- Comparative study of the efficacies of nine assay methods for the dextransucrase synthesis of dextran
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A comparative study of nine assay methods for dextransucrase and related enzymes has been made. A relatively widespread method for the reaction of dextransucrase with sucrose is the measurement of the reducing value of D-fructose by alkaline 3,5-dinitrosalicylate (DNS) and thereby the amount of D-glucose incorporated into dextran. Another method is the reaction with 14C-sucrose with the addition of an aliquot to Whatman 3MM paper squares that are washed three times with methanol to remove 14C-D- fructose and unreacted 14C-sucrose, followed by counting of 14C-dextran on the paper by liquid scintillation counting (LSC). It is shown that both methods give erroneous results. The DNS reducing value method gives extremely high values due to over-oxidation of both D-fructose and dextran, and the 14C-paper square method gives significantly low values due to the removal of some of the 14C-dextran from the paper by methanol washes. In the present study, we have examined nine methods and find two that give values that are identical and are an accurate measurement of the dextransucrase reaction. They are (1) a 14C-sucrose/dextransucrase digest in which dextran is precipitated three times with three volumes of ethanol, dissolved in water, and added to paper and counted in a toluene cocktail by LSC; and (2) precipitation of dextran three times with three volumes of ethanol from a sucrose/dextransucrase digest, dried, and weighed. Four reducing value methods were examined to measure the amount of D-fructose. Three of the four (two DNS methods, one with both dextran and D-fructose and the other with only D-fructose, and the ferricyanide/arsenomolybdate method with D-fructose) gave extremely high values due to over-oxidation of D-fructose, D-glucose, leucrose, and dextran.
- Vettori, Mary Helen P.B.,Mukerjea, Rupendra,Robyt, John F.
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- Entrapment of purified novel dextransucrase obtained from newly isolated Acetobacter tropicalis and its comparative study of kinetic parameters with free enzyme
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Purified Acetobacter tropicalis dextransucrase was immobilized in different matrices viz. calcium-alginate, κ-carrageenan, agar, agarose and polyacrylamide. Calcium-alginate was proved to be superior to the other matrices for immobilization of dextransucrase enzyme. Standardization of immobilization conditions in calcium-alginate resulted in 99.5% relative activity of dextransucrase. This is the first report with such a large amount of relative activity as compared to the previous reports. The immobilized enzyme retained activity for 11 batch reactions without a decrease in activity which suggested that enzyme can be used repetitively for 11 cycles. The dextransucrase was also characterized, which revealed that enzyme worked best at pH 5.5 and 37 °C for 30 min in both the free as well as immobilized state. Calcium-alginate immobilized dextransucrase of A. tropicalis showed the Km and Vmax values of 29 mM and 5000 U/mg, respectively. Free and immobilized enzyme produced 5.7 mg/mL and 2.6 mg/mL of dextran in 2 L bench scale fermenter under optimum reaction conditions. This immobilization method is very unconventional for purified large molecular weight dextran-free dextransucrase of A. tropicalis as this method is used usually for cells. Such reports on entrapment of purified enzyme are rarely documented.
- Nisha,Azmi, Wamik
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- Bioengineering of Leuconostoc mesenteroides glucansucrases that gives selected bond formation for glucan synthesis and/or acceptor-product synthesis
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The variations in glucosidic linkage specificity observed in products of different glucansucrases appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. Various amino acid mutations near active sites of DSRBCB4 dextransucrase from Leuconostoc mesenteroides B-1299CB4 were constructed. A triple amino acid mutation (S642N/E643N/V644S) immediately next to the catalytic D641 (putative transition state stabilizing residue) converted DSRBCB4 enzyme from the synthesis of mainly α-(1→6) dextran to the synthesis of α-(1→6) glucan containing branches of α-(1→3) and α-(1→4) glucosidic linkages. The subsequent introduction of mutation V532P/V535I, located next to the catalytic D530 (nucleophile), resulted in the synthesis of an α-glucan containing increased branched α-(1→4) glucosidic linkages (approximately 11%). The results indicate that mutagenesis can guide glucansucrase toward the synthesis of various oligosaccharides or novel polysaccharides with completely altered linkages without compromising high transglycosylation activity and efficiency.
- Kang, Hee Kyoung,Kimura, Atsuo,Kim, Doman
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experimental part
p. 4148 - 4155
(2011/10/30)
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- STILBENE-BASED COMPOSITIONS AND METHODS OF USE THEREFOR
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Disclosed are compositions, formulations and methods relating to one or more stilbene-based compounds for use in humans. In particular, compositions and formulations comprising an effective amount of the stilbene-based insulinogenic compound can improve athletic performance, lower blood glucose levels, and increase lean muscle mass when administered (e.g., orally) to a human.
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- COMPOSITIONS COMPRISING AMINO ACID BICARBONATE AND METHODS OF USE THEREOF
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The invention relates to compositions comprising one or more ionic salts, each of said ionic salts consisting of a bicarbonate anion and a cation selected from the group consisting of an amino acid, an amino acid derivative, a di-peptide and a tri-peptide, and to methods of making and using said compositions.
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- NOVEL FORMULATION OF DEHYDRATED LIPID VESICLES FOR CONTROLLED RELEASE OF ACTIVE PHARMACEUTICAL INGREDIENT VIA INHALATION
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A new formulation of dehydrated lipid vesicles employs a vesicle preserver and permits the control of release and delivery of active pharmaceutical ingredients into the respiratory system for treatment in particular of asthma. The typical formulation provides controlled release of the active pharmaceutical ingredient from 0% to 100% from 0 to 72 hours after inhalation, changes the systemic administration to topical administration, allows prolonged therapeutic period for one administration, increased stability, with reduced dose, reduced systemic side effects, reduced toxicity.
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- Dextrans - Potential polymeric drug carriers for flurbiprofen
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Dextrans have been used as carrier for flurbiprofen. Conjugates of flurbiprofen were synthesized by preparing their acylimidazol derivatives which were condensed in situ with dextrans of different molecular weight (40 000, 60 000, 110 000 and 200 000). The structures of the synthesized conjugates were confirmed by IR and NMR spectroscopy. The degrees of substitution were obtained between 8.0 to 9.5% and molecular weight was determined by Mark-Howin Sakurada viscosity equation. A hydrolysis study was performed in different buffer solutions (pH 1.2, 7.4, 9.0) and 80% human plasma (pH 7.4). The hydrolysis followed first order kinetics. Much faster hydrolysis was observed at pH 9.0 compared to buffer solution pH 7.4 and 80% human plasma (pH 7.4). The biological evaluation for acute and chronic anti-inflammatory activity was performed and the results were found to be comparable with the parent drug. The conjugates showed remarkable reduction in ulcerogenicity compared to parent flurbiprofen.
- Shrivastava,Jain,Trivedi
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p. 389 - 391
(2007/10/03)
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- Mechanism of the action of Leuconostoc mesenteroides B-512FMC dextransucrase: Kinetics of the transfer of D-glucose to maltose and the effects of enzyme and substrate concentrations
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The kinetics of the reaction of Leuconostoc mesenteroides B-512FMC dextransucrase with sucrose were studied. This enzyme catalyzes the synthesis of dextran from sucrose with a k(cat) of 641 s-1 and the transfer of D-glucose from sucrose to maltose with a k(cat) of 1070 s-1. The enzyme was also found to catalyze two new reactions in the absence of sucrose, using dextran as the substrate; D-glucose was transferred from the non-reducing ends of dextran chains to maltose with a relatively low k(cat) of 3.2 s-1; and D-glucose was hydrolyzed from the non-reducing ends of dextran chains with a very low k(cat) of 0.085 s-1. Ping-pong/bi-bi kinetics of these reactions are consistent with the formation of a glucosyl-enzyme covalent intermediate. It is shown that an increase in the concentrations of both maltose and sucrose in the D-glucose transfer reaction to maltose gives an exponential decrease in the amount of dextran and a concomitant increase in the amount of acceptor products. It is further shown that increasing the amount of dextransucrase gives a decrease in the amount of dextran and an increase in the amount of acceptor products, after the sucrose has been consumed. This anomaly occurs because the relatively high amounts of enzyme catalyze the transfer of D-glucose from the non-reducing ends of the dextran chains to maltose, giving a decrease in the amount of dextran and an increase in the amount of acceptor product. Further, the high amounts of enzyme catalyze the hydrolysis of the D-glucose residues from the ends of the dextran chains, giving a decrease in the amount of dextran. These reactions are not observed when lower amounts of enzyme are used, as the reactions are much slower than the synthesis of dextran and the usual acceptor transfer reactions of D-glucose from sucrose to acceptor. Copyright (C) 1999 Elsevier Science Ltd.
- Kitaoka, Motomitsu,Robyt, John F.
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p. 183 - 191
(2007/10/03)
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- Macromolecular microparticles and methods of production and use
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Microparticles formed by mixing a macromolecule with a polymer at a pH near the isoelectric point of the macromolecule and incubating the mixture in the presence of an energy source for a predetermined length of time. The microparticles are composed of homogeneously distributed, intertwined macromolecule and polymer. Each microparticle allows aqueous fluids to enter and allows solubilized macromolecule and polymer to exit the microparticle and may be formulated to provide a sustained release of macromolecule and polymer from the interior of the microparticle when placed in an appropriate aqueous medium, such as under physiological conditions. Methods of production and methods of use for research, diagnostics and therapeutics are provided.
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- Macromolecular microparticles and methods of production and use
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Microparticles formed by mixing a macromolecule with a polymer at a pH near the isoelectric point of the macromolecule and incubating the mixture in the presence of an energy source for a predetermined length of time. The microparticles are composed of homogeneously distributed, intertwined macromolecule and polymer. Each microparticle allows aqueous fluids to enter and allows solubilized macromolecule and polymer to exit the microparticle and may be formulated to provide a sustained release of macromolecule and polymer from the interior of the microparticle when placed in an appropriate aqueous medium, such as under physiological conditions. Methods of production and methods of use for research, diagnostics and therapeutics are provided.
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- Method of preserving ophthalmic solutions and compositions therefor
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A preservative for ophthalmic solutions having an active ingredient is provided, having a hydrogen peroxide content of about 0.001% to about 0.10% by weight; and diethylene triamine penta(methylene phosphonic acid) or a physiologically compatible salt thereof, present at about 0.002% to 0.03% by weight and/or 0.005% to about 0.20% by weight of 1-hydroxyethylidene-1,1-diphosphonic acid, or physiologically acceptable salt thereof.
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- Method for apparatus for a defined serumfree medical solution useful for corneal preservation
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A defined serumfree medical solution for applications in Ophthalmology, that contains one or more cell nutrient supplements which maintains and enhances the preservation of eye tissues, including human corneal tissues at low temperatures (2° C. to 15° C.). This solution is composed of a defined aqueous nutrient and electrolyte solution, supplemented with a glycosaminoglycan(s), a deturgescent agent(s), an energy source(s), a buffer system(s), an antioxidant(s), membrane stabilizing components, antibiotic(s), ATP precursors and nutrient cell supplements.
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- Method and apparatus of a serumfree medical solution
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A serumfree medical solution for applications in Ophthalmology, that contains one or more growth factors which maintains and enhances the preservation of eye tissues, including human corneal tissues at low temperatures (2°C to 15°C). This solution is composed of a an aqueous nutrient and electrolyte solution, supplemented with a glycosaminoglycan(s), a deturgescent agent(s), a buffer system(s), an energy source(s), an antioxidant(s), membrane stabilizing components, and containing one or more growth factors.
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