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dc.contributor.advisorSmit, M. S.
dc.contributor.advisorSteyn, L.
dc.contributor.authorMaseme, Mpeyake Jacob
dc.date.accessioned2015-11-16T12:21:18Z
dc.date.available2015-11-16T12:21:18Z
dc.date.issued2014-07
dc.identifier.urihttp://hdl.handle.net/11660/1670
dc.description.abstractEnglish: Different oxidoreductases are being investigated for hydroxylation reactions to satisfy the global need for environmentally friendly industrial processes. These enzymes are found in microorganisms, animals and plants. Comparative study of different yeasts has shown the best heterologous expression of the fatty-acid hydroxylase (CYP505A1) and vanillyl-alcohol oxidase (VAO) in B. adeninivorans UOFS Y-1220. In an attempt to improve this, we studied the effect of cultivation conditions on biomass production and the expression of both genes. Medium constituents and bioreactor conditions that play significant roles and the nature of those roles on CYP505A1 and VAO expressions were investigated. Plackett-Burman shake-flask studies revealed that vitamins, substrate concentration, inoculum size, buffer concentration, initial pH and culture volume had significant effects on biomass production, while factors significant for VAO expression were buffer concentration and initial pH. Higher culture volume and vitamins had a negative effect on biomass production while substrate concentration, inoculum size, buffer concentration and initial pH showed a positive effect. The yeast preferred potassium phosphate buffer, glycerol as carbon source and longer cultivation times but not δ-aminolevulinic acid although these were statistically insignificant. CYP505A1 expression was better when using MES buffer, glycerol and shorter cultivation times but not δ-aminolevulinic acid. In addition, we observed that substrate concentration and culture volume had negative and positive effect, respectively. Higher buffer concentration and initial pH had a positive effect on VAO expression. Moreover, though not statistically significant, substrate concentration and culture volume had negative effect while vitamins, metals, basal salts and inoculum size showed positive effect. Lastly, high DOT, in the bioreactors, had positive impact on biomass production while it decreased the level of VAO expression. In conclusion, the importance of cultivation conditions for gene expression has been highlighted and thus, based on this study, further research on medium composition and bioreactor cultivation can be pursued for optimization purposes.en_ZA
dc.description.abstractAfrikaans: Verskillende oksidoreduktases word ondersoek vir hidroksileringsreaksies om die globale behoefte vir omgewingsvriendelike industriële prosesse te bevredig. Hierdie ensieme is teenwoordig in mikroörganismes, diere en plante. Vergelykende studies van verskillende giste het die beste heteroloë uitdrukking van die vetsuur hidroksilase (CYP505A1) en vanilliel-alkohol oksidase (VAO) in Blastobotrys adeninivorans UOVS Y-1220 gelewer. In 'n poging om dit te verbeter, het ons die effek van kweektoestande op biomassa produksie en die uitdrukking van beide gene ondersoek. Medium bestanddele en bioreaktor toestande wat 'n belangrike rol speel en die aard van hierdie rolle op CYP505A1 en VAO uitdrukking is ondersoek. Plackett-Burman skud-vles studies het getoon dat vitamiene, substraatkonsentrasie, inokulum grootte, buffer konsentrasie, aanvanklike pH en kultuur volume bepalend was vir biomassa produksie, terwyl buffer konsentrasie en aanvanklike pH bepalend was vir VAO uitdrukking. Hoër kultuur volume en vitamiene konsentrasie het 'n negatiewe effek op biomassa produksie gehad, terwyl substraatkonsentrasie, inokulum grootte, buffer konsentrasie en aanvanklike pH 'n positiewe effek gehad het. Alhoewel nie statisties betekenisvol nie, wou dit voorkom asof biomassaproduksie bevorder is deur kaliumfosfaat buffer, gliserol as koolstofbron en langer inkubasietyd, maar benadeel is deur δ-aminolevuliniese suur. CYP505A1 uitdrukking was beter met MES buffer, gliserol en korter verbouing, maar nie met δ-aminolevuliniese suur nie. Daarbenewens, het ons opgemerk dat substraatkonsentrasie en kultuur volume onderskeidelik 'n negatiewe en positiewe uitwerking gehad het. Hoër buffer konsentrasie en aanvanklike pH het 'n positiewe uitwerking op VAO uitdrukking gehad. Verder, hoewel nie statisties beduidend nie, het substraatkonsentrasie en kultuur volume 'n negatiewe effek gehad terwyl vitamiene, metale, basale soute en inokulum grootte'n positiewe effek gehad het. Laastens, het 'n hoë DOT in die bioreaktore, 'n positiewe effek op die biomassa produksie gehad, terwyl dit die vlak van VAO uitdrukking verlaag het. Ten slotte, is die belangrikheid van groeitoestande vir die uitdrukking van gene uitgelig en dus, gebaseer op hierdie studie, kan verdere navorsing oor medium samestelling en bioreaktor verbouing ondersoek word vir optimisering.af
dc.description.sponsorshipNational Research Foundation (NRF)en_ZA
dc.description.sponsorshipc*change, South Africaen_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectOxidoreductasesen_ZA
dc.subjectCultivation conditionsen_ZA
dc.subjectMedium compositionen_ZA
dc.subjectPlackett-Burman factorial designen_ZA
dc.subjectBlastobotrys adeninivoransen_ZA
dc.subjectVanillyl-alcohol oxidaseen_ZA
dc.subjectSelf-sufficient fatty acid hydroxylaseen_ZA
dc.subjectDissolved oxygen tensionen_ZA
dc.subjectTranslation elongation factoren_ZA
dc.subjectHeterologous gene expressionen_ZA
dc.subjectMonooxygenases.en_ZA
dc.subjectCytochrome P-450.en_ZA
dc.subjectDissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2014en_ZA
dc.titleEffect of cultivation conditions on heterologous expression of oxidoreductases by the yeast Blastobotrys adeninivoransen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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