Дисертації: "Endo-(1,4)-β-glucanase"

1

Woolley, Lindsey C. "The role of endo-#beta#-1,4-glucanase in strawberry fruit development." Thesis, Open University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340705.

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2

Akiba, Shunichi. "Studies on Protease-Resistant Endo-β-1, 4-glucanase from Aspergillus niger". Kyoto University, 1999. http://hdl.handle.net/2433/181918.

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3

Robert, Stéphanie. "Etude de la protéine KORRIGAN, une endo-β-1,4-glucanase impliquée dans la synthèse de cellulose chez Arabidopsis thaliana et Gossypium hirsutum". Paris 11, 2005. http://www.theses.fr/2005PA112007.

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Afin de mieux comprendre les mécanismes moléculaires régissant la synthèse de cellulose chez les végétaux, je me suis intéressée à la protéine KORRIGAN1, une endo--1,4-glucanase. La première partie de ma thèse a été consacrée à l’étude de KOR1 au sein d’un complexe multiprotéique membranaire chez Arabidopsis et le cotonnier. La mise en évidence d’une différence de taille de ce complexe en fonction du niveau de synthèse de la cellulose laisse supposer un lien étroit entre la formation de ce complexe et la fonction de KOR. Différents partenaires potentiels de KOR1 au sein de ce complexe ont été identifiés, révélant de potentielles interactions entre KOR1 et le cytosquelette. Le rôle de KOR1 a aussi été abordé, et notamment son implication dans le recyclage d’amorces lipidiques. Cette fonction hypothétique appuyée par des résultats obtenus chez le cotonnier, n’a pas été confortée par études réalisées chez Arabidopsis. Je me suis enfin intéressée à la localisation subcellulaire de KOR1. KOR1 est localisée dans des compartiments intracellulaires, distincts de l’appareil de Golgi, et qui colocalisent partiellement avec les endosomes précoces. Ces compartiments sont mobiles et cette motilité dépend d’un réseau de microtubules intact mettant à nouveau en évidence un lien entre KOR1 et les éléments du cytosquelette. Lorsque la synthèse de cellulose est inhibée par de l’isoxabène, les compartiments contenant KOR1 sont plus près de la membrane plasmique et distincts des endosomes précoces. Ceci suggère donc que la synthèse de cellulose est fortement contrôlée, notamment à travers la régulation de la localisation et des interactions des protéines enzymatiques y participant
In order to better understand the molecular mechanisms that govern cellulose synthesis, KORRIGAN1, an endo--1,4-glucanase involved in this process, has been studied. In the first part of my thesis, KOR1 was shown to be present in high molecular weight complexes in Arabidopsis thaliana and cotton fibers. The weight of this complex changes in function of the level of cellulose synthesis, demonstrating that cellulose synthesis and KOR1 function are linked. Different putative protein partners have been identified which suggest a close interaction between KOR1 and the cytoskeleton. It has been suggested that KOR1 may recycle sitosterol primers involved in the initiation of celullose synthesis in cotton fibers. In the second part of my thesis, I tested this hypothesis and the results obtained suggest that this hypothetic function is not real. Finally, I focussed on the subcellular localization of KOR1. Using a variety of methods, we showed that KOR1 was present in intracellular compartments, distinct from the Golgi apparatus and showing some colocalization with endosomal compartments. These compartments were very motile in a microtubules integrity dependent manner, linking KOR1 to the cytoskeleton again. When cellulose synthesis was inhibited, the KOR1 compartments were closer to the plasma membrane and distinct from the endosomal compartments. These results suggest that cellulose synthesis is highly regulated through the control of the localisation and interaction of the enzymes involved in this process

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4

Phan, Minh Thi Tuyet, Viet Quoc Nguyen, Hy Gia Le, Thoa Kim Nguyen та Man Dinh Tran. "Molecular cloning gene and nucleotide sequence of the gene encoding an endo-1,4-β-glucanase from Bacillus sp VLSH08 strain applying to biomass hydrolysis". Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-99522.

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Bacillus sp VLSH08 screened from sea wetland in Nam Dinh province produces an extracellular endo-1,4-beta-glucanase. According to the results of the classified Kit API 50/CHB as well as sequence of 1500 bp fragment coding for 16S rRNA gene of the Bacillus sp VLSH 08 strain showed that the taxonomical characteristics between the strain VLSH 08 and Bacillus amyloliquefaciene JN999857 are similar of 98%. Culture supernatant of this strain showed optimal cellulase activity at pH 5.8 and 60 Celsius degree and that was enhanced 2.03 times in the presence of 5 mM Co2+. Moreover, the gene encoding endo-1,4-beta-glucanase from this strain was cloned in Escherichia coli using pCR2.1 vector. The entire gene for the enzyme contained a 1500-bp single open reading frame encoding 500 amino acids, including a 29-amino acid signal peptide. The amino acid sequence of this enzyme is very close to that of an EG of Bacillus subtilis (EU022560.1) and an EG of Bacillus amyloliquefaciene (EU022559.1) which all belong to the cellulase family E2. A cocktail of enzyme containing this endo-1,4-beta-glucanase used for biomass hydrolysis indicated that the cellulose conversion attained to 72.76% cellulose after 48 hours
Chủng vi khuẩn Bacillus sp VLSH08 được tuyển chọn từ tập hợp chủng vi khuẩn phân lập ở vùng ngập mặn tỉnh Nam Định có khả năng sinh tổng hợp enzyme endo-1,4-beta-glucanase ngoại bào. Kết quả phân loại chủng vi khuẩn Bacillus sp VLSH08 bằng Kit hóa sinh API 50/CHB cũng như trình tự gen mã hóa 16S rRNA cho thấy độ tương đồng của chủng Bacillus sp VLSH08 và chủng Bacillus amyloliquefaciene JN999857 đạt 98%. Dịch lên men của chủng được sử dụng làm nguồn enzyme thô để nghiên cứu hoạt độ tối ưu của enzyme ở pH 5,8 và nhiệt đô 60oC. Hoạt tính enzyme tăng 2,03 lần khi có mặt 5 mM ion Co2+. Đồng thời, gen mã hóa cho enzyme endo-1,4-betaglucanase cũng được tách dòng trong tế bào Escherichia coli sử dụng vector pCR 2.1. Gen mã hóa cho enzyme này có chiều dài 1500 bp, mã hóa cho 500 axit amin, bao gồm 29 axit amin của chuỗi peptid tín hiệu. So sánh cho thấy trình tự gen endo-1,4-beta-glucanase của chủng Bacillus sp VLSH08 có độ tương đồng cao với enzyme này của chủng Bacillus subtilis (EU022560.1) và của chủng Bacillus amyloliquefaciene (EU022559.1). Tất cả các enzyme nhóm này đều thuộc họ cellulase E2. Enzyme của chủng này cũng đã được phối trộn với các enzyme khác tạo thành cocktail để thủy phân sinh khối cho kết quả cellulose bị thủy phân 72,76% sau 48 giờ

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Louw, Maureen Elizabeth. "Characterization of an alkalophilic Bacillus brevis isolate with respect to its endo-(1,3-1,4)-β-glucanase gene, protein hyperproduction and the degS-degU operon". Doctoral thesis, University of Cape Town, 1994. http://hdl.handle.net/11427/21696.

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Bibliography: pages 127-144.
Bacillus brevis Alk 36 was isolated from soil during a screening programme for the selection of extracellular enzyme producing strains. A gene coding for an endo(1,3- 1,4 )-.8-glucanase (or lichenase) was cloned from B. brevis Alk 36 and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and found to encode a protein of 252 amino acid residues. The amino acid sequence of the B. brevis lichenase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-β-glucanases. The enzyme exhibited some unique properties. The optimum temperature and pH for enzyme activity were 65-70°C and 8-10, respectively. When held at 75°C for 1 h, 75% residual activity was measured. The molecular mass was estimated to be 29 kDa and the enzyme was found to be resistant to sodium dodecyl sulphate (SDS). B. brevis Alk 36 was evaluated as a potential host strain for the efficient production and secretion of foreign proteins and was found to grow optimally between pH 8.0 and pH 9.5 and between 42°C and 52°C. B. brevis was successfully transformed using vector DNA and was found to produce relatively low levels of protease. In addition, it was evaluated as a possible protein hyper-secreting strain. However, using PCR technology, the highly conserved cell wall protein genes could not be positively identified in B. brevis Alk 36.

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6

Tarrés, Farrés Joaquim Agustí. "Endo-β-1,4-glucanasa para la fabricación de micro/nanocelulosa: propiedades y aplicaciones". Doctoral thesis, Universitat de Girona, 2017. http://hdl.handle.net/10803/456211.

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In the recent years, significant interest on the production, characterization and application of cellulose nanofibers has brought out among scientific and technic authorities. This special attention mainly comes from their biodegradability, renewability and versatility, making them able to be used for several applications and fields. This topic outbreak took place about ten years ago, fact that is confirmed by the exponential increase on scientific publications and patents since then. In a nutshell, the present thesis aims to demonstrate that enzymatically hydrolyzed cellulose nanofibers can be successfully produced and used in several sectors. Their versatility, availability, low cost and low environmental impact justify the ongoing research in this field
En els darrers anys, entre la comunitat científica i tecnològica s’ha despertat un gran interès en la producció, caracterització i utilització de nanofibres de cel·lulosa. Aquesta especial atenció es deu, principalment, al seu caràcter biodegradable, el seu origen renovable i la versatilitat que presenten, fent-les aptes per ser utilitzades en multitud d’aplicacions. L’esclat d’aquesta temàtica de recerca va tenir lloc aproximadament deu anys enrere, doncs només cal observar el creixement exponencial de publicacions científiques i patents des d’aleshores. De manera general, la present tesi pretén demostrar que les nanofibres de cel·lulosa obtingudes mitjançant un pretractament d’hidròlisi enzimàtica poden ser produïdes de forma efectiva, de manera que pugin ser aplicades en una gran varietat de camps científics i tecnològics. La seva gran versatilitat, disponibilitat, baix cost i baix impacte mediambiental, justifiquen que en el futur es continuï amb la seva investigació sobre noves aplicacions

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Stronach, Morag Shona. "The molecular cell biology of a xyloglucan specific endo 1,4 #beta#-D-glucanase from Tropaeolum majus L. cotyledons." Thesis, University of Stirling, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294012.

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8

Silva, Caio de Oliveira Gorgulho. "Emericella nidulans e bagaço de cana-de-açucar : ferramentas para produção de endo-β-1,4-xilanase". reponame:Repositório Institucional da UnB, 2014. http://repositorio.unb.br/handle/10482/15669.

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Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2014.
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O bagaço de cana-de-açúcar é um importante resíduo agroindustrial brasileiro que apresenta grande potencial para ser utilizado como fonte de carbono para produção de holocelulases de interesse industrial por microrganismos. As xilanases, objeto de estudo deste trabalho, são enzimas que apresentam uma série de aplicações biotecnológicas que incluem a produção de etanol de segunda geração, o branqueamento de papel, a produção de sucos e pães e o uso como aditivo em rações animais. O objetivo desta pesquisa foi purificar e caracterizar uma xilanase produzida pelo fungo Emericella nidulans quando cultivado em bagaço de cana, visando o aproveitamento deste resíduo e a avaliação do potencial biotecnológico da enzima. O fungo foi capaz de secretar xilanases a partir do primeiro dia de cultivo sob fermentação submersa utilizando o bagaço. Uma xilanase de 22 kDa foi purificada a partir do extrato bruto obtido no cultivo através de ultrafiltração, precipitação com sulfato de amônio e cromatografias de filtração em gel e troca aniônica. A enzima apresentou alta homologia com endo-β-1,4-xilanase A (XynA) de E. nidulans e desta forma foi chamada. A enzima XynA apresentou maior atividade a 55°C e na faixa de pH 3,0 – 6,5. A enzima se mostrou pouco termoestável, com meiasvidas de 40, 10 e 7 minutos a 28, 50 e 55°C, respectivamente. XynA foi mais ativa sobre a porção solúvel da xilana, com valores de KM e Vmáx 3,39 mg/mL e 0,502 UI/mL, respectivamente. A hidrólise da xilana por XynA gerou xilooligossacarídeos, indicando ação tipo endo. Diferentes compostos fenólicos comumente liberados durante o pré-tratamento de biomassa lignocelulósica causaram efeitos variados sobre XynA. Os ácidos tânico e cinâmico inibiram a enzima, enquanto o ácido 4-hidroxi-benzóico aumentou sua atividade e os ácidos ferúlico, p-cumárico e vanilina não mostraram efeito. O etanol aumentou a atividade, estabilidade e Vmáx da enzima, indicando potencial para aplicação em processos de sacarificação e fermentação simultâneas de biomassa. O ultrafiltrado (uma fração semipurificada de xilanases) foi capaz de hidrolisar polpas de celulose em diferentes etapas do processo Kraft, resultando na liberação de açúcares redutores, cromóforos, pentoses e produtos de hidrólise de xilana sem concomitante liberação de glicose. O extrato bruto se mostrou capaz de degradar bagaço de cana-de-açúcar não-tratado ou sumetido a explosão a vapor, liberando açúcares redutores e produtos de hidrólise de xilana. ______________________________________________________________________________ ABSTRACT
Sugarcane bagasse is a major lignocellulosic agroindustrial residue in Brazil with great potencial for utilization as carbon source for production of industrial holocellulases by microrganisms. Xylanases present several biotechnological applications such as in ethanol production, paper bleaching, juice and bread production and utilization as feed additive. The goal of this reseach was to purify and characterize one xylanase produced by the fungus Emericella nidulans when grown on sugarcane bagasse, aiming the use of this residue and the investigation of biotechnological potencials of the enzyme. E. nidulans secreted xylanases from the first day of growth on liquid media containing bagasse as sole carbon source. One 22 kDa xylanase was purified from crude extract through ultrafiltration, ammonium sulphate precipitation, gelfiltration and anion-exchange chromatographies. The enzyme showed significant homology with endo-β-1,4-xylanase A (XynA) from E. nidulans, and was named that way. XynA was most active at 55°C and pH 3,0 – 6,5. Considering its thermostability, XynA presented half-lives of 40, 10 and 7 minutes at 28, 50 and 55°C, respectively. The enzyme was more active on the soluble portion of xylan, with Km and Vmax values 3,39 mg.mL¯ ¹ and 0,502 UI.mL¯ ¹, respectively. Xylan hydrolysis by XynA produced xylooligossacharides, indicating endo-type action. Phenolic compounds released during pre-treatment of lignocellulosic biomass caused different effects on XynA. Tannic and cinnamic acids inhibited XynA, while 4-hidroxibenzoic acid enhanced its activity and ferulic and p-coumaric acids and vanillin caused no effect. Ethanol increased XynA activity, thermostability and Vmax, suggesting potencial for aplication in simultaneous sacharification and fermentation processes. Ultrafiltrate sample (partialy purified xylanases) was able to hydrolyse celullose pulps obtained from different stages of Kraft process, resulting in release of reducing sugars, chromophores, pentoses and xylans degradation products with apparent no release of glucose. E. nidulans crude extract was able to hydrolyse untreated or steam-explosion pretreated sugarcane bagasses, releasing reducing sugars and xylans degradation products.

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Miotto, Lis Schwartz. "Estudos moleculares, estruturais e funcionais da Cel12A de Gloeophyllum trabeum, uma endo-1,4-β-glucanase da família 12 de hidrolases de glicosídeos." Universidade Federal de São Carlos, 2014. https://repositorio.ufscar.br/handle/ufscar/272.

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Universidade Federal de Minas Gerais
The production of second-generation ethanol by enzymatic hydrolysis of biomass is considered a viable and promising alternative to face the global energy crisis and to decrease our dependence on fossil fuels. Therefore, it is necessary to degrade the constituent molecules of the plant cell wall such as lignin, cellulose and hemicellulose to fermentable sugars. However, the use of enzymes for this purpose is still expensive, leading to the increase on studies seeking to make them more feasible economically and technically. The present study aimed the molecular, structural and functional characterization of the endoglucanase Cel12A from the fungus Gloeophyllum trabeum by different techniques. Biochemical data revealed the substrate specificity for the enzyme and showed that β-glucan is the best substrate for its activity (239.2 ± 9.1 U mg-1). Optimal conditions for activity were pH 4.5 and temperature of 50 oC. Thermal stability assay indicated a half-life of 84.6 ± 3.6 hours at 50 oC. The kinetic parameters Km (3.2 ± 0.5 mg mL-1) and Vmax (0,40 ± 0,02 μmol min-1) were determined using β-glucan as substrate. Analysis of scanning electron microscopy of oat spelts and filter paper samples submitted to the hydrolysis by GtCel12A evidenced the degradation effects of these substrates compared to control samples. Moreover, the low-resolution envelope and the crystallographic structure for GtCel12A were determined. The structure revealed a β-sandwich fold with two β-sheets (A and B) and three α-helices, while sheet A showed five strands and sheet B nine strands. The comparative analysis of the amino acid sequence and homologous structures prompted us to identify the catalytic residues, Glu142 and Glu227 in the active site of the enzyme. These results are important for understanding and elucidating the enzyme molecular mechanism of action and other glycoside hydrolase family 12 endoglucanases.
A produção de etanol de segunda geração, a partir da hidrólise enzimática da biomassa vegetal é considerada uma alternativa viável e promissora para enfrentarmos a crise energética mundial e diminuirmos a dependência das fontes fósseis de energia. Para isso, é necessário que ocorra a degradação das moléculas constituintes da parede celular como a lignina, a celulose e a hemicelulose a açúcares fermentescíveis. No entanto, a utilização de enzimas para esse fim ainda apresenta um custo elevado, o que tem desencadeado, cada vez mais, estudos que busquem torná-las mais viáveis econômica e tecnicamente. O presente estudo visou à caracterização molecular, estrutural e funcional da endoglucanase Cel12A do fungo Gloeophyllum trabeum por diferentes técnicas. Os dados bioquímicos revelaram a especificidade por substratos da enzima, sendo que o melhor substrato para a atividade foi o β-glucano (239,2 ± 9,1 U mg-1). As condições ótimas para a atividade foram pH 4,5 e temperatura de 50 oC. Os ensaios de estabilidade térmica indicaram uma meia-vida de 84,6 ± 3,6 horas a 50 oC. Os parâmetros cinéticos Km (3,2 ± 0,5 mg mL-1)) e Vmax (0,40 ± 0,02 μmol min-1) foram determinados utilizando-se β-glucano como substrato. Análises de microscopia eletrônica de varredura de amostras de papel de filtro e aveia submetidos à hidrólise pela GtCel12A evidenciaram os efeitos de degradação dos substratos em relação às amostras controle. Adicionalmente, o envelope de baixa resolução e a estrutura cristalográfica da GtCel12A foram obtidos. O modelo de alta resolução revelou um enovelamento do tipo sanduíche β, com duas folhas β (A e B) e três hélices α, sendo que a folha A apresentou cinco fitas e a B, nove fitas. Por meio de análises comparativas da sequência de aminoácidos e de estruturas homólogas identificamos os resíduos catalíticos Glu142 e Glu227 no sítio ativo da enzima. Tais resultados são importantes para a elucidação e compreensão do mecanismo molecular de atuação dessa enzima e de outras endoglucanases da família GH12.

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Menu-Bouaouiche, Laurence. "Relations structure-fonction et propriétés de surface de protéines PR5 "thaumatin-like" de fruits comestibles." Toulouse 3, 2003. http://www.theses.fr/2003TOU30171.

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Les protéines PR5 ou thaumatin-like (TLP) forment une famille structurale et phylogénique très homogène, mais elles possèdent in vitro des propriétés disparates : antifongiques, antigel, liaison et/ou hydrolyse de glucane. Certaines d'entre elles sont induites par des stress biotiques ou abiotiques et semblent intervenir dans la protection plus ou moins efficace des plantes vis à vis de pathogènes fongiques. D'autre part, certains fruits accumulent des protéines similaires au cours de leur maturation, dont celles de cerise et de pomme, allergéniques. Une meilleure connaissance des propriétés fonctionnelles et structurales des TLP spécifiques de fruits devrait permettre d'élaborer de nouveaux diagnostics d'allergènes et d'orienter, par rapport aux éventuels risques d'allergie, l'amélioration de la résistance naturelle des plantes. Afin de définir les relations structure-fonction de ces protéines, nous avons, tout d'abord, réalisé l'analyse de la phylogénie, de la structure et des propriétés électrostatiques et hydrophobes de surface de différents membres de la famille, par des techniques d'analyse de séquence et de modélisation moléculaire. .
PR-5 or Thaumatin-like proteins (TLP) are structurally and phylogenetically related but their activities in vitro are very diverse: antifungal, antifreeze, glucan affinity and/or hydrolysis. Some of them are induced by biotic or abiotic stresses and could be involved in plant's defense against pathogenic fungi. In contrast, some fruits accumulate similar proteins during their maturation, like these of cherry or apple that are allergenic A better understanding of functional and structural properties of fruit specific's PR-5 proteins could allow new allergy diagnostics elaboration and new orientations' definition, beware of allergy risks, for natural plant defense amelioration. For the definition of TLP structure-function relationships, we have, first, analyzed different members of this family, for their phylogeny, their structure and their surface electrostatics and hydrophobic properties, by sequence analysis and molecular modeling methodologies. Thereafter, cherry, apple and banana's TLP and Thaumatin were compared for their enzymatic and antifungal activities. .

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Brutus, Alexandre. "Etude de deux endo β-(1,4) xylanases de famille 11 provenant des champignons Penicillium funiculosum et Botrytis cinerea – Interaction avec des inhibiteurs protéiques du blé, XIP-I, TAXI-I et II". Aix-Marseille 3, 2005. http://www.theses.fr/2005AIX30019.

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Nous avons exprimé dans différents systèmes hétérologues l'ADNc de la xylanase (XYNB) du champignon filamenteux Penicillium funiculosum ainsi que celle du phytopathogène Botrytis cinerea (XynBc) appartenant toutes deux à la famille des GH11 des glycosylhydrolases. Les propriétés physico-chimiques des enzymes recombinantes ont été déterminées. XYNB et XynBc montrent les mêmes spectres d'inhibitions en présence d'inhibiteurs protéiques probablement impliqués dans des mécanismes de défenses des plantes et issus du blé ; les activités sont fortement inhibées par XIP-I et TAXI-I tandis que TAXI-II n'a aucun effet. Des essais de RT-PCR semi-quantitatives ont montré la présence des ARNm au cours des 72 premières heures de l'infection de feuilles de tabacs par B. Cinerea
The phytopathogen fungus Botrytis cinerea and the filamentous fungus Penicillium funiculosum produce various glycosidases with xylanase activity. In the present study, we report the heterologous expression, purification and characterization of two family 11 xylanase produced by these fungi. These enzymes present similar activities on different substrates and identical suceptibilities to different proteinaceous inhibitors from wheat. These xylanases were sensitive to XIP-I, TAXI-I but not to TAXI-II. The inhibition was competitive and the inhibition constant were in the nanomolar range indicting a great affinity between these xylanases and the wheat inhibitors. Moreover, we have shown that the presence of glycosylation or of a carbohydrate binding module in the xylanase, did not affect the interaction of the enzyme with the inhibitor

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Pellet, Alain. "Réactivité des pyridylacétonitrile, aminonitrile et éther de cyanhydrine vis à vis de composés carbonylés α, β [alpha-béta] éthyléniques : synthèse et stéréochimie de γ [gamma] cétonitriles et de γ [gamma] cétoaldéhydes". Paris 11, 1988. http://www.theses.fr/1988PA112130.

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Ce travail porte sur l'étude de la régiosélectivité d'addition de carbanions en alpha de nitrile sur des composés carbonylés alpha-bêta éthyléniques dans le but de réaliser la synthèse de composés carbonylés gamma fonctionnalisés. Dans le premier chapitre, nous avons montré que l'addition du dérivé lithié du pyridyl-3 acétonitrile sur diverses énones conduit dans le tétrahydrofuranne après protonation exclusivement aux adduits 1,4. Les cétones bicycliques obtenues présentent une jonction de cycle cis tandis que la stéréochimie cis ou trans des cyclanones-2,3 disubstituées est orientée par le contrôle cinétique ou thermodynamique de la protonation des énolates. L'addition 1,2 de ce carbanion sur le méthyl-3 butène-2 al est réalisée dans le tétrahydrofuranne tandis que l'addition 1,4 est observée sous contrôle thermodynamique en présence d'hexaméthylphosphorotriamide ou d'éthérate de trifluorure de bore. Dans le second chapitre, nous avons réalisé les additions régiosélectives des anions lithié et potassé du N-diméthylammophénylacétonitrile et lithié de l'(éthoxy-éthoxy) phénylacétonitrile (équivalents de benzoyle) sur divers aldéhydes insaturés. Notre étude a porté sur 1'tnfluence de la nature des réactifs, du solvant, du cation, des acides de Lewis et des conditions réactionnelles sur la régiosélectivité et la stéréosélectivité de cette addition. Nous avons mis au point une méthodologie permettant d'accéder aux cétoaldéhydes et aux cétoalcools allyliques, d'obtention généralement délicate, que nous avions appliquée à la synthèse de composés optiquement actifs dont la formation est discutée. Dans le troisième chapitre, l'étude de la décyanation réductrice d'un aminonitrile modèle par divers borohydrures a été réalisée afin d'accéder aux aminoaldéhydes et ammoalcools allyliques. Suivant l'agent réducteur, l'amine attendue est accompagnée du complexe stable amine-borane en proportions variables. L'application de cette méthodologie aux adduits 1,4 a conduit à l'aminoalcool correspondant ; l'adduit 1,2 subit une rétrocondensation
This work deals with the regioselectivity of addition of alpha nitrile carbanions on unsaturated carbonyl compounds in the aim of synthesizing gamma functionalized carbonyl compounds. In the first chapter, we have shawn that the addiion of lithiated 3-pyridylacetonitrile to various enones in tetrahydrofurane, followed by protonation, leads exclusively to 1,4 adducts, the bicylcic ketones are Cis ring fused. The cis or trans stereochemistry of the 2,3-disubstituted cyclanones depends on the protonation of enolates under kinetic or thermodynamic control. The 1,2 addition of this anion on 3-methyl 2-butene 1-al is performed in tetrahydrofurane whereas 1,4 addition is observed under thermodynamic control in the presence of hexamethylphosphoramide or trifluoroboron etherate. In the second chapter, we have realized regioselective additions of lithiated and potassiated dimethylaminophenylacetonitrile and lithiated (ethoxy-ethoxy) phenylacetonitrile (latent benzoyl equivalents) on various unsaturated aldehydes. Our study centers on the influence of the nature of the reactants, solvents, cations, Lewis acids, and experimental conditions on the regio and stereoselectivities of these additions. A methodology leading to ketoaldehydes and keto allylic alcohols, usually difficult to obtain, was improved and applied to the synthesis of optically active compounds. In the third chapter, the reductive decyanation by different borohydrides was studied on an aminonitrile model with the aim of accessing to aminoaldehydes and aminoallylic alcohols. The desired free amine is accompanied by various amounts of stable amine-borane complexe according to the reducing agent. This methodology, applied to 1,4 adducts, leads to the corresponding aminoalcohols while retrocondensation occurs from the1,2 adducts

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13

Chao, Shih-hsien, та 趙士賢. "Cloning and Expression of Arabidopsis endo-1,4-β-glucanase in Pichia pastoris". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/57115889957522041717.

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碩士
國立中山大學
生物科學系研究所
98
In recent years. as industrialized society developed. people have made a lot of environment pollution problems owing to overusing fossil fuel, moreover. fossil fuel is going to deplete. As the result. the study of the substitute energy is promoted. Ligonocellulose (lignin, cellulose and hemicelluloses are included) are the most plentiful renewable resources in the nature, and it have high economic values which extensively use on food, paper and energy. Recent years, it is a hot issue that decomposing cellulose into the minimum unit called glucose, and further, fermenting into alcohol to generate biomass energy. This research goal is cloning Arabidopsis endo-1,4-β-glucanase and transfer to Escherichia coli(DH5α). Selection pPICZαA the success transferr DNA Fragment. Then to sequence and because of cell in pPICZαA have expression in Pichia. pastoris. AOX1 promoter have Mass productions Arabidopsis endo-1,4-β-glucanase gene in Pichia pastoris. Carries on the extracellular expression by the methyl alcohol induction way. Can obtain recombinant DNA protein. However the Western blotting analysis demonstration has this enzyme active protein At4g11050 pellet Molecular weight about 89 KDa. Again by way of congo red and Dye-CMC assay activeness of the examination enzyme protein. The result discovers At4g11050 in the pellet cell activity compares with negative control has the obvious activity.

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14

Ye, Ting-Juan, та 葉婷娟. "Basic properties and characterizations of endo-β-1,4-glucanase from Meiothermus taiwanensis WR220". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/87sgae.

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碩士
國立臺灣大學
生化科學研究所
103
In nature, bacteria, fungi and insects often utilize some cellulases (e.g., endo-β-1,4-glucanase), to acquire their carbon sources from hydrolyzing plants’ cell walls. Industrially, these enzymes are often applied to renewable energy production or paper industry, and can also be used as additives for animal feeds and alcohol fermentation. From whole genome sequence of Meiothermus taiwanensis WR220, we found a protein annotated as endo-β-1,4-glucanase (Mta-Glucanase), which is less commonly observed in Thermus species. Interestingly, the result from sequence alignments, an uncommon region (269-379) was identified, sharing no homology to any other known glucanases. Therefore, in order to elucidate the function of that extra region, we constructed both wild type (GlucanaseWT) and deletion mutant (Glucanase269-379). Based on circular dichroism, although they shared similar CD spectrum, GlucanaseWT contained more percentage of beta-sheet than that of Glucanase269-379. Not only the thermal stability but also the activity of GlucanaseWT is better than those of the mutant. To gain more insight into the mechanism underlying β-1,4-glucan (e.g., CMC), we compared the basic enzyme kinetic parameters of them. According to their Km and kcat, we thought the function of that extra region seems to play a role in binding to the substrate. By homology modeling, we found that region might be similar to the domain in lectin-like proteins responsible for carbohydrate-binding. Together with the results from isothermal titration calorimetry (VP-ITC), we demonstrated the extra region as a sugar-binding domain essential for CMC binding. Taken together, the extra region of GlucanaseWT plays important roles in not only thermal stability but also the affinity to the substrate. These findings are of vital importance in future industrial applications.

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15

Din, Neena. "Characterisation of the cellulose binding domain of endo-β-1,4-glucanase A from Cellulomonas fimi". Thesis, 1994. http://hdl.handle.net/2429/7076.

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Endoglucanase A (CenA) from the bacterium Cellulomonasfimi comprises a catalytic domain and a non-hydrolytic cellulose binding domain (CBD) which can function independently. The DNA fragment encoding CBD[sub CenA] was cloned and expressed in E. coli and the polypeptide produced was characterised in an attempt to examine the mechanisms by which CBDs adsorb to their substrate and to determine the role of CBDs in the process of cellulose hydrolysis. CBD[sub CenA] and related CBDs from other bacterial glycanases have a characteristic motif of four highly conserved tryptophan residues. Each of two of these residues (W14 and W68) was mutated in CBD[sub CenA] to give the mutant polypeptides CBD [sub CenA]W14A and CBD[sub CenA]W68A. The binding affinities of CBD[sub CenA]W14A and CBD[sub CenA]W68A for crystalline cellulose were reduced 50 and 30 fold respectively compared with CBD[sub CenA]. Data obtained from CD, ¹H NMR and tryptophan fluorescence spectroscopy indicated that the mutant polypeptides have a similar conformation to CBDp. Tryptophan fluorescence data also suggested that W14 and W68 are exposed on the surface of CBD[sub CenA] and hence positioned to interact with residues on the cellulose surface upon adsorption of the polypeptide. Tryptophan fluorescence spectroscopy was used to examine the interaction of CBD CenA with the soluble glucans cellohexaose, carboxymethylcellulose and hydroxyethylcellulose and the insoluble substrate, bacterial microcrystalline cellulose (BMCC). No interaction of CBD[sub CenA] with the soluble glucans was detected, however fluorescence quenching of CBD[sub CenA] was observed upon addition of BMCC. The isolated binding domain of CenA released small particles from cotton and disrupted the structure of cellulose fibres. This is the first demonstration of the dispersive effects of a CBD and implies art active role for some CBDs in cellulose hydrolysis. The related CBD[sub Cex] showed similar properties to CBD[sub CenA] in disrupting the structure of cellulose fibres; however the mutant polypeptides CBD[sub CenA]W14A and CBD[sub CenA]W68A did not release small particles from cotton. A co-operative interaction between the isolated binding and catalytic domains of CenA in the release of soluble and insoluble sugar from cotton fibres was observed. The two domains interacted synergistically in the hydrolysis of cotton fibres. This intramolecular synergism is distinct from the previously described intermolecular synergism observed between individual cellulases.

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16

Buchanan, Margaret. "Cellulose, stem strength and the Endo-(1,4)-β-Glucanase gene family in barley and maize". Thesis, 2012. http://hdl.handle.net/2440/78870.

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Failure of the stem to support the grain head in cereals is a serious cause of economic loss to farmers worldwide and increasing the cellulose content of the cell wall has the potential to increase stem strength. The glycosyl hydrolase family 9 endo-(1,4)-β-glucanases are believed to have a role in cellulose synthesis. Plants containing mutants of the KORRIGAN-like endo-(1,4)-β-glucanase genes have perturbed cell walls and reduced cellulose. Also growth enhancement occurs in poplar when overexpressing a soluble endo-(1,4)-β-glucanase. However, little is known of the gene family in cereals. Examination of the GH9 endo-(1,4)-β-glucanase gene family in barley and maize was undertaken with exploration of the potential biological correlation between cellulase activity and stem strength. A detailed phylogeny of the endo-(1,4)-β-glucanase family for barley, maize, sorghum, rice and Arabidopsis was performed using sequence data from various sources. Notwithstanding that the maize genome contains 29 endo-(1,4)-β-glucanase genes, including homoeologues and duplicates, the number of genes across the different species only varies between 23 and 25. The phylogenetic tree showed variations in clade structure between the cereals and Arabidopsis and indicated some differential gene loss and gain. Where information on map position was available, synteny between the cereals was examined and found to be strong. Along with comparative intron and exon gene structure studies, orthologous genes across barley, maize, sorghum and rice were identified. An evolutionary analysis provided evidence that sorghum was the donor genome in an allotetraploid event in maize that occurred 10-20 million years ago. It was also possible to differentiate between homoeologues resulting from the tetraploidisation of maize and gene duplications. A transcript analysis for barley and maize was performed and indicated that some members of the endo-β-1,4-D-glucanase gene family are transcribed across a wide range of tissues while other genes are specific to one tissue. Furthermore, there are strong correlations between several members of the endo-(1,4)-β-glucanase family at the transcriptional level. Similar correlations were also shown to exist between members of the endo-(1,4)-β-glucanase family and other genes known to be involved in cell wall synthesis. This data also suggested that evolutionary conservation of transcription exists between orthologues in barley and maize. Analysis of protein activity in barley was undertaken on plants that had been transformed with endo-(1,4)-β-glucanase genes using the 35s overexpression promoter. Stem strength of these transgenic plants was found to be positively correlated with cellulose levels. Plants of the T₁ generation showed strong phenotypic variation. A number of plants were dwarfed and had much reduced levels of cellulose. It was speculated that gene inactivation of both transgene and the endogenous gene was producing ”knockdown” plants. On the other hand, some overexpressing plants had higher levels of cellulose than the control plants, however, this was not strongly correlated with stronger stems. Heterologous expression of barley endo-(1,4)-β-glucanases in microbial systems was attempted. In E.coli large amounts of protein were produced, but it was largely insoluble and inactive.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 2012

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17

Robson, Lori Marlénè. "Endo-[beta]-1,4-glucanase production by Bacillus subtilis DLG." 1986. http://catalog.hathitrust.org/api/volumes/oclc/15536490.html.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1986.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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18

Kung, Ching-Yu, та 孔慶宇. "Purification and characterization of a chimeric1,3-1,4-β-D-glucanase and β-1,4-xylanase enzyme". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/y9x8z3.

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碩士
國立臺北科技大學
有機高分子研究所
97
Truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (TG) specifically hydrolyzes 1,4-β-D-glucosidic bonds adjacent to 1,3-β-linkages in mix-linked β-glucans or lichenan. Truncated uncultured Neocallimastigales xylanase (TX) is a hydrolytic enzyme which cleaves the β-1,4 glycosidic bonds of the complex plant cell wall polysaccharide xylan. A chimeric TG and TX was expressed successfully as a 6xHis tagged recombinant protein in Escherichia coli BL21 (DE3). The purified recombinant chimeric protein exhibited both TG and TX catalytic activities. Optimal temperature 45 ℃ and pH 7.5 were found for the TG activity, whereas 55 ℃ and pH 7.0 were determined for the TX activity. Compared with the wild type single enzyme activity, the TG increased 1.8-fold and TX activity decreased 0.9-fold in catalytic efficiency value (kcat/Km), respectively. Recombinant chimeric enzyme was stable when incubated in 30-55 ℃ and pH 3-10. This is the first successful attempt to fuse glucanase and xylanase as one chimera while retaining individual enzymatic activities. X-ray crystallographic study for the recombinant protein is in progress.

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19

Hsu, Li-Chuan, та 許力川. "Isolation, purification and characterization of endo-β-glucanase of Xylaria regalis". Thesis, 2003. http://ndltd.ncl.edu.tw/handle/16711462006163520059.

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碩士
國立陽明大學
生物化學研究所
91
Abstract Cellulose is a linear polysaccharide of glucose residues connected by β-1,4- glucosidic linkages. Native crystalline cellulose is insoluble and occurs as fibers of densely packed, hydrogen bonded chains of 15 to 10,000 glucose units. Its density and complexity make it very resistant to hydrolysis without chemical or mechanical degradation. As a result, utilization of cellulase is the most important step during energy conversion from cellulosic biomass. A cellulosic enzyme system consists of three major components: endo-β- glucanase, exo-β-glucanase andβ-glucosidase. Endo-β-glucanase makes random scission of cellulose chains yielding glucose and cello-oligo saccharides. Exo-β- glucanase commits exo-attack on the non-reducing end of cellulose with cellobiose as the primary structure. β-glucosidase hydrolyzes cellobiose to glucose. These three enzymes act synergistically to degrade cellulose. In this study, we isolate, purify and characterize endo-β-glucanase by Ascomycete Xylaria regalis. The cellulose-containing medium is used to induce the production of the cellulose-degrading enzymes. When these enzymes are produced at maximum level, we harvest and filtrate the culture medium. The filtrate is purified by anion exchanger column (QAE) and hydrophobic interaction column (phenyl-650M). The purification fold of endo-β- glucanase increases 4.3 times, the specific activity approaches 3.7 unit/mg, and the recovery is 27.6%, respectively. SDS-PAGE shows that the molecular weight of endo-β-glucanase is 47 kDa. The optimal pH of the enzyme is 5 and the optimal temperature of the enzyme is 50℃. The pH stability of enzyme is between 4 and 5. The thermostability of enzyme is 40℃. Some metal ions, like Mg2+, Zn2+, Cu2+, Fe3+, Hg2+ and Ba2+, slightly enhance the enzyme activity; however, Co2+ ion suppresses it.

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20

Wei-ChengHuang and 黃偉晟. "Characterization of an endo-1,4-beta-glucanase from an anaerobic ruminal bacterium in Nubian goat." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/31281509889234562167.

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21

Huang, Hsiao-Chuan, та 黃曉娟. "Crystal Structure of Truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase Mutant F40I". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/97w3k4.

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碩士
國立臺北科技大學
有機高分子研究所
95
Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase, lichenase; E.C. 3.2.1.73) belongs to glycosyl hydrolases family 16 (GHF 16). Fsβ-glucanase specifically hydrolyzes the β-1,4-glycosidic bonds when β-1,3-glycosidic linkages are located prior to β-1,4-glycosidic bond in lichenan or β-D-glucans. It has been suggested that the truncated Fsβ-glucanase (designated TFsβ-glucanase) could accommodate five glucose rings into its active site upon enzyme-substrate interaction and 20 residues involved with substrate binding. Phe40 was proposed to make stacked interaction with subunit -1 of carbohydrate substrate in our previous study of enzyme complex structure. The mutant F40I crystal was found to be orthorhombic, space group P212121, with cell dimension a = 60.3 Å, b = 68.0 Å and c = 70.5 Å, to a 1.8 Å resolution. The overall structure folding of F40I mutant remained in a nearly identical jellyroll β-sandwich as that of the wild type enzyme. Moreover, an additional Ca+2 ion binding region coordinated with seven coordinations is present. The F40I structure and kinetic data suggest that Phe40 play an important role in stabilizing substrate binding and catalytic function.

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22

Chiang, Yuan-Neng, та 江元能. "Crystal Structure of Truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase Mutant E85I". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/4r3zv6.

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碩士
國立臺北科技大學
有機高分子研究所
96
Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase, EC 3.2.1.73) belongs to glycosyl hydrolases family 16. Fsβ-glucanase specifically hydrolyzes the β-1,4-glycosidic bonds when β-1,3-glycosidic linkages are located prior to β-1,4-glycosidic bond in lichenan or β-D-glucans. It has been suggested that the truncated Fsβ-glucanase could accommodate five glucose rings into its active site upon enzyme-substrate interaction and many residues involved with substrate binding. Glu85 was proposed to make stacked interaction with subunit +1 of carbohydrate substrate in our previous study of enzyme complex structure. Here we describe the mutant E85I crystal structure to a 2.2 Å resolution. The overall structure of E85I mutant was similar to the jellyroll β-sandwich structure of the wild type enzyme. One Ca2+ ion and one Cs+ ion were found on the surface of the E85I structure with six- and five- ligands, respectively. Further analysis of the E85I mutant structure revealed that the catalytic residue Glu60 shifted away because the loop located at the concave site moved approximately 2 Å from its position in the native enzyme complex. The E85I structure and kinetic data suggest that Glu85 plays an important role in catalytic function.

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23

Wang, Shih-Hsiang, та 汪詩祥. "Purification, crystallization and survey of potential inhibitors for endo-1,3-β-glucanase from Streptomyces sioyaensis". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/53050266561896847566.

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碩士
國立中興大學
生物化學研究所
94
β-1,3-glucanases specially hydrolyze β-1,3-D-glucosidic linkages of β-1,3-glucans. Based on the product and the hydrolysis reactions catalyzed by the glucanase, β-1,3-glucanases are classified into exo-1,3-β-glucanase (EC 3.2.1.58) and endo-1,3-β-glucanase (EC 3.2.1.39). In plants, these enzymes are thought to be a type of defense system against fungal pathogens. In fungi, β-1,3-glucanases involved in fungal pathogen-plant interactions during pathogen attack. In bacteria, it is related to the assimilation of fungal cell walls as a food source. In this project, the endo-1,3-β-glucanase N-termainal catalytic domain from Streptomyces sioyaensis shares sequence similarity with bacterial endo-1,3-β- glucanases which is classified as glycosyl hydrolase family 16 (GHF 16). The protein fragment is 273 a.a. in lengh and the molecular weight is 29.4 kDa. The other homologous GHF 16 endo-1,3-1,4-β-glucanase (EC 3.2.1.73) catalyze the hydrolysis of β-1,4- D- glucosidic linkages in β-D-glucans with mixed 1,3- and 1,4-linkages. The overall structure of endo-1,3-β-glucanase N-terminal catalytic domain and endo-1,3-1,4-β-glucanase share a similar jellyroll β-sandwich fold. Yet, their substrate specificity is very different. Here, we aim to investigate the substrate binding specificity of endo-1,3-β-glucanase through structural determination of the β-1,3- glucan and substrate co-crystal. The catalytic domain of endo-1,3-β-glucanase from Streptomyces sioyaensis was purified in large quantity and reproduce good quality crystals by using the vapor diffusion and microseeding techniques. We examined four derivatized mono saccharide compounds, 4-Nitrophenyl β-D- glucopyranoside, 4-Nitrophenyl α-D-glucopyranoside, 4-Nitrophenyl β-D- galactopyranoside, 4-Nitrophenyl β-D-xylopyranoside of endo-1,3-β-glucanase for potential inhibitors. Although we found all of the result did not show promising candidate for endo-1,3-β-glucanase co-crystals, the developed method can be used to screen for possible inhibitor candidates.

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24

Lin, Hsia-Ting, та 林夏亭. "Molecular cloning and analysis of 1,3-1,4-β-glucanase in Bacillus subtilis WL-A12". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/01287828775151304762.

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25

Chou, Jiun-Shian, та 周峻賢. "Expression of exogenous protein β-1,3-1,4-glucanase and xylanase in Lactobacillus and its application". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/m86e6k.

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26

Hung, Yu-Lung, та 洪玉龍. "The cloning and biochemical characterization of β-1,3-1,4-glucanase genes from Neocallimastix frontalis J11". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/81447005465634943536.

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Анотація:

碩士
國立屏東科技大學
生物科技研究所
95
Abstract Student ID：M9318015 Title of Thesis:：The cloning and biochemical characterization of β-1,3-1,4-glucanase genes from Neocallimastix frontalis J11 Total Page：66 Name of Institute:National Pingtung University of Science Technoogy Name of Department:Graduate Institute of Biotechnology Graduate Date：July, 2007 Degree Conferred：Master Name of Student：Yu-Lung Hung Adviser：Yo-Chia Chen The Contents of Abstract in This Thesis： β-1,3-1,4-glucans constitute a non-substituted group of hemicellulosess. Structurally, they are linear glucans of up to about 1200 β-D-glucosyl residues linked through β-1,3 and β-1,4 glycosidic bonds, with variations in the proportion of both types of linkages. β-1,3-1,4-glucanase exhibits a strict substrate specificity for cleavage of β-1,4 glycosidic bonds in 3-O-substituted glucopyranose units, and the main final hydrolysis products of barley β-glucans are the trisaccharide (3-O-β-cellobiosyl-D-glucopyranose) and the tetrasaccharide (3-O-β-cellotriosyl-D-glucopyranose), and are widely applied in the beer brewing and animal feedstuff industries. In this study, four β-1,3-1,4-glucanase genes, m2, a13, a16 and a51 , were isolated from cDNA libraries of an anaerobic rumen fungus Neocallimastix frontalis J11 and sorted by three- dimensional protein structure from seven β-1,3-1,4-glucanase genes . Based upon the results of the DNA sequence alignment, the most similar sequence with the four recombinant enzymes was Orpinomyces sp. PC-2 licA gene and it showed 81.8 to 82.3 percent sequence similarity to the known licA gene. The four β-1,3-1,4-glucanase genes, m2, a13 a16 and a51, expressed in Escherichia coli BL21(DE3) had the same molecular weight about 27 kDa, and the specific activities which using barley β-glucans as substrate were 41,209, 32,095, 39,524 and 26,529 U/mg, respectively. Optimal temperature for enzyme activity was 45℃ except for M2 which was 50℃, and optimal pH was 6 besides A51 which was 6.5. A13 had better thermal stability than others, and M2 was the best pH stability enzyme. The effect of various metal ions on the activity indicated that the activities of all of the recombinant enzymes were significant increased by Co2+. The results of substrate specificity analysis showed that the four recombinant enzymes were only able to hydrolyse barley β-glucans and lichenans. Comparing with the diversities of protein structures and biochemical properties within the four recombinant enzymes, there was probably no direct relationship between them. Key words: β-1,3-1,4-glucan, β-1,3-1,4-glucanase , Neocallimastix frontalis

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27

Hsiao, Ching-Hua, та 蕭敬樺. "Structural and functional analysis of truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase mutant W203F". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/8fv5uz.

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Анотація:

碩士
國立臺北科技大學
有機高分子研究所
97
1,3-1,4-β-D-glucanases hydrolyze and cleave β-1,4-glycosidic bonds precisely where β-1,3-glycosidic linkages are located prior to β-1,4-glycosidic linkages in lichenan or β-D-glucans. The truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (TFsβ-glucanase) in complex with its product has revealed that the β-1,3-1,4-cellotriose product spans subsites -3 to -1 in its active cleft in interaction with 14 amino acid residues. The four aromatic residues Phe40, Tyr42, Phe205, and Trp203 were found to make hydrophobic interactions with subsites -1, -2, and -3 of β-1,3-1,4-cellotriose, respectively. In order to understand the structural and functional roles of the aromatic residues, many mutants have been produced. The structure of the W203F mutant diffraction data were collected to 1.4 Å resolution. The active site topology was similar to that of the native complex structure, due to the presence in the structure of Tris molecules which occupied the place normally taken by the -1 subsite of the substrate, bound to the catalytic residues Glu56 and Glu60. In addition, two extra calcium ions were found in both mutant structures. Kinetic experiments revealed that Tris is a competitive inhibitor of the W203F mutant. This study suggests that aromatic residues in the active site of TFsβ-D-glucanase play critical roles in protein-carbohydrate binding, stabilization and catalysis.

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28

Li, Wei-Ru, та 李韋儒. "Tris and Calcium ion inhibitory effect on the truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/43t2xc.

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Анотація:

碩士
國立臺北科技大學
有機高分子研究所
98
Fibrobacter succinogenes 1,3-1,4-β-D-glucanase hydrolyzes and cleaves β-1,4-glycosidic bonds precisely where β-1,3-glycosidic linkages are located prior to β-1,4-glycosidic linkages in lichenan or β-D-glucans. Four aromatic residues, Phe40, Tyr42, Trp203 and Phe205 in the catalytic domain of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (TFs-β-glucanase) interact with sugar units of the product via hydrophobic stacking interactions. For the purpose of understanding of the structural and functional roles of the active site residues, many mutants have been characterized. The crystal structures of the F40I and W203F mutants revealed that two extra calcium ions and a Tris molecule have been identified. The Tris molecule was found at the position normally taken by substrate binding to the -1 subsite and bound to the catalytic residues Glu56 and Glu60. In addition, a second Ca2+ ion was found near residues Phe152 and Glu154, and a third near the active site. In order to understand the kinetic inhibition properties of Tris and the calcium ion, kinetic experiments were performed. Further kinetics analysis revealed that Tris is a competitive inhibitor of the enzyme, while calcium ion is a non-competitive inhibitor.

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29

Chang, Wen-Chin, та 張文進. "Structural and functional analysis of the truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase mutant Y42L and inhibitor complex". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/uye5f6.

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Анотація:

碩士
國立臺北科技大學
有機高分子研究所
100
Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase, E.C.3.2.1.73) specifically hydrolyze the β-1,4 bonds when β-1,3 linkages are located prior to the β-1,4 bonds in β-D-glucan or lichenan. The final hydrolyzed products are tri- and penta- saccharides. Three calcium ions and two tris molecules are found in the truncated Fsβ-glucanase mutant Y42L structure. The first calcium ion is located at the same position as that of wild type. The second Ca2+ ion was found near the residues Phe152 and Glu154 on the protein’s surface, and the third one near the active site residue Asp202. Moreover, a tris molecule interacts with the catalytic residues Glu56 and Glu60 at subunit -1 of substrate. Based on the kinetic data, it is shown that the third Ca2+ ion and tris molecule are non-competitive and competitive inhibitors for the enzyme, respectively.

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30

Lin, Chun-Jung, та 林君蓉. "Structural insights into the substrate specificity and transglycosylation activity of an endo-1,4-β-mannanase from soybean (Glycine max)". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/2p29up.

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Анотація:

碩士
國立臺灣大學
農業化學研究所
105
Endo-1,4-β-mannanase (β-mannanase, EC. 3.2.1.78) is a hydrloase that catalyzes cleavage of β-1-4 bonds in the mannan polymer. This enzyme family is involved in soften of the mannan-rich cell walls and consumption of endosperm, which is benifical to radicle protrusion upon seed germination. However, there is limited information about the structural and functional relationship of plant-type β-mannanase. In this study, plant-type β-mannanases from soybean (Glycine max) were studied, since soybean is not only a globally important commercial crops, but also a potential material for use in food, feed or industrial applications. Using genome mining, we find out that there are 21 types of β-mannanase gene in the genome of soybean, and GmMAN19-1 was selected as primiary target due to its unique position on phylogentic tree. RT-PCR data showed GmMAN19-1 was expressed only in the cotyledons tissue after 7-day germination. Purified recombinant GmMAN19-1 was acidophilic with a pH optimum of 4.6, and exhibited a higher activity to linear polysaccharides. For detailed information, crystal sturctures of GmMAN19-1 in apo form and in complex with mannopentose were determined. Intriguingly, the complex structure existed two distinct binding modes of mannopentaose, presented as the substrates for glycohydrolase (subsides -3, -2, -1, +1, +2) and transglycohydrolase (subsides -5, -4, -3, -2, -1), respectively. In addition, structural comparison of GmMAN19-1 with other β-mannanases from fungus reveals that GmMAN19-1 has two extended loops, producing a narrower active site cleft. Based on the solved structure of GmMAN19-1/pentaose complex, rational design was conducted to engineer GmMAN19-1 in an attempt to alter the substrate selectivity toward branched mannans. Among the 5 mutants we constructed, the most promising Q267W showed a 50% increase in specific activity toward the branched-mannan guar gum by comparison with the wild-type enzyme. GmMAN19-1-Q267W, GmMAN19-1-Y264W and its complex were also structurally characterized. Taken together, our findings provide structural insights into the substrate specificity and transglycosylation activity of GmMAN19-1 and demonstrate the structural differences between plant-type and fungal β-mannanase. In particular, Q267W mutant shows potential to hydrolysis branched substrate, which providing a basis for further enzymatic engineering of GmMAN19-1 and molecular breeding of soybean.

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31

Willis, Jonathan Duran. "Identification and Characterization of Novel Cellulases from Dissosteira carolina (Orthoptera: Acrididae) and Molecular Cloning and Expression of an Endo-beta-1,4-Glucanase from Tribolium castaneum (Coleoptera: Tenebrionidae)." 2009. http://trace.tennessee.edu/utk_gradthes/573.

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Дисертації з теми "Endo-(1,4)-β-glucanase"

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