Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 81-84). Also available via the Internet.

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.

Thesis (Ph.D.)--Ohio University, August, 2009. / Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (leaves 111-128)

Thesis (Ph.D.)--Ohio University, August, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (leaves 111-128)

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Plant biomass, the most abundant renewable resource in nature, consists of matrices of mainly lignin, cellulose, hemicellulose as well as inorganic components. Xylan, the major hemicellulose component in plant cell walls, is the most abundant polysaccharide after cellulose. This makes the main constituent sugar of xylan, D-xylose, the second most abundant renewable monosaccharide in nature. Very few hemicelluloses are either homopolymeric or entirely linear. Therefore, the variety of enzymes involved in their hydrolysis is more complex than the enzyme group responsible for the hydrolysis of cellulose. Although the ability to degrade xylan is common among bacteria and filamentous fungi, this trait is relatively rare among yeasts. However, some strains of the yeast Pichia stipitis are, amongst others, able to degrade xylan. As P. stipitis is also one of the best D-xylose fermenting yeasts thus far described, this yeast has the potential of fermenting polymeric xylan directly to ethanol. However, it was shown that the natural xylanolytic ability of this yeast is very weak. In this study, xylanolytic genes were expressed in P. stipitis to test the ability of the yeast to produce heterologous proteins, and to determine the enhancement of xylan utilisation by the recombinant strain. The native xylose reductase gene (XYLl) and transketolase gene (TKL) and the heterologous Saccharomyces cerevisiae phosphoglycerate kinase (PGKl) gene promoter were cloned into P. stipitis transformation vectors and used to express the Trichoderma reesei ~-xylanase encoding gene (xyn2) as reporter gene. It was shown that the XYLl promoter was induced in the presence of D-xylose and that the TKL promoter was constitutively expressed. The PGKl promoter of S. cerevisiae did not function in P. stipitis . When the T reesei xyn2 gene and the Aspergillus kawachii ~-xylanase encoding gene (xynC) were expressed under control of the XYLl promoter, extracellular ~-xylanase activity of up to 136 nkat/ml and 171 nkatlml was observed, respectively. This activity declined over time due to the presence of extracellular proteases, secreted by P. stipitis. Growing the cultures in a fermentor and controlling the pH level to pH 6 did not alleviate the reduction of heterologous l3-xylanase activity. When the Aspergillus niger l3-xylosidase encoding gene (xlnD) was expressed as a fusion gene (designated XL02) with the S. cerevisiae mating factor secretion signal (MFal) under control of the P. stipitis TKL promoter, extracellular l3-xylosidase activity of 0.132 nkatlml was observed. Co-expression of the xyn2 and XL02 genes led to B-xylanase and l3-xylosidase activities of 128 nkatlml and 0.113 nkat/ml, respectively. Co-expression of the xynC and XL02 genes led to l3-xylanase and l3-xylosidase activities of 165 nkat/ml and 0.124 nkatlml, respectively. The expression of the fungal xylanolytic genes in P. stipitis also led to an increased biomass yield when the recombinant strains were cultured on birchwood xylan as sole carbon source. The strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding genes was the most successful, yielding a 3.2-fold higher biomass level than the control strain. Biomass levels of the recombinant strains were further improved on average by 85% by growing them in a fermentor under conditions of high oxygenation. The strains were also tested for direct conversion of xylan to ethanol and the strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding genes produced 1.35 giL ethanol, which represents a 3.6-fold increase in ethanol yield over the reference strain. These strains represent a step towards the efficient degradation and utilisation of hemicellulosic materials by ethanol-producing yeasts. / AFRIKAANSE OPSOMMING: Plant biomassa, die volopste hernubare koolstotbron in die natuur, bestaan uit matrikse van lignien, sellulose en hemisellulose. Xilaan, die hoof hemisellulose komponent in plantselwande, is na sellulose die volopste polisakkaried. Gevolglik is die hoof suikerkomponent van xilaan, naamlik D-xilose, die tweede volopste hernubare monosakkaried in die natuur. Baie min hemisellulose molekules is homopolimere of heeltemal linieêr. Daarom is die ensieme betrokke by die atbraak van hemiselluloses meer kompleks as die ensieme betrokke by die atbraak van sellulose. Bakterieë en filamentagtige fungi wat oor die vermoë om xilaan af te breek beskik, kom wydversprei voor maar relatief min giste kan xilaan benut. Sommige rasse van die gisspesie Pichia stipitis het egter beperkte vermoë om xilaan af te breek. P. stipitis is ook een van die beste D-xilose fermenterende giste wat tot dusver beskryf is en het dus die potensiaalom etanol vanafpolimeriese xilaan te produseer. In hierdie studie is gene wat kodeer vir xilaanatbrekende ensieme in P. stipitis uitgedruk om die vermoë van die gis as heteroloë uitdrukking sisteem te evalueer. Verder is die effek van die heteroloë xilaanatbrekende ensieme tydens groei op xilaan as enigste koolstotbron getoets. Die promoters van die xilosereduktasegeen (XYLl), die transketolasegeen (TKL) van P. stipitis en die fosfogliseraatkinasegeen (PGKl) van Saccharomyces cerevisiae is in P. stipitis transformasie vektore gekloneer en gebruik om die Trichoderma reesei ~-xilanasegeen (xyn2) as verklikkergeen uit te druk. Dit het bewys dat die XYLI promotor induseerbaar is in die teenwoordigheid van D-xilose terwyl die TKL geen konstant uitgedruk was. Die PGKI promotor van S. cerevisiae was nie funksioneel in P. stipitis nie. Ekstrasellulêre ~-xilanase aktiwiteit van onderskeidelik 136 nkatlml en 171 nkatlml kon waargeneem word wanneer die T reesei xyn2 geen of die Aspergillus kawachii ~-xilanasegeen (xynC) onder beheer van die XYLI promotor uitgedruk is. Hierdie aktiwiteit het afgeneem na gelang van tyd a.g.v. die teenwoordigheid van ekstrasellulêre proteases wat deur P. stipitis uitgeskei word. Die afname van ekstrasellulêre ~-xilanase aktiwiteit kon nie voorkom word deur die kulture in 'n fermentor te groei en die pH vlak tot pH 6 te beheer nie. Tydens uitdrukking van die Aspergillus niger ~-xilosidase geen (xlnD) as 'n fusiegeen (genoem XL02) met die paringsfaktor sekresiesein (MFal) van S. cerevisiae onder transkripsionele beheer van die P. stipitis TKL promotor, kon ekstrasellulêre ~-xilosidase aktiwiteit van 0.132 nkatlml waargeneem word. Gesamentlike uitdrukking van die xyn2 en XL02 gene het gelei tot ~-xilanase en ~-xilosidase aktiwiteite van 128 nkatlml and 0.113 nkat/ml, onderskeidelik. Gesamentlike uitdrukking van die xynC en XL02 gene het gelei tot ~-xilanase en ~-xilosidase aktiwiteite van 165 nkatlml and 0.124 nkatlml, onderskeidelik. Die uitdrukking van xilaanatbrekende ensieme III P. stipitis het verhoogbe biomassaproduksie teweeg gebring wanneer die rekombinante gisrasse op birchwood xilaan as enigste koolstotbron gegroei het. Die rekombinante ras wat die A. kawachii ~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitdruk, was die mees suksesvolle ras en het 3.2-voudig hoër biomassa as die kontrole ras opgelewer. Die biomassa van die rekombinante rasse tydens groei op xilaan as enigste koolstotbron kon gemiddeld met 85% verhoog word deur die giste onder hoë suurstotkonsentrase in 'n fermentor te kweek. Die rekombinante rasse is verder ook getoets vir hul vermoë om xilaan direk tot etanol om te skakel. Die rekombinante ras wat die A. kawachii ~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitgedruk het, het 'n 3.6- voudige verhoging in etanolproduksie getoon en 1.35 gIL ethanol gelewer. Hierdie rekombinante gisrasse verteenwoordig 'n stap nader aan die doeltreffende atbraak en benutting van hemisellulose deur etanolproduserende giste.

No description available.

No description available.

Thesis (PhD)--University of Stellenbosch, 1999. / ENGLISH ABSTRACT: Hemicellulose, consisting mainly of xylan, ranks after cellulose, as the most abundant group of renewable polysaccharides in agricultural biomass. Xylan is a complex polymer consisting of a β D 1,4 linked xylopyranoside backbone, which may contain substituents. Enzymatic hydrolysis of xylan involves the action of a number of different hydrolytic enzymes. The yeast Saccharomyces cerevisiae has been used extensively in traditional food and beverage processes (baking, brewing and winemaking), as well as for the production of ethanol (potable alcohol and fuel extenders) and single-cell protein (protein supplements in food and animal feed). S. cerevisiae therefore has complete GRAS (Generally Regarded as Safe) status. However, the yeast S. cerevisiae can neither degrade nor utilize complex polysaccharides, including xylan. Through recombinant DNA technology, S. cerevisiae can be complemented by heterologous polysaccharase-encoding genes, thereby broadening its substrate range and facilitating a direct bioconversion of polysaccharides to valuable commodities, such as potable ethanol, protein supplements and industrial enzymes. In this study, the successful expression and co-expression of a β xylanase gene (Trichoderma reesei xyn2) and two β xylosidase genes (Bacillus pumilus xynB and A. niger xlnD) in S. cerevisiae, is described. Expression of these genes was obtained with the aid of multi-copy episomal yeast plasmids pJC1, pDLG1, pDLG4 and pRLR1. These plasmids contain either the derepressible alcohol dehydrogenase 2 (ADH2) or the constitutive phosphoglycerate kinase 1 (PGK1) promoter and terminator sequences. The enhanced production of recombinant enzymes by S. cerevisiae in a rich medium, without the risk of losing the episomal vector, was obtained by disrupting the uracil phosphoribosyltransferase (FUR1) gene in the plasmid-containing S. cerevisiae strains. This step ensured auto-selection of the URA3-bearing expression plasmids in rich growth medium. High level expression of the T. reesei β xylanase gene in S. cerevisiae enabled the yeast to degrade xylan to short xylo-oligosaccharides, but very little monomeric D xylose was formed. Both β xylosidase genes enabled S. cerevisiae to degrade short xylo-oligosaccharides like xylobiose and xylotriose. Co-expression of the β xylanase and the B. pumilus β xylosidase led to a small increase in the β xylanase activity, but a substantial decrease in the amount of β xylosidase activity. This recombinant yeast strain was unable to degrade xylan to D xylose. Expression of the T. reesei β xylanase with the A. niger β xylosidase gene enabled this strain to completely degrade xylan to its monomeric constituents, D xylose. / AFRIKAANSE OPSOMMING: Hemisellulose, wat hoofsaaklik uit xilaan bestaan, is ná sellulose, die volopste hernubare polisakkaried in landbouafval. Xilaan is 'n komplekse polimeer wat bestaan uit 'n β-D-1,4-gekoppelde xilopiranoseruggraat wat in sommige gevalle ook sykettings bevat. Ensimatiese afbraak van xilaan benodig die werking van hele aantal hidrolitiese ensieme. Die gis Saccharomyces cerevisiae word al vir baie jare in die voedsel- en drankbedryf (bak van brood en die maak van bier en wyn), asook vir die produksie van etanol (vir menslik gebruik en as brandstof aanvuller) en enkelselproteïene (proteïenaanvulling vir mens en dier) gebruik en het daarom volledige GRAS (Generally Regarded As Safe) status. Ongelukkig kan S. cerevisiae nie komplekse polisakkariede, xilaan ingesluit, afbreek of as koolstofbron benut nie. Met behulp van rekombinante-DNA-tegnologie kan S. cerevisiae gekomplementeer word met die nodige gene wat kodeer vir polisakkariedafbrekende ensieme om sodoende die gis in staat te stel om 'n wyer verskeidenheid van substrate af te breek en te benut. Dit sal lei tot die direkte bio-omskakeling van polisakkariede na bruikbare produkte soos etanol, proteïenaanvullers en ensieme vir industriële gebruik. In hierdie proefskrif word die suksesvolle uitdrukking asook die gesamentlike uitdrukking van 'n xilanasegeen (Trichoderma reesei xyn2) en twee β-xilosidasegene (Bacillus pumilus xynB en A. niger xlnD) in S. cerevisiae beskryf. Multikopie episomale plasmiede pJC1, pDLG1, pDLG4 en pRLR1 met die glukose onderdrukbare alkoholdehidrogenase 2 (ADH2) of die konstitutiewe fosfogliseraatkinase 1 (PGK1)- promoter en -termineerder is vir hierdie doel gebruik. Verhoogde produksie van die rekombinante ensieme deur S. cerevisiae in 'n ryk medium, sonder dat die gis die episomale plasmiedvektore verloor is moontlik gemaak deur die urasielfosforibosieltransferasegeen (FUR1) van hierdie giste te onderbreek met behulp van die LEU2-geen. Op hierdie manier word daar outomaties vir giste wat die URA3-uitdrukkingsplasmiede bevat geselekteer, selfs in ryk medium. Hoë vlak uitdrukking van T. reesei se xilanasegeen het S. cerevisiae in staat gestel om xilaan tot kort xilo-oligosakkariede af te breek, maar byna geen monomeriese D-xilose is gevorm nie. Albei die β-xilosidasegene het die gis in staat gestel om kort xilo-oligosakkariede soos xilobiose en xilotriose na D-xilose af te breek. Die gesamentlike uitdrukking van die xilanasegeen en B. pumilus se β-xilosidase geen het 'n klein toename in die xilanase-aktiwiteit tot gevolg gehad, maar 'n drastiese afname in die β-xilosidase-aktiwiteit. Hierdie rekombinante ras kon dus nie xilaan tot xilose afbreek nie. Uitdrukking van T. reesei se β-xilanasegeen saam met die β-xilosidasegeen van A. niger, het S. cerevisiae in staat gestel om xilaan tot sy monomeriese boustene, D-xilose, af te breek.