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1
Charuel, Jean-Luc. "Amylases et tumeurs." Paris 5, 1991. http://www.theses.fr/1991PA05P082.
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Ramachandran, Nivetha. "Development of improved [alpha]-amylases /." Link to the online version, 2005. http://hdl.handle.net/10019.1/1102.
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Ramachandran, Nivetha. "Development of improved α-amylases." Thesis, Stellenbosch : University of Stellenbosch, 2005. http://hdl.handle.net/10019.1/1102.
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Thesis (DSc (Microbiology))--University of Stellenbosch, 2005.The technological advancement of modern human civilisation has, until recently, depended on extensive exploitation of fossil fuels, such as oil, coal and gas, as sources of energy. Over the last few decades, greater efforts have been made to economise on the use of these nonrenewable energy resources, and to reduce the environmental pollution caused by their consumption. In a quest for new sources of energy that will be compatible with a more sustainable world economy, increased emphasis has been place on researching and developing alternative sources of energy that are renewable and safer for the environment. Fuel ethanol, which has a higher octane rating than gasoline, makes up approximately two-thirds of the world’s total annual ethanol production. Uncertainty surrounding the longterm sustainability of fuel ethanol as an energy source has prompted consideration for the use of bioethanol (ethanol from biomass) as an energy source. Factors compromising the continued availability of fuel ethanol as an energy source include the inevitable exhaustion of the world’s fossil oil resources, a possible interruption in oil supply caused by political interference, the superior net performance of biofuel ethanol in comparison to gasoline, and a significant reduction in pollution levels. It is to be expected that the demand for inexpensive, renewable substrates and cost-effective ethanol production processes will become increasingly urgent. Plant biomass (including so-called ‘energy crops’, agricultural surplus products, and waste material) is the only foreseeable sustainable source of fuel ethanol because it is relatively low in cost and in plentiful supply. The principal impediment to more widespread utilisation of this important resource is the general absence of low cost technology for overcoming the difficulties of degrading the recalcitrant polysaccharides in plant biomass to fermentable sugars from ethanol can be produced. A promising strategy for dealing with this obstacle involves the genetic modification of Saccharomyces cerevisiae yeast strains for use in an integrated process, known as direct microbial conversion (DMC) or consolidated bioprocessing (CBP). This integrated process differs from the earlier strategies of SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation, in which enzymes from external sources are used) in that the production of polysaccharide-degrading enzymes, the hydrolysis of biomass and the fermentation of the resulting sugars to ethanol all take place in a single process by means of a polysaccharidefermenting yeast strain. The CBP strategy offers a substantial reduction in cost if S. cerevisiae strains can be developed that possess the required combination of substrate utilisation and product formation properties. S. cerevisiae strains with the ability to efficiently utilise polysaccharides such as starch for the production of high ethanol yields have not been described to date. However, significant progress towards the development of such amylolytic strains has been made over the past decade. With the aim of developing an efficient starch-degrading, high ethanol-yielding yeast strain, our laboratory has expressed a wide variety of heterologous amylase-encoding genes in S. cerevisiae. This study forms part of a large research programme aimed at improving these amylolytic ‘prototype’ strains of S. cerevisiae. More specifically, this study investigated the LKA1- and LKA2-encoded α-amylases (Lka1p and Lka2p) from the yeast Lipomyces kononenkoae. These α-amylases belong to the family of glycosyl hydrolases (EC 3.2.1.1) and are considered to be two of the most efficient raw-starch-degrading enzymes. Lka1p functions primarily on the α-1,4 linkages of starch, but is also active on the α-1,6 linkages. In addition, it is capable of degrading pullulan. Lka2p acts on the α-1,4 linkages. The purpose of this study was two-fold. The first goal was to characterise the molecular structure of Lka1p and Lka2p in order to better understand the structure-function relationships and role of specific amino acids in protein function with the aim of improving their substrate specificity in raw starch hydrolysis. The second aim was to determine the effect of yeast cell flocculence on the efficiency of starch fermentation, the possible development of high-flocculating, LKA1-expressing S. cerevisiae strains as ‘whole-cell biocatalysts’, and the production of high yields of ethanol from raw starch. In order to understand the structure-function relationships in Lka1p and Lka2p, standard computational and bioinformatics techniques were used to analyse the primary structure. On the basis of the primary structure and the prediction of the secondary structure, an N-terminal region (1-132 amino acids) was identified in Lka1p, the truncation of which led to the loss of raw starch adsorption and also rendered the protein less thermostable. Lka1p and Lka2p share a similar catalytic TIM barrel, consisting of four highly conserved regions previously observed in other α-amylase members. Furthermore, the unique Q414 of Lka1p located in the catalytic domain in place of the invariant H296 (TAKA amylase), which offers transition state stabilisation in α-amylases, was found to be involved in the substrate specificity of Lka1p. Mutational analysis of Q414 performed in the current study provides a basis for understanding the various properties of Lka1p in relation to the structural differences observed in this molecule. Knowing which molecular features of Lka1p contribute to its biochemical properties provides us with the potential to expand the substrate specificity properties of this α-amylase towards more effective processing of its starch and related substrates. In attempting to develop ‘whole-cell biocatalysts’, the yeast’s capacity for flocculation was used to improve raw starch hydrolysis by S. cerevisiae expressing LKA1. It was evident that the flocculent cells exhibited physicochemical properties that led to a better interaction with the starch matrix. This, in turn, led to a decrease in the time interval for interaction between the enzyme and the substrate, thus facilitating faster substrate degradation in flocculent cells. The use of flocculation serves as a promising strategy to best exploit the expression of LKA1 in S. cerevisiae for raw starch hydrolysis. This thesis describes the approaches taken to investigate the molecular features involved in the function of the L. kononenkoae α-amylases, and to improve their properties for the efficient hydrolysis of raw starch. This study contributes to the development of amylolytic S. cerevisiae strains for their potential use in single-step, cost-effective production of fuel ethanol from inexpensive starch-rich materials.
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Nahoum, Virginie. "Alpha-amylases de mammifères et d'insectes, relation structure/fonction." Aix-Marseille 3, 2000. http://www.theses.fr/2000AIX30010.
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Les -amylases (-1,4-glucan-4-glucanohydrolase, ec 3. 2. 1. 1) catalysent l'hydrolyse des liaisons -(1,4) glycosidiques des composes de l'amidon. Dans cette these je presente l'analyse structurale de deux classes d'enzymes, les -amylases de mammiferes et d'insectes. Malgre l'abondance de donnees biochimiques sur ces complexes, les complexes entre l'-amylase humaine et des oligosaccharides avaient echappe a toute caracterisation structurale. Dans cette etude les structures cristallographiques de l'-amylase pancreatique humaine (hpa) en complexe avec des inhibiteurs naturels ont ete resolues. Le tetrasaccharide acarbose et un pseudo-pentasccharide appartenant a la famille des trestatines ont montre des densites electroniques continues similaires correspondant a un pentasaccharide, occupant les sous-sites 3 a +2 de l'enzyme, et probablement resultant d'une transglycosylation. La fixation du cur acarviosine lie a une unite glucose aux sous-sites 1 a +2 apparait etre une etape critique du processus d'interaction entre les -amylases et les inhibiteurs derives des trestatines. Deux formes cristallines du complexe entre hpa et un inhibiteur proteique (-ai) issu du grain de haricot phaseolus vulgaris, obtenues a differents ph, ont ete resolues. La boucle flexible, typique des -amylases de mammiferes, montre deux conformations differentes, suggerant une sensibilite de cette boucle au ph. Une information structurale est procuree pour le residu arg 74 qui n'etait pas visible dans les precedentes analyses structurales. L'-amylase de la larve de tenebrio molitor (tma) a ete cristallisee en complexe avec l'inhibiteur -ai. Tma presente la structure commune aux -amylases, de grandes differences avec les -amylases de mammiferes se produisant dans les boucles. Malgre ces differences dans les boucles impliquees dans l'interaction, l'inhibiteur du haricot est capable d'inhiber a la fois les -amylases de mammiferes et d'insectes. La boucle flexible des -amylases de mammiferes, tronquee dans l'-amylase d'insecte montre une conformation differente dans les structures de tma native et de tma en complexe avec -ai. La modelisation de l'attachement des isoformes de -ai dans le site actif de differentes -amylases jette un regard sur la specificite de ces inhibiteurs.
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Talamond, Pascale. "Etude de l' α-amylase de lactobacillus fermentum : purification, caractérisation et propriétés. Comparaison avec les α-amylases de Lb. Plantarum et Lb. Manihotivorans." Aix-Marseille 3, 2002. http://www.theses.fr/2002AIX30087.
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Une nouvelle a-amylase de Lb. Fermentum (FERMENTA) obtenue par fermentation à partir de céréales a été purifiée, étudiée au niveau structural et fonctionnel et comparée avec deux autres a-amylases de Lb. Plantarum (PLANTAA) et de Lb. Manihotivorans (MANIHOA). Les trois a-amylases présentent une masse moléculaire voisinant les 100 kDa et des pI du même ordre de grandeur (3,5±0,3). Comme PLANTAA et MANIHOA, la structure de FERMENTA contient des séquences répétées en C-terminal. Les propriétés physico-chimiques (pH 5 et température 40ʿC) ont été déterminées et l'étude cinétique avec l'amylose pour substrat et l'acarbose pour inhibiteur a été effectuée. L'inhibition non-compétitive mixte pour FERMENTA (K1i = 5,3 æM et L1i = 1,7 æM) et incompétitive pour PLANTAA et MANIHOA révèlent que l'acarbose est un puissant inhibiteur pour ces trois a-amylases. Ces études cinétiques montrent l'existence d'un site secondaire de fixation des carbohydrates distinct du site actif et nécessaire pour l'hydrolyse des substrats longsA new a-amylase from Lactobacillus fermentum (FERMENTA) was purified. The structural and functional characteristics were studied and compared with Lb. Plantarum (PLANTAA) and Lb. Manihotivorans (MANIHOA) a-amylases. FERMENTA molecular mass (100 kDa) is in the same range than those determined for PLANTAA and MANIHOA. Structure of FERMENTA indicates that the sequence contains two equal parts with the C-terminal repeats. Isoelectric point of the three a-amylases are about the same (3. 5). The functional properties of FERMENTA are studied : optimal pH (5. 0) and temperature (40ʿC). Kinetics of the three a-amylases with amylose and acarbose were carried out. Inhibition of FERMENTA is of mixed noncompetitive type while the inhibition of PLANTAA and MANIHOA is of uncompetitive type. Whatever the inhibition type, acarbose is a strong inhibitor of these amylases. These results indicate that they contain, in addition to the active site, a soluble carbohydrate (substrate or product) binding site
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Rumbak, Elaine. "The cloning and characterization of an α-amylase and a branching enzyme from Butyrivibrio fibrisolvens H17c and their expression in Escherichia coli." Doctoral thesis, University of Cape Town, 1991. http://hdl.handle.net/11427/22555.
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Bibliography: pages 145-172.Butyrivibrio fibrisolvens H17c is an important anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to establish a genebank of B. fibrisolvens H17c DNA in E.coli and to isolate and characterize genes encoding enzymes involved in the degradation of the major plant polysaccharides. A library of chromosomal DNA fragments from B. fibrisolvens was established in the E. coli-Bacillus subtilis shuttle vector pEBl. The library was screened for the expression of B. fibrisolvens genes in E. coli. E. coli clones expressing glutamine synthetase, carboxymethylcellulase, β-glucosidase and amylolytic-type activities were isolated. A gene (amyA) expressing amylolytic activity and encoding an α-amylase was located on a 5.0 kb DNA fragment and expressed from its own promoter in E. coli. It was shown that more than 86% of the amylolytic actvity was located in the periplasm of the E.coli host and TnphoA mutagenesis indicated the presence of a functional signal peptide. The nucleotide sequence of amyA was determined and encoded a protein of 976 amino acids with a calculated Mr of 106,964. High sequence similarity was demonstrated between the B. fibrisolvens α-amylase and other α-amylases in the three highly conserved regions which constitute the active centre. Conserved regions were all located in the N-terminal half of the B. fibrisolvens amylase and no homology to other amylases was detected for the remainder of the protein. Approximately 40% of the C-terminal region of the protein could be deleted without loss of enzymatic activity. The B. fibrisolvens α-amylase degraded amylose, amylopectin and soluble starch with maltotriose as the major initial hydrolysis product. A gene (glgB) encoding a glycogen branching enzyme, the activity of which produced clearing on starch azure plates, was isolated. The glgB gene was expressed from its own promoter in the host E.coli and encoded a protein of 639 amino acids with a calculated Mr of 73,875. The deduced amino acid sequence of the glgB gene showed high sequence homology (46-50%) to other branching enzymes. The branching enzyme was purified to homogeneity and the properties of the purified enzyme were investigated. Optimal activity of the branching enzyme was at pH 7.2 and 37°C. The branching enzyme was shown to transfer chains of between 5 to 10 glucose units using α-1,4 glucans as substrates, and to stimulate the "de novo" synthesis of a polysaccharide similar to glycogen.
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Cottaz, Sylvain. "Synthèses chimiques et enzymatiques de maltodextrines modifiées : étude du centre actif de la cyclodextrine-glucosyltransférase." Grenoble 1, 1989. http://www.theses.fr/1989GRE10136.
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Des enzymes de la super-famille des amylases peuvent etre etudiees par la methode aux analogues de substrat. Les thiomaltodextrines lineaires et cycliques ont ete obtenues par synthese chimique. Les syntheses enzymatiques de nouveaux substrats specifiques des alpha-amylases ont ete realisees soit par esterification de maltodextrines, soit par reaction de couplage avec une cyclodextrine modifiee catalysee par la cyclodextrine-glucosyltransferase (cgtase). L'utilisation par la cgtase de fluorures de maltosyle modifies a apporte de nouvelles informations sur la specificite de son centre actif, et permis la synthese de cyclodextrines modifiees
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Pan, Oscar Chi-Chien. "In search of peptide inhibitors for alpha-amylases." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0027/MQ51441.pdf.
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LECOMMANDEUR, DIDIER. "Etude moleculaire des alpha-amylases de differentes cereales." Paris 6, 1989. http://www.theses.fr/1989PA066295.
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Une etude comparative des alpha-amylases de grains en germination de plusieurs cereales a ete effectuee. Un microdosage specifique utilise comme substrat le benzilidene p-nitrophenyl maltoheptaoside. Les alpha-amylases de mais, sorgho, avoine et riz ont ete purifiees par precipitation au glycogene et chromatographie d'interaction hydrophobe, les deux isoformes de l'alpha-amylase d'orge parchromatofocalisation et chromatographie d'affinite. Une caracterisation immunochimique, utilisant les reactions croisees avec des anticorps polyclonaux anti-alpha-amylase 1 et anti-alpha-amylase 2 d'orge, montre que chez l'avoine et le sorgho deux antigenes sont presents, et trois chez le mais. Leurs tailles sont voisines (43000 a 47000). Les alpha-amylases d'orge, de sorgho et d'avoine ne semblent pas glycosylees. Une forme de l'alpha-amylase de mais est o-glycosylee. La n-glycosylation de l'alpha-amylase de riz produit, a partir de deux polypeptides de taille differente, des glycoproteines de meme taille. Sept anticorps monoclonaux anti-alpha-amylase 1 d'orge ont ete produits et caracterises. Six sont specifiques de cette forme, le spetieme reconnait egalement la forme 2. Certains anticorps reconnaissent des alpha-amylases de riz, mais ou sorgho
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Valantin-Rollet, Carole. "Etude du non parallélisme de la composition en 4 hydrolases digestives du pancréas de rat et de sa sécrétion : influence de la synthèse, du "turnover", du transport et de l'excrétion : effets de l'âge et d'une malnutrition protéique (...) suivie." Dijon, 1985. http://www.theses.fr/1985DIJOS015.
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Pohu, Anne. "Formation des amidons résistants au cours des traitements thermiques et enzymatiques." Nantes, 2002. http://www.theses.fr/2002NANT2067.
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Un nouveau type d'amidon résistant fabriqué par déramification de maltodextrines en solutions concentrées a été étudié. Cette étude a porté essentiellement sur l'organisation structurale de ces amidons résistants, les mécanismes impliqués dans leur formation et leur hydrolyse partielle par les a-amylases. Au cours du procédé de fabrication, deux structures bien distinctes apparaissent successivement. La première se présente sous forme de réseaux peu denses et la seconde sous forme d'agrégats de cristaux de type A. Ces deux structures ont des comportements à l'hydrolyse par les a-amylases très distincts avec une très faible résistance pour les réseaux de type B et une forte résistance pour les agrégats de cristaux de type A. La résistance de ces derniers a été attribuée à la morphologie particulièrement dense et compacte résultant de l'agrégation de plaquettes cristallines élémentaires et offrant peu de sites accessibles aux amylases au contraire des réseaux de type BA new type of resistant starches obtained after debranching in concentrated maltodextrin solutions was studied. This study particularly focused on the structural organisation of these resistant starches, the processes involved during their synthesis and their partial hydrolysis by a-amylases. Two distinct structural states appeared at different stages of the reaction. The first one is made out of a loose fibre network and the second one of type A crystal aggregates. These two structures respond very differently when tested in a-amylases hydrolysis assays : type B structures do not resist well to hydrolysis while the opposite is true for the type A crystal aggregates. We attributed aggregates resistance to enzyme hydrolysis to their particularly dense and compact morphology, resulting from the aggregation of elementary crystalline plates. The resulting structure tends to present few accessible sites to amylases, whereas type B network tend to expose more carbon chains to enzymes
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Lauro, Marianna. "[Alpha]-amylolysis of barley starch /." Espoo [Finland] : Technical Research Centre of Finland, 2001. http://www.vtt.fi/inf/pdf/publications/2001/P433.pdf.
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Carter, Meredith Diane. "Genome-level studies on late maturity alpha amylase and boron tolerance in wheat." Thesis, Carter, Meredith Diane (2006) Genome-level studies on late maturity alpha amylase and boron tolerance in wheat. PhD thesis, Murdoch University, 2006. https://researchrepository.murdoch.edu.au/id/eprint/514/.
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Under certain environmental conditions, some varieties of wheat synthesize the enzyme alpha amylase late in grain ripening, even in the absence of rain or sprouting. The resulting grain has a sound appearance but can be unsuitable for end-product applications due to the presence of late maturity alpha amylase (LMA) activity. Reduction of LMA and the development of cultivars tolerant to boron toxic soils are high priority traits in the WA wheat breeding program and the use of molecular markers closely linked to these traits for marker assisted selection (MAS) is highly desirable. The aims of this study were to take a genomics approach to provide detailed structural information for the region on wheat chromosome 7BL in which quantitative trait loci (QTLs) for LMA and boron tolerance (Bo1) have been mapped. Once the structure had been determined, this then laid the foundation for further studies to investigate the function of putative candidate genes identified within this region. The research involved the use of bioinformatic tools and rice/wheat synteny to investigate the structure of this chromosome region, followed by the use of molecular probes to isolate genomic DNA clones (BAC clones) corresponding to this region.
A two-step bioinformatics strategy was used, involving (1) alignment of portions of the wheat and rice genomes, to identify rice genomic regions syntenic to wheat group 7L and (2) selection of candidate genes from those regions of the rice genome. The selected candidate genes included an anion transporter, as a candidate gene for boron tolerance, and GAMYB-like genes, as candidate genes for LMA. The GAMYB class of transcription factors identified were of particular interest because of published literature indicating its importance in controlling alpha-amylase levels in cereal grains. The key phenotype of interest in this thesis is LMA and different levels of expression of ±-amylase are a key feature of this phenotype.
Molecular markers and candidate genes were then used to screen two BAC libraries, one derived from the French cultivar, 'Renan' and the other derived from Aegilops tauschii (the source of the D genome of wheat). About 300 BAC clones corresponding to the chromosome region of interest were obtained. Of these, 8 BAC clones (6 chosen through hybridization to a GAMYB-like probe, and 2 from wheat ESTs anchored to the rice genome) were selected for sequencing, allowing for the development of new microsatellite and single-nucleotide polymorphism (SNP) markers and for the discovery of novel transposable elements that provide a rich source of polymorphism for the development of additional markers. Novel microsatellite and SNP markers that were identified from the BAC clone sequence were mapped on the Cranbrook/Halberd doubled haploid (DH) mapping population. Markers were located to chromosomes 7AL, 7BL and 7DL. New markers derived from the BAC sequence information were used to anchor the BAC clones to the genetic map and develop a framework physical-genetic map. An automated annotation pipeline has been established and was used to annotate selected contigs of the sequenced BAC clones.
A new marker assisted selection strategy, termed Multiplex Trait Signature (MuTs) analysis, was developed and tested on 39 wheat cultivars of known LMA phenotype. MuTs provides a graphical genotype of individuals for a particular chromosomal region and is a convenient tool for interrogating genetic similarity in the individuals surveyed. Based on assays of 22 markers (12 spanning the LMA QTL on chromosome 7BL and 10 spanning the LMA QTL on chromosome 3BS) on these 39 wheat cultivars, it was found that the varieties can be grouped according to pedigree and provides a tool for interpreting LMA status for a variety. Validation of the 7BL LMA and boron tolerance (Bo1) QTL regions was achieved using a targeted mapping approach using the doubled haploid population Pastor/RAC891 using published molecular markers and markers developed in this thesis. The main outcome of this study is that the genomic organisation of this region on chromosome 7BL is complex, and that the identification of candidate genes in wheat controlling 1) tolerance of cultivars to boron toxic soils and 2) pathways regulating the expression of LMA, is likely to involve the interplay of a network of regulatory genes.
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Carter, Meredith Diane. "Genome-level studies on late maturity alpha amylase and boron tolerance in wheat." Carter, Meredith Diane (2006) Genome-level studies on late maturity alpha amylase and boron tolerance in wheat. PhD thesis, Murdoch University, 2006. http://researchrepository.murdoch.edu.au/514/.
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Under certain environmental conditions, some varieties of wheat synthesize the enzyme alpha amylase late in grain ripening, even in the absence of rain or sprouting. The resulting grain has a sound appearance but can be unsuitable for end-product applications due to the presence of late maturity alpha amylase (LMA) activity. Reduction of LMA and the development of cultivars tolerant to boron toxic soils are high priority traits in the WA wheat breeding program and the use of molecular markers closely linked to these traits for marker assisted selection (MAS) is highly desirable. The aims of this study were to take a genomics approach to provide detailed structural information for the region on wheat chromosome 7BL in which quantitative trait loci (QTLs) for LMA and boron tolerance (Bo1) have been mapped. Once the structure had been determined, this then laid the foundation for further studies to investigate the function of putative candidate genes identified within this region. The research involved the use of bioinformatic tools and rice/wheat synteny to investigate the structure of this chromosome region, followed by the use of molecular probes to isolate genomic DNA clones (BAC clones) corresponding to this region.
A two-step bioinformatics strategy was used, involving (1) alignment of portions of the wheat and rice genomes, to identify rice genomic regions syntenic to wheat group 7L and (2) selection of candidate genes from those regions of the rice genome. The selected candidate genes included an anion transporter, as a candidate gene for boron tolerance, and GAMYB-like genes, as candidate genes for LMA. The GAMYB class of transcription factors identified were of particular interest because of published literature indicating its importance in controlling alpha-amylase levels in cereal grains. The key phenotype of interest in this thesis is LMA and different levels of expression of ±-amylase are a key feature of this phenotype.
Molecular markers and candidate genes were then used to screen two BAC libraries, one derived from the French cultivar, 'Renan' and the other derived from Aegilops tauschii (the source of the D genome of wheat). About 300 BAC clones corresponding to the chromosome region of interest were obtained. Of these, 8 BAC clones (6 chosen through hybridization to a GAMYB-like probe, and 2 from wheat ESTs anchored to the rice genome) were selected for sequencing, allowing for the development of new microsatellite and single-nucleotide polymorphism (SNP) markers and for the discovery of novel transposable elements that provide a rich source of polymorphism for the development of additional markers. Novel microsatellite and SNP markers that were identified from the BAC clone sequence were mapped on the Cranbrook/Halberd doubled haploid (DH) mapping population. Markers were located to chromosomes 7AL, 7BL and 7DL. New markers derived from the BAC sequence information were used to anchor the BAC clones to the genetic map and develop a framework physical-genetic map. An automated annotation pipeline has been established and was used to annotate selected contigs of the sequenced BAC clones.
A new marker assisted selection strategy, termed Multiplex Trait Signature (MuTs) analysis, was developed and tested on 39 wheat cultivars of known LMA phenotype. MuTs provides a graphical genotype of individuals for a particular chromosomal region and is a convenient tool for interrogating genetic similarity in the individuals surveyed. Based on assays of 22 markers (12 spanning the LMA QTL on chromosome 7BL and 10 spanning the LMA QTL on chromosome 3BS) on these 39 wheat cultivars, it was found that the varieties can be grouped according to pedigree and provides a tool for interpreting LMA status for a variety. Validation of the 7BL LMA and boron tolerance (Bo1) QTL regions was achieved using a targeted mapping approach using the doubled haploid population Pastor/RAC891 using published molecular markers and markers developed in this thesis. The main outcome of this study is that the genomic organisation of this region on chromosome 7BL is complex, and that the identification of candidate genes in wheat controlling 1) tolerance of cultivars to boron toxic soils and 2) pathways regulating the expression of LMA, is likely to involve the interplay of a network of regulatory genes.
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Planchot, Véronique. "Alpha-amylases d'aspergillus fumigatus. Mecanismes d'action en phase heterogene." Nantes, 1993. http://www.theses.fr/1993NANT2056.
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L'alpha-amylolyse des substrats amylaces en phase heterogene a ete abordee selon une approche de type mecanistique, par un developpement prealable d'outils biochimiques. La demarche experimentale presentee dans ce memoire comporte deux volets: un suivi phenomenologique de l'hydrolyse tant par les enzymes que par les substrats, et une analyse des differentes etapes de l'alpha-amylolyse, plus particulierement l'adsorption et l'hydrolyse sensu stricto. La premiere partie a concerne la preparation de substrats references et la recherche d'une nouvelle source d'alpha-amylase efficace sur les amidons peu susceptibles a l'hydrolyse enzymatique (amidons de pomme de terre et de mais riche en amylose). La seconde partie consacree a l'alpha-amylolyse de substrats cristallins permet d'apprehender les phenomenes d'adsorption et d'hydrolyse sensu stricto sur des substrats references moins complexes que le grain d'amidon. L'importance de la cristallinite a ete cernee selon ses differentes composantes: type cristallin, morphologie et mode d'association des cristaux. Outre les methodes mises au point et les resultats experimentaux accumules, ce travail a conduit a proposer un mecanisme d'interaction entre une alpha-amylase et des substrats cristallins. Cette hypothese possede des utilisations potentielles dans les technologies de fermentation et la comprehension des processus digestifs
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Barrett, Tracey Elaine. "Structural studies on amylases and a cyanogenic beta-glucosidase." Thesis, University of York, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333724.
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Gibbs, Bernard F. "Characteristics of isolated and synthetic a-amylase inhibitors." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=24006.
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Effective inhibition of starch digestion in vivo may diminish glucose formation and absorption by the small intestine and increase the amount of undigested starch reaching the colon. The enzyme involved in the digestion of starch, alpha-amylase, has been identified and crystallized several years ago. Controversy exists as to whether effective inhibition can decrease starch digestion sufficiently to result in weight loss. The objective of this project is (a) to isolate and characterize alpha-amylase inhibitors from Phaseolus species (b) to synthesize and characterize known inhibitors in an effort to understand their mechanism of action.The supernatant from ground beans was subjected to reverse phase chromatography. The separated peaks were lyophilized and assayed for alpha-amylase inhibitory activity. The inhibitor with the highest activity (peak 6) was repurified and fully characterized. It was exposed to physiological amounts of endoproteases to check its stability.
A known inhibitor of alpha-amylase was synthesized and studied. Its binding constant has not been previously reported. (Abstract shortened by UMI.)
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Dauvillée, David. "Implication des enzymes de débranchement dans la biosynthèse de l'amylopectine." Lille 1, 2001. https://pepite-depot.univ-lille.fr/LIBRE/Th_Num/2001/50376-2001-1.pdf.
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L'implication des enzymes de débranchement, tout particulièrement de l'isoamylase, dans la biosynthèse de l'amylopectine semble désormais acquise. Une mutation au locus sta7 de l'algue verte unicellulaire chlamydomonas reinhardtii entraîne un arrêt total de la synthèse d'amidon et l'apparition d'un polysaccharide soluble hyperbranché : le phytoglycogène. A l'instar des végétaux supérieurs, la présence de phytoglycogène laisse présager la défectuosite d'une enzyme de débranchement. Dans ce travail, nous déterminons dans un premier temps quelles sont les enzymes responsables de la production du phytoglycogène. La découverte d'un nouveau locus (STA8) nous ouvre une nouvelle voie vers la compréhension du rôle de l'isoamylase dans la biosynthèse de l'amylopectine. Ce locus, lorsqu'il est muté, provoque non pas la disparition (comme c'est le cas chez le mutant sta7) mais une réduction de l'activité de l'enzyme. Ceci a pour conséquence une diminution de la quantité d'amidon accumulée avec encore une fois apparition concomitante de phytoglycogène. Cette diminution d'activité n'explique pas a elle seule le phénotype observé. Les analyses génétiques et biochimiques detaillées des deux mutations sta7 et sta8 révèlent la présence d'un complexe enzymatique de haute masse responsable de l'activité isoamylasique chez Chlamydomonas. La mutation sta8 entraîne un défaut d'architecture de ce complexe qui reste malgré tout actif alors que la mutation sta7 provoque sa disparition. La structure du complexe isoamylasique s'avère donc être un facteur déterminant pour la biosynthèse correcte de l'amylopectine.
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Gomes, Maria Regina Araujo. "Effect of high pressure treatment on polyphenoloxidases, papain and amylases." Thesis, University of Reading, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363398.
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Mrisho, Latifa Mbwana. "Production and characterization of alkaliphilic amylases from Bacillus halodurans Alk36." Master's thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/20089.
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Amylases are hydrolytic enzymes that cause the breakdown of starch and related polysaccharides to simple sugars. Amylases are applied in brewing, food, detergent and textile industries. Most commercial amylases are derived from fungi or bacteria. Bacterial amylases are desired for commercial use, due to their thermo-stability and faster production rates. Bacteria of the genus, Bacillus, are considered to be a good source of extracellular proteins because they have high growth rates and have a naturally high capacity for secretion of extracellular proteins. Bacillus halodurans Alk36 is an alkaliphilic, thermotolerant isolate that can grow over a wide pH and temperature range. Preliminary studies have shown that B. halodurans Alk36 can grown in EnBase® medium (at pH 8.5) containing starch as the carbon source, without the addition of a commercial amylase. The ability to grow on starch, in the absence of an external amylase, indicated that this strain produces endogenous alkaliphilic amylases, which may be exploited for a number of industrial applications. In the present study, the physiological and biochemical characterisation of B. halodurans Alk36 and its endogenous amylases were investigated.
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Bueso, Ucles Francisco Javier. "Antistaling properties of amylases, wheat gluten and CMC on corn tortilla." [College Station, Tex. : Texas A&M University, 2003. http://hdl.handle.net/1969.1/19.
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Thesis (Ph.D.)--Texas A&M University, 2003."Major Subject: Food Science and Technology" Title from author supplied metadata (record created on Jul. 18, 2005.) Vita. Abstract. Includes bibliographical references.
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Hudečková, Helena. "Studie možnosti využití odpadního pečiva k bioprodukci vybraných metabolitů." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2014. http://www.nusl.cz/ntk/nusl-217057.
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The aim of this diploma thesis was to study the possibility of using waste bread to bioproduction of selected metabolites. As bakery waste was used waste bread that came from coffee-house “Zastávka”. Bread was pre-treated by grinding into small particles and then it was made to form 15% w/v suspension, which was subjected to enzymatic hydrolysis. For the hydrolysis has been used the -amylase for liquefaction of the substrate and that was followed by a glucoamylase which sacharificated the substrate. There have been several methods of hydrolysis from which was chosen the optimal method for pre-treatment of the substrate prior to fermentation. The effectivity and a process of hydrolysis were determined spectrophotometrically by Somogyi-Nelson method. Final yields of glucose from hydrolysis were determined by HPLC method. Enzymatic hydrolysis was followed by fermentation, which was carried out in two ways, namely by adjusting the pH of the hydrolyzate to pH 5, and without pH adjustment. During the fermentation was carried out sampling in which was determined the content of glucose, glycerol and ethanol by HPLC method. The yeasts Saccharomyces cerevisiae were used for the fermentation which was performed at 30 °C. High yield of glucose was achieved by hydrolysis in two steps (70,28 gl-1), but for the fermentation was used mixed hydrolysis (second method of mixed hydrolysis) with yield 67,94 gl-1. High ethanol yield was achieved during fermentation without treatment pH, namely 31,5 gl-1.
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Chang, Siu-Chi 1962. "Influence of manganese on amylase gene expression." Thesis, The University of Arizona, 1989. http://hdl.handle.net/10150/291616.
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Previous studies have suggested that manganese (Mn) deficiency is associated with increased pancreatic amylase activity in rats. The present study investigated whether this increase in amylase activity is a result of increased pancreatic amylase messenger RNA (mRNA) levels. Weanling rats were fed a high carbohydrate diet containing either 39.6 ppm (control) or 0.5 ppm (deficient) manganese for 4 to 8 weeks. Manganese deficiency was confirmed by determining hepatic manganese content which was significantly lower in Mn-deficient rats than in the respective controls. Pancreatic RNA was size-fractionated on formaldehyde gels, and hybridized with 32P-labeled complementary DNAs (cDNA) for amylase and trypsinogen. Amylase mRNA levels were increased significantly in both 4 week (200%) and 8 week (250%) Mn-deficient rats when compared with their respective controls. In contrast, manganese deficiency was not associated with alternations in trypsinogen mRNA levels. Moreover, in vitro translation of the pancreatic mRNA indicated that manganese deficiency increased amylase mRNA levels supporting the Northern Blot analysis. Insulin and corticosterone, hormones known to increase amylase mRNA levels, were not affected by Mn-deficiency. These observations suggest that manganese may participate in the regulation of amylase gene expression.
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Ferey-Roux, Geneviève. "L'amylase pancréatique humaine. Purification des isoformes. Etudes structurales, immunologique et cinétique (inhibition par l'acarbose)." Aix-Marseille 3, 1998. http://www.theses.fr/1998AIX30097.
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Duong, Khanh Linh. "Amylases and Aspergillus Fumigatus cell wall synthesis: new roles for classical enzymes." Thesis, University of Iowa, 2010. https://ir.uiowa.edu/etd/796.
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Dainou, Ogoubi. "Polymorphisme et rôle physiologique de l'Amylase chez Drosophila melanogaster et espèces affinés." Paris 7, 1985. http://www.theses.fr/1985PA07F045.
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Ce travail aborde les problèmes de polymorphisme de l'amylase dans les populations naturelles des espèces de Drosophila appartenant au sous groupe melanoqaster et de la relation entre l'enzyme et l'utilisation métabolique de l'amidon par les drosophiles. L'étude des populations naturelles de D. Melanogaster a révélé que cette espèce est très polymorphe (12 allèles trouvés) et qu'elle présente des variations géographiques importantes du taux de polymorphisme… L'utilisation d!une souche amylase nulle (qui n'exprime pas cette enzyme) a permis d'étudier la nature et la fréquence des haplotypes, c'est-à-dire des allèles situés côte à côte sur le même chromosome. . Cette étude, effectuée sur environ 2000 chromosomes prélevés dans 4 populations naturelles d'Afrique et d'Amérique tropicale, a fourni des résultats concordants. Aucun déséquilibre zygotique n'a été observé, c'est-à-dire qu'il existe une panmixie des haplotypes. En revanche, il existe toujours un déséquilibre de liaison. Dans les différentes populations étudiées, ce sont les mêmes haplotypes qui sont en excès et les mêmes qui sont déficients. Le rôle physiologique de l'amylase a enfin été analysé, en relation avec l'activité de cette enzyme. Un test biologique mis au point sur les adultes a permis de mesurer l'utilisation digestive de l'amidon. Le mutant dépourvu d'enzyme est incapable d'utiliser ce produit comme source d'énergie. D. Melanoqaster, qui a une forte activité, utilise bien l'amidon comme nourriture. Au contraire, D. Sechellia , où l'activité est faible, utilise mal ce substrat. L'étude de D. Mauritiana a enfin mis en évidence un phénomène surprenant, à savoir une très grande différence entre les mâles et les femelles, phénomène qui n'existe pas chez les autres espèces. En définitive, l’amylase de D. M e 1 a n o g a s t e r est une protéine particulièrement intéressante à étudier en raison de sa structure génétique (duplication), de son haut degré de polymorphisme, de sa valeur phylogénétique et enfin de son rôle physiologique identifiable. Les caractéristiques des populations naturelles s'expliquent à la fois par des processus stochastiques (effet de fondation, dérive) et par la sélection naturelle, en particulier la proportion d'amidon existant dans les ressources.
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Sacco, Laís Postai [UNESP]. "Isolamento de bactérias produtoras de enzimas de interesse em processos biotecnológicos." Universidade Estadual Paulista (UNESP), 2013. http://hdl.handle.net/11449/94874.
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000737304.pdf: 599788 bytes, checksum: 3c0e4ba9cbd16debc1aaf8235e004268 (MD5)Os micro-organismos representam uma importante fonte de produtos naturais bioativos diversos. Esses organismos produzem enzimas que exercem papel importante em processos industriais com alto valor econômico agregado ou ambientalmente desejado. Apresentam uma série de vantagens em seu uso e produção. No presente trabalho foram utilizados consórcios degradadores de resíduos da biomassa lignocelulolítica, visando o isolamento e identificação de micro-organismos de interesse biotecnológico. De acordo com os resultados obtidos é possível verificar que apesar dos consórcios produzirem celulase, nenhum isolado produziu esse tipo de enzima. Foram isoladas bactérias produtoras de amilase, protease, lipase e solubilizam fosfato. Utilizando o sequenciamento do 16s rRNA foi identificado isolados pertencentes aos gêneros Burkholderia, Pandoraea, Pseudomonas, Bacillus, Asticcacaulis.
Microorganisms represent an important source of many bioactive natural products. These organisms produce enzymes that are important in industrial processes with high value aggregate economic or environmentally desired. The use of enzymes of microbial origin has been widely explored due to a series of industrial and environmental advantagens.In this present work, a degrading consortium of lignocellulose biomass residues was used aiming the isolation and identification of the bacteria that produce enzymes with biotechnological interest. According to the observed results it can be said that although the consortia produces cellulose, isolated bacteria do not then produced no such enzyme. Bacteria isolated produced amylase, protease, lipase and phosphatase. With the sequencing of the 16S rRNA it was possible to determine that the isolates belong to the genus Burkholderia, Pandoraea, Pseudomonas, Bacillus, Asticcacaulis.
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Sacco, Laís Postai. "Isolamento de bactérias produtoras de enzimas de interesse em processos biotecnológicos /." Jaboticabal, 2013. http://hdl.handle.net/11449/94874.
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Orientadora: Lucia Maria Carareto AlvesBanca: Tiago Santana Balbuena
Banca: Mariana Carina Frigieri
Resumo: Os micro-organismos representam uma importante fonte de produtos naturais bioativos diversos. Esses organismos produzem enzimas que exercem papel importante em processos industriais com alto valor econômico agregado ou ambientalmente desejado. Apresentam uma série de vantagens em seu uso e produção. No presente trabalho foram utilizados consórcios degradadores de resíduos da biomassa lignocelulolítica, visando o isolamento e identificação de micro-organismos de interesse biotecnológico. De acordo com os resultados obtidos é possível verificar que apesar dos consórcios produzirem celulase, nenhum isolado produziu esse tipo de enzima. Foram isoladas bactérias produtoras de amilase, protease, lipase e solubilizam fosfato. Utilizando o sequenciamento do 16s rRNA foi identificado isolados pertencentes aos gêneros Burkholderia, Pandoraea, Pseudomonas, Bacillus, Asticcacaulis.
Abstract: Microorganisms represent an important source of many bioactive natural products. These organisms produce enzymes that are important in industrial processes with high value aggregate economic or environmentally desired. The use of enzymes of microbial origin has been widely explored due to a series of industrial and environmental advantagens.In this present work, a degrading consortium of lignocellulose biomass residues was used aiming the isolation and identification of the bacteria that produce enzymes with biotechnological interest. According to the observed results it can be said that although the consortia produces cellulose, isolated bacteria do not then produced no such enzyme. Bacteria isolated produced amylase, protease, lipase and phosphatase. With the sequencing of the 16S rRNA it was possible to determine that the isolates belong to the genus Burkholderia, Pandoraea, Pseudomonas, Bacillus, Asticcacaulis.
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Lara, Erika Christina. "Lamb meat diet as influenced by microbial inoculant and amylolytic enzyme /." Jaboticabal, 2017. http://hdl.handle.net/11449/151167.
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Orientador: Ricardo Andrade ReisCoorientador: Juliana Duarte Messana
Banca: Marcia Helena Machado da Rocha Fernandes
Banca: Giovani Fiorentini
Banca: Thiago Fernandes Bernardes
Banca: Odilon Gomes Pereira
Resumo: Objetivou-se neste trabalho avaliar os efeitos de dietas contendo silagem inoculada com Lactobacillus plantarum e Bacillus subtilis e suplementadas ou não com amilase sobre a digestibilidade aparente, fermentação ruminal e síntese de proteína microbiana em carneiros, assim como o desempenho e qualidade de carne de cordeiros. Para tanto, dois estudos foram conduzidos, no quais os animais receberam um dos quatro tratamentos (dietas): 1) silagem de milho não inoculada sem adição de amilase na mistura total da ração (MTR); 2) silagem de milho não inoculada e amilase adicionada na MRT; 3) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis, sem adição de amilase; 4) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis e amilase adicionada na MRT. A enzima utilizada foi a amilase numa taxa de aplicação de 2 g de produto / kg de matéria seca (MS) da dieta (602 unidade dextrinizante (UD) / kg de MS da dieta). A suplementação com amilase em dietas contendo silagem não inoculada aumentou (P=0,045) o consumo de matéria seca dos carneiros quando comparados com aqueles alimentados com silagem não inoculada sem suplementação com amilase (1,311 vs. 1,066 g/d), mas não diferiu dos outros tratamentos. A digestibilidade aparente da MS, MO, PB, FDN e EB aumentou (P<0,01) nos carneiros alimentos com silagem inoculada ou suplementados com amilase, sem interações entre os tratamentos. Os animais alimentados com dietas contendo ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Objetivou-se neste trabalho avaliar os efeitos de dietas contendo silagem inoculada com Lactobacillus plantarum e Bacillus subtilis e suplementadas ou não com amilase sobre a digestibilidade aparente, fermentação ruminal e síntese de proteína microbiana em carneiros, assim como o desempenho e qualidade de carne de cordeiros. Para tanto, dois estudos foram conduzidos, no quais os animais receberam um dos quatro tratamentos (dietas): 1) silagem de milho não inoculada sem adição de amilase na mistura total da ração (MTR); 2) silagem de milho não inoculada e amilase adicionada na MRT; 3) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis, sem adição de amilase; 4) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis e amilase adicionada na MRT. A enzima utilizada foi a amilase numa taxa de aplicação de 2 g de produto / kg de matéria seca (MS) da dieta (602 unidade dextrinizante (UD) / kg de MS da dieta). A suplementação com amilase em dietas contendo silagem não inoculada aumentou (P=0,045) o consumo de matéria seca dos carneiros quando comparados com aqueles alimentados com silagem não inoculada sem suplementação com amilase (1,311 vs. 1,066 g/d), mas não diferiu dos outros tratamentos. A digestibilidade aparente da MS, MO, PB, FDN e EB aumentou (P<0,01) nos carneiros alimentos com silagem inoculada ou suplementados com amilase, sem interações entre os tratamentos. Os animais alimentados com dietas contendo silagem não inoculada e suplementados com amilase apresentaram alta proporção de ácido propiônico e baixa de ácido acético, e consequentemente baixa relação de aceitoc:propiônico. A síntese de proteína microbiana tendeu a ser maior (P=0,097) nos carneiros alimentados com silagem não inoculada e suplementados com a... (Complete abstract click electronic access below)
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Combes, Didier. "Influence du microenvironnement sur l'activité, la spécificité et la stabilité des enzymes : [thèse en partie soutenue sur un ensemble de travaux]." Toulouse 3, 1990. http://www.theses.fr/1990TOU30232.
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Le comportement de quatre enzymes, l'-amylase d'aspergillus oryzae, l'invertase, le lysozyme et la glucose oxydase a ete etudie du point de vue de leur activite, de leur specificite et de leur stabilite dans des conditions de microenvironnement se rapprochant des conditions industrielles: milieux concentres en substrats, milieux modeles concentres en additifs depresseurs de l'activite de l'eau ou bien en presence de limitations diffusionnelles. L'evolution de l'activite catalytique des enzymes dans ce type de milieu faiblement hydrate ne repond pas a une regle generale et depend a la fois de la nature de l'enzyme (sa conformation, ses sous-unites) et de la nature du milieu de reaction (structure du solvant et du substrat, diffusion et viscosite). Une grande variete d'additifs (polyols, halogenures alcalins, polyethylene glycols) augmente de maniere notable le temps de demi-vie des enzymes, vis-a-vis de la denaturation thermique. Cet effet protecteur correspond a un renforcement des interactions hydrophobes a l'interieur de la proteine. Il a ete montre que l'action de ces molecules est d'autant plus important que la surface de la proteine est hydrophile. La mesure de la balance hydrophile/hydrophobe de cette surface, par chromatographie sur colonne hydrophobe, est un parametre essentiel pour determiner l'efficacite d'un additif. Enfin, l'etude par spectroscopie raman de la dynamique de l'eau montre que les polyols ont une influence plus faible que les sels (halogenures alcalins) et, que les anions, par leur sphere d'hydratation induisent des modifications de structure du solvant plus importante que les cations. Le mecanisme de stabilisation des enzymes par modification de leur microenvironnement qui a ete etabli, prend en compte l'effet direct des additifs sur l'enzyme et leur effet indirect sur le degre d'association des molecules de solvant
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Desseaux, Véronique. "Structure et fonction des [alpha]-amylases de porc et d'orge : étude par protéolyse limitée et action des anticorps polyclonaux." Aix-Marseille 3, 1991. http://www.theses.fr/1991AIX30036.
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Les alpha-amylases catalysent l'hydrolyse des liaisons alpha-1,4 internes de l'amidon et de ses derives. Nous nous sommes attaches a comprendre les evenements moleculaires qui precedent (attachement, introduction dans le site catalytique), et suivent l'hydrolyse proprement dite. La structure de l'amylase de porc est connue a 2-2,9 a de resolution. Cette molecule (55 kd) comprend un corps central ab(tonneau) et un petit domaine c distinct. Le tonneau contient le site catalytique. Le domaine c participe avec (a) a un 2eme site de fixation du substrat (site surface). Notre but a ete d'etudier le role du site surface et du domaine c dans l'action amylolytique. Nous avons utilise 3 approches: 1) l'etude de la vitesse d'hydrolyse du p. Nitrophenylmaltoside en fonction du ph; 2) la proteolyse; 3) l'effet des anticorps sur l'activite enzymatique. Nos resultats sont en faveur de la participation de residus carboxylates au site catalytique. L'amylase d'orge est plus resistante a la proteolyse que l'amylase de porc. La coupure proteolytique ne se fait pas a la charniere comme attendu mais sur la derniere boucle du tonneau. La proteolyse provoque une perte d'activite et les fragments separes sont inactifs. Des anticorps anti-domaine c et anti-ppa ont ete purifies. Les anti-ppa inhibent l'hydrolyse des longs substrats tandis que l'hydrolyse du p. Nitrophenylmaltose n'est pas inhibee. Notre conclusion est que l'attaque proteolytique peut modifier l'environnement du site surface et la structure du centre actif. L'inhibition par les anticorps indique l'importance du site surface dans l'hydrolyse des chaines polyglucosidiques devant se fixer avant de se positionner correctement au centre actif. Cette etude montre egalement que les deux domaines aba et c sont fortement lies
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Tawil, Georges. "Compréhension et modélisation des mécanismes physico-chimiques impliqués lors de l'hydrolyse enzymatique de l'amidon natif." Nantes, 2011. http://www.theses.fr/2011NANT2003.
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Cette étude porte sur la compréhension des mécanismes physico-chimiques impliqués dans l’hydrolyse enzymatique de l’amidon natif. Deux nouvelles α-amylases d’origine Rhizomucor sp. (RA) et Anoxybacillus flavothermus (AFA), optimisées respectivement pour la production de bioéthanol et de sirop de glucose à basse température, ont été étudiées. Leur mode d’action a été déterminé à partir des transformations structurales de l’amidon au cours de l’hydrolyse et des cinétiques d’hydrolyse propres aux deux enzymes. Les paramètres limitant l’hydrolyse totale ont aussi été déterminés. La très grande efficacité de RA et AFA pour l’hydrolyse de l’amidon concentré (31 %) a été mise en évidence. RA présente de plus une grande aptitude à hydrolyser préférentiellement les domaines cristallins et AFA des vitesses d’hydrolyses particulièrement élevées. Le taux d’hydrolyse final est dépendant de la concentration pour RA. L’action de AFA est limitée par la baisse rapide de son activité. Remarquablement RA ne libère que du glucose ce qui la rend très performante pour la fabrication de bioéthanol alors que AFA libère des oligomères de DP variable. La forte baisse de la structure cristalline native de type A au cours des premières étapes de l’hydrolyse s’accompagne d’une forte diminution de l’amylopectine. Dans le même temps les complexes amylose-lipides présents dans l’amidon sont résistant à l’hydrolyse. Dans les étapes finales d’hydrolyse, les fragments d’amylose libérés recristallisent en structures de type B qui provoquent l’arrêt de l’hydrolyse. Les mécanismes d’hydrolyse des différents niveaux de structure du grain sont discutés pour ces enzymes au comportement exceptionnelThe main purpose of this study was to elucidate the physico-chemical mechanisms involved in enzymatic hydrolysis of concentrated raw starch. Two new α-amylases from Rhizomucor sp. (RA) and Anoxybacillus flavothermus (AFA) optimized for bioethanol and low temperature glucose syrup production respectively, were studied in detail. Their mode of action was determined by monitoring the change in the starch structure during hydrolysis and also from the kinetics curves in different conditions. The limiting factors for incomplete hydrolysis were also determined. RA and AFA hydrolyze very efficiently concentrated suspensions of native starch (31%). RA has a remarkable ability to degrade preferentially the crystalline domains in maize starch, while AFA is characterized by a very high initial velocity. Final hydrolysis rate is dependent on starch concentration for RA, while the action of AFA is more limited by its rapid loss of activity. On contrary to AFA which releases a broad range of oligomers, RA yields only glucose. This behaviour makes it well suited to bioethanol production. The decrease of the native A-type crystalline structure observed during the first stages of hydrolysis is in agreement with the large decrease of the amylopectin fraction. At the same time, the amylose-lipid complex present in maize starch is more resistant to hydrolysis and hydrolyzed in further stages releasing amylose fragments. Then, these fragments rearrange into very resistant Btype structure which prevent from a complete hydrolysis. The mechanisms involved in the hydrolysis of the different levels of structure present in the starch granule are discussed for these two original enzymes
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Justice, Jill Diane 1963. "Effects of diet on amylase content and synthesis in cultured rat acinar cells." Thesis, The University of Arizona, 1989. http://hdl.handle.net/10150/276962.
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To study adaptation of pancreatic amylase to diet, an affinity adsorbent, alpha-GHI-AH-Sepharose 4B, was used to determine amylase synthesis in cultured pancreatic acinar cells. This adsorbent exhibited a consistent binding capacity and was specific for amylase. Acinar cells from rats fed high fat (HF) or carbohydrate (HC) diets for 7 d were cultured 1-48 h in serum-free medium. Amylase activity remained significantly higher in HC cells than in HF cells through 24 h in culture, despite its decrease with time in culture. The relative synthesis of amylase (3H-phe amylase/3H-phe total protein x 100) was significantly higher in HC than in HF cells at isolation and remained higher during culture. These results demonstrate that this affinity adsorbent can be used to determine amylase synthesis and suggest that the effect of diet on amylase activity and relative synthesis persists in cultured pancreatic acinar cells.
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Mishra, Ravi Shankar. "Amylases From A Thermophilic Fungus Thermomyces Lanuginosus Iisc 91 :Their Purification And Properties." Thesis, Indian Institute of Science, 1994. http://hdl.handle.net/2005/90.
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A knowledge of molecular properties and structure of heat-stable enzymes is important for the understanding of basic principles governing thermo stability of proteins and evolution of life at high temperatures. Information on functional characteristics of thermo stable enzymes is necessary also for improving existing biotechnologies and developing new ones. Because of these reasons enzymes from thermophilic organisms are being exploited. In this context, amylolytic enzymes represent a useful choice for investigation from both basic and applied points of view.
a-Amylase and glucoamylase hydrolyse starch into oligosaccharides and glucose, respectively. In the present study a thermophilic fungus, Thermomyces lanuginosus, was selected as a source of thermostable starch-degrading enzymes. The main objectives of this research were to understand the physicochemical properties, mechanism of starch utilization by T. lanuginosus and effect of heat, on amylo1ytic enzymes.
Purification of amylolytic enzymes
A strain of T, lanuginosus, IlSc 91, isolated from a manure heap in our laboratory was found to produce higher levels of extra cellular amylolytic enzymes than strains obtained from culture collection in U.S.A, and Europe. This strain produced 4 units of glucoamylase and 40 units of a-amylase per ml of culture filtrate when grown on 2% starch at 50 C. Culture filtrate was used as the starting material for purification of these enzymes. Glucoamylase and a-amylase were purified by ultrafiltration and a combination of ion exchange and gel-filtration chromatography 93- and 112-fold with 30 and 41% recovery, respectively; Homogeneity of purified enzymes was established by the criteria of native- PAGE, SDS-PAGE, gel-filtration on HPLC and N-terminal amino acid analysis. Some of the physicochernical properties of these enzymes were studied.
Physicochemical characteristics
Glucoernylase is a monomeric glycoprotien (carbohydrate content 11 %, w/w) and has a molecular weight, of 45 kDa. It produces only glucose from starch. Km and Vmax, for soluble potato starch are 0.04 mg ml-1 and 666 p mole glucose min-1 mg protein-1, respectively. The enzyme is optimally active at 70 C at pH 6.0. Its activation energy is 14.0 kCal mole-1. It has melting temperature of 73 C. Molar extinction coefficient of glucoamylase is 5.5 x 104 mole-1 cm-l. It is stable at 60°C for > 7h. The enzyme is rich in alanine, serine and aspartate/ asparagine. Glucoamylase contains alanine as the N-terminal amino acid. It does not contain cysteine.
Purified a-amylase is a homodimeric protein of 40 kDa and contains 5% (w/w) carbohydrate. It liberates oligosaccharides from starch with maltose being the principle product of hydrolysis. The Km for soluble starch is 2.5 mg ml-1. A high Vmax, of 8000 mg starch min-1 mg protein-1 was found. The enzyme is optimally active at 65°C at pH 5.6. The activation energy is 10.9 kCal mole-1. At 50DC, which is the optimal temperature of growth of T. lanuginosus, purified a-amylase is completely stable for over 6 h. Ca2+ increases the melting temperature of a-amylase from 66°C to 73°C. a-Amylase requires Ca2+ for its activity and structural stabi1it.y The molar extinction coefficient of the enzyme is 4.7 x 10' mole-1 cm-1 a- Amylase is rich in aspartate / asparagine, glutamatme /glutamine, alanine, glycine and leucine. It does not contain cysteine. a- Amylase contains alanine as the N-t.ermina1 amino acid.
Hydrolysis of starch by a-amylase and glucoamylase
Experiments were done to understand the role of a-amylase and glucoamylase in the
utilization of starch by T. lanuginosus. Crude and purified amylase preparations hydrolyse raw potato starch slightly more efficiently than soluble potato starch. The extent of starch hydrolysis by a mixture of a-amylase and glucoamylase is equal to that by culture filtrate containing the same amount of enzyme activities, Electrophoresis of crude culture filtrate proteins on native-PAGE and activity staining on gel showed the presence of one species each of a-amylase and glucoamylase. This suggests that in T. lanuginosus hydrolysis of starch is mediated by one species each of extracellular a-amylase and glucoamylase. The hydrolysis of starch by a mixture of a-amylase and glucoamylase is equal to the arithmetic sum of hydrolysis by individual enzyme showing that the enzymes do not act synergistically. a-Amylase is the main starch depolymerizing enzyme. Conversion of starch into glucose by glucoamylase does not require the presence of a-amylase. Starch is hydralysed to a maximum of 72 and 97% by glucoamylase and a-amylase, respectively.
Effect of heat on a-amylase
The effect, of heat, on a-amylase and glucoamylase was studied with the view to obtain information on the thermal inactivation of these proteins. Five-min heat treatment of the native a-amylase (40 kDa) results in the specific conversion of all protein molecules into partially active (approximately 50% residual activity) and SDS-undissociable dimer of 45 kDa. a-Amylase (45 kDa) after 5-min heat treatment. is partially active and can he rendered completely active by incubation at 37°C for 3 h. This altered form of a-amylase is not due to the formation of disulfide linkage in protein because the enzyme does not contain cysteine and b mercaptoethanol does not prevent heat-induced structural change. Heat, treatment, for 20 min or more results in further structural changes which result in the irreversible inactivation of the enzyme. Prolonged heating (>40 min) probably causes the degradation of protein. Reactivation of 20-min heat-inactivated a-amylase occurs specifically at 37°C or 50°C within 3 h but not at lower temperatures (0°C or 4°C). Native-PAGE analysis of the native and 20-min heated-reactivated a-amylase shows that the reactivated sample is comprised of two protein species of different charge and/or mass. Activity staining shows that only one of these protein band is active and it has electrophoretic mobility identical to that of the native enzyme. Native and the active fraction of 20-min heated-reactivated a-amylase possess similar specific activity. This suggests that it is cat8alytmically and perhaps structurally similar to the native enzyme. The native and the reactivated a-amylase are resist ant to trypsin digestion. However, heat- inactivated a-amylase is degraded into low molecular weight, peptides. These observation suggest that heat-inactivated a-amylase is partially unfolded, Unlike the native, the heat-treated (94"C, 5 min) a-amylase can not be stained with AgN03 while both forms can be stained with Coomassie brilliant blue R and by Schiff's base. On the basis of these observations a tentative model was proposed for the effect of heat on a-amylase (Fig.)
Staining by + +
Staining by AgNO3, + +
Staining by Schiff 's base + +
Sensitivity to trypsin + +
Figure : Schematic represent ation of heat-induced changes in a-amylase
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Ishikawa, Kazuhiko. "STUDIES ON FUNCTIONAL AMINO ACID RESIDUES AND CATALYTIC MECHANISM OF MAMMALIAN α-AMYLASES." Kyoto University, 1992. http://hdl.handle.net/2433/78041.
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Sebesta, Dawn Wilson. "Glycosidase induction in Pseudomonas stutzeri and properties of one of its amylases, maltotetraohydrolase." Thesis, Royal Holloway, University of London, 1987. http://repository.royalholloway.ac.uk/items/6583eec2-ceaa-4212-a383-ab490a3121f6/1/.
Full textAbstract:
Pseudomonas stutzeri has been shown to produce extracellular amylases of different specificity, as well as a carbohydrase which hydrolyzed oligosaccharide but not starch. Pseudomonas stutzeri NCIB 11359 and NRRL B-3389 were adapted to, and then cultured in five different growth media. Both culture collections showed similar patterns in cell morphology and glucosidase secretion. Maltotetraohydrolase was produced in several of the media, a 'typical' alpha-amylase was exclusively produced in one of the defined media, and no glucanase in yet another medium. The endo-a-amylase which produced primarily maltose was present during early exponential cell growth and no longer detected by late exponential growth. When maltotetraohydrolase was produced, it was found throughout exponential growth, and in one of the media remained at maximum levels of activity into stationary phase. Maltotetraohydrolase hydrolyzed a suspension of commercial amylose in water producing 71-99% maltotetraose in the oligosaccharide fraction. Studies with maltotetraohydrolase demonstrated endo-amylase activity with the substrates blue amylose and with several 0-amylase limit dextrins. A limit dextrin was produced which could be further hydrolyzed by a-amylase. 1H-nmr studies showed that no a-1,6-glycosidic linkages were hydrolyzed and that the a-anomer was produced from the hydrolysis of a-1,4-linkages. Between 18-24% of the glycosidic linkages in amylopectin and 11-20% in B-limit dextrin were hydrolyzed. Maltotetraose was the primary product from hydrolysis of glucans but from the B-limit dextrins a high proportion of the smaller oligosaccharides was produced. Maltotetraohydrolase limit dextrin representing 38-54% of wheat amylopectin was purified and it had a molecular weight of 7000-15000; a degree of branching of 15%; ca. 13 branches per molecule; and an average chain length of 7. HPLC showed it to have a narrow weight distribution. Comparison of wheat and potato amylopectin indicated potato amylopectin to have more evenly distributed branching and probably less densely branched clusters.
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Erra, i. pujada Marta. "Isolement, clonage et expression du gène codant pour la pullulanase hyperthermostable de thermococcus hydrothermalis." Reims, 2000. http://www.theses.fr/2000REIMS033.
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James, Jennylynd Arlene. "Production, characterization and cloning of glucoamylase from Lactobacillus amylovorus ATCC 33621." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=42060.
Full textAbstract:
Glucoamylase, a saccharifying enzyme, is applied in the brewing industry to hydrolyse the dextrins of malted barley into simple sugars which can then be fermented by brewer's yeast. In order to establish the potential of glucoamylase from Lactobacillus amylovorus for application in the brewing industry, the main objectives of this study were: (1) to determine the cultural conditions for growth and glucoamylase production, (2) to purify the enzyme to homogeneity using chromatography and electrophoretic techniques, (3) to study biochemical properties of the purified enzyme, and (4) to clone the gene coding for glucoamylase, and characterize the recombinant glucoamylase.The actively amylolytic Lactobacillus amylovorus ATCC 33621 produced an intracellular glucoamylase activity. Conditions for growth and glucoamylase production were maximized by using dextrose free MRS medium supplemented with 1% dextrin, at pH 5.5 and 37$ sp circ$C. Enzyme production was maximal during the late logarithmic phase of growth from 16-18 h. Crude cell extract showed optimal activity at pH 6.0 and 55$ sp circ$C.
Native and SDS-PAGE of the purified enzyme showed a monomeric protein of 47 kD. Glucoamylase activity was confirmed by activity staining using a starch/polyacrylamide gel where a zone of clearing was visible on a blue/black background stained with Kl/I$ sb2.$ Optimal pH, pl and temperature of purified glucoamylase were 4.5, 4.39 and 45$ sp circ$C, respectively. The enzyme was rapidly inactivated by temperatures above 55$ sp circ$C and was inhibited by heavy metals, e.g. Pb$ sp{2+}$ and Cu$ sp{2+}$ at 1.0 mM. EDTA did not inhibit the enzyme activity at a final concentration of 10 mM. Enzyme inhibition by 1 mM of p-chloromercuribenzoic acid (pCMB) and iodoacetate suggested that a sulfhydryl group was present in the enzyme active site. Kinetic studies of glucoamylase confirmed that the enzyme reacted preferentially with polysaccharides. HPLC analyses of the end products of enzyme action showed that glucose was the major end product of enzyme action and this glucose was responsible for end product inhibition.
The gene coding for glucoamylase was cloned into Escherichia coli using the STA2 glucoamylase gene of Saccharomyces diastaticus as a probe. Three glucoamylase producing transformants were identified as the insert sizes of about 5.2 Kb, 6.4 Kb and 5.9 Kb, respectively. When the characteristics of both recombinant and purified wild type glucoamylases were compared, both enzymes showed a similar pH range of 3.0-8.0, and temperature optimum of 45$ sp circ$C. The recombinant enzyme pH profiles were broader than that of the wild type and an optimum pH of 6.0 was obtained. This study has shown that glucoamylase from Lb. amylovorus is less heat stable than other bacterial glucoamylases and thus may be suitable for application in the brewing industry. Successful cloning of this gene coding for glucoamylase in brewer's yeast, Saccharomyces cerevisiae, would reap the advantageous properties of the enzyme while eliminating the costs of adding commercial enzymes.
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Robert, Xavier. "Etude cristallographique et fonctionnelle des isoenzymes de l'α-amylase d'orge : leurs structures 3D en action révèlent des modes distincts de reconnaissance des polysaccharides et de nouvelles molécules inhibitrices." Lyon 1, 2002. http://www.theses.fr/2002LYO10059.
Full textAbstract:
Le grain d'orge contient deux isoenzymes de la famille des alpha-amylases (AMY1 et AMY2) qui sont impliqués dans la dégradation de l'amidon nécessaire à la croissance de l'embryon de plante. La structure tridimensionnelle de AMY2 (résolue par cristallographie et diffraction des rayons X) est connue dans son état natif, mais aussi en complexe, d'une part avec l'acarbose (un pseudo-tétrasaccharide inhibiteur) et d'autre part avec l'inhibiteur protéique bifonctionnel endogène BASI. Malgré leur très grande similitude de séquence, AMY1 et AMY2 se distinguent à plusieurs niveaux : point isoélectrique, stabilité à pH acide ou à haute température, affinité pour les ions calcium et pour les substrats solubles, efficacité différente pour l'hydrolyse des granules d'amidon. Enfin, seul AMY2 est inhibé par BASI. Cette étude présente la structure native de AMY1, résolue par cristallographie, et l'analyse comparée des deux isoenzymes, en vue d'expliquer leurs remarquables différences de propriétés. La résolution des structures des complexes avec l'acarbose et des analogues de substrat fournit également des informations sur les mécanismes d'hydrolyse et d'inhibition de AMY1. De plus, la structure d'un complexe entre AMY1 et un substrat naturel (maltoheptaose) nous a permis d'étudier en détail les différents sous-sites de fixation du substrat à l'enzyme et d'identifier un nouveau site de surface pouvant lier des polysaccharides. Ce site, présent uniquement chez AMY1, révèle pour la première fois, le rôle du domaine C d'une alpha-amylase. Enfin, la découverte d'une nouvelle classe potentielle d'inhibiteurs de glycosidases de type polyamine est présentée. Un complexe AMY1/spermidine a montré que la polyamine se fixait préférentiellement dans le site actif, même en présence d'acarbose. Cette propriété semble pouvoir s'étendre à d'autres alpha-amylases (notamment humaine) et servir de base à la conception de nouveaux inhibiteurs à visées thérapeutiques.
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Kleiber, Didier. "Les tanins du sorgho : accumulation dans la graine et évaluation de leur pouvoir antinutritionnel." Toulouse, INPT, 1990. http://www.theses.fr/1990INPT014A.
Full textAbstract:
Les tanins condenses (proanthocyanidines) dans les graines de sorgho (sorghum bicolor (l) moench) entrainent une baisse de la valeur alimentaire. Les dosages par les methodes: folin ciocalteu, vanilline hcl, daiber ou la reaction de bate-smith, conduisent a une appreciation globale sans differencier les tanins des autres composes phenoliques de la graine. La separation sur gel sephadex lh20 a permis leur isolement et a montre que les varietes francaises divergent plus par la proportion que par la nature des composes presents. La richesse en polyphenols des varietes est directement liee a la proportion de proanthocyanidines representant jusqu'a 70% des composes phenoliques de la graine a maturite; la majorite des polymeres atteignent 6 a 7 monomeres. Le suivi de l'accumulation des polyphenols dans la graine revele l'apparition des premieres proanthocyanidines dans les trois premiers jours suivant l'anthese. Il existe une polymerisation non enzymatique, qui pourrait se poursuivre au-dela de la recolte. Un test d'inhibition d'une alpha-amylase a ete mis au point et applique aux fractions separees sur sephadex. Etant bien correle aux pertes d'energie metabolisable, il permet d'evaluer in vitro les effets antinutritionnels lies a la presence de tanins dans le sorgho
41
Stephenson, Keith. "Construction and use of chimeric α-amylases to study protein secretion in Bacillus subtilis." Thesis, University of Newcastle Upon Tyne, 1996. http://hdl.handle.net/10443/702.
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To investigate the influence of exoprotein charge on protein secretion, genes encoding a range of chimeric α-amylases with altered net charge were constructed by a PCR-based technique and expressed in B. subtilis. The chimeric α-amylases were based on AmyL and contained specific regions from two related Bacillus α-amylases, AmyQ and AmyS. The engineered changes were introduced into amyL to increase the net positive charge of the chimeric enzymes, when compared to wild type AmyL. Isoelectric focusing confirmed that chimeric α-amylases possessed considerable positive charge and the temperature and pH optima of the most basic chimeric protein, AmyLQS50.5, were largely unaffected by the engineered changes. However, the structural stability, thermostability and the specific activity of this chimeric α-amylase were adversely affected. In general, lower amounts of chimeric α-amylases were released into the culture supernatants. Pulse-chase experiments revealed that the rate of processing of AmyLQS50.5 was reduced when compared to AmyL and also that the mature forms of both α-amylases were subjected to degradation during or shortly after translocation, although the chimeric enzyme was affected most. When compared to AmyL, the rate of refolding of AmyLQS50.5 was reduced approximately 3-fold, maintaining this protein in a protease-sensitive conformation for an increased period of time. Therefore, it is proposed that the extensive co- or post-translocational degradation of the chimeric enzyme was a consequence of reduced folding kinetics on the outer surface of the cytoplasmic membrane since, when in its native conformation, AmyLQS50.5 is highly resistant to the activity of B. sublifis extracellular proteases. These observations have important implications for the use of B. subtilis as a host for the secretion of heterologous proteins. The most positively charged chimera, AmyLQS50.5, was shown to bind significantly to cell walls isolated from B. sublifis 168, whereas AmyL and human serum albumin did not. This suggests that protein charge can influence the degree to which exoproteins interact with, and bind to, the cell wall as a consequence of electrostatic interactions with the anionic polymers.
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Grewal, Navneet Kaur. "Structural changes induced in waxy maize starch and normal wheat starch by maltogenic amylases." Thesis, Kansas State University, 2012. http://hdl.handle.net/2097/18145.
Full textAbstract:
Master of ScienceDepartment of Grain Science and Industry
Yong Cheng Shi
Maltogenic amylases are widely being used as an antistaling agent in baking industry. However, their action on starch in granular, swelled and dispersed forms, important components formed during bread baking, is largely unknown. Actions of two maltogenic amylases- A and -B on waxy maize starch (WMS) (100% amylopectin) and normal wheat starch (NWS) (~25% amylose) were studied and compared. For any given starch type, starch form, and hydrolysis time, maltogenic amylase-B hydrolyzed both starches more than maltogenic amylase-A as seen through sugar profile analysis indicating its higher degree of multiple attack action (DMA). Their action on non reducing ends blocked compound, p nitrophenol maltoheptaoside, confirmed their endo action. Maltogenic amylase-B showed a higher endo to total enzyme activity ratio than maltogenic amylase-A at any given enzyme weight. Greater MW reduction of dispersed starches by maltogenic amylase-B indicates its higher level of inner chain attack (LICA). Interestingly, MW distributions profiles of swelled starch hydrolysates did not show significant differences irrespective of swelling temperatures. Both enzymes showed differences in oligosaccharides compositions in dispersed and swelled starches’ reaction mixtures with sugars of degree of polymerization (DP) > 2 being degraded to glucose and maltose during later stages. For granular starches, enzymes followed a random pattern of formation and degradation of sugars with DP >2. MW distributions of hydrolyzed granular starches did not show significant shift until at the end of 24h when a low MW peak was observed. Morphological study of granular starches showed that maltogenic amylase-A mainly caused pinholes on WMS while maltogenic amylase-B caused surface corrosion with fewer pinholes. For NWS, both enzymes degraded A granules with deep cavities formation during later stages. A decrease in crystallinity of granular starches means that enzymes were able to hydrolyze both amorphous and crystalline regions. These results indicate that maltogenic amylase-B with a high LICA and high DMA possesses a better starch binding domain which can decrease the starch MW without affecting bread resilience. Strucuture of maltogenic amylase-A modified amylopectin (AP) in relation to its retrogradation was also studied. AP retrogradation was completely inhibited at % DH ≥ 20. MW and chain length distributions of debranched residual AP indicated with increase in % DH, a high proportion of unit chains with DP ≤ 9 and low proportion of unit chains with DP ≥ 17 were formed. Higher proportion of short outer AP chains which cannot participate in double helices formation supports the decrease and eventually complete inhibition of retrogradation. Thus, maltogenic amylase-A can play a very powerful role in inhibiting starch retrogradation even at limited DH (%).
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Hirata, Akira. "Studies on the Structure and Function of Mutant β-Amylases with Altered pH Optimum." Kyoto University, 2004. http://hdl.handle.net/2433/147766.
Full textAbstract:
Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第10917号
農博第1423号
新制||農||892(附属図書館)
学位論文||H16||N3928(農学部図書室)
UT51-2004-G764
京都大学大学院農学研究科農学専攻
(主査)教授 内海 成, 教授 廣瀬 正明, 助教授 三上 文三
学位規則第4条第1項該当
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Ritz, Casey Warren. "Effects of amylase supplementation upon the growth, endogenous amylase activity, and intestinal morphology of male turkey poults." Diss., Virginia Tech, 1993. http://hdl.handle.net/10919/40107.
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Lévêque, Emmanuel. "Clonage d'un gene codant pour une alpha-amylase a partir de l'archaebacterie thermophile thermococcus hydrothermalis al662 (collection ifremer-brest). Etude des parentes phylogenetiques avec les autres alpha-amylases." Reims, 1998. http://www.theses.fr/1998REIMS029.
Full textAbstract:
Nous avons cherche a cloner le gene codant pour l'-amylase de l'archaebacterie thermococcus hydrothermalis chez escherichia coli. Pour cela, des banques genomiques ont ete construites a partir de l'adn de la souche t. Hydrothermalis et criblees a l'aide de techniques biochimiques et microbiologiques. Exception faite d'un clone, les activites exprimees par les genes clones n'ont pu etre maintenues. De plus, cette colonie etait porteuse au niveau du plasmide qu'elle renfermait d'un transposon et apparemment le gene codant l'activite amylasique clone avait ete insere dans le genome de la bacterie receptrice. Il nous a donc fallu changer notre approche experimentale. Un amplifiat de 221 pb a alors ete construit par pcr a partir de l'adn genomique de t. Hydrothermalis puis clone et marque. Il a ensuite ete utilise pour isoler le gene recherche par la technique de southern-blot. Ainsi, un fragment de 4 kpb a ete isole et clone. Le gene recherche a ete localise a l'interieur de ce fragment, puis sous-clone apres construction du plasmide p662el100. L'insert de ce plasmide (2,7 kpb) a alors ete sequence. Le gene de l'-amylase de t. Hydrothermalis est long de 1374 pb. Il code pour une enzyme de 457 acides amines fortement homologue aux autres -amylases issues de thermococcales connues. En amont et en aval de l'orf ont ete retrouvees des sequences promotrices et terminatrices typiques des archaebacteries. Apres production de l'enzyme recombinante (thamy), ses proprietes physico-chimiques ont ete recherchees. Ces etudes ont montre qu'elle avait conserve ses proprietes initiales (temperature optimale : 75-85c ; ph optimal : 5,0-5,5) et que c'etait une enzyme liquefiante. L'effet de differents ions metalliques sur l'activite de thamy a egalement ete etudie. Une etude des relations evolutives entre les -amylases a egalement ete realisee. Cette etude a montre que les -amylases issues de thermococcales etaient phylogenetiquement tres proches des enzymes issues de plantes.
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Kaur, Harkirat, and h_harkiratkaur@student rmit edu au. "Baking enzymes and microencapsulation strategies for retardation of staling." RMIT University. Applied Sciences, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20081203.133339.
Full textAbstract:
The staling of baked products remains a significant cause of economic loss due to the loss of enjoyment seen as crumb firming occurs. The aims of the current project have been to investigate the stability of amylases in bakery formulations. In addition, the impact of partial hydrolysis products of starch on staling is investigated. Specific assays were used to measure Ñ-amylase and Ò-amylase, in the presence of the other potentially interfering activity. Ñ-Amylase activity levels appeared to gradually increase during the proofing stages and then to decline upon heating of the dough. However, the activity remaining in the final baked loaf was readily measurable indicating that not all of the enzyme had been inactivated. Free and total Ò-amylase activities were also measured and most was found to be in the free form. Ò-Amylase was unstable with only relatively low activities remaining in the final baked loaf. It appears that of the two amylolytic enzymes, Ñ-a mylase is sufficiently stable that it may exert some impact on the crumb characteristics in the freshly baked product and during subsequent storage. In order to assess the likelihood that amylolysis is of significance to crumb characteristics, HPLC was used to analyse aqueous extracts for sugars. Commercial flours were found to contain low levels of sugars with maltose being the predominant sugar present. A number of commercial breads were also analysed and the composition found to vary between the different samples. Typically maltose was present at higher levels than the other sugars. When experimental loaves were analysed, the patterns showed that other sugars declined during proofing whereas maltose remained at readily measurable levels. Upon baking and subsequent storage the amounts of maltose increased. These results are consistent with the findings that some amylolytic activity remains in the baked product. In the third phase of this study, a potential means of investigating the role of particular carb ohydrates in product textures and staling rates was examined. The approach of spray drying was used to prepare microencapsulated maltodextrin. The encapsulating agents used were based upon rice starch and guar galactomannan. When these microcapsules were incorporated into the breadmaking formulation and baked, it appeared that softer crumb characteristics were achieved. The data also indicates an effect of delay in the staling rates. In a preliminary evaluation of the potential of two X-ray scattering methods, it was found that both techniques appear useful. The differences seen for samples of bread crumb analysed at various stages of storage did not show large differences in the intensity patterns. Of the two approaches, small angle analysis (SAXS) appears to show greater potential for application in ongoing studies of staling. In conclusion, cereal grain Ñ-amylase may be more stable during breadmaking than previously thought. There appears to be an increase in the level of some low molecular weight sugars in the final, baked product. Microencapsulation may offer a useful technique for the study of the role of specific carbohydrates during baking and storage of breads.
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Tong, Zhen 1970. "Evaluation of conventional and microwave heating systems for food processing based on TTI kinetics." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=29481.
Full textAbstract:
Thermal kinetics of enzymatic time-temperature integrators (TTIs) were experimentally evaluated under both conventional and microwave heating systems in the pasteurization temperature range (50 to 90°C). Recent developments of process evaluation methodologies have Shown that standardized enzymatic time-temperature integrators (TTIs) could be successfully used for fast and correct quantification of thermal processes. Promising results have been reported for the alpha-amylase based TTI from Bacillus subtilis (BAA), which was chosen in this study as the TTI to compare the effectiveness of continuous-flow heating systems with microwave and conventional heating modes. Thermal inactivation kinetics of alpha-amylase was studied by measuring the residual activity of heat treated samples in isothermal conditions in a temperature range of 50 to 95°C and pH range, 5.0 to 6.9. Based on a first order rate of inactivation kinetics, kinetic parameters, decimal reduction time, D, and temperature sensitivity indicator, z, were calculated. (Abstract shortened by UMI.)
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Ara, Andleeb. "Development of NASBA-primer search software for designing forensic saliva tandem repeat markers for mucin and amylase." Muncie, Ind. : Ball State University, 2009. http://cardinalscholar.bsu.edu/635.
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Koukiekolo, Roger Patrick. "Mode d'action de l'α-amylase pancréatique de porc. Inhinition par les cyclodextrines et par l'inhibiteur protéique de Phaseolus, α-AI, mise en évidence et caractérisation de sites secondaires." Aix-Marseille 3, 2001. http://www.theses.fr/2001AIX30037.
Full textAbstract:
L'α-amylase pancréatique de porc (APP) catalyse l'hydrolyse des liaisons glycosidiques α-(1,4) internes dans les composants de l'amidon, du glycogène et des maltodextrines. La configuration α est conservée chez les produits ; l'APP appartient à la famille 13 des glycosyls hydrolases. La séquence peptidique et la structure 3-D à 2 Å de résolution sont connues. Le site actif de APP est localisé dans une faille et contient 5 sous-sites. Afin de mieux comprendre le mécanisme enzymatique, l'inhibition par des pseudosaccharides et des composants protéiques du type lectine a été étudiée. Faisant suite à des études cinétiques sur l'acarbose réalisées dans notre laboratoire, l'effet de l'inhibiteur protéique du haricot (α-AI) et celui des α-, β- et γ-cyclodextrines (CD) sont ici présentés. Spectre différentiel et sensibilité aux protéases des complexes APP-inhibiteurs ont également fait l'objet de notre étude. L'inhibition par α-AI est de type non-compétitif mixte quel que soit le substrat hydrolysé (amylose/maltopentaose). Le modèle correspondant contient les formes enzymatiques ES, EI, ESI, EI2 et ESI2. La sensibilité du complexe α-AI-APP à la subtilisine est augmentée par rapport à celle de l'enzyme seule suggérant un changement de conformation d'ailleurs établi par cristallographie. .Porcine pancreatic α-amylase (PPA) catalysed the hydrolysis of internal α-(1,4) glycosidic linkages in starch components, glycogen and maltodextrins, giving products with retaining conformation (α). It belongs to family 13 glycosyl-hydrolases. The peptidic sequence and the 2-Å tertiary structure have been reported. Following the use of acarbose as inhibitor in a previous work, the effect of the red bean protein inhibitor (α-AI) and α-, β- and γ-cyclodextrins (CD) have been reported. Differential spectrum analysis and protease sensitivity to the inhibitor amylase complexes have also been performed. α-AI inhibition is of the mixed non-competitive type whatever amylose or maltopentaose is used as substrate. The corresponding model model contains the ES, EI, ESI, EI2 and ESI2 enzymatic forms. The substilisin sensitivity of the α-AI-APP complex is higher than one of free PPA (no inhibitor present). .
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Zimmerman, Rosalind Kane. "Maize alpha-amylase: purification and properties and induction by gibberellic acid." Thesis, Virginia Polytechnic Institute and State University, 1987. http://hdl.handle.net/10919/80140.
Full textAbstract:
Alpha-amylase synthesis can be induced in wheat and barley half-seeds by addition of gibberellic acid (GA) to the incubation medium. In maize, induction in de-embryonated kernels by exogenous GA has been reported in some studies but not others.
Alpha-amylase induction was investigated in maize by measuring activity in extracts from whole and de-embryonated kernels incubated with and without GA during germination. Alpha-amylase activity was first detected on the 3rd day of germination in whole kernels and GA-treated endosperms and on the 4th day in the controls. Thereafter both whole kernels and GA-treated endosperms followed approximately the same time course in α-amylase activity with the control lagging a day behind. Studies indicated that maximum α-amylase activity occurred on the 7th day in whole kernels and GA-treated endosperms and the 8th in control endosperms.
Maize α-amylase was purified using differential solubility, column chromatography, glycogen precipitation and polyacrylamide gel electrophoresis, of these, the best purification method was glycogen precipitation.
Maize α-amylase exhibited isozymes. The isozyme patterns were qualitatively similar in all samples and throughout incubation. Wheat and barley α-amylase isozymes have been divided into two groups on the basis of a number of characteristics. Genetics analysis revealed these isozymes to be the result of two multigene families. To shed light on the genetic basis of the maize α-amylase isozymes, physicochemical characterization was initiated. Studies of pH and temperature profiles and optima showed no differences between maize isozymes. The pH optima was pH 5 and the temperature optima was about 37°C.Master of Science