The purification and study of a honey bee abdominal sucrase exhibiting unusual solubility and kinetic properties.

A sucrase from honey bee abdomens was purified to a high state of homogeneity. It was unusual in that it was completely soluble in high concentrations of ammonium sulfate and because curved rather than rectilinear lines were obtained when initial velocity data for at least two substrates were plotted. The action of the enzyme towards a large number of glycosides showed that the enzyme was able to hydrolyze all α-glucosides tested except trehalose and starch. pH Optima of sucrose and p-nitrophenyl-α-d-glucopyranoside differed by 1.0 pH unit. The unusual kinetic patterns which were found seem to be unique to this disaccharidase and were shown to be the result of a combination of hydrolytic and transferolytic activity in which the initial substrate is also a very good acceptor molecule for the transferolytic process. The K_m value for hydrolysis was found to be about an order of magnitude lower than for other insect sucrases with the more usual type of kinetic action. Amino acid and amino sugar analyses showed that the sucrase was a glycoprotein which contained glucosamine and either mannosamine or galactosamine. The molecular weight of the enzyme was estimated to be 70,000 or higher and there was no evidence that the enzyme had subunit structure. An s⁰_20,w value of 5.3S was determined. The enzyme was quite stable to a series of denaturing conditions and sulfhydryl reacting agents had little effect on the activity.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Sucrose acceptance,discrimination and proboscis responses of honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) in the field and the laboratory

Laboratory studies in honey bees have shown positive correlations between sucrose responsiveness, division of labour and learning. We tested the relationships between sucrose acceptance and discrimination in the field and responsiveness in the laboratory. Based on acceptance in the field three groups of bees were differentiated: (1) bees that accept sucrose concentrations >10%, (2) bees that accept some but not all of the sucrose concentrations <10% and water, and (3) bees that accept water and all offered sucrose concentrations. Sucrose acceptance can be described in a model in which sucrose- and water-dependent responses interact additively. Responsiveness to sucrose was tested in the same bees in the laboratory by measuring the proboscis extension response (PER). The experiments demonstrated that PER responsiveness is lower than acceptance in the field and that it is not possible to infer from the PER measurements in the laboratory those concentrations the respective bees accepted in the field. Discrimination between sucrose concentrations was tested in three groups of free-flying bees collecting low, intermediate or high concentrations of sucrose. The experiments demonstrated that bees can discriminate between concentrations differences down to 0.2 relative log units. There exist only partial correlations between discrimination, acceptance and PER responsiveness.     

Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli.

The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli with the strong, inducible tac promoter. The enzyme was purified from crude E. coli cell lysates by salting out with ammonium sulfate and chromatography on DEAE-Sepharose CL-6B, S-Sepharose, and MonoQ-Sepharose. The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence. Levanase was active on levan, inulin, and sucrose with Km values of 1.2 microM, 6.8 mM, and 65 mM, respectively. The pH optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55 degrees C. Levanase was rapidly inactivated at 60 degrees C, but activity could be retained for longer times by adding fructose or glycerol. The enzyme activity was completely inactivated by Ag+ and Hg2+ ions, indicating that a sulfhydryl group is involved. A ratio of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50 mg/ml. The mechanism of enzyme action was investigated. No liberation of fructo-oligomers from inulin and levan could be observed by thin-layer chromatography and size exclusion chromatography-low-angle laser light scattering-interferometric differential refractive index techniques. This indicates that levanase is an exoenzyme acting by the single-chain mode.     

Identification, preliminary characterization, and evidence for regulation of invertase in Streptococcus mutans

Sucrose dissimilation was studied in five strains of Streptococcus mutans. Glucose-adapted strain SL-1 makes acid more slowly from sucrose than from glucose; glucose-adapted strain SL-1 gives diauxie growth kinetics in broth containing limiting amounts of both glucose and sucrose. Thus, at least part of the sucrose dissimilative system appears inducible. Sucrase activity was identified in the 37,000 x g soluble cell fraction of five strains. Its intracellular location implies the presence of sucrose permease. The specific activity of the sucrase is higher in sucrose-adapted cells than in cells adapted to glucose or other sugars, further suggesting its inducibility. The enzyme from strain SL-1 was partially purified by diethylaminoethyl-cellulose chromatography and shown to be a single molecule with a molecular weight of about 48,000. The partially purified enzyme is specific for sucrose and produces equimolar glucose and fructose. Since it degrades raffinose, but not melezitose or other alpha-glucosides, it is an invertase. The invertase has a relatively high K(m) for its substrate and a pH optimum of 5.5 to 6.2. It is activated by inorganic orthophosphate (P(i)), P(i) functioning as a positive effector. Arsenate can substitute for phosphate. Neither the crude cell-free extract nor the partially purified enzyme preparations has detectable sucrose phosphorylase activity. A possible potent role of the invertase in the regulation of sucrose carbon flow in S. mutans is discussed.     

Influence of nutritional factors on the nature, yield, and composition of exopolysaccharides produced by Gluconacetobacter xylinus I-2281

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.     

Living on a high sugar diet: the fate of sucrose ingested by a phloem-feeding insect,the pea aphid Acyrthosiphon pisum

The natural diet of aphids, plant phloem sap, generally contains high concentrations of sucrose. When pea aphids (Acyrthosiphon pisum) were fed on chemically defined diets containing sucrose radiolabelled in the glucose or fructose moiety, 2 to 12-fold and 87 to 110-fold more radioactivity was recovered from the tissues and honeydew, respectively, of aphids that ingested [U-(14)C-glucose]-sucrose than from those ingesting [U-(14)C-fructose]-sucrose. The total radioactivity recovered was 70% of the ingested [U-(14)C-glucose]-sucrose and <5% of ingested [U-(14)C-fructose]-sucrose. The dominant honeydew sugars produced by aphids feeding on 0.75 M sucrose diets were oligosaccharides comprising glucose. In vitro the guts of pea aphids had high sucrase activity, 1-5 U mg(-1) protein, generating equimolar glucose and fructose except at high sucrose concentrations where glucose production was inhibited (K(si)=0.1 M). These data suggest that the fructose moiety of ingested sucrose is assimilated very efficiently and may be preferentially respired by the aphid, and that the glucose moiety of sucrose is incorporated into oligosaccharides by the transglucosidase activity of the gut sucrase at high sucrose concentrations. These differences in the fate of sucrose-derived glucose and fructose are important elements in both the carbon nutrition and osmoregulation of aphids.     

Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides.

Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures. Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.     

Influence of Nutritional Factors on the Nature, Yield, and Composition of Exopolysaccharides Produced by Gluconacetobacter xylinus I-2281

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.     

Effects of inoculum concentration,temperature, and carbon sources on tannase production during solid state fermentation of cashew apple bagasse

In this study, the optimization of tannase production by solid state fermentation was investigated using cashew apple bagasse (CAB), an inexpensive residue produced by the cashew apple agroindustry, as a substrate. To accomplish this, CAB was enriched with 2.5% (w/w) tannic acid and 2.5% (w/w) ammonium sulphate and then moistened with water (60 mL/100 g of dry CAB). The influence of inoculum concentration (10⁴ to 10⁷ spores/g), temperature (20, 25, 30, and 35°C) and several additional carbon sources (glucose, starch, sucrose, maltose, analytical grade glycerol, and glycerol produced during biodiesel production) on enzyme production by Aspergillus oryzae was then evaluated. Supplementation with maltose and glycerol inhibited tannase synthesis, which resulted in lower enzyme activity. Starch and sucrose supplementation increased enzyme production, but decreased the enzyme productivity. The maximum tannase activity (4.63 units/g of dry substrate) was obtained at 30°C, using 10⁷ spores/g and 1.0% (w/v) sucrose as an additional carbon source.     

Proboscis behavioral response of four honey bee Apis species towards different concentrations of sucrose,glucose, and fructose

Honey bees forage for pollen and nectar. Sugar is an important stimulus for foraging and a major source of energy for honey bees. Any differential response of bees to different concentrations of sugary nectar can affect their foraging. The sugar responsiveness of Apis species (Apis dorsata, Apis florea, and Apis cerana) was determined in comparison to that of Apis mellifera by evaluating the proboscis extension response (PER) with eight serial concentrations (0.00001, 0.0001, 0.001, 0.01, 0.1, 0.5, 1.0, and 1.5 M) of sucrose, glucose and fructose. Nectar foragers of bee species (A. dorsata, A. florea, A. cerana, and A. mellifera) exhibited an equal response for sucrose, glucose, and fructose, with no significant differences in their PER at all tested concentrations of these sugars within the same species. The inter-species comparison between Apis species revealed the differential responsiveness to the different concentrations of sugars, and the lowest concentration at which a response occurs was considered as the response threshold of these bee species for sugar solutions. A. mellifera presented significantly higher responsiveness than A. dorsata to low concentrations (0.00001, 0.0001, 0.001, 0.01, and 0.1 M) of sucrose, glucose and fructose. A. mellifera displayed a significantly higher response to water than A. dorsata. A. florea and A. mellifera presented no significant difference in their responsiveness to sucrose, glucose, and fructose at all tested concentrations, and their water responsiveness was also significantly at par but relatively higher in A. mellifera than in A. florea. Likewise, the responsiveness of A. cerana and A. mellifera to different concentrations of sucrose, glucose and fructose was significantly at par with no difference in their water responsiveness. This study represents preliminary research comparing the response of different honey bee species to three sugar types at different concentrations. The results imply that the native species are all better adapted than A. mellifera under local climate conditions.     

Properties of a free and a solubilized form of bound alpha,alpha-trehalase purified from honey bee thorax.

The free and bound forms of alpha,alpha-trehalase (EC 3.2.1.28) of the honey bee thorax were separated and the bound enzyme was solubilized by raising the pH to 8.0 for 10 h. Both enzymes were purified. They were homogeneous as determined by several electrophoretic criteria. It was found that the two enzymes had very similar Km's (each about 0.89 mM), Vm's (53.2 and 54.3 U/mg for free and solubilized, respectively), inhibition characteristics, specificities (both only hydrolyzed alpha,alpha-trehalose), pH maxima (each had maxima at about 3.5 and 6.5), molecular weights (65,000), isoelectric points (5.1), reactivities to sulfhydryl reagents, electrophoretic mobilities, activation energies (about 12.8 kcal/mol), and similar stabilities to heat, pH, and urea. Some significant differences between the two enzymes were, however, found: the solubilized alpha,alpha-trehalase floated at 70% saturation of ammonium sulfate while the free alpha,alpha-trehalase did not; the solubilized alpha,alpha-trehalase did not dissociate into subunits as readily as did the free one; and the solubilized alpha,alpha-trehalase was found to bind more readily to a hydrophobic grouping than the free enzyme. In addition to these comparisons, three new findings relating to thorax alpha,alpha-trehalases are reported. (1) Thorax alpha,alpha-trehalases are strongly inhibited by beta-glucosides (Ki values of about 8 x 10(-4) M); (2) under certain conditions thorax alpha,alpha-trehalases from honey bees dissociated into subunits of one-half the normal molecular weight; (3) honey bee thorax alpha,alpha-trehalases have unusual biphasic pH activity profiles.     

Biochemical effects of high intensity constant magnetic fields on worker honey bees

Hemolymph samples from adult bees that had completed their pupal development and emergence in a 7 Tesla field contained a lower percentage of glucose than controls, indicating that trehalase enzyme activity in honey bees is reduced in strong magnetic fields. Significantly more phospholipids were found in the intestines of magnetic field-exposed bees than in controls. No significant differences were found for fatty acids, triacylglycerols, or steroids.     

Honey bee (Hymenoptera: Apidae) foraging in response to preconditioning with onion flower scent compounds

Onion (Allium cepa L.) seed production has long been plagued with yield problems because of lack of pollination by the honey bee, Apis mellifera L. To attempt to attract more pollinators to the onion seed production field, honey bees were conditioned to associate onion floral odor components with a reward. Isolated nucleus hives of honey bees were fed 30% sucrose solutions scented with a 0.2% solution of onion floral odor compounds. After feeding on these solutions for 6 wk, bees were not found to prefer onion flowers to two competing food sources, carrot and alfalfa flowers, at the 5% significance level. However, there was an overall trend indicating a change in honey bee behavior, with fewer "trained" bees visiting alfalfa and carrot and more visiting onion. Thus, it may be possible to alter honey bee behavior with preconditioning but probably not to a degree that would be economically significant.     

Shigella dysenteriae 60R strain adheres to and invades tissue culture cells in the absence of virulence plasmid

The influence of various carbon and nitrogen sources on fusarin C synthesis was examined in submerged cultures of Fusarium moniliforme NRRL 13616. Using a zinc-deficient, synthetic medium, highest levels of fusarin C were produced by cultures grown with urea or ammonium sulfate as the nitrogen source and fructose, sucrose, or glucose as the carbon source. In media supplemented with various concentrations of glucose and ammonium sulfate, glucose concentrations which provided excess carbohydrate significantly increased fusarin C synthesis, regardless of the ammonium sulfate concentration.     

The occurrence and intracellular localization of sucrase activity in rat liver

The presence of an enzyme in rat liver which hydroluzes sucrose is demonstrated in this report. The hydrolysis of sucrose was studied in vivo after injecting [¹⁴C]sucrose into rats, and a method was developed for the extraction and analysis of the radioactive sugars stored within subcellular particles. The results show that, besides sucrose, glucose and fructose are also found in the lysosomal fraction of the liver homogenate. In vitro studies reveal the presence of a sucrase, although the activity of the enzyme is very low. Intracellular distribution studies indicate that sucrase is present in the lysosomes as well as in the microsomes, the microsomal enzyme having a pH optimum different from that of the lysosomal enzyme.     

Characterization of a novel low molecular weight sucrase from filamentous fungus Termitomyces clypeatus

An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%, w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties, molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) medium in the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 °C and pH 5.0. K_m and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity.     

Colour association influences honey bee choice between sucrose concentrations

Certain colours associated with floral food resources are more quickly learned by honey bees (Apis mellifera) than are other colours. But the impact of colour, and other floral cues, on bee choice behaviour has not yet been determined. In these experiments, colour association and sugar concentration of reward were varied to assess how they interact to affect bee choice behaviour. Thirty-five bees were individually given binary choices between blue and yellow artificial flowers that contained either the same rewards or rewards of different sucrose concentrations. Honey bee choice between sucrose concentrations was affected by colour association and this effect was greatest when absolute difference between rewards was the lowest. The honey bee's ability to maximize energetic profitability during foraging is constrained by floral cue effectiveness.