Thèses (Ph.D.)--Université de Sherbrooke (Canada), 1998. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 120-148).

Yuan Zhao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 89-96). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Lignocelluloses --- p.2 / Chapter 1.1.1 --- component of lignocellulose --- p.2 / Chapter 1.1.2 --- Xylans --- p.3 / Chapter 1.2 --- Degradation of lignocellulose --- p.9 / Chapter 1.3 --- "Endo-β-1,4- xylanase" --- p.12 / Chapter 1.3.1 --- Structure of xylanase --- p.12 / Chapter 1.3.2 --- Mode of action --- p.17 / Chapter 1.3.3 --- Appications of xylanase --- p.20 / Chapter 1.4 --- Aims of my study --- p.24 / Chapter Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Cloning of xylanase genes --- p.26 / Chapter 2.1.1 --- Materials --- p.26 / Chapter 2.1.1.1 --- Bacterial and fungal strains --- p.26 / Chapter 2.1.1.2 --- Growth media --- p.26 / Chapter 2.1.1.3 --- Vector --- p.26 / Chapter 2.1.1.4 --- Reagents for agarose gel electrophoresis --- p.27 / Chapter 2.1.1.5 --- Reagents for preparation of competent cells --- p.27 / Chapter 2.1.2 --- Methods --- p.29 / Chapter 2.1.2.1 --- Isolation of chromosomal DNA --- p.29 / Chapter 2.1.2.2 --- Amplification of exons of xylanase genes --- p.29 / Chapter 2.1.2.3 --- Agarose gel electrophoresis of DNA --- p.37 / Chapter 2.1.2.4 --- DNA recovery from agarose gel --- p.37 / Chapter 2.1.2.5 --- Assemble and amplify the full length genes --- p.38 / Chapter 2.1.2.6 --- Restriction endonuclease digestion --- p.39 / Chapter 2.1.2.7 --- Ligation of purified DNA fragment into vector --- p.39 / Chapter 2.1.2.8 --- Transformation --- p.40 / Chapter 2.1.2.9 --- Methods for making competent cells --- p.40 / Chapter 2.1.2.10 --- Plasmid DNA preparation --- p.40 / Chapter 2.1.2.11 --- DNA sequencing --- p.41 / Chapter 2.2 --- Mutagenesis of xylanase --- p.43 / Chapter 2.2.1 --- Amplification of xylanases genes --- p.47 / Chapter 2.2.2 --- DNA random mutagenesis --- p.48 / Chapter 2.2.2.1 --- DNase digestion --- p.48 / Chapter 2.2.2.2 --- Reassembly of DNA fragments --- p.48 / Chapter 2.2.2.3 --- Amplification of full-length genes --- p.48 / Chapter 2.2.2.4 --- Construction of library --- p.49 / Chapter 2.2.3 --- Screening of mutants --- p.49 / Chapter 2.2.3.1 --- Preparation of RBB-xylan --- p.49 / Chapter 2.2.3.2 --- Plate assay for screening of mutants --- p.50 / Chapter 2.3 --- Expression of xylanase genes --- p.51 / Chapter 2.4 --- Enzyme assays --- p.52 / Chapter 2.4.1 --- Xylanase assay with RBB-xylan --- p.52 / Chapter 2.4.2 --- Xylanase assay with DNS-method --- p.52 / Chapter 2.4.2.1 --- Reagents --- p.53 / Chapter 2.4.2.2 --- Xylose standard curve --- p.53 / Chapter 2.4.2. 3 --- Activity assay --- p.54 / Chapter 2.4.2. 4 --- Thermostability assay --- p.54 / Chapter Chapter 3 --- Results --- p.55 / Chapter 3.1 --- Cloning of xylanase genes --- p.56 / Chapter 3.2 --- Mutagenesis of xylanase --- p.59 / Chapter 3.2.1 --- DNA random mutagenesis --- p.59 / Chapter 3.2.2 --- Screening of mutants --- p.67 / Chapter 3.3 --- Enzyme assays --- p.69 / Chapter Chapter 4 --- Discussions --- p.76 / Chapter 4.1 --- Gene shuffling --- p.77 / Chapter 4.2 --- Screening method and activity assay --- p.78 / Chapter 4.3 --- Sequence analysis --- p.80 / Chapter 4.4 --- Future work --- p.88 / Bibliography --- p.89

Bu Su. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 139-158). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Peijun Zuo. / "November 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 188-212). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

No description available.

No description available.

Submitted in complete fulfillment for the Degree of Master of Science (Applied Science), Durban University of Technology, Durban, South Africa, 2016. / β-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant. / M

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-15Bitstream added on 2014-06-13T18:41:24Z : No. of bitstreams: 1 aragon_cc_dr_araiq_parcial.pdf: 352399 bytes, checksum: 4041cfc92fd5c40daad9f3969d15d31d (MD5) Bitstreams deleted on 2015-02-23T17:35:46Z: aragon_cc_dr_araiq_parcial.pdf,Bitstream added on 2015-02-23T17:36:21Z : No. of bitstreams: 1 000718951.pdf: 1019999 bytes, checksum: b74f94cf1bb93fd6c428e6e6426f5372 (MD5) / As xilanases são glicosidases que catalisam a hidrólise das ligações 1,4-β-xilosídicas da xilana e que possuem potencial biotecnológico em vários processos industriais, como na clarificação de sucos e vinhos, na fabricação de pães, na filtração da cerveja e no tratamento das polpas celulósicas. Recentemente, recebem atenção pela produção dos xilooligossacarídeos como ingredientes prebióticos. Embora possuam diversas vantagens sobre os métodos químicos, as enzimas são, geralmente, limitadas para uso industrial. A imobilização em suportes sólidos melhora a estabilidade dos biocatalisadores e o controle operacional, promovendo a recuperação do produto sem a contaminação pela enzima. Assim, os objetivos deste trabalho foram: produzir e caracterizar a xilanase de Aspergillus niger; imobilizar covalentemente, em suportes sólidos, a xilanase de A. niger e quatro outras, provenientes de diferentes fontes (Aspergillus versicolor, Trichoderma longibrachiatum, Thermomyces lanuginosus e Streptomyces halstedii); caracterizar os derivados obtidos; analisar o produto de hidrólise da xilana pelos derivados. A xilanase de A. niger mostrou ótima estabilidade até 55°C, com meia-vida de 15 minutos a 60°C, e sua produção foi induzida por pequenas concentrações de xilose. O fungo secretou pelo menos duas isoformas com atividade xilanolítica. A xilanase I foi purificada em uma única etapa, por adsorção em suporte ativado com quelatos, assim como a enzima de S. halstedii, contendo cauda de histidina. A xilanase de T. longibrachiatum (comercial) foi parcialmente purificada com suportes iônicos, e as de T. lanuginosus (comercial) e A. versicolor já apresentavam alto grau de pureza. As enzimas foram imobilizadas em agarose ativada com diferentes grupos reativos, com fatores de estabilização entre 12 (T. lanuginosus) e 600... / Xylanases are glycosidases that catalyze the hydrolytic cleavage of β-1,4-linked polymers of D-xylose and they have biotechnological potential in various industrial processes such as in the clarification of juices and wines, in the manufacturing of breads, in beer filtration and in the treatment of cellulose pulps. Recently, attention is given to the production of xylo-oligosaccharides as prebiotic ingredients. While enzymes have several advantages over chemical methods, they are generally limited for industrial use. Immobilization on solid supports improves the stability of the biocatalyst and the operational control, and promotes the easy recovery of the product without contamination by the enzyme. The objectives of this study were: to produce and characterize xylanases from Aspergillus niger; to immobilize the xylanase of Aspergillus niger and four others from different sources (Aspergillus versicolor, Trichoderma longibrachiatum, Thermomyces lanuginosus and Streptomyces halstedii) covalently on solid supports; to characterize the derivatives obtained; to analyze the products profile of xylan hydrolysis by the derivatives. The xylanase of A. niger showed excellent stability up to 55°C, with a half-life of 15 minutes at 60°C, and its production was induced by low concentrations of xylose. The fungus produced at least two isoforms with xylanolytic activity. Xylanase I was purified in one step by adsorption on support activated with chelates, as well as the histidine-tagged enzyme of S. halstedii. The xylanase of T. longibrachiatum (commercial) was partially purified with ionic supports, and those from T. lanuginosus (commercial) and A. versicolor already showed high degree of purity. The xylanases were then immobilized on agarose activated with different reactive groups, and presented stabilization factors between 12 (T. lanuginosus)... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-10-24Bitstream added on 2014-06-13T19:28:58Z : No. of bitstreams: 1 zaneti_vm_me_araiq.pdf: 906459 bytes, checksum: 31326d4c24892f34266c70f06d8d025a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho foi utilizada a metodologia de superfície de resposta, por meio de delineamento composto central rotacional para investigar as melhores condições de produção de xilanase pelo fungo filamentoso Aspergillus niger. Este micro-organismo é considerado um bom produtor de enzimas xilanases, sendo que estas enzimas têm a capacidade de hidrolisar xilana em xilooligossacarídeos e xilose. Produtos assim obtidos estão sendo cada vez mais utilizados em rações animais para melhoria da flora intestinal; para a produção de xilitol e também para a produção de álcool de segunda geração. A análise estatística dos resultados obtidos neste trabalho mostrou que as melhores condições de produção da enzima extracelular foram: pH 5,0, temperatura de 37 ºC, agitação de 80 rpm, e concentração de fonte de carbono de 2 % (p/v). Após a determinação das condições ideais, o extrato foi clarificado por filtração em caulim, e as proteínas assim obtidas foram precipitadas com acetona ocorrendo uma melhora sensível na atividade específica. Após filtração em Sephadex G-75 foi mostrada a presença de atividade xilanolítica em dois picos, e as frações referentes ao segundo pico foram reunidas e submetidas à coluna de troca iônica DEAE-Trisacryl, na qual se constatou uma fração sendo eluída com 0,06 mol/L de NaCl, contendo atividade de xilanase. A SDS-PAGE da fração majoritária revelou uma única banda protéica com massa molar aparente de 34 kDa. A cromatografia em sílica gel P60 revelou que os produtos de hidrólise foram constituídos de xilooligossacarídeos, após 120 min de hidrólise / In the present work, response surface methodology was utilized, through appliance of rotational central composite design to investigate the best conditions of production of xylanase by the filamentous fungus Aspergillus niger. This microorganism is considered a good producer of xylanase enzyme, which has the ability to hydrolyze hemicelluloses in xylooligosaccharides and xylose. Products obtained this way are being increasingly utilized in animal feeding to improve the intestinal flora, to produce xylitol and also to produce second generation ethanol. The statistical analysis of the obtained results showed that the best conditions for the extracellular enzyme production were: pH 5.0, 37 ºC, 80 rpm shaking, and 2 % (w/v) carbon source concentration. After the determination of the ideal production conditions, the enzyme extract was clarified through filtration in kaolin, and the protein obtained were precipitated with acetone, with a sensitive increase in the specific activity. After molecular exclusion chromatography in Sephadex G-75 the presence of xylanolitic activity was shown in two peaks, and the fractions related to the second peak were collected and submitted to DEAE-Trisacryl ion exchange column, in which were observed fractions showing xylanase activity that was eluted with 0.06 mol/L NaCl. SDS-PAGE of the majority fraction revealed only one proteic band with apparent molar mass of 34 kDa. P60 silica gel chromatography revealed that the product of hydrolysis was constituted of small xylooligosaccharides released after 120 min of hydrolysis