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La [bêta] (1,6)-glucanase de Streptomyces sp. EF-14, un actinomycète antagoniste a Phytophthora spp. production, purification et caractérisation /Fayad, Karine-Paule. January 1997 (has links)
Thèses (M.Sc.)--Université de Sherbrooke (Canada), 1997. / Titre de l'écran-titre (visionné le 26 juin 2008). Publié aussi en version papier.
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Recherches physico-chimiques sur l'amylase et la maltase /Philoche, Ch., January 1908 (has links)
Thèse de doctorat--Sciences naturelles--Faculté des sciences de Paris, 1908. N°: 1285. / Extrait du Journal de Chimie-Physique, Tome VI, 1908. Notes bibliogr.
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Control of a-glucosidase in Bacillus subtilis during growth and sporulationHansen, Marc Frederick. January 1983 (has links)
Thesis (M.S.)--University of Wisconsin--Madison, 1983. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 90-97).
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A mechanistic investigation of Agrobacterium [beta]-glucosidaseKempton, Julie B. January 1990 (has links)
The mechanism of glucoside hydrolysis by Agrobacterium β-glucosidase has been investigated through the study of linear free energy relationships and a-secondary deuterium kinetic isotope effects. A two-step mechanism has previously been proposed for this process, consisting of;
(1) cleavage of the glucosidic bond and formation of a covalent glucosyl-enzyme
intermediate ("glucosylation"), and
(2) hydrolysis of the intermediate to yield free enzyme and glucose
("deglucosylation").
Values of kcat and Km were determined for enzymic hydrolysis of fifteen substituted phenyl β-D-glucopyranosides with leaving group pKa's ranging from 3.96 to 10.34. A linear free energy correlation of log(kcat) vs. leaving group pKa resulted in a concave-downward plot with a break near pKa 8, indicating a change in rate-determining step of a multistep reaction. The rates of hydrolysis of substrates with leaving group pKa's < 8 are independent of phenol structure, indicating that deglucosylation is rate-limiting. Glucosides with leaving group pKa's > 8 exhibit a linear dependence of hydrolysis rate upon pKa, and thus it is proposed that glucosylation is the rate-determining step for these substrates. The value of the Hammett reaction constant, ρ, is 1.6, indicating that cleavage of the glucosidic bond is significantly advanced at the transition state.
The α-secondary deuterium kinetic isotope effects on hydrolysis of five substituted phenyl β-D-glucopyranosides were determined (deuterium substitution at the anomeric center), and the values were found to be segregated into two groups. The faster substrates (leaving group pKa < 8) exhibited kH/kD values of approximately 1.11, while values for the slower substrates averaged 1.06. These results support the hypothesis of a change in rate-determining step as leaving group pKa decreases. The magnitude of the isotope effect on glucosylation indicates a small amount of sp³ to sp² rehybridization at the transitionstate, which combined with the ρ value for this process suggests a substantial degree of nucleophilic participation of the enzymic carboxylate.
2-Deoxy-2-fluoro-D-glucosides with highly activated leaving groups are potent covalent inactivators of Agrobacterium β-glucosidase which operate by trapping the
enzyme as its glucosyl-enzyme intermediate. Values of ki and Ki were determined for six substituted phenyl 2-deoxy-2-fluoro-β-D-glucopyranosides whose leaving group pKa's
ranged from 3.96 to 7.18. A linear correlation was observed for both log(ki) and log(ki/Ki) vs. leaving group pKa, with ρ values of 2.0 and 2.7, respectively, which
indicates that cleavage of the glycosidic bond is virtually complete at the transition state. Such an observation of a linear free energy relationship between the rate of enzyme inactivation and the electronic structure of the inactivator is rarely accomplished in enzymology. Preliminary investigation of the α-secondary deuterium kinetic isotope effect on enzyme inactivation by 2',4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside indicates that the effect is quite small, probably 0-5%. These results suggest that the inactivation proceeds via an essentially concerted mechanism and that the transition state has little oxocarbonium ion character. / Science, Faculty of / Chemistry, Department of / Graduate
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Metabolic control of [beta]-glucosidase synthesis in yeastMacQuillan, Anthony Mullens, January 1962 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. Includes 2 reprints of articles from Biochemical and biophysical research communications: The control of enzyme synthesis by glucose and the repressor hypothesis / A.M. MacQuillan, S. Winderman and H.O. Halvorson. Vol. 2, no. 6 (June 1960), p. 77-80 -- The effect of glucose repression on the level of ribosomal-bound [beta]-glucosidase / J.G. Hauge, A.M. MacQuillan, A.L. Cline and H.O. Halvorson. Vol. 5, no. 4 (1961), p. 267-269. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 136-148).
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The properties and induction of a yeast [beta]-glucosidaseDuerksen, Jacob Dietrich, January 1958 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 92-99).
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Genetic control of [beta]-glucosidase synthesis in yeastHerman, Alberta Irene, January 1963 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 135-143).
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Purification and preliminary characterisation of β-glucosidase from Alcaligenes faecalis (ATCC 21400)Day, Anthony George January 1985 (has links)
A β-glucosidase was isolated from A. faecalis and purified 880 fold by a combination of classical and medium pressure chromatographic techniques to a specific activity of 31.6 units/mg. The protein was homogeneous
by the criteria of SDS-PAGE and gel chromatography.
The sub-unit molecular weight was determined to be 51,000 by SDS-PAGE. The apparent oligomeric molecular weight was determined to be 75,000 by Superose gel chromatography and 98,000 by Waters 1-250 gel chromatography, suggesting that the enzyme is a dimer.
The enzyme was shown to be a retaining β-glucosidase with exo-glucanase activity only.
The kinetic parameters of a number of substrates and inhibitors were determined allowing deductions to be made about the nature of the active site and catalytic mechanism. The Km's determined for cellobiose and PNPG were low for a bacterial β-glucosidase, 0.70 mM and 0.083 mM respectively. In the cellodextrin series, cellobiose to cellopentaose, the enzyme was most efficient (as defined by Vm/Km) with cellotriose as substrate.
In common with other cellulolytic β-glucosidases, the glycone site showed a high specificity for glucose (although it would tolerate some modifications) and poor specificity at the aglycone site. Catalytic activity
was (unusually) observed with p-nitrophenyl-β-D-mannopyranoside as substrate.
Activation energies were determined by means of Arrhenius plots. / Science, Faculty of / Chemistry, Department of / Graduate
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Synthetic studies towards acarbose: syntheses of N,O,O,O,O-pentaacetylvalienamine and N,O,O,O,O-pentaacety1-2-epi-valienamine.January 1996 (has links)
by Li Tin Yau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 56-60). / Acknowledgment --- p.i / Table of Contents --- p.ii / Abstract --- p.iii / Abbreviation --- p.iv / Chapter I --- Introduction --- p.1 / Chapter I-1 --- General Background --- p.1 / Chapter I-2 --- Mechanistic Aspect of Glycosidase Inhibition --- p.4 / Chapter I-3 --- Previous Syntheses of Valienamine and its Diastereomers --- p.7 / Chapter II --- Results and Discussion --- p.16 / Chapter II-1 --- General Strategy --- p.16 / Chapter II-2 --- Synthesis of Tribenzyl ether69 --- p.18 / Chapter II-3 --- Synthesis of Diol67 --- p.20 / Chapter II-4 --- "Synthesis of N,O,O, O, O-Pentaacetylvalienamine30 and its 2-Epimer62" --- p.22 / Chapter II-5 --- Synthesis of Diacetate66 --- p.26 / Chapter II-6 --- Synthesis of the Valienamine Derivatives 63 and64 --- p.30 / Chapter III --- Conclusion --- p.34 / Chapter IV --- Experimental --- p.36 / Chapter V --- Reference --- p.56
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Glucocerebrosidase expression and analysisCampbell, Tessa Nicole. 10 April 2008 (has links)
Gaucher disease, an autosomal recessive disorder, is characterized by a heterogeneous set of signs and symptoms caused by a deficiency in the lysosomal enzyme glucocerebrosidase. As a single gene enzyme deficiency, Gaucher disease is a prime candidate for enzyme replacement therapy. Such therapy exists, though the exorbitant cost prevents many fiom receivii treatment. Thus, a more cost-effective method of producing glucocerebrosidase was examined. The Pichiapastoris yeast system was chosen, but resultant production levels were low. Two variants of green fluorescent protein (GFP), red-shifted GFP (RSGFP) and enhanced GFP (EGFP), were employed as molecular reporters to track enzyme production and isolation. No expression of glucocerebrosidase was evident, indicating that the P. pastoris system was not an appropriate choice for glucocerebrosidase production. Both GFP variants were successfully expressed, with EGFP levels apparently greater than RSGFP levels. To study glucocerebrosidase production and trafficking in a higher eukaryotic system, EGFP-tagged glucocerebrosidase constructs were expressed in HeLa cells. Though EGFP was readily visualized, few cells expressing glucocerebrosidase constructs were present. No co-localization with organelle markers was evident. Examination at the RNA level indicated successfid transcription, however, an apparent translational inefficiency was encountered. To shed light on the possible cause of this inefficiency, two approaches were taken: one examined expression of truncated glucocerebrosidase constructs in HeLa cells, the other included co-transfection with small interfering RNAs (siRNAs) in both HeLa and COS- 1 cells. In the first approach, greater expression was seen itom the EGFPtagged construct devoid of the proposed inhibitory binding site than itom the EGFPtagged construct containing the binding site. Expression of both truncated constructs was greater than that of EGFP-tagged glucocerebrosidase starting at either initiation codon, indicating a more complex mechanism of translational control than strictly inhibition fiom the proposed site. In the second approach, a siRNA was designed to block TCP80, which has been suggested to inhibit glucocerebrosidase translation. Co-transfection studies of siJ3NAs (control, EGFP and TCP80) and glucocerebrosidasel EGFP plasmids were performed in HeLa and COS-1 cells. In both cell types, all constructs were successfblly expressed when co-transfected with control siRNA, as indicated by RNA and protein examination. Introduction of TCP80 siRNA in both cell types did not serve to increase glucocerebrosidase expression as expected, but instead decreased such expression. EGFP expression was readily knocked down in HeLa and COS-1 cells by GF'P-targeted siRNk Knockdown was evident in the expression of glucocerebrosidase/EGFP constructs, indicating that hsion with EGFP may serve as a means to introduce a foreign gene, then knock its expression down at a desired time by introduction of a GFP-targeted siRNA.
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