Dissertationen zum Thema „Glycosides Analysis“

Human angiotensin-converting enzyme (ACE) is a key enzyme in the regulation of blood pressure via the renin-angiotensin and kallikrein-kinin systems. A number of orally active drugs have been developed over the years that target somatic ACE, for the treatment of hypertension, myocardial infarction and congestive heart failure. Protein structural information about ACE is an important key for the understanding of the mechanism and substrate-specificity of the enzyme. However, this information has only begun to be elucidated in the past year, with the solution of crystal structures of human testis ACE (tACE), and homologues Drosophila AnCE and human ACE2. tACE is identical to the C-terminal domain of somatic ACE, which consists of two homologous domains, each having a slightly different substrate-specificity. This thesis describes the purification, crystallisation and X-ray crystal structure-determination of a glycosylation-deficient mutant of tACE, tACEG1,3, to 2.9 Å
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Includes a list of publications co-authored by the author during the preparation of this thesis. Bibliography: leaves 103-111. Studies the links between grape composition, wine composition and wine sensory properties. Developed a new method of measuring glycoside concentration in grapes (glycosyl-glucose assay) and a modified measurement of wine colour density in red wine. These were used to analyse samples of Shiraz grapes and wines from a comprehensive vineyard irrigation trial. Glycosyl-glucose concentrations shows promise for the prediction of wine composition and flavor intensity.

The aim of this study has been to explore the site specific phosphorylation pattern of rat heart glycogen synthase paying particular attention to phosphorylations that are important to the in vivo control of enzyme activity. This problem has been approached using techniques of immuneprecipitation of 32P labeled synthase from hormonally responsive, freshly isolated adult rat cardiomyocytes. Identification of the active subunit of rat heart glycogen synthase was accomplished by immuneprecipitating synthase from 32P-labeled cardiomyocytes and performing Western blot analysis on DEAE-cellulose fractions containing synthase activity. Using these methods, glycogen synthase activity has been localized to a protein of 88,000 daltons. Reverse phase HPLC analysis of synthase tryptic peptides from either hormone responsive cardiomyocytes or synthase treated in vitro with cAMP-dependent protein kinase and protein phosphatase-1 (PP-1) resulted in finding six reproducible peaks of phosphopeptides. The incorporation of radioactivity into peaks 1 and 2 was associated with both the treatment of cardiomyocytes with epinephrine and the in vitro phosphorylation of rat heart synthase with cAMP-dependent protein kinase. These same two peaks are selectively dephosphorylated when cAMP-dependent kinase treated synthase is incubated with protein phosphatase-1. This dephosphorylation of peaks 1 and 2 are coincident with the conversion of synthase from the D to the I form. Peak 3 is dephosphorylated upon treatment of cardiomyocytes with insulin and hyperphosphorylated in cardiomyocytes derived from alloxan diabetic animals. Taken together these results demonstrates the direct relationship between the phosphopeptides in peaks 1 and 2 and the inhibition of synthase activity in response to epinephrine treatment in the cell. This inhibition can be explained by the activity of cAMP-dependent protein kinase which can duplicate the intracellular, epinephrine-stimulated synthase phosphopeptide pattern. This inhibition can be relieved in vitro by protein phosphatase-1 which dephosphorylates peaks 1 and 2. The effect of insulin and alloxan diabetes is localized to peak 3 whose phosphorylation is unaffected in vitro by either cAMP-dependent protein kinase or protein phosphatase-1.

L’objectif de ce travail est de contribuer à l'amélioration des connaissances phytochimiques et biologiques des plantes medicinales, afin d’en valoriser et d’en promouvoir l'usage en médicine traditionnelle au Vietnam. Dans ce travail de thèse, nous avons mené une étude phytochimique sur trois espèces végétales: Cleome chelidonii (Cleomaceae), Dolichandrone spathacea (Bignoniaceae) et Flacourtia rukam (Salicaceae). 90 composés ont été isolés et leurs structures ont été déterminées à l’aide des techniques spectroscopiques de RMN 1D et 2D, par la spectrométrie de masse ESI-MS, des données spectrales en UV, IR, la mesure du pouvoir rotatoire et des courbes de CD, et par comparaison avec les données de la littérature. Parmi ces composés, 29 correspondent à des molécules nouvellement décrites. Les composés isolés peuvent être classés en plusieurs groupes : flavonoïdes, iridoides, saponosides, mégastigmanes, glycosides phénoliques, alcaloïdes, …L’évaluation de l’activité anti-radicalaire (test DPPH) a été effectuée sur les flavonoides de C. chelidonii, et les activités antimicrobiennes des extraits et composés de D. spathacea et F. rukam ont été mesurées. Parmi les composés testés, le glycoside de quercétol nouveau CF-3 est le seul à posséder une activité anti-radicalaire importante (CI50 = 17,74 µM) et le glucoside phénolique nouveau FRP-4 possède l’activité antibactérienne la plus importante contre trois bactéries à Gram positif (CMI = 31,2 µg/ml) et deux bactéries à Gram négatif (CMI = 125 µg/ml)
The objective of this work is to contribute to the improvement of phytochemical and biological knowledge of medicinal plants, in order to enhance and promote their uses in traditional medicine in Vietnam. In this thesis, we carried out a phytochemical study on three plants: Cleome chelidonii (Cleomaceae) Dolichandrone spathacea (Bignoniaceae) and Flacourtia rukam (Salicaceae). 90 compounds were isolated and their structures were determinated using the spectroscopic techniques of 1D & 2D NMR and by the ESI-MS mass spectrometry, spectral data UV, IR, measurement of optical rotation and CD, and by comparison with the literature data. Among them, 29 are new molecules. The isolated compounds may be classified into many groups: flavonoids, iridoids, saponins, megastigmanes, phenolic glycosides, alkaloids...The antiradical activity of the flavonoids of C. Chelidonii was evaluated by the DPPH test, and the antimicrobial activity were examinated on all extracts and compounds of D. spathacea and F. rukam. Among the tested compounds, the new flavonoid CF-3 has a significant anti-radical activity (IC50 = 17.74 µM) and the new phenolic glucoside FRP-4 has the most significant antibacterial activity against three Gram-positive bacteria (MIC = 31.2 µg / ml) and two gram-negative bacteria (MIC = 125 µg /ml)

The Compounds 2-deoxy-2-fluoro-β-D-glucosyl fluoride (1), 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2), 2-deoxy-2-fluoro-β-D-galactosyl fluoride (3) and 2-deoxy-2-fluoro-β-D-mannosyl fluoride (4) were all found to be potent covalent inhibitors of β-glucosidase from Alcaligenes faecalis (pABG5 β-glucosidase), which function through the accumulation of a stable glycosyl-enzyme intermediate. The mechanism of action for these inhibitors was investigated and found to be both specific and active site directed, involving a 1:1 stoichiometric formation of an enzyme inhibitor complex. Investigation of the pre-steady state kinetics for the inhibition reaction provided values for the rates of formation (kon) and hydrolysis (koff) of the glycosylated-enzyme intermediate. The values of kon determined in this manner are 5.9, 25, 3.6 and 5.6 min.⁻¹ for compounds 1,2,3 and 4 respectively, while the values of koff were found to be much smaller; 1.5x10⁻⁵, 5.4 x 10⁻³ and 1.0 x 10⁻³ min.⁻¹, respectively for the intermediates formed from compounds 1, 3 and 4. The isolated intermediates were also found to be competent in the catalysis of transglycosylation reactions and evidence for the participation of a specific binding site for the acceptor compound in this process was gained. The results of these kinetic experiments were corroborated by data obtained from ¹⁹F-NMR spectra of the glycosyl-enzyme intermediate and by isolation and subsequent characterization of the transglycosylation products. The stereochemistry of the inhibition reaction was investigated by a simple ¹⁹F-NMR experiment, and was found to be consistent with a double displacement reaction mechanism, as would be expected from the generally accepted reaction mechanism for this type of enzyme. The compound D-glucal was found to be a substrate for pABG5 β-glucosidase (kcat = 2.28 min.⁻¹ Km = 0.85 mM). Hydration of this compound by pABG5 β-glucosidase in deuterated buffers demonstrated that the double bond of D-glucal was deuterated stereo-specifically from below the α-face. The compound 2-fluoro-D-glucal was found to be a weak competitive inhibitor (Ki = 30 mM) of pABG5 β-glucosidase. Glycogen phosphorylase catalyses the reversible phosphorolysis of glycogen. A series of deoxy analogues of the enzymes natural substrate, α-D-glucose 1-phosphate have been prepared and along with a number of deoxyfluoro analogues tested as substrates. All were found to act as substrates but at exceedingly slow rates. The large rate reductions when compared with the normal substrate can be attributed to a deleterious combination of electronic and binding effects in the modified substrates reducing the stability of the enzymic transition states. A linear free energy relationship between kcat and the first order rate constant for the acid catalysed hydrolysis of the same series of deoxy and deoxyfluoro glucopyranosyl phosphates was demonstrated, suggesting similar transition states for the two reactions and implicating an oxocarbonium ion-like transition state in the enzymic reaction. The binding data obtained from the steady state kinetics of these analogues suggests that hydrogen bonding interactions are qualitatively conserved in the glucopyranose binding site during the T- to R-state conformational transition of the enzyme and that interactions between the enzyme and the hydroxyl groups at the 3- and 6-positions of the glucopyranose ring of the substrate are potentially important for stabilization of the enzymic transition state. The specificity of the substrate phosphate binding site has been probed using the compounds 2-deoxy-2-fluoro-α-D-glucopyranosyl phosphate (5), (1-deoxy-α-D-glucopyranosyl) methylphosphonate (6) and 2-deoxy-2-fluoro-α-D-glucopyranosyl phosphofluoridate (7). The results suggested that phosphorylase b can bind both the monanionic and dianionic forms of its substrate with approximately equal affinity. NMR studies of the ternary enzyme-ligand complexes formed with glycogen phosphorylase b and 5 or 6 indicated that no proton donation occurred in the ground-state active site complex. A preliminary investigation into the ability of the cellulase complex from a number of different wood-degrading fungi to hydrolyse p-nitrophenyl β-glucoside has been carried out. This work is aimed at producing environmentally safe fungicides, which are activated by the β-glucosidase component of the cellulase complex in these organisms and this study was carried out in conjunction with Forintek Canada.
Science, Faculty of
Chemistry, Department of
Graduate

The present study presents the research findings of the first ever multi-modular Paenibacillus mucilaginosus glycoside hydrolase (PmGH). Furthermore, we report the successful crystallisation of a multi-modular GH. The GH is composed of two catalytic modules (GH5 and GH6) and two carbohydrate binding modules (both CBM3). Functional analysis demonstrated that the cellulase, mannanase and xylanase activities of PmGH (130 kDa) were attributed to the GH5 catalytic domain. The presence of the GH6 catalytic domain resulted in slightly increased cellulase activity in PmGH. Optimal PmGH activity and functional stability was highest at pH 4-6 and at 40-60°C The structural properties of PmGH that determine its robust nature were further investigated. Homology modelling of PmGH showed the GH5 and GH6 domains to be independent but provided no structural information for the CBMs and linker regions. However, successful homology modelling of the individual domains indicated that the combination of the modules makes PmGH structurally and functionally novel. Glycoside hydrolases occur as independent modules or as part of a multi-modular protein with other catalytic and/or non-catalytic modules. Multiple combinations of these modules can occur in nature resulting in novel proteins such as PmGH. In an attempt to determine the PmGH crystal structure, a range of crystallisation conditions were tested. After extensive screening and optimisation, multiple PmGH crystals were diffracted, using both local diffraction and Synchrotron radiation sources (ESRF, France). Overall ~90% of the PmGH protein crystals did not diffract and of the remaining ~10% yielded unsatisfactory data. Phasing by molecular replacement also yielded no structural solutions. Alternative phasing methods such as multi-wavelength anomalous dispersion were also unsuccessful due to the quality of the diffraction data collected. Given the severe lack of multi-modular GH crystal structures in protein structure databases, the present study highlights the major limitations in structural studies of these important enzymes.
Thesis (PhD)--University of Pretoria, 2018.
Genetics
PhD
Unrestricted

The GM2 gangliosidoses are a group of recessive disorders, which lead to the accumulation of GM2 ganglioside in neuronal cells. The genes responsible for these disorders are HEXA (Tay-Sachs disease and variants), HEXB (Sandhoff disease and variants) and GM2A (AB variant of GM2 gangliosidosis). We have established three relational locus-specific databases recording allelic variation at the HEXA, HEXB and GM2A genes, and these can be accessed through the G M2 Gangliosidoses home page (http://data.mch.mcgill.ca/gm2-gangliosidoses/). The purpose of these databases is to collect and distribute information on mutations in the genes responsible for GM2 gangliosidosis. These databases are available online for users to search and retrieve information about specific mutations either by mutation, phenotype or author(s). In addition, submission forms are available for the addition of new mutations to the databases.
In order to provide information concerning the effects of mutations on the manifestations of disease, we proceeded to model on the theoretical model of the alpha subunit a few missense mutations. (Abstract shortened by UMI.)

The major beta-hexosaminidase isozymes in humans are Hex A (deficient in Tay-Sachs disease, TSD), an alphabeta heterodimer and Hex B (deficient in Sandhoff disease) a betabeta homodimer. Hex S, the alphaalpha homodimer is physiologically unstable. Mature alpha and beta subunits share 60% sequence identity. The beta subunit active site hydrolyzes neutral substrates. The alpha subunit active site hydrolyzes neutral (4MUG) and charged substrates (4MUGS, GM2 ganglioside). Only Hex A hydrolyzes the natural substrate, GM2 ganglioside, in the presence of the GM2 activator protein (AP).
We investigated regions of the alpha and beta subunits involved in AP binding, subunit dimerization, and substrate specificity. We constructed four chimeric cDNAs: alpha1--259beta292--544 , alpha1--118beta152--544, beta 1--418alpha387--529, and beta1--151 alpha119--259beta292--544 (subscripts refer to amino acid residues). Chimeric cDNAs were expressed in a TSD neuroglial cell line, which produces no endogenous alpha subunits. The chimeric isozymes were chromatofocused and assayed for hydrolysis of (a) 4MUG, (b) 4MUGS and (c) GM2 ganglioside.
Transfection of the cDNA constructs lead to expression of homodimeric and heterodimeric chimeric proteins, albeit at lower yields than transfection of wild alpha-cDNA. All of the chimeric proteins hydrolyzed 4MUG but none were active towards 4MUGS or GM2 ganglioside. These results suggest that (a) all constructs contained sufficient information to form both heterodimeric and homodimeric chimeric proteins, (b) the chimeras lacked the alpha-subunit sequence necessary for the hydrolysis of charged substrates.

Carbohydrates are one of the most important biochemical molecules due to their role as energetic resource for non-photosynthetic organisms, cell-cell recognition and adhesion, protection of cell membranes of bacteria and plants and ensure the proper functionality of several enzymes. Glycoproteins are important components of cell surfaces and the extracellular medium where molecular-cell interactions take place. Defects in the activity of glycoproteins are the cause of several human diseases, such as diabetes and lysosomal storage diseases. Glycoside hydrolases (GHs) are one of the most relevant glycoproteins. They catalyse the cleavage of glycosidic bonds of oligosaccharides to generate smaller oligosaccharides or monosaccharides with relevant biological roles. To know the proper way to modulate the activity of these enzymes, it is crucial to understand their molecular mechanisms of action. Sugars are very flexible molecules, most of them formed by 6- membered rings whose conformation changes during the reaction catalysed by GHs. Capturing these conformations, in particular the one at the reaction transition state, is important in inhibitor design. Experiments aimed at characterizing (indirectly) the transition state conformation of GHs include crystallizing the Michaelis complex (MC), which usually is done using thioglycoside derivatives of the natural substrate (i.e. the glycosidic oxygen is substituted by sulfur) or enzyme mutants (e.g. the catalytic acid/base residue mutated to glutamine or asparagine). However, in some cases, the degree of resemblance of the sugar conformation in thioglycosides or enzyme mutants with respect to the natural/wild type counterparts unclear. In last years, our group has demonstrated that the conformational free energy landscape (FEL) of natural sugars (e.g. β-glucose, α,β-mannose and β-xylose) can be used to predict catalytic pathways of GHs. The conformational FEL is explored using Cremer & Pople puckering coordinates as collective variables in the metadynamics method. A natural extension of these studies is to explore the conformational FEL of 7-membered sugar rings (septanosides), which are currently the focus of great interest as potential GH substrates. In this Thesis, we applied Car-Parrinello molecular dynamics methods, within the QM/MM approach, to elucidate the catalytic mechanism of a family 13 retaining GH, (amylosucrase) and a family 125 inverting GH (exo-1,6-α-mannosidase). In both cases, prior structural information was available from enzyme mutants and/or thioderivative substrates. In addition, we have extended previous ring conformational analyses of pyranoses to septanosides in order to assess their potential as new substrates of selected GHs. The Thesis is organised as follows: Chapters I and II focuses on the conformational flexibility of carbohydrates, GH mechanisms and experimental techniques aimed to trap the MC, as well as the methodology used in this Thesis. In Chapter III, we investigate the conformational landscape of α-glucose and the conformational itinerary that an α-glucoside (fructose) follows during catalysis by GH13 amylosucrase (both hydrolysis and polymerization have been investigated). In Chapter IV, we present the results of conformational study of a thioglycoside vs the natural substrate in a GH125 α-mannosidase, including a mechanistic analysis that uncovered the conformational catalytic itinerary for family 125 GHs. In Chapter V, we investigate four MC structures of a promiscuous GH3 β- glucohydrolase, able to cleave several types of glycosidic linkages, for which only structures with thioglycosides are available. The reconstruction of the structure with the natural substrate allowed us to predict the mechanism of action of these enzymes. In Chapter VI, we apply the Cremer & Pople puckering coordinates for 7-membered rings as collective variable for metadynamics on several septanoside molecules (both isolated and “on-enzyme”) and assess their potential as substrate/inhibitors of GHs. Finally, Chapter VII contains the main conclusions of this work.
Los carbohidratos son una de las biomoléculas más importantes debido a su papel como fuente de energía para organismos no-fotosintéticos, reconocimiento célula-célula, protección de la membrana celular de bacterias y plantas y por asegurar la buena funcionalidad de algunas enzimas. Las glicoproteínas son componentes esenciales en la superficie de las células y en el medio extracelular. Defectos en la actividad de estas proteínas son la causa de varias enfermedades humanas, como la diabetes o problemas en el lisosoma. Las glicosil hidrolasas (GHs) son una de las glicoproteínas más relevantes. Catalizan la rotura de enlaces glicosídicos de oligosacáridos para generar monosacáridos o cadenas más pequeñas de azúcares con relevancia biológica. Para encontrar una manera adecuada de modular la actividad de estas enzimas, es crucial entender sus mecanismos moleculares. Los azúcares son moléculas muy flexibles, la mayoría formados por anillos de 6 átomos cuya conformación cambia durante la reacción en GHs. Capturar estas conformaciones, en particular la del estado de transición, es clave para diseñar inhibidores. La manera de caracterizar indirectamente la conformación de ese estado de transición es cristalizando el complejo de Michaelis de la GH con un tioderivado del sustrato natural (el oxígeno glicosídico es substituido por azufre) o usando mutantes de la enzima. No obstante, en algunos casos, la semejanza a nivel conformacional del mímico y el sustrato natural no queda suficientemente clara. En los últimos años, nuestro grupo ha demostrado el uso para predecir itinerarios catalíticos de GHs a partir de las superficies de energía libre conformacional de los azúcares naturales (glucosa, manosa y xilosa). Estas superficies se exploran utilizando las coordenadas de empaquetamiento de Cremer y Pople como variables colectivas en el método de metadinámica. Una extensión de estos estudios es el análisis conformacional de azúcares formados por anillos de 7 átomos (septanósidos), que están actualmente en el foco de interés como sustratos de GHs. En la presente Tesis, aplicamos métodos basados en dinámica molecular Car- Parrinello, dentro de la aproximación QM/MM, para estudiar el mecanismo catalítico de las GHs de la familia 13 (amilosucrasa), la familia 125 (exo-1,6-α-manosidasa) y la familia 3 (enzima HvExoI). Además, hemos extendido el estudio conformacional de anillos a los septanósidos para poder predecir su potencial como nuevos sustratos de GHs concretas.

Includes bibliographical references.
Carbohydrates consist of one or more sub-units usually various 5- and 6-membered cycles (furanoses and pyranoses) which can twist, bend or flip into a variety of conformers that differ in strain - this is ring puckering. These puckers notably the strained puckering conformers are observed during enzymatically assisted bond formation or cleavage of the glycosidic bonds of carbohydrate substrates. In this thesis, the free energy of ring puckering is calculated by implementing the Hill-Reilly reduced coordinate pucker description into the sampling enhancing Free Energies from Adaptive Reaction Coordinate Forces (FEARCF) method. FEARCF non-Boltzmann simulations of prototypical sugars β-Dribose and β-D-glucose converged to yield free energy pucker surfaces and volumes when using several semi-empirical QM methods - AM1, PM3, PM3CARB-1 and SCC-DFTB. From this, the accessible puckering conformations and minimum free energy paths of puckering were reasoned An analysis of the furanose and pyranose free energy pucker surfaces and volumes compared with both Density Functional Theory RB3LYP/6-311++G** optimised structures and a Hartree-Fock free energy surface revealed that SCC-DFTB provides the best semi-empirical description of 5- and 6- membered carbohydrate ring deformation. This illustrates that necessary high energy ring conformations observed in enzymatic binding sites requires the enzyme to induce and preserve high energy conformations required for successful hydrolyses and synthesis of the glycosidic bond. To further test this hypothesis, a 5- and 6-membered cycle were studied within enzymatic environments. The polysaccharide cellulose contains β 1-4 linked glucose subunit and is degraded by cellulase, a glycosidase. Specifically, the retaining cellobiohydrolase I (CBHI) of Trichoderma Reesei which cleaves cellobiose units from crystalline cellulose.The free energy volumes of puckering for the glucose sub-unit (in the catalytic position of an 8 unit cellulosic fragment - cellooctaose) were calculated and explored in vacuum, water and in the active site of CBHI. It was observed that the binding pocket of enzymes limits the ring pucker and that the active site amino acids preferentially stabilise certain puckering conformations. For CBHI, the first part of the glycosidase reaction is the glycosylation step. This was driven to completion during SCC-DFTB QM/MD FEARCF calculations where GLU212, ASP214 and GLU217 and part of the substrate were treated quantum mechanically. The general hybrid orbital method was used to connect the QM and MM regions. The free energy barriers of glycosylation were computed and the puckering statistics during the conversion of cellooctaose to products were correlated with this. Guanosine, a 5-membered ribose derivative is phosphorylated by Purine Nucleoside Phosphorylase (PNP) in order to salvage the guanine base. The effect of the PNP protein environment on ring pucker was studied by using FEARCF SCC-DFTB QM/MD non Boltzmann free energy calculations to quantify the pucker change induced in guanosine when changing environment from vacuum, to water and to the protein. In vacuo, the E4 and E1 pucker conformers were observed as minima. Upon solvation, the puckering phase space became less restricted with the 3T4 and 2T3 pucker conformers as minima. In the PNP active site pucker became restricted with only the 4E conformer observed.

Des glycoside hydrolases (EC. 3. 2. 1. X) (GH) lysosomales dont la fonction consiste en l'hydrolyse de composés glycosylés cellulaires sont impliquées dans des maladies génétiques humaines regroupées sous le terme de "maladies lysosomales". Nous avons effectué des études structurales et fonctionnelles sur certaines de ces GH lysosomales afin de mieux comprendre la physiopathologie des maladies de surcharge métabolique très invalidantes les impliquants. Une vaste étude sur différentes GH, utilisant un même mécanisme catalytique (rétention de la configuration anomérique), incluant des GH lysosomales, a été menée en collaboration avec des spécialistes de modélisation moléculaire. L'analyse de la séquence primaire des protéines par la méthode HCA (Hydrophobic Cluster Analysis) de prédiction 2D, et l'exploitation des banques de donnnées sur des GH cristallisées, ont conduit à la proposition d'un modèle structural de type tonneau (α/ß)8, commun à toutes les enzymes dans le clan des brins ß4 et ß7 du tonneau ont été localisés. Les éléments structuraux et fonctionnels du site catalytique de cinq GH lysosomales : la glucocérébrosidase (maladie de gaucher), l'α-L-iduronidase (mucopolysacharidose de type I), la ß-galactosidase (ganglyosidose GM1), la ß-glucuronidase (syndrome de Sly) et a ß-mannosidase (ß-mannosidose) ont été caractérisés. L'impact structural et fonctionnel de mutations génétiques a été analysé. Nous avons ensuite exploité les prédictions de modélisation effectuées sur ces enzymes pour mieux caractériser les acides aminés, catalyseurs acide-bases et nucléophile, de la glucocérébrosidase et l'α-L-iduronidase humaine ; des protéines pour lesquelles une thérapie enzymatique est développée. A l'aide d'une stratégie de biologie cellulaire basée sur une mutagénèse substitutive des acides aminés suspectés et une expression des protéines recombinantes, mutées et non mutées, dans un système cellulaire hétérologue, nous avons obtenu une invalidation totale de l'activité catalytique des enzymes. Nous avons montré que la perte d'activité n'était pas liée à un défaut de biosynthèse et de maturation des protéines mutées, mais des mutations introduites. Aussi, nous avons été en mesure d'impliquer très fortement les acides glutamiques 235 et 340 de glucocérébrosidase et les acides glutamiques 182 et 299 de l'α-L-iduronidase dans la fonction catalytique de ces GH. Nous avons ainsi apporté une validation expérimentale à nos prédictions. L'ensemble de nos travaux de modélisation moléculaire et d'analyse de relation structure-fonction souligne ici l'importance d'une complémentarité entre les approches de prédictions de structure et les approches de vérifications expérimentales pour la connaissance du fonctionnement catalytique de GH lysosomales impliquées dans des maladies génétiques
Glycosyl hydrolases (GH) are a widespread group of enzymes. Several lysosomal strorage diseases characterized by a severe handicap are due to deficiences in GH. A structural biology study concerning the numerous enzymes belonging to clan GH-A of GH was performed. Clan GH-A includes 5 human enzymes implicated in lysosomal diseases : glucocerebrosidase, α-L-iduronidase, ß-galactosidase, ß-glucuronisade and ß-mannosidase. Predictions concerning the active site structure of these enzymes were made by Hydrophobic Cluster Analysis (HCA), a bidimensional analytical method permitting to compare highly divergent protein sequences. We found that all the active sites may have a similar 3D structures consisting af an (α/β)8 barrel. In particular, a pair of glutamic acid residues, presumed to directly participate in the enzymatic hydrolysis, was identified. Finally, analysis of mutations described in patients was in agreement with the predictions. Next, we performed site-directed mutagenesis studies to obtain experimental evidence supporting our HCA predictions. These studies concerned the glutamatic acid residues supposed to be involved in the catalytic activity of glucocerebrosidase and α-L-iduronidase. Substitution of these glutamatic acids by alanine residues led to complete inactivity of the mutant proteins without affecting their folding/processing. These data further support that Glu235/Glu340 and Glu182/Glu299 play a key role in th enzymatic activity of glucocerebrosidase and α-L-iduronidase, respectively. In conclusion, our work shows that strutural biology and mutagenesis studies may be highly complementary for a better understanding of the structure/function relationship in enzymes involved in severe genetic diseases

As part of a program directed toward the synthesis of novel glycosyl transferase inhibitors possessing a sugar-CH₂-SO₂-CH₂-SO₂-CH₂-nucleoside structure, β-C-glycoside sulfones have been prepared with high stereoselectivity. Both glucose and fucose derivatives were prepared. Sulfur incorporation was achieved by free radical addition of thiolacetic acid to exocyclic glycals. As part of a program directed toward the preparation of amide-linked sialic acid oligomers, a strategy was developed for the synthesis of sialic acid derivatives possessing either a free amine or a free acid functionality. Solution phase coupling of these monomers using standard peptide coupling techniques resulted in the synthesis of (1 → 5)-amide linked sialic acid dimers. As part of a program directed toward the identification of novel helical structures, the solution phase conformation of the polylactone of colominic acid was examined by NMR and molecular modeling. The two structures generated from molecular modeling that were consistent with the NOE data were both helical.

Sea salt is a natural product obtained from the evaporation of seawater in saltpans due to the combined effect of wind and sunlight. Nowadays, there is a growing interest for protection and re-valorisation of saltpans intrinsically associated to the quality of sea salt that can be evaluated by its physico-chemical properties. These man-made systems can be located in different geographical areas presenting different environmental surroundings. During the crystallization process, organic compounds coming from these surroundings can be incorporated into sea salt crystals, influencing their final composition. The organic matter associated to sea salt arises from three main sources: algae, surrounding bacterial community, and anthropogenic activity. Based on the hypothesis that sea salt contains associated organic compounds that can be used as markers of the product, including saltpans surrounding environment, the aim of this PhD thesis was to identify these compounds. With this purpose, this work comprised: 1) a deep characterisation of the volatile composition of sea salt by headspace solid phase microextraction combined with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (HS-SPME/GCGC–ToFMS) methodology, in search of potential sea salt volatile markers; 2) the development of a methodology to isolate the polymeric material potentially present in sea salt, in amounts that allow its characterisation in terms of polysaccharides and protein; and 3) to explore the possible presence of triacylglycerides. The high chromatographic resolution and sensitivity of GC×GC–ToFMS enabled the separation and identification of a higher number of volatile compounds from sea salt, about three folds, compared to unidimentional chromatography (GC–qMS). The chromatographic contour plots obtained revealed the complexity of marine salt volatile composition and confirmed the relevance of GC×GC–ToFMS for this type of analysis. The structured bidimentional chromatographic profile arising from 1D volatility and 2D polarity was demonstrated, allowing more reliable identifications. Results obtained for analysis of salt from two locations in Aveiro and harvested over three years suggest the loss of volatile compounds along the time of storage of the salt. From Atlantic Ocean salts of seven different geographical origins, all produced in 2007, it was possible to identify a sub-set of ten compounds present in all salts, namely 6-methyl-5-hepten-2-one, 2,2,6-trimethylcyclohexanone, isophorone, ketoisophorone, β-ionone-5,6-epoxide, dihydroactinidiolide, 6,10,14-trimethyl-2-pentadecanone, 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate, 2,4,4-trimethylpentane-1,3-diyl bis(2-methylpropanoate), and 2-ethyl-1-hexanol. These ten compounds were considered potential volatile markers of sea salt. Seven of these compounds are carotenoid-derived compounds, and the other three may result from the integration of compounds from anthropogenic activity as metabolites of marine organisms. The present PhD work also allowed the isolation and characterisation, for the first time, of polymeric material from sea salt, using 16 Atlantic Ocean salts. A dialysis-based methodology was developed to isolate the polymeric material from sea salt in amounts that allowed its characterisation. The median content of polymeric material isolated from the 16 salts was 144 mg per kg of salt, e.g. 0.014% (w/w). Mid-infrared spectroscopy and thermogravimetry revealed the main occurrence of sulfated polysaccharides, as well as the presence of protein in the polymeric material from sea salt. Sea salt polysaccharides were found to be rich in uronic acid residues (21 mol%), glucose (18), galactose (16), and fucose (13). Sulfate content represented a median of 45 mol%, being the median content of sulfated polysaccharides 461 mg/g of polymeric material, which accounted for 66 mg/kg of dry salt. Glycosidic linkage composition indicates that the main sugar residues that could carry one or more sulfate groups were identified as fucose and galactose. This fact allowed to infer that the polysaccharides from sea salt arise mainly from algae, due to their abundance and composition. The amino acid profile of the polymeric material from the 16 Atlantic Ocean salts showed as main residues, as medians, alanine (25 mol%), leucine (14), and valine (14), which are hydrophobic, being the median protein content 35 mg/g, i.e. 4,9 mg per kg of dry salt. Beside the occurrence of hydrophobic volatile compounds in sea salt, hydrophobic non-volatile compounds were also detected. Triacylglycerides were obtained from sea salt by soxhlet extraction with n-hexane. Fatty acid composition revealed palmitic acid as the major residue (43 mol%), followed by stearic (13), linolenic (13), oleic (12), and linoleic (9). Sea salt triacylglycerides median content was 1.5 mg per kg of dry salt. Both protein and triacylglycerides seem to arise from macro and microalgae, phytoplankton and cyanobacteria, due to their abundance and composition. Despite the variability resulting from saltpans surrounding environment, this PhD thesis allowed the identification of a sea salt characteristic organic compounds profile based on volatile compounds, polysaccharides, protein, and triacylglycerides.
O sal marinho é um produto natural que deriva da evaporação da água do mar nas salinas pela ação do vento e da luz solar. Atualmente existe um interesse crescente na proteção e revalorização das salinas, o que está intrinsecamente associado à qualidade do sal, que pode ser avaliada pelas suas características físico-químicas. Estes sistemas construídos pelo homem podem encontrar-se em diferentes áreas geográficas, apresentando diferentes envolventes ambientais. Durante o processo de cristalização, compostos orgânicos provenientes da envolvente ambiental podem ficar incorporados nos cristais de sal, influenciando a sua composição. A matéria orgânica associada ao sal marinho é proveniente de três principais origens: algas, comunidade bacteriana envolvente e atividade antropogénica. Com base na hipótese de que o sal marinho contém compostos orgânicos associados que podem ser utilizados como marcadores do próprio sal marinho, incluindo as condições ambientais envolventes das salinas, o objetivo desta tese de doutoramento foi identificar estes compostos no sal. Para tal, procedeu-se a: 1) uma caracterização detalhada da composição volátil do sal marinho por microextração em fase sólida do espaço de cabeça combinada com cromatografia em fase gasosa bidimensional abrangente acoplada à espectrometria de massa por tempo de voo (HS-SPME/GCGC–ToFMS), proporcionando a identificação de potenciais marcadores voláteis; 2) ao desenvolvimento de uma metodologia que permitisse isolar material polimérico potencialmente presente no sal marinho, em quantidades suficientes para a sua caracterização em termos de polissacarídeos e proteína; e 3) à exploração da possível presença de triacilglicerídeos. A elevada resolução cromatográfica e sensibilidade da GC×GC–ToFMS possibilitou a separação e identificação de um maior número de compostos voláteis do sal marinho, cerca do triplo, em comparação com a cromatografia unidimensional (GC–qMS). Os cromatogramas bidimensionais revelaram a presença de 165 compostos, pertencentes a 11 famílias químicas, o que mostra a complexidade da composição volátil do sal marinho. O perfil cromatográfico em duas dimensões, resultante da separação por volatilidade em 1D e por polaridade em 2D, permite a dispersão dos compostos no espaço cromatográfico bidimensional em que os compostos estruturalmente similares ocupam o mesmo espaço cromatográfico (cromatograma estruturado), o que permitiu maior fiabilidade nas identificações de acordo os tempos de retenção (1tR e 2tR) e espetro de massa. Os resultados da análise de sal proveniente de dois locais do salgado de Aveiro, recolhido ao longo de três anos, sugerem a perda de compostos voláteis ao longo do tempo de armazenamento do sal. A partir de sais do Oceano Atlântico provenientes de sete origens geográficas, todos produzidos em 2007, foi possível identificar um conjunto de dez compostos presentes em todos os sais, nomeadamente: 6-metil-5-hepteno-2-ona, 2,2,6-trimetil-ciclo-hexanona, isoforona, cetoisoforona, 5,6-epóxi-β-ionona, di-hidroactinidiolida, 6,10,14-trimetil-2-pentadecanona, 2-metilpropanoato de 3-hidroxi-2,4,4-trimetilpentilo, bis(2-metilpropanoato) de 2,4,4-trimetilpentano-1,3-diilo e 2-etil-1-hexanol. Estes dez compostos foram considerados como potenciais marcadores voláteis do sal marinho. Sete destes compostos são derivados dos carotenoides e os restantes três poderão resultar da integração de compostos provenientes da atividade antropogénica no metabolismo dos organismos marinhos. Com o presente trabalho foi isolado e caracterizado, pela primeira vez, o material polimérico presente no sal marinho. Foram usados 16 sais do Oceano Atlântico. Foi desenvolvida uma metodologia baseada num processo de diálise, que permitiu isolar material polimérico a partir de sal marinho em quantidades suficientes que permitissem a sua caracterização. O conteúdo de material polimérico isolado a partir dos 16 sais foi, em mediana, 144 mg por kg de sal seco, i.e. 0,014% (m/m). A espetroscopia de infravermelho médio e a termogravimetria revelaram a presença maioritária de polissacarídeos sulfatados no material polimérico do sal marinho e também a presença de proteína. Os polissacarídeos do sal marinho mostraram ser ricos em resíduos de ácido urónico (21 mol%), glucose (18), galactose (15) e fucose (13). O conteúdo em sulfato representou, em mediana, 45 mol%, sendo a mediana do conteúdo em polissacarídeos sulfatados de 461 mg/g de material polimérico, o que representa 66 mg/kg de sal seco. As ligações glicosídicas encontradas indicaram que os resíduos de açúcares maioritários que poderiam conter um ou mais grupos sulfato eram a fucose e a galactose. Este facto leva a propor que os polissacarídeos do sal marinho tenham origem principalmente em algas, tendo em conta a sua composição e abundância. O perfil em aminoácidos do material polimérico dos 16 sais em estudo revelou como resíduos maioritários, em mediana, a alanina (25 mol%), a leucina (14) e a valina (14), que têm em comum a sua hidrofobicidade, sendo a mediana do conteúdo em proteína de 35 mg/g, i.e. 4,9 mg por kg de sal seco. Para além da fração hidrofóbica volátil, também foram detetados compostos hidrofóbicos não voláteis no sal marinho. Foram isolados triacilglicerídeos por extração com soxhlet, usando n-hexano. A composição em resíduos de ácidos gordos revelou como ácido maioritário o ácido palmítico (43 mol%), seguido do esteárico (13), linolénico (13), oleico (12) e linoleico (9). O conteúdo, em mediana, de triacilglicerídeos foi de 1,5 mg por kg de sal seco. Dada a sua composição e abundância, tanto a proteína como os triacilglicerídeos parecem ter origem em macro e microalgas, fitoplacton e cianobactérias. Apesar da variabilidade resultante do ambiente envolvente das salinas, a presente tese de doutoramento permitiu identificar um perfil em compostos orgânicos característico do sal marinho com base nos compostos voláteis, polissacarídeos, proteína e triacilglicerídeos.

Chez Arabidopsis thaliana, deux gènes codent des enzymes de branchement (BE) BE2.2 et BE2.1 responsables de la formation des liaisons α-1,6 de l’amidon transitoire synthétisé dans la feuille de la plante. Ces enzymes, appartenant à la famille GH13_8 de la classification CAZy, agissent en clivant une liaison α-1,4 d’un glucane puis en transférant la chaîne clivée en position α-1,6 selon un mécanisme d’action qui peut être intra ou intermoléculaire. Dans ce travail, une caractérisation enzymatique et structurale des BEs d’A. thaliana (classées de type II d’après leur séquence en acides aminés) a été réalisée et les résultats comparés à ceux de la BE d’E. coli (GlgB, enzyme de type I).L'état oligomérique, la forme en solution et l’organisation structurale des BEs ont été évalués par une approche SAXS. Par des analyses spectrophotométriques, le pH, la température, mais aussi le KM pour l’amylose et l’amylopectine ont été déterminés. Une analyse sur gel de polyacrylamide, en conditions natives, a permis d’évaluer le comportement électrophorétique des BEs en présence ou en absence de ces substrats et d’en déterminer leur constante d’affinité (Ks). Notre étude révèle que les BEs d’ A. thaliana ont plus d'affinité pour l’amylopectine que pour l’amylose contrairement à GlgB. En présence d’un substrat branché, des changements d’oligomérie et/ou de la conformation des BEs d’A. thaliana ont été observés. Finalement, des analyses en chromatographie échangeuse d'anions ont permis de déterminer la taille minimale du substrat nécessaire à l’activité des BEs et la taille des chaînes transférées. Les résultats obtenus pointent vers un mécanisme d’action intramoléculaire de BE2.2
BE2.2 and BE2.1 are the two genetically independent branching enzymes (BE) isoforms involved in transitory starch synthesis in A. thaliana and belong to family GH13_8 (according to CAZy database). Both are classified as type II BE due to their amino acid sequence. In Arabidopsis leaves, they are the only enzymes that catalyze the formation of α-1,6 branch points by cleaving α-1,4 linkages and transferring the newly formed reducing end in α-1,6 position through an intra or intermolecular mechanism. In this work, we report in vitro enzymatic characterization and structural analysis of A. thaliana BEs, these results were compared to E. coli BE enzymatic analysis (GlgB, type I enzyme).Structural analysis using SAXS approach was used to evaluate A. thaliana BEs oligomeric state, shape in solution and to determine BE organization. In vitro enzymatic analyses were performed using spectrophotometry assays to establish their catalytic parameters such as pH, temperature and also KM for amylose and amylopectin. Native PAGE analyses were also used to assess BEs behaviour in the presence or absence of substrates and to determine their affinity constant (Ks) for amylopectin and amylose. Enzymatic characterization reveals that both A. thaliana BEs have more affinity for amylopectin than for amylose, contrary to GlgB. Moreover, interaction of A. thaliana BEs with branched substrates induces protein oligomerization and/or conformational changes. Finally, determination of the minimal length of their substrate and characterization of reaction products were performed using anions exchange chromatography analyses.Taken together, our data point to an intramolecular mechanism of action of BE2.2

En las especies bovina y porcina se ha comunicado la presencia de glicosidasas ligadas a los espermatozoides (Hayashi et al., 2004). Otros autores han propuesto diferentes glicosidasas espermáticas que interactúan con carbohidratos específicos en la ZP en otras especies animales incluyendo al hombre (Tulsiani et al., 1989, 1990; Avilés et al., 1996; Song et al., 2000; Venditti et al., 2007, 2010).En el presente trabajo se llevó a cabo un estudio de las principales glicosidasas espermáticas para establecer su ubicación en el espermatozoide, el origen de su síntesis, la expresión génica y mediante FIV porcina valorar el papel de aquellas glicosidasas con mayor actividad enzimática (AE) en el reconocimiento de gametos, fecundación y desarrollo embrionario temprano.
In bovine and porcine species, the presence of sperm bound glycosidases has been reported (Hayashi et al., 2004). Others studies have proposed different sperm glycosidases that interact with specific ZP carbohydrates in other species, including men (Tulsiani et al., 1989, 1990; Avilés et al., 1996; Song et al., 2000; Venditti et al., 2007, 2010). In the present work, the major sperm glycosidases were studied to establish their localization in the spermatozoon, the origin of their synthesis, their gene expression and through porcine IVF; we sought to define the role of the glycosidases with greater activity during gamete interaction, fertilization and early embryo development.

Doutoramento em Bioquímica
Os polissacarídeos são os componentes maioritários dos grãos de café verde e torrado e da bebida de café. Os mais abundantes são as galactomananas, seguindo-se as arabinogalactanas. Durante o processo de torra, as galactomananas e arabinogalactanas sofrem modificações estruturais, as quais estão longe de estar completamente elucidadas devido à sua diversidade e à complexidade estrutural dos compostos formados. Durante o processo de torra, as galactomananas e arabinogalactanas reagem com proteínas, ácidos clorogénicos e sacarose, originando compostos castanhos de alto peso molecular contendo nitrogénio, designados de melanoidinas. As melanoidinas do café apresentam diversas atividades biológicas e efeitos benéficos para a saúde. No entanto, a sua estrutura exata e os mecanismos envolvidos na sua formação permanecem desconhecidos, bem como a relação estrutura-atividade biológica. A utilização de sistemas modelo e a análise por espectrometria de massa permitem obter uma visão global e, simultaneamente, detalhada das modificações estruturais nos polissacarídeos do café promovidas pela torra, contribuindo para a elucidação das estruturas e mecanismos de formação das melanoidinas. Com base nesta tese, oligossacarídeos estruturalmente relacionados com a cadeia principal das galactomananas, (β1→4)-Dmanotriose (Man3), e as cadeias laterais das arabinogalactanas, (α1→5)-Larabinotriose (Ara3), isoladamente ou em misturas com ácido 5-Ocafeoilquínico (5-CQA), o ácido clorogénico mais abundante nos grãos de café verde, e péptidos compostos por tirosina e leucina, usados como modelos das proteínas, foram sujeitos a tratamento térmico a seco, mimetizando o processo de torra. A oxidação induzida por radicais hidroxilo (HO•) foi também estudada, uma vez que estes radicais parecem estar envolvidos na modificação dos polissacarídeos durante a torra. A identificação das modificações estruturais induzidas por tratamento térmico e oxidativo dos compostos modelo foi feita por estratégias analíticas baseadas principalmente em espectrometria de massa, mas também em cromatografia líquida. A cromatografia de gás foi usada na análise de açúcares neutros e ligações glicosídicas. Para validar as conclusões obtidas com os compostos modelo, foram também analisadas amostras de polissacarídeos do café obtidas a partir de resíduo de café e café instantâneo. Os resultados obtidos a partir dos oligossacarídeos modelo quando submetidos a tratamento térmico (seco), assim como à oxidação induzida por HO• (em solução), indicam a ocorrência de despolimerização, o que está de acordo com estudos anteriores que reportam a despolimerização das galactomananas e arabinogalactanas do café durante a torra. Foram ainda identificados outros compostos resultantes da quebra do anel de açúcares formados durante o tratamento térmico e oxidativo da Ara3. Por outro lado, o tratamento térmico a seco dos oligossacarídeos modelo (individualmente ou quando misturados) promoveu a formação de oligossacarídeos com um maior grau de polimerização, e também polissacarídeos com novos tipos de ligações glicosídicas, evidenciando a ocorrência de polimerização através reações de transglicosilação não enzimática induzidas por tratamento térmico a seco. As reações de transglicosilação induzidas por tratamento térmico a seco podem ocorrer entre resíduos de açúcares provenientes da mesma origem, mas também de origens diferentes com formação de estruturas híbridas, contendo arabinose e manose como observado nos casos dos compostos modelo usados. Os resultados obtidos a partir de amostras do resíduo de café e de café instantâneo sugerem a presença de polissacarídeos híbridos nestas amostras de café processado, corroborando a ocorrência de transglicosilação durante o processo de torra. Além disso, o estudo de misturas contendo diferentes proporções de cada oligossacarídeo modelo, mimetizando regiões do grão de café com composição distinta em polissacarídeos, sujeitos a diferentes períodos de tratamento térmico, permitiu inferir que diferentes estruturas híbridas e não híbridas podem ser formadas a partir das arabinogalactanas e galactomananas, dependendo da sua distribuição nas paredes celulares do grão e das condições de torra. Estes resultados podem explicar a heterogeneidade de estruturas de melanoidinas formadas durante a torra do café. Os resultados obtidos a partir de misturas modelo contendo um oligossacarídeo (Ara3 ou Man3) e 5-CQA sujeitas a tratamento térmico a seco, assim como de amostras provenientes do resíduo de café, mostraram a formação de compostos híbridos compostos por moléculas de CQA ligadas covalentemente a um número variável de resíduos de açúcar. Além disso, os resultados obtidos a partir da mistura contendo Man3 e 5-CQA mostraram que o CQA atua como catalisador das reações de transglicosilação. Por outro lado, nas misturas modelo contendo um péptido, mesmo contendo também 5-CQA e sujeitas ao mesmo tratamento, observou-se uma diminuição na extensão das reações transglicosilação. Este resultado pode explicar a baixa extensão das reações de transglicosilação não enzimáticas durante a torra nas regiões do grão de café mais ricas em proteínas, apesar dos polissacarídeos serem os componentes maioritários dos grãos de café. A diminuição das reações de transglicosilação na presença de péptidos/proteínas pode dever-se ao facto de os resíduos de açúcares redutores reagirem preferencialmente com os grupos amina de péptidos/proteínas por reação de Maillard, diminuindo o número de resíduos de açúcares redutores disponíveis para as reações de transglicosilação. Além dos compostos já descritos, uma diversidade de outros compostos foram formados a partir dos sistemas modelo, nomeadamente derivados de desidratação formados durante o tratamento térmico a seco. Em conclusão, a tipificação das modificações estruturais promovidas pela torra nos polissacarídeos do café abre o caminho para a compreensão dos mecanismos de formação das melanoidinas e da relação estrutura-atividade destes compostos.
Polysaccharides are the major components of green and roasted coffee beans, and coffee brew. The most abundant ones are galactomannans, followed by arabinogalactans. During the roasting process, galactomannans and arabinogalactans undergo structural modifications that are far to be completely elucidated due to their diversity and complexity of the compounds formed. During the roasting process, galactomannans and arabinogalactans react with proteins, chlorogenic acids, and sucrose, originating high molecular weight brown compounds containing nitrogen, known as melanoidins. Several biological activities and beneficial health effects have been attributed to coffee melanoidins. However, their exact structures and the mechanisms involved in their formation remain unknown, as well as the structure-biological activity relationship. The use of model systems and mass spectrometry analysis allow to obtain an overall view and, simultaneously, detailed, of the structural modifications in coffee polysaccharides promoted by roasting, contributing to the elucidation of the structures and formation mechanisms of melanoidins. Based on this thesis, oligosaccharides structurally related to the backbone of galactomannans, (β1→4)-D-mannotriose, and the side chains of arabinogalactans, (α1→5)-Larabinotriose, alone or in mixtures with 5-O-caffeoylquinic acid, the most abundant chlorogenic acid in green coffee beans, and dipeptides composed by tyrosine and leucine, used as models of proteins, were submitted to dry thermal treatments, mimicking the coffee roasting process. The oxidation induced by hydroxyl radicals (HO•) was also studied, since these radicals seem to be involved in the modification of the polysaccharides during roasting. The identification of the structural modifications induced by thermal and oxidative treatment of the model compounds was performed mostly by mass spectrometry-based analytical strategies, but also using liquid chromatography. Gas chromatography was used in the analysis of neutral sugars and glycosidic linkages. To validate the conclusions achieved with the model compounds, coffee polysaccharide samples obtained from spent coffee grounds and instant coffee were also analysed. The results obtained from the model oligosaccharides when submitted to thermal treatment (dry) or oxidation induced by HO• (in solution) indicate the occurrence of depolymerization, which is in line with previous studies reporting the depolymerization of coffee galactomannans and arabinogalactans during roasting. Compounds resulting from sugar ring cleavage were also formed during thermal treatment and oxidative treatment of Ara3. On the other hand, the dry thermal treatment of the model oligosaccharides (alone or when mixed) promoted the formation of oligosaccharides with a higher degree of polymerization, and also polysaccharides with new type of glycosidic linkages, evidencing the occurrence of polymerization via non-enzymatic transglycosylation reactions induced by dry thermal treatment. The transglycosylation reactions induced by dry thermal treatment can occur between sugar residues from the same origin, but also of different origins, with formation of hybrid structures, containing arabinose and mannose in the case of the model compounds used. The results obtained from spent coffee grounds and instant coffee samples suggest the presence of hybrid polysaccharides in these processed coffee samples, corroborating the occurrence of transglycosylation during the roasting process. Furthermore, the study of mixtures containing different proportions of each model oligosaccharide, mimicking coffee bean regions with distinct polysaccharide composition, subjected to different periods of thermal treatment, allowed to infer that different hybrid and non-hybrid structures may be formed from arabinogalactans and galactomannans, depending on their distribution in the bean cell walls and on roasting conditions. These results may explain the heterogeneity of melanoidins structures formed during coffee roasting. The results obtained from model mixtures containing an oligosaccharide (Ara3 or Man3) and 5-CQA and subjected to dry thermal treatment, as well as samples derived from spent coffee grounds, showed the formation of hybrid compounds composed by CQA molecules covalently linked to a variable number of sugar residues. Moreover, the results obtained from the mixture containing Man3 and 5-CQA showed that CQA acts as catalyst of transglycosylation reactions. On the other hand, in the model mixtures containing a peptide, even if containing 5-CQA and subjected to the same treatment, it was observed a decrease in the extent of transglycosylation reactions. This outcome can explain the low extent of non-enzymatic transglycosylation reactions during roasting in coffee bean regions enriched in proteins, although polysaccharides are the major components of the coffee beans. The decrease of transglycosylation reactions in the presence of peptides/proteins can be related with the preferential reactivity of reducing residues with the amino groups of peptides/proteins by Maillard reaction, decreasing the number of reducing residues available to be directly involved in the transglycosylation reactions. In addition to the compounds already described, a diversity of other compounds were formed from model systems, namely dehydrated derivatives formed during dry thermal treatment. In conclusion, the identification of the structural modifications in coffee polysaccharides promoted by roasting pave the way to the understanding of the mechanisms of formation of melanoidins and structure-activity relationship of these compounds.

Ying Zheng.
Thesis submitted in: October 2001.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 84-90).
Abstracts in English and Chinese.
ABSTRACT --- p.ii
中文摘要 --- p.iv
ACKNOWLEDGEMENTS --- p.vi
TABLE OF CONTENTS --- p.vii
LIST OF FIGURES --- p.x
LIST OF TABLES --- p.xii
ABBREVIATIONS --- p.xiii
Chapter CHAPTER 1. --- Introduction --- p.1
Chapter 1.1. --- Rationale of the Study --- p.2
Chapter 1.2. --- Flavonoids --- p.3
Chapter 1.2.1. --- Introduction --- p.3
Chapter 1.2.2. --- Potential Health Effects --- p.5
Chapter 1.2.3. --- Absorption Studies --- p.6
Chapter 1.3. --- Drug Absorption --- p.9
Chapter 1.3.1. --- Pathways and Mechanisms of Intestinal Absorption --- p.9
Chapter 1.3.2. --- Transporters Potentially Involved in the Absorption of Flavonoids --- p.11
Chapter 1.3.2.1. --- Glucose Transporters --- p.11
Chapter 1.3.2.2. --- Multidrug Resistance Systems --- p.13
Chapter 1.3.2.2.1. --- P-glycoprotein --- p.13
Chapter 1.3.2.2.2. --- Non-P-glycoprotein Efflux Mechanisms --- p.15
Chapter 1.4. --- In vitro Models to Study Absorption --- p.15
Chapter 1.4.1. --- Ussing Chamber --- p.16
Chapter 1.4.2. --- Cultured Cells --- p.17
Chapter 1.4.2.1. --- Choice of Cells --- p.17
Chapter 1.4.2.2. --- Caco-2 Cell Monolayers as in vitro Model --- p.18
Chapter 1.4.2.3. --- Correlation Between in vivo Absorption and in vitro Permeability Coefficients --- p.19
Chapter 1.4.3. --- Everted Gut Sacs --- p.20
Chapter 1.4.4. --- Brush Border Membrane Vesicles (BBMVs) --- p.20
Chapter 1.4.5. --- In situ Experiments --- p.21
Chapter 1.5. --- Aims and Scope of the Present Study --- p.23
Chapter CHAPTER 2. --- Materials & Methods --- p.25
Chapter 2.1. --- Materials --- p.26
Chapter 2.1.1. --- Chemicals --- p.26
Chapter 2.1.2. --- Materials for Cell Culture --- p.27
Chapter 2.1.3. --- Instruments --- p.28
Chapter 2.1.4. --- Animals --- p.28
Chapter 2.2. --- Methods --- p.29
Chapter 2.2.1. --- Preformulation Studies on Selected Flavonoids --- p.29
Chapter 2.2.1.1. --- Determination of Stability --- p.29
Chapter 2.2.1.2. --- Thermal Analysis --- p.29
Chapter 2.2.1.3. --- Determination of Solubility --- p.29
Chapter 2.2.1.4. --- Determination of Partition Coefficient --- p.30
Chapter 2.2.2. --- Validation of in vitro Models --- p.30
Chapter 2.2.2.1. --- Ussing Chamber --- p.30
Chapter 2.2.2.1.1. --- Tissue Preparation --- p.30
Chapter 2.2.2.1.2. --- Electrical Measurements --- p.31
Chapter 2.2.2.1.3. --- Experimental Protocols --- p.31
Chapter 2.2.2.1.4. --- Calculations of Permeability --- p.32
Chapter 2.2.2.2. --- Caco-2 Cell Monolayers --- p.32
Chapter 2.2.2.2.1. --- Preparation of Caco-2 Cell Monolayers --- p.32
Chapter 2.2.2.2.2. --- Validation of Caco-2 Cell Monolayers --- p.32
Chapter 2.2.2.2.3. --- Calculation of Permeability --- p.34
Chapter 2.2.3. --- Transport Studies of Selected Flavonoids --- p.34
Chapter 2.2.4. --- Brush Border Membrane Vesicles (BBMVs) --- p.35
Chapter 2.2.4.1. --- Preparation of BBMVs --- p.35
Chapter 2.2.4.2. --- Uptake of D-glucose by BBMVs --- p.38
Chapter 2.2.4.3. --- Counting of 3H-D-glucose in BBMVs --- p.39
Chapter 2.2.4.4. --- Calculation of Glucose Uptake --- p.39
Chapter 2.2.4.5. --- Total Protein Assay --- p.40
Chapter 2.2.5. --- Analytical Methods --- p.41
Chapter 2.2.5.1. --- HPLC Analysis --- p.41
Chapter 2.2.5.1.1. --- HPLC Analysis of Quercetin and Related Glycosides --- p.41
Chapter 2.2.5.1.2. --- HPLC-MS Analysis of Degradation Products --- p.41
Chapter 2.2.5.1.3. --- HPLC Analysis of Propranolol --- p.42
Chapter 2.2.5.2. --- UV Analysis --- p.42
Chapter 2.2.5.3. --- Fluorescence Analysis --- p.42
Chapter 2.2.5.4. --- Analysis of Radiolabeled Markers --- p.42
Chapter 2.2.6. --- Statistical Analysis --- p.42
Chapter CHAPTER 3. --- Results & Discussions --- p.44
Chapter 3.1. --- Preformulation Studies on Selected Flavonoids --- p.45
Chapter 3.1.1. --- Stability --- p.45
Chapter 3.1.2. --- Thermal Analysis --- p.52
Chapter 3.1.3. --- Aqueous Solubility --- p.58
Chapter 3.1.4. --- Partition Coefficient --- p.61
Chapter 3.2. --- Validation of in vitro Models --- p.62
Chapter 3.2.1. --- Selection of Marker Compounds --- p.62
Chapter 3.2.2. --- Validation of Ussing Chamber --- p.63
Chapter 3.2.3. --- Validation of Caco-2 Cell Monolayers --- p.64
Chapter 3.2.3.1. --- Integrity of Caco-2 Cell Monolayers --- p.64
Chapter 3.2.3.2. --- Permeabilities of Marker Compounds --- p.65
Chapter 3.2.3.3. --- Selection of in vitro Models --- p.66
Chapter 3.2.3.3. --- Validation of Sodium/Glucose Cotransporter (SGLT1) --- p.66
Chapter 3.3. --- Transport Studies of Quercetin and Related Flavonoids --- p.67
Chapter 3.3.1. --- Direction of Transport --- p.67
Chapter 3.3.2. --- Concentration Dependence --- p.69
Chapter 3.3.3. --- Inhibition of P-gp by Verapamil --- p.71
Chapter 3.3.4. --- Metabolism of Quercetin in Caco-2 Cells --- p.72
Chapter 3.3.5. --- Studies of Quercetin-3-glucoside with Sugar Transporters --- p.73
Chapter 3.4. --- Uptake of D-glucose by Brush Border Membrane Vesicles (BBMVs) --- p.75
Chapter CHAPTER 4. --- Conclusions --- p.80
References --- p.83

博士
國立臺灣大學
食品科技研究所
89
Abstract Most of the sesame oil is produced by roasting and pressing of the sesame seeds. After high temperature of roasting, the sesame meal is bitter tasted and the protein was highly denatured. Therefore, the sesame meal is generally used as feeds or fertilizers. However, lignans and lignan glycosides of sesame meal are found to have several functional effects. The purpose of this project was to study (1) the change of the contents of lignans and lignan glycosides versus the ingluence of the antioxidative activity during the roasting process (2) the antioxidative activity of the unroasted defatted sesame meal, aiming at finding the components responsible for its antioxidative activity (3) the effect of enzymatic and acid hydrolysis on the antioxidative activity of lignan glycosides crude extract of unroasted sesame meal. We hope that we can increase the amount of functional components and enhance the utilization of sesame meal. Burma black sesame was used as the experimental material in this study. In sesame lignans, the contents of sesamin and sesamolin were 2.164 and 1.380 mg/g seed, respectively; in sesame lignan glycosides, the contents of sesaminol triglucoside and sesaminol diglucoside were 1.731 and 0.084 mg/g seed, respectively. Sesaminol monoglucoside was not detected in sesame seeds. Increasing the roasting temperature from 160℃ to 240℃, the content of sesamin didn’t change significantly. The content of sesamolin was decreased from 7.04 mg/g oil (unroasted) to 0.14 mg/g oil (roasted at 240℃ fro 20min), with a decreasing rate of 93.145. Meanwhile, the content of sesamol was significantly increased. The oxidative stability of sesame oil was also significantly increased while the roasting temperature was increasing. It might be due to the increasing of sesamol and maillard reaction products. With regard to the DPPH free radical scavenging effect and Cu+2-induced oxidation of human LDL assay, the unroasted crude extract of lignan glycosides had the poorest antioxidative activity. Under the roasting temperature from 160℃ to 240℃, the antioxidative activity were increased with increasing temperature. The increment of the antioxidative activity of crude extract of lignan glycosides was probably due to the total phenolics contents and maillard reaction products formed during the roasting process. The most abundant lignan glycosides of Burma black sesame were sesaminol triglucoside and sesaminol diglucoside. Neither sesaminol triglucoside nor sesaminol diglucoside showed good antioxidative activity toward DPPH free radical scavenging effect and inhibition of LDL oxidation. In this study, we found that the brown materials isolated from Fr2 showed excellent DPPH free radical scavenging effect and also had pronounced inhibitory effect toward the oxidation of human LDL. It was suggested that the brown materials plays a prominent role in the antioxidative activity of the lignan glycosides crude extract. Identification of the responsible components is underway. 10 mg of lignan glycosides crude extract (from unroasted sesame meal) was hydrolyzed with 7 mg of cellulase and 6 mg of β-glucosidase at 50℃ for 48 hour. The hydrolysates showed a better DPPH free radical scavenging effect. At the concentration of 100 μg/mL, the scavenging effect was 2.12 fold higher than that of the unhydrolyzed one. In the case of acid hydrolysis with HCl aqueous solution from 0 to 2N, we found that when the lignan glycosides crude extract was hydrolyzed with 0.5N HCl for 30 min, the hydrolysates showed the best DPPH free radical scavenging effect. The scavenging effect was 1.55 fold higher than that of the unhydrolyzed one when the concentration of hydrolysates was 100μg/mL. The possible might be due to the formation of sesaminol during the hydrolysis process.

Includes a list of publications co-authored by the author during the preparation of this thesis.
Bibliography: leaves 103-111.
vi, 111 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Studies the links between grape composition, wine composition and wine sensory properties. Developed a new method of measuring glycoside concentration in grapes (glycosyl-glucose assay) and a modified measurement of wine colour density in red wine. These were used to analyse samples of Shiraz grapes and wines from a comprehensive vineyard irrigation trial. Glycosyl-glucose concentrations shows promise for the prediction of wine composition and flavor intensity.
Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 2001

Includes a list of publications co-authored by the author during the preparation of this thesis.
Bibliography: leaves 103-111.
vi, 111 leaves : ill. ; 30 cm.
Studies the links between grape composition, wine composition and wine sensory properties. Developed a new method of measuring glycoside concentration in grapes (glycosyl-glucose assay) and a modified measurement of wine colour density in red wine. These were used to analyse samples of Shiraz grapes and wines from a comprehensive vineyard irrigation trial. Glycosyl-glucose concentrations shows promise for the prediction of wine composition and flavor intensity.
Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 2001

博士
國立臺灣大學
藥學研究所
105
Part I. Chemical investigation of Planchonella obovata leaf (III): isolation, analysis of triterpenoid glycosides and confirmation of their glycon moieties Planchonella obovata is an evergreen tree, one of the three Sapotaceae plants in Taiwan. This thesis is a continuation of our previous study on the chemical investigation of the polar components in the n-BuOH soluble fraction. Further separation through Sephadex LH-20 and reversed-phase C-18 columns led to the isolation of two new compounds, 6β-hydroxy-conyzasaponin G (8) and glycerol 1,2-disinapate 3-α-glucuronide (9), and five known compounds. The monosaccharide compositions in these glycosides were determined by TLC analysis of the acid hydrolysates of Mi-saponins B and C. The absolute configurations of these monosaccharides was determined by GC analysis of the corresponding trimethylsilyl L-cysteine derivative. These monosaccharides are determined as D-apiose, L-arabinose, D-xylose, D-glucose, and L-rhamnose. Part II Bioactive constituents from Palaquium formosanum kernel Palaquium formosanum (Sapotaceae) is a tall tree indigenous to Philippines and Taiwan. The n-BuOH-soluble fraction of the ethanol extact of the P. formosanum kernel being cytotoxic against PC-3 prostate cancer cell line was further fractionated by Sephadex LH-20. The fractions rich in saponins showed cytotoxicity were further separated mainly by RP-18 columns.This effort led to the isolation of one new, 3''-O-rhamnopyranosyl-arganin C (10), and 11 known oleanane-type saponins (11–21). The 1H and 13C NMR spectra of these compounds are quite complex and their assignments were established by analysis of 1D NMR (1H and 13C), 2D NMR (1H−1H COSY, NOESY, HSQC, and HMBC), and selectively excited 1D TOCSY spectra. This work revised some reported 1H and 13C NMR assignments, and these data being recorded in methanol-d4 are friendly to the user as a reference for identifying the related triterpenoid saponins. Bioassay of these isolated compounds and five from Planchonella obovata leaf indicated arganin C to be most potent against PC-3 prostate cancer cells with the IC50 value of 13.8 μM. Some structure and activity relationships were also drawn. The n-BuOH-soluble fraction also displayed potent inhibitory activity against α-glucosidase (87.8% at 10 μg/mL). Further investigation of this fraction via the bioassay-guide approach yielded ten flavonoids (22–31). Four of them (28–31) are dimeric proanthocyanidins with 4α→8 linkage, arising rotational isomers from axial rotation. Thus, the 1H and 13C NMR spectra of prodelphinidin B3 (28), catechin-(4α→8)-gallocatechin (29), gallocatechin-(4α→8)-gallocatechin (30), and gallocatechin-(4α→8)-epigallocatechin gallate (31) were quite complex. The conformations of these rotamers were identified through analysis of 2D ROESY spectra. The complete 1H and 13C NMR assignments of these rotamers were made by extensive analysis of 2D NMR (1H−1H COSY, ROESY, HSQC, and HMBC), and this is the first report for such assignments.

碩士
國立臺灣海洋大學
食品科學系
93
Abstract Plant sterol not only can decrease cholesterol absorption and thus protect against atherosclerosis but also have a beneficial effect against cancer. In addition, steryl glycosides have immunomodulating activities. Total plant sterol contents were usually determined in different plant materials which were the total amounts of free sterols (FS), steryl esters (SE), steryl glycosides (SG), and acylated steryl glycosides (ASG). Recently, reliable data on concentrations of phytosterol species in various plant-based foods were highly desired. Sterols in more complex sample were determined by using a method that comprised of acid hydrolysis and alkaline hydrolysis to provide the amount of total sterols. SG were determined by the deduction the total sterols contents by the sterol contents resulted by acid hydrolysis treatment. In this study, sitosterol-��-glucoside in different plants (Dioscorea pseudojaponica, and marine algae, such as Spirulina spp., Ulva lactuca, Gracilaria coronopifolia and Gloiopeltis tenax) were analyzed by RP-HPLC. Quantification of sitosterol-��-glucoside by RP-HPLC analysis was higher than using traditional method. Although the retention times of sitosterol-��-glucoside of some materials were closed to the authentic standard, it was unlikely that the signal represented to sitosterol-��-glucoside after a closer examination of their UV spectra.

This thesis deals with the sorting and use of sweeteners in the production of fruit spreads. The theoretical part focuses on the sorting of sweetening agents and their classification. Furthermore, it presents procedures of production of fruit spreads and their assortment. The main goal is to compare sensory attributes of jams that I produced according to an assigned recipe, which is described in the practical part of this thesis. These jams were made from regional fruits (strawberries, apricots, sour-cherries) and sweetened with both natural (sucrose, sorbitol, Steviol glycosides) and artificial (sucralose) sweetening agents. The control group of this research were the students and employees of the University of South Bohemia in České Budějovice in the age of 24-60 years. The evaluators filled in a sequence test protocol and a questionnaire for consumers. The results of the sensory analysis were processed into tables and graphs with the aid of statistical methods.

In an attempt to identify agents that specifically target neuroblastoma (NB) tumour-initiating cells (TIC) we performed drug screens using libraries of bioactive compounds. Cardiac glycosides (CGs) were the largest class of drugs identified with antitumour activity. At high CG doses inhibitory effects on the Na+/K+-ATPase induce cardiotoxicity; therefore, CG analogues were designed in an attempt to separate the effects on NB cells from cardiotoxicity. We identified RIDK34 as our lead compound from a structure-activity-relationship analysis (IC50 8 nM). RIDK34 contains a unique oxime group and shows increasing potency against NB TICs. The Na+/K+-ATPase is a target for the apoptotic activity of digoxin and RIDK34, whereby a signaling cascade involving Src and ERK may induce apoptosis. Furthermore, we predict that signaling activation does not require inactivation of the Na+/K+-ATPase and subsequent deregulation of [Na+]i and [K+]I gradients. Thus CGs and particularly RIDK34 may be expected to display diminished cardiotoxicity and greater therapeutic potential.

碩士
國立臺北科技大學
有機高分子研究所
100
The three-dimensional (3D) structure of glycoside hydrolase family (GHF) 7, 11, 12 and 16 are collectively referred to as a jellyroll β-sandwich fold with a similar cleft catalytic active site,although the amino acid sequences of these four families are diverse. Based on the results of primary sequence alignment and 3D structural comparison, GHF 7 and 16 possess a conserved catalytic motif of RYYDQDNExDxEHFWWYP and ExDxE/ExDxxE, whereas GHF11 and 12 share a general active site motif of YWEnYPFQEn+(88~94) and NWYEnMWPEn+(83~97), respectively. The first and last glutamyl residues found in the catalytic motifs have been clearly identified as catalytic nucleophile and general acid/base for retention hydrolytic mechanism, respectively. A detailed structural comparison among the known structures reveals that they share a low level of amino acid sequence identity about 22%, but the enzymes have a high degree of structural conservation at the active sites.

碩士
國立清華大學
資訊工程學系
99
In this research, the objective is to analyze the residues, which are around the active site of Glycoside Hydrolases where the ligands bind, from sequence and structure. Currently, there are about 74,300 structures in Structural Classification of Proteins (SCOP)[1]. The structure of the protein can provide the information of different folds and the position where substrates bind with proteins in space. We consider the catalytic domains of the proteins, and construct the templates library which includes ligands and structures. Through mapping the relative positions, where ligands and proteins bind, to the corresponding sequences, there are some segments of the residues, and analysis of these segments. Utilized the flow chart, the analysis of the GH families is used to determine whether the protein with similar structure has the function of catalyzed glycoside or not.

Carbohydrates are important in a large number of cellular, physiological, and pathological processes. Carbohydrates often function as the human host’s first line of defence against pathogen invasion by coating surfaces of epithelial cells and as glycan-rich mucins which line the entrances to the body. Various pathogenic bacteria exploit their hosts by modifying their glycans through the production of carbohydrate-active enzymes. Two kinds of pathogenic bacteria that are notable for their production of carbohydrate-active enzymes are Streptococcus pneumoniae and Clostridium perfringens. Both S. pneumoniae and C. perfringens inhabit glycan-rich niches in the human body, the respiratory and gastrointestinal tracts, respectively. To properly colonize their human hosts both bacteria have developed an extensive repertoire of glycoside hydrolases (GHs) which are enzymes responsible for the breakdown of carbohydrates. These GHs have known or predicted specificities for human glycans, specifically those found in mucins. We chose C. perfringens and S. pneumoniae as model systems to study these enzymes due to their large complements of GHs, many of which are known virulence factors. The objectives are to probe the key features of the GHs from these two different kinds of bacteria that inhabit similar human niches and to study catalysis, modularity and overall enzyme structure. This work uses a multidisciplinary approach and provides molecular level insight into the S. pneumoniae and C. perfringens host-pathogen interaction.
Graduate

碩士
國立臺南大學
生物科技學系碩士班
106
This thesis comprises two parts to study two proteins responsible for redox reactions: The first part describes the biotransformation of a commercially available rude extract of soy isoflavones by recombinant Escherichia coli expressing a tyrosinase from Bacillus megaterium. The original crude soy extracts contains significant amounts of the isoflavone glycosides daidzin and genistin. After biotransformation, two major products were isolated and identified as 3'-hydroxydaidzin and 3'-hydroxygenistin, respectively. The free radical scavenging activities of the two 3'-hydroxyisoflavone glycosides were, respectively, higher than the activity of their precursors. This is the first demonstration of the bio-production and potential antioxidant applications of both 3'-hydroxydaidzin and 3'-hydroxygenistin. In the second part, the unicellular organism Chlamydomonas reinhardtii was used to analyze the function of nucleoredoxin (NRX) genes. There are three members in the NRX family in Chlamydomonas, but their physiological roles and functional differentiation remains unclear. An exogenous DNA insertion was mapped to the 3’UTR of the NRX2 gene in a flagellum-defected mutant HKCW19, but RT-qPCR showed that the NRX2 transcript was not affected. In another nrx2-1 mutant, the NRX2 expression was abolished, but it contained normal flagella and can swim. Therefore, the disruption of NRX2 does not affect flagellar growth. The nrx2-1 mutant contained a higher ratio of large cells and was more sensitive to H2O2 treatment. The absence of NRX2 does not affect the transcript and protein levels of catalase (CAT), an enzyme responsible for H2O2 clearance. However, the activity of CAT is severely reduced in nrx2-1. Interestingly, loss of NRX2 caused the increase of NRX3 mRNA expression while NRX1 was not affected. Both NRX2 and NRX3 mRNA were induced after pH-shock treatment while NRX1 was not. These expression analyses suggest that NRX2 and NRX3 share similar functions or are involved in the same pathway.

碩士
國立臺北科技大學
有機高分子研究所
105
The β-glucosidase identify from Neocallimastix frontalis (NfrBGS), belongs to the glycoside hydrolase family three (GH3), and has 769 amino acids with the molecular weight of 84.246 kDa, which hydrolyzes the cellobiose into glucose. The enzymatic products were analyzed by using liquid chromatography with refractive index detector (LC-RI). The reaction time was 10 minutes to 48 hours in pH 6.0, 0.1M sodium phosphate buffer solution at 40°C. It was found that cellobiose had been hydrolyzed to glucose after 16 hours of reaction time. Based on the amount of glucose from the LC-RI product, we analyzed the Km was 4.62±0.23 mg·ml-1 and the kcat was 1275.87±33.97 s-1. In addition, we built a structural model of NfrBGS based on the results from sequence alignment analyses analyzed by BLAST (Basic Local Alignment Search Tool), using β-glucosidase of Trichoderma reesei QM9414 (pdb: 3ZYZ) as a model. The modeled NfrBGS structure is composed mainly of three domains, two derived catalytic modules (Glyco_Hydro_3: 68-342 , Glyco_Hydro_3C : 343-570 ) and an acceptor module (sigma70 : 704-765 ). Homologous structural modeling of NfrBGS suggested that Glu446 as proton acceptor and Asp244 as nucleophilic amino acid are at the cleft site located between the two derived catalytic modules, which are the putative substrate-binding site and catalytic center. We also discussed the hydrolysis of lactose by NfrBGS .

The human gut is home to a significant number of microorganisms including the dominant symbiont Bacteroides thetaiotaomicron. This microbe is predicted to possess an array of glycoside hydrolases, majority of which are involved in starch utilization. Presented here is a comparative functional analysis of two alpha-glucosidases, Family 31 Glycoside Hydrolases from Bacteroides thetaiotaomicron. Enzymatic kinetics revealed these enzymes both preferentially cleave alpha-(1,6) linkage in comparison to the predicted alpha-(1,4) and favour maltose derived substrates of longer length.

碩士
東海大學
食品科學系
105
Diabetes is associated with deficiencies in insulin secretion or activity. Excess reactive oxygen species are building up with chronic hyperglycemia, and are subsequently contributing to oxidative stress and complications. Antioxidants have important roles in disorders involving oxidative stress. Glycosidases are not only involved in human digestion, but also affect the occurring of many metabolic diseases such as diabetes. The most important glycosidases in human digest system are α-amylase and α-glucosidase. Lonicera japonica Thunb., is an herbaceous plant of Caprifoliaceae family. Previous studies have demonstrated that Lonicera japonica Thunb. has antibacterial activity, antiviral activity, anti-inflammatory, neuroprotective, antioxidative and hepatoprotective activities. However, possible roles of Lonicera japonica Thunb. in hypoglycemic regulation have not yet been explored. In the present study, glycosidases inhibitions of Lonicera japonica Thunb. were investigated. Lonicera japonica Thunb. was extracted with methanol first, and then methanol extracts were extracted by water with n-hexane, ethyl acetate and n-butanol successively. These extracts were analysed for their potential health promoting properties including their inhibitory effect on starch digestive enzymes and antioxidant activities. Ethyl acetate extract of Lonicera japonica Thunb. showed better antioxidant and inhibitory properties against α-glucosidase. The extract was further separated successively by Amberlite XAD-7, Comosil 75C18-OPN and reversed HPLC to obtain five components. Due to the contents of isolated components were too little to get enough spectral information for 1H-NMR, by using liquid chromatography tandem-mass spectrometry( LC-MS/MS), the components from ethyl acetate fraction were identified to be three caffeoylquinic acids: 5-O-caffeoylquinic acid (1), 3,5-di-O-caffeoylquinic acid (2) and 4,5-di-O-caffeoylquinic acid (3). Based on antioxidant test and glycosidase inhibition, LC-MS / MS structure identification and reference from literature, the active substances of Lonicera japonica Thunb. for antioxidation and glycosidase inhibition may be mainly derived from polyphenolic compounds of the caffeoylquinic acids.