' product information, American Hospital Formulary Service (AHFS) Drug Information 91, and Facts and Comparisons Drug Information. We then contacted each manufacturer by mail or telephone to verify the information. The study was conducted at the University of Cincinnati Hospital, a tertiary-care, teaching hospital. A total of 129 products (98 chemical entities) were reviewed. Fifty-four (42 percent) of the products examined contained sorbitol. The frequency of sorbitol presence by liquid type was: solutions (33 percent), suspensions (43 percent), syrups (59 percent), elixirs (43 percent), concentrates (67 percent), drops (33 percent), tinctures (0 percent), and emulsions (0 percent). The percentage of listings indicating the presence of sorbitol was: manufacturer's product information (79 percent), Facts and Comparisons (52 percent), and AHFS Drug Information 91 (13 percent). Only three of the 54 products had the exact sorbitol content stated in any source.

Cryopreservation and Plant Regeneration from Embryogenic Cultures of Larch (Larix×eurolepis) and Black Spruce (Picea mariana)

A method has been developed for the routine cryopreservation of embryogenic cultures of hybrid larch (Larix×eurolepis) and black spruce (Picea mariana Mill.). The method involves growing the cultures in the presence of sorbitol and then briefly exposing them to DMSO followed by controlled cooling to −40°C. The cultures were then submerged and stored in liquid nitrogen. Growth of the embryogenic cultures was monitored for 14 d after rapid thawing and plating on to media. The highest relative increase in the tissue fresh weight, after storage in liquid nitrogen, was observed when embryogenic cultures of both species were pregrown for 24 h in a medium with 0·4 M sorbitol and then treated with 10% DMSO. This pretreatment also ensured the shortest lag phase in resuming the growth. The post-thaw cultures gave rise to mature somatic embryos which developed into plants

A novel strategy for production of a highly expressed recombinant protein in an active form

Under standard growth conditions, E. coli transformed with the high-level expression vector pMON5525 produces recombinant DMAPP/AMP transferase in inactive, insoluble complexes. We have produced large amounts of active, soluble protein by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine. This caused an increase of up to 427-fold in the active yield, and the disappearance of the protein from the pelletable fraction of cell extracts. This treatment may have wide applicability.

The Effect of sorbitol on acid phosphatase deactivation

Acid phosphatase thermal deactivation follows a complex path: an initial decay toward an equilibrium distribution of at least two intermediate structures, mutually at the equilibrium, followed by a final breakdown toward a completely inactive enzyme configuration. The results obtained in the presence of sorbitol have been compared to those produced in the course of purely thermal deactivation of the native enzyme. For any sobitol concentration, an equivalent temperature is calculated that results in exactly the same activity-versus-time profile. This suggests enzyme deactivation to be controlled by a single, unchanging step. Immobilized enzyme runs have been performed, as well, by entrapping acid phosphates within a polymeric network formed onto the upstream surface of an ultrafiltration membrane. The stabilizing effect of entrapment cumulates with that produced by sorbitol. In this case, however, an equivalent temperature cannot be determined, thus indicating that a different deactivation mechanism is followed.

The effect of aluminum ions and sorbitol on collagen and skin: A thermally stimulated current spectroscopy study

The influence of aluminum ions introduced either directly or with sorbitol, in collagen and skin, has been investigated by Thermally Stimulated Current/TSC spectroscopy. The intramolecular mobility of collagen has been found to be significantly reduced by aluminum ions. The substitution of water molecules by aluminum ions on intramolecular hydrophilic sites is suggested to be responsible for this evolution. The presence of sorbitol minimizes this effect. The intermolecular mobility of collagen is decreased upon introduction of aluminum ions in presence of sorbitol. This result can be explained by the interaction of sorbitol-aluminum entities with the telopeptidic regions of collagen molecules. In skin samples, the intra- and intermolecular mobilities are restored due to the combined actions of sorbitol and noncollagenous proteins and other macromolecules.

Sorbitol transport by Streptococcus sanguis 160

Sorbitol metabolism was examined with a sorbitol-fermenting strain (160) of Streptococcus sanguis isolated from the dental plaque of a subject using sorbitol-containing chewing-gum for 4 years. S. sanguis 160 was grown in continuous culture (pH, 7.0; dilution rate, 0.1 h-1) with glucose, sorbitol and nitrogen (sorbitol-excess) limitations. Cells grown with a glucose limitation exhibited low, but detectable, uptake of [14C]-sorbitol and transition to medium limiting in sorbitol resulted in a 5-fold increase in sorbitol uptake. Kinetic data revealed that both glucose and sorbitol-limited cells possessed 2 transport systems for sorbitol (Ks = 3.3-6.7 and 36-64 microM), but continued growth of the organism on limiting sorbitol resulted in the loss of the high-affinity system. Decryptified, sorbitol-limited cells phosphorylated sorbitol in the presence of phosphoenolpyruvate (PEP), but not with ATP, indicating sorbitol transport solely via the PEP phosphotransferase (PTS) system. PEP-dependent activity in glucose-limited and sorbitol-excess cells was 6- and 4-fold lower than that of the sorbitol-limited cells. Uptake of [14C]-sorbitol and activity for Ell for sorbitol [Ellsor] of the PTS in cells in transition from a glucose to sorbitol limitation confirmed the induction of the sorbitol-PTS and the repression of the glucose-PTS in the presence of sorbitol. Cells grown with an excess of sorbitol exhibited very low Ellsor activity. A crossover experiment with membranes and soluble fractions from glucose-, sorbitol- and nitrogen-limited cells of S. sanguis 160 demonstrated the induction of a soluble PTS component in sorbitol-limited cells essential for sorbitol transport via the PTS.(ABSTRACT TRUNCATED AT 250 WORDS)

Sorbitol inhibition of glucose metabolism by Streptococcus sanguis 160

Clinical studies in Sweden have shown that the proportion of sorbitol-utilizing strains of Streptococcus sanguis increases in dental plaque from individuals using sorbitol-containing products for prolonged periods. We have undertaken to study the metabolism of glucose and sorbitol by S. sanguis 160, isolated from a subject consuming sorbitol-containing chewing-gum 4 times a day for 4 years. Growth on glucose was inhibited by the presence of sorbitol in the growth medium and sorbitol was utilized in the presence of glucose, albeit, at a slower rate than glucose. In addition, pulses of glucose added to cultures growing on sorbitol resulted in the expulsion of sorbitol from the cell. In order to examine further the relationship of sorbitol and glucose, uptake assays were carried out with S. sanguis 160 grown in continuous culture (pH 7.0, dilution rate = 0.1 h-1) with glucose, sorbitol or nitrogen (sorbitol excess) limitations. The uptake of [14C]-glucose by sorbitol-limited cells, but not by glucose-limited cells, was inhibited by sorbitol, as was glycolysis. Kinetic experiments with glucose-limited cells showed 2 transport systems for glucose with Ks values of 5.2 and 40 microM, and glucose phosphorylation activity by decryptified cells indicated transport by the P-enolpyruvate (PEP) phosphotransferase system (PTS) with lesser activity for an ATP-dependent transport process. Transition from glucose-limited growth to sorbitol-limited growth revealed repression of total [14C]-glucose uptake by intact cells and activity for Enzyme II for glucose (Ellglc) of the PTS measured in membrane preparations in the presence of an excess of the soluble PTS proteins in crude cell-free supernatant fractions.(ABSTRACT TRUNCATED AT 250 WORDS)