The absorption of protons with alpha-methyl glucoside and alpha-thioethyl glucoside by the yeast N.C.Y.C. 240. Evidence against the phosphorylation hypothesis.

1. When yeast N.C.Y.C. 240 was grown with maltose in a complex medium based on yeast extract and peptone, washed cell preparations fermented alpha-methyl glucoside much more slowly than maltose. 2. The yeast absorbed alpha-methyl[14C]glucoside from a 10mM solution in the presence of antimycin and iodoacetamide, producing [14C]glucose, which accumulated outside the cells. The yeast itself contained hexose phosphates, trehalose, alpha-methyl glucoside and other products labelled with 14C, but no alpha-methyl glucoside phosphate. 3. About 1 equiv. of protons was absorbed with each equivalent of alpha-methylglucoside, and 1 equiv. of K+ ions left the yeast. 4. alpha-Thioethyl glucoside was also absorbed along with protons. Studies by g.l.c. showed that the yeast concentrated the compound without metabolizing it. 5. The presence of trehalose, sucrose, maltose, L-sorbose, glucose or alpha-phenyl glucoside in each case immediately stimulated proton uptake, whereas fructose, 3-O-methylglucose and 2-deoxyglucose failed to do so. 6. The observations support the conclusion that alpha-thioethyl glucoside, alpha-methyl glucoside and maltose are substrates of one or more proton symports, whereas they seem inconsistent with the notion that the absorption of alpha-methyl glucoside involves the phosphorylation of the carbohydrate [Van Stevenick (1970) Biochim. Biophys. Acta 203, 376-384].     

The genetics of melezitose fermentation in Saccharomyces

Summary A multiple series of alleles of the gene MZ exist inSaccharomyces. The members of this series are differentiated from one another by their adaptive response to maltose, turanose, sucrose, melezitose and alpha-methyl glucoside. The more capable members of the series can ferment all five sugars while various multiple alleles are characterized by loss of ability to act on alpha-methyl glucoside, melezitose or sucrose. MZ is linked to the gene MA which controls the production of a specific maltase and to the gene MG which controls the production of a specific alpha-methyl glucosidase. All members of the series of multiple alleles produce the same enzyme showing that the gene and the enzyme are not identical. The enzyme is produced in three different steps, the initial one being a specific reaction of gene with substrate and the final one being a non-specific elicitation of enzyme.This work has been supported by research grants from The National Cancer Institute of the National Institutes of Health, U.S. Public Health Service and Anheuser-Busch, Inc.     

Purification of the glycoprotein allergen Ag7 from mugwort pollen by concanavalin A affinity chromatography.

Two affinity columns comprising immobilized concanavalin A (Con A), Con A-Sepharose and Con A-XP3507, were evaluated for their purifying ability for the glycoprotein allergen Ag7 from a partially purified extract of mugwort pollen. The most pronounced difference between the two columns was the nature of their nonspecific interactions; hydrophobic interactions were dominant with Con A-XP3507, whereas ionic interactions were dominant with Con A-Sepharose. Both Con A-columns were effective for purifying Ag7 with a recovery of 50% after specific elution with displacing sugars. The inclusion of 1.0 M NaCl and 20% ethylene glycol in the elution medium was useful for desorbing nonspecifically bound material, prior to specific elution of adsorbed Ag7 in the presence of the displacing sugars, alpha-methyl glucoside and alpha-methyl mannoside. The most efficient purification of Ag7 was achieved with Con A-Sepharose at room temperature rather than at 4 degrees C. Affinity chromatography with Con A-XP3507 resulted in a slightly more contaminated product (purity 54%) than with Con A-Sepharose (purity 64%).     

Substrate specificity of sugar transport by rabbit SGLT1: single-molecule atomic force microscopy versus transport studies

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/d-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of d-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-d-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-d-glucose >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-methyl glucoside transport assays, where the ranking of inhibition was as follows: d-glucose > d-galactose > 6-deoxy-d-glucose; no uptake inhibition by d-xylose, 3-deoxy-d-glucose, 2-deoxy-d-glucose, or l-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway.     

Evidence for the glycoprotein nature of radish beta-fructosidase.

Concanavalin A (Con A) was utilized free, bound to Sepharose 4 B or cross-linked to glutaraldehyde to investigate the possibility of binding this lectin to radish beta-fructosidase (E.C.3.2.1.26). The choice of cross-linked Con A as affinoadsorbent is discussed and standard conditions for binding are defined. Specificity of precipitation of this enzyme by the lectin was especially investigated. Thus, the possibility of binding was tested in the presence of high ionic strength, ethylene glycol, alpha-methyl mannoside, alpha-methyl glucoside and during periodate oxidation of the enzyme. Based on the interactions observed between beta-fructosidase and Con A under these conditions it is concluded that the saccharide binding site of the lectin is primarily involved with a secondary contribution from the hydrophobic site. The specificity of binding and the complete precipitation of beta-fructosidase activity by the insolubilized lectin imply that all beta-fructosidase activity measured in Raphanus sativus seedling extracts is linked to (a) glycoprotein form(s) of this enzyme.     

Isolation of cells from rabbit renal proximal tubules by using a hyperosmolar intracellular-like solution.

A novel method of isolation of cells from rabbit kidney proximal tubules by using an intracellular-like solution (ICS) and gentle mechanical agitation in the absence of enzymes or chelators is described. Metabolic and functional characteristics of these cells were studied after washing and resuspension in modified Hanks medium, and the results were compared with those obtained in cells similarly prepared in extra-cellular-like solution (ECS). Trypan Blue exclusion and protein content were not different between the two preparations. However, oxygen consumption, ATP content and time- and concentration-dependent rates of uptake of phosphate, alpha-methyl glucoside and L-alanine were severalfold higher in cells prepared in ICS. Na+-dependent uptake of these solutes was 95% and 80% of total uptake in cells prepared in ICS and ECS respectively. Maximum transport rates (Tmax.) of phosphate, alpha-methyl glucoside and L-alanine were significantly higher in cells prepared in ICS. We propose that the use of ICS in the isolation procedure would yield a functionally more viable cell preparation, and therefore provides an ideal model for transport and metabolic studies at a cellular level.     

Studies of the cell surface of Dictyostelium discoideum during differentiation. The binding of 125I-concanavalin A to the cell surface.

125I-concanavalin A (125I-Con A) was found to be equally effective as native Con A in binding to and agglutinating cells of Dictyostelium discoideum, suggesting that iodination of the molecule had no effect on the interaction of the protein with the cell surface. Almost all of the 125I-Con A binding to the cells was inhibited by alpha-methyl glucoside. The binding of 125I-Con A to the cells was extremely rapid, and once bound, the molecule was not readily displaced by prolonged incubation or by the addition of excess native concanavalin A (Con A). In contrast, the 125I-Con A was displaced rapidly from the cell surface by alpha-methyl glucoside. The binding of 125I-Con A to D. discoideum was identical at 22 degrees and 4 degrees, and was unaffected by metabolic inhibitors, suggesting that the protein was not subject to endocytosis. The cell surface Con A binding sites became saturated at high 125I-Con A concentrations. Scatchard plots of the data indicated that growing cells possessed 4 X 10(7) sites/cell, all of equal affinity. Similar plots for "aggregation phase" cells indicated at least two classes of binding sites. A small proportion of the sites had an affinity close to that for the sites on growing cells, but the majority of the sites had a markedly decreased affinity. The total number of binding sites increased only slightly during aggregation to 5.6 X 10(7) sites/cell.     

Specificity of maltase to maltose in three different directions of reaction: Hydrolytic,vanillyl alcohol glucoside and vanillyl alcohol isomaltoside synthesis

Vanillyl alcohol glucoside is very attractive molecule due to its very powerful physiological activity. In this article, a detailed kinetic study of transglucosylation of vanillyl alcohol was performed. It was demonstrated that this reaction is very efficient (selectivity factor is 149) and occurred by a ping‐pong mechanism with inhibition by glucose acceptor. At low concentration of vanillyl alcohol one additional transglucosylation product was detected. Its structure was determined to be α‐isomaltoside of vanillyl alcohol, indicating that vanillyl alcohol glucoside is a product of the first transglucosylation reaction and a substrate for second, so the whole reaction mechanism was proposed. It was demonstrated that the rate of isomaltoside synthesis is two orders of magnitude smaller than glucoside synthesis, and that maltase has interestingly high K_m value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The use of octyl beta-D-glucoside as detergent for hog kidney brush border membrane

Octyl beta-D-glucoside was synthetized from alpha-acetobromoglucose with an improved method yielding a very pure product with a sharp melting point (108-109 degrees C) and free of intermediate products as judged by IR and NMR spectra. The yield of the synthesis is 66% when referred to alpha-acetobromoglucose. The potency of this compound as a detergent on hog kidney brush border membranes was compared to the action of Triton X-100. Octyl glucoside preferentially extracts aminopeptidase M and gamma-glutamyltranspeptidase in a concentration-dependent manner. The more deeply imbedded membrane enzyme, alkaline phosphatase, was relatively resistent to the action of octyl glucoside. In contrast, Triton X-100 extracted all membrane proteins to about the same extent. Additionally it was found that octyl glucoside can be removed from membrane extracts by Biobead SM 2. The capacity of the beads is about 170 mg detergent/g of dry Biobead SM 2. Thus octyl glucoside seems to be a useful tool for solubilization and purification of brush border membranes proteins.     

Pathways of degradation of organic components of waste water of (meth)acrylate-producing factories to methane by communities of microorganisms of adapted and unadapted sludge]

Pathways of the degradation of the main compounds of (meth)acrylate-producing factories wastewater (methyl methacrylate, methyl and butyl acrylate, acrylate and methacrylate, acetone, isopropanol, butanol and methanol) by the anaerobic microbial consortium of mesophilic unadapted granulated sludge from the "UASB" reactor and of adapted activated sludge from the contact reactor were comparatively studied. It was shown that the degradation of fatty acids and alcohols took place in both types of sludge. Methacrylate, acrylate and acetone degradation occurred only in adapted sludge. Both types of sludge were characterized by the reversible conversion of acetone and isopropanol and by the presence of the isomeric transition of butyrate and isobutyrate too. The present results allow to suggest that the adaptation of activated sludge to substrate includes the accumulation of biomass of microorganisms capable of hydrolyze specific substrates into such general intermediates as low-molecular-weight fatty acid and alcohols further metabolized to methane and carbon dioxide.     

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata.

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

The Biosynthesis of Steryl Glucosides in Plants

Mitochondrial preparations from pea root (Pisum sativum L. var. Alaska) cauliflower inflorescence (Brassica cauliflora Gars.) and avocado inner mesocarp (Persea americana Mill. var. Fuerte), and chloroplast preparations from spinach leaf (Spinacia oleracea L. var. Bloomsdale) incorporate glucose into steryl glucoside and acylated steryl glucoside when either uridine diphosphate-glucose or uridine diphosphate-galactose is supplied as precursor. In the case of pea root mitochondria, galactosyl diglycerides are not formed from either nucleotide sugar. In the case of spinach chloroplasts only 3% of the metabolized uridine diphosphate-galactose is found as steryl glycosides. Time course experiments indicate that the steryl glucoside is the precursor of the acylated steryl glucoside. The effect of pH on the over-all reaction and analysis of the reaction products suggest that the glucosylation of the sterol has a pH optimum of 8 to 9, and the pH optimum for the acylation of the steryl glucoside is 6.5 to 7. The synthesis of steryl glucoside and acylated steryl glucoside, catalyzed by acetone powders of pea root mitochondria, is stimuated by added sitosterol and stigmasterol.     

Inducible System for the Utilization of β-Glucosides in Escherichia coli I. Active Transport and Utilization of β-Glucosides

Wild-type Escherichia coli strains (beta-gl(-)) do not split beta-glucosides, but inducible mutants (beta-gl(+)) can be isolated which do so. This inducible system consists of a beta-glucoside permease and an aryl beta-glucoside splitting enzyme. Both can be induced by aryl and alkyl beta-glucosides. In beta-gl(-) and noninduced beta-gl(+) cells, C(14)-labeled thioethyl beta-glucoside (TEG) is taken up by a constitutive permease, apparently identical with a glucose permease (GP). This permease has a high affinity for alpha-methyl glucoside and a low affinity for aryl beta-glucosides. No accumulation of TEG occurs in a beta-gl(-) strain lacking glucose permease (GP(-)). In induced beta-gl(+) strains, there appears a second beta-glucoside permease with low affinity for alpha-methyl glucoside and high affinity for aryl beta-glucosides. Autoradiography shows that TEG is accumulated by the beta-glucoside permease and glucose permease in two different forms (one being identical with TEG, the other probably phosphorylated TEG). In GP(+) beta-gl(+) strains with high GP activity, alkyl beta-glucosides induce the enzyme and the beta-glucoside permease after a prolonged induction lag, and they competitively inhibit the induction by aryl beta-glucosides. The induction lag and competition do not exist in GP(-) beta-gl(+) strains. It is assumed that phosphorylated alkyl and thioalkyl beta-glucosides inhibit the induction, and that this inhibition is responsible for the induction lag.     

Optimization of production and downstream processing of the almond beta-glucosidase-mediated glucosylation of glycerol.

This article describes the synthesis of glyceryl glucoside from glycerol and glucose with almond beta-glucosidase as the catalyst. A yield of 54% (0.45 mmol/g) was obtained. The influence of the enzyme stability, the water concentration, and the water activity on the glucoside yield were determined. A molar fraction-based equilibrium constant of 2.4 +/- 0.6 was found, with which the glucoside yield could be calculated for all possible combinations of initial substrate and water fractions in the reaction mixture. A model was used to optimize the glucoside yield while minimizing one of the substrate concentrations at equilibrium. This straightforward model gives a good prediction of the measured glucoside yield, according to a parity plot.     

Production of alkyl glucoside from cellooligosaccharides using yeast strains displaying Aspergillus aculeatus β-glucosidase 1

For efficient alkyl glucoside production from cellooligosaccharides, we constructed a yeast strain for alkyl glucoside synthesis by genetically inducing the display of β-glucosidase 1 (BGL1) from the filamentous fungus Aspergillus aculeatus No. F-50 on the cell surface. The localization of BGL1 on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying BGL1 catalyzed alkyl glucoside synthesis from p-nitrophenyl β-d-glucoside and primary alcohols. The highest yield of alkyl glucoside was 27.3% of the total sugar. The substrate specificities of the BGL1-displaying yeast strain and almond β-glucosidase were compared using different-chain-length cellooligosaccharides. The BGL1-displaying yeast showed efficient alkyl glucoside production from not only glucose but also cellohexaose. This yeast is applicable as a whole-cell biocatalyst for alkyl glucoside production from cellulose hydrolysates.     

Enzymatic synthesis of nuatigenin 3β-D-glucoside in oat (Avena sativa) leaves

Crude homogenates or acetone powder preparations from oat leaves efficiently catalyse the glucosylation of a steroidal sapogenin, nuatigenin [22,25-epoxy-(20S)(22S)(25S)-furost-5-en-3β,26-diol], using UDP-glucose as the sugar donor. The reaction product was identified as nuatigenin 3β-D-monoglucoside. In contrast to the glucosylation of phytosterols, which is also catalysed by enzyme preparations from oat leaves, the formation of nuatigenin glucoside is not stimulated by Triton X-100. This result suggests that glucosyltransferases with different specifirity patterns are involved in sterol and nuatigenin glucosylation in oat leaves. Enzymatic acylation of nuatigenin glucoside to its monoacyl derivative with the use of an endogenous acyl source was also observed with a crude homogenate or a crude membranous fraction as the enzyme preparation.     

Regulation of superoxide dismutase synthesis in Escherichia coli: glucose effect.

Growth of Escherichia coli, based upon the fermentation of glucose, is associated with a low intracellular level of superoxide dismutase. Exhaustion of glucose, or depression of the pH due to accumulation of organic acids, causes these organisms to then obtain energy from the oxidative degradation of other substances present in a rich medium. This shift in metabolism is associated with a marked increase in the rate of synthesis of superoxide dismutase. Depression of the synthesis of superoxide dismutase by glucose is not due to catabolite repression since it is not eliminated by cyclic adenosine 3',5'-monophosphate and since alpha-methyl glucoside does not mimic the effect of glucose. Moreover, glucose itself no longer depresses superoxide dismutase synthesis when the pH has fallen low enough to cause a shift to a non-fermentative metabolism. It appears likely that superoxide dismutase is controlled directly or indirectly by the intracellular level of O2- and that glucose depressed the level of this enzyme because glucose metabolism is not associated with as rapid a production of O2- as is the metabolsim of many other substances. In accord with this view is the observation that paraquat, which can increase the rate of production of O2- by redox cycling, caused a rapid and marked increase in superoxide dismutase.     

Downstream processing of enzymatically produced geranyl glucoside

Geraniol plays an important role in the fragrance and flavor industry. The corresponding glucoside has interesting properties as a "slow release" aroma compound. Therefore, the enzymatic production and downstream processing of geranyl glucoside were investigated. Geranyl glucoside was produced in a spray column reactor with an initial production rate of 0.58 mg x U(-1) x h(-1). A pretreated hydrophobic microfiltration membrane was used to prevent migration of the aqueous, enzyme-containing phase to the downstream process. No retention of the glucoside, which accumulated in the geraniol phase, was found. On the basis of examples from the literature, four downstream processes were tested on their viability for this system. Extraction with water and foaming were not suitable to recover geranyl glucoside from geraniol. In the first case, the glucoside selectivity for the geraniol phase was found to be high, which made extraction with water unsuccessful. In the second case it was possible to obtain a stable foam, but significant enrichment of the foam with glucoside did not occur. Adsorption on alumina and distillation under reduced pressure were applied successfully and tested in-line with the bioreactor. A maximum glucoside adsorption of 7.86 mg x g(-1) was achieved on alumina. After desorption and evaporation of the extractant the pure glucoside was obtained quantitatively. A pure product could not be obtained after distillation because a small amount of glucose was present in the permeate as well, which accumulated in the bottom fraction. It was shown that with this reactor system a production of 1 kg of geranyl glucoside in 2 days is possible using an initial amount of 50,000 units of enzyme.