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dc.contributor.advisorDu Preez, J. C.
dc.contributor.advisorKilian, S. G.
dc.contributor.authorMare, Jacques Ettienne
dc.date.accessioned2018-04-20T06:19:28Z
dc.date.available2018-04-20T06:19:28Z
dc.date.issued1999-09
dc.identifier.urihttp://hdl.handle.net/11660/8219
dc.description.abstractEnglish: Endo-β-I,4-xylanase production by a recombinant strain of Saccharomyces cerevisiae Y294, transformed with a Trichoderma reesei xylanase gene (XYN2), was evaluated in batch and continuous cultures. Expression of the heterologous xylanase gene by this yeast was under control of a promoter-terminator expression cassette derived from the alcohol dehydrogenase II gene (ADH2) of S. cerevisiae, which is mainly regulated through carbon catabolite repression. The 3,5-dinitrosalicylic acid and Sornogyi-Nelson assays for endo-β-I,4-xylanase activity (hereafter referred to as xylanase) in supernatants from cultures of the recombinant S. cerevisiae strain and in a commercial xylanase preparation were compared. These two assays gave widely differing activity values, even more so than anticipated. These huge discrepancies in activity values obtained indicated that these assays did not allow a reliable comparison of the activities of xylanolytic enzyme mixtures and preparations containing a single xylanase without accessory enzymes. In aerobic batch culture, the highest specific rate of xylanase production (20.8 nkat.mg biomassi-1.h-1) and activity (1 590 nkat.ml-1) were recorded with glucose as carbon source. The inclusion of ethanol as carbon source, which is a standard procedure for the derepression of ADH2, resulted in poor xylanase production. The influence of glucose flux, glucose concentration and growth rate on xylanase production by this recombinant strain of S. cerevisiae Y294 was investigated in carbon-limited continuous culture and compared to results obtained with recombinant xylanase-producing S. cerevisiae strains HI58 and CEN.PK Il 0-6C. The latter two strains harboured the same expression cassette and xylanase gene (XYN2) as the recombinant S. cerevisiae Y294 strain. Xylanase production by strains Y294, HI58 and CEN.PK110-6C was markedly reduced at glucose flux values greater than 0.98, l.44 and l.68 mmol glucose.g biomass-1.h-1, respectively. Despite the obvious strain differences, with all three yeast strains the glucose flux appeared to contribute to a greater extent towards the regulation of xylanase production than did the glucose concentration in the culture. Xylanase production by recombinant S. cerevisiae strains Y294 and CEN.PKII0-6C was found to be transcriptionally repressed by ethanol at and above 5.5 g ethanol.I". This finding was surprising, since the xylanase gene was under the regulation of the ADH2 promoter and alcohol dehydrogenase II is associated with the assimilation of ethanol by effecting the catalysis of ethanol to acetaldehyde. This finding adds a new perspective to the regulation of alchohol dehydrogenase II. In a fed-batch culture of recombinant S. cerevisiae Y294, the specific rate of xylanase production was increased 2.5-fold as compared to batch cultures, through control of the growth rate at 0.1 h-I by using an exponentially increasing feed rate. These observations also indicate the important role of the growth rate, and thus possibly the carbon flux, in regulating ADH2-mediated xylanase production.en_ZA
dc.description.abstractAfrikaans: Endo-β-1,4-xilanase produksie deur 'n rekombinante Saccharomyces cerevisiae Y294 stam, wat getransformeer is met 'n xilanase geen (XYN2) afkomstig vanaf Trichoderma reesei, is bestudeer in lot- en kontinue kulture. Uitdrukking van die xilanase-geen in hierdie rekombinant was onder die invloed van 'n inisieerder-termineerder uitdrukkingsvektor afgelei vanaf die alkoholdehidrogenase II (ADH2) geen. ADH2 word hoofsaaklik deur koolstof kataboliet repressie gereguleer. Die 3,5-dinitrosalisiliese suur en Somogi-Nelson metodes vir bepaling van endo-β-1,4- xilanase aktiwiteit in bostand van kulture van die rekombinante S. cerevisiae stam en 'n kommersiële xilanase preparaat is met mekaar vergelyk. Hierdie twee metodes het aktiwiteitwaardes wat baie van mekaar verskil het, gelewer, selfs meer as wat verwag is. Hierdie groot afwyking in aktiwiteitswaardes het aangedui dat hierdie twee metodes me 'n vertrouenswaardige vergelyking tussen die xilanase-aktiwiteit van xilanolitiese ensiemmengsels en preparate wat 'n enkele xilanase ensiem, sonder bykomende xilanolitiese hulpensieme, kon maak nie. In aërobiese lotkulture is die hoogste spesifieke snelheid van xilanase-produksie (20.8 nkat.mg biomassa-1.h-1) en -aktiwiteit (1590 nkat.ml-1) met glukose as koolstofbron verkry. Die insluiting van etanol as koolstofbron, wat 'n standaardprosedure vir die derepessie van ADH2 is, het tot swak xilanase-produksie gelei. Die invloed van glukose-vloei, glukosekonsentrasie en groeisnelheid op xilanase-produksie deur hierdie rekombinante S. cerevisiae Y294 stam is in koolstof-beperkte kontinue kulture ondersoek en met resultate verkry vir rekombinante xilanase-produserende S. cerevisiae stamme HI58 en CEN.PK110-6C, vergelyk. Die laasgenoemde twee stamme het dieselfde uitdrukkingsvektor en xilanase-geen as S. cerevisiae Y294 gehuisves. Xilanase-produksie deur stamme Y294, HI58 en CEN.PK110-6C is merkwaardig verlaag wanneer die glukose vloei 0.98, 1.44 en 1.68 mmol glukose.g biornassa-1.h-1, respektiewelik, oorskry het. Ten spyte van die oogmerklike verskille tussen die stamme, het die glukose-vloei, eerder as die glukose-konsentrasie, in al drie die stamme oorwegend tot die regulering van xilanase produksie bygedra. Xilanase-produksie deur die rekombinante S. cerevisiae stamme Y294 en CEN.PK110-6C is transkripsioneel deur etanol konsentrasies van 5.5 g.1-1 of hoër onderdruk. Hierdie bevinding was onverwags, aangesien die xilanase-geen onder regulering van die ADH2 promoter is. Alkoholdehidrogenase II word geassosieer met die opname van etanol deur die katalise van etanol na asethaldehied te fasiliteer. Hierdie bevinding voeg 'n nuwe dimensie toe tot die regulering van die alkohol dehidrogenase II geen. In gevoerde lotkulture van die rekombinante S. cerevisiae Y294 is die spesifieke snelheid van xilanase-produksie 2.5-voudig verhoog in vergelyking met lotkulture, deur die groeisnelheid te beheer by 0.1 h-I , deur gebruik te maak van 'n eksponensieel-toenemende voersnelheid. Hierdie bevindings dui daarop dat die groeisnelheid, en dus ook moontlik die glukose-vloei, 'n belangrike rol gespeel het in die regulering van ADH2-gefasiliteerde xilanase produksie.en_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectXylanasesen_ZA
dc.subjectSaccharomycesen_ZA
dc.subjectEnzymesen_ZA
dc.subjectDissertation (M.Sc. (Microbiology and Biochemistry))--University of the Free State, 1999en_ZA
dc.titleXylanase production by a recombinant strain of Saccharomyces cerevisiae: the effect of carbon catabolite repression and ethanol concentration in batch and continuous culturesen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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