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dc.contributor.advisorSmit, M. S.
dc.contributor.advisorOpperman, D. J.
dc.contributor.authorVan Rooyen, Newlandé
dc.date.accessioned2016-01-07T12:15:52Z
dc.date.available2016-01-07T12:15:52Z
dc.date.copyright2010-01
dc.date.issued2012-01
dc.date.submitted2012-01
dc.identifier.urihttp://hdl.handle.net/11660/2055
dc.description.abstractEnglish:There are currently only two confirmed fungal vanillyl-alcohol oxidases (VAOs), one from Penicillium simplicissimum (here called PsVAO) and one from Byssochlamys fulva. Only the gene sequence of PsVAO is available. Fusarium spp. was targeted as a source of more VAOs, because they are plant pathogens known for production of lignolytic enzymes and utilization of aromatic compounds. BLAST searches of the databases of the Fungal Genome Initiative of the Broad Institute using PsVAO as query supported this choice. The predicted protein (called FvVAO) of one hit, gene number FVEG 03424 from Fusarium verticillioides, shared 63% amino acid identity with PsVAO and grouped with PsVAO in a phylogenetic analysis. Seven Fusarium strains from three species F. verticilliodes (synonym Fusarium moniliforme), Fusarium graminearum and Fusarium oxysporum were investigated for VAO activity. F. moniliforme MRC 6155 consistently displayed VAO activity in cell-free extracts with 0.036 U/mg protein obtained after veratryl alcohol induction. Primers based on the FvVAO gene were used to amplify the VAO gene (called FmVAO) from F. moniliforme MRC 6155 from both genomic DNA and mRNA. Comparison of the genomic sequences of FvVAO and FmVAO, which both have the same four introns, revealed a total of 42 nucleotide differences while the deduced amino acid sequences differed by seven amino acids. The sequences of the new FmVAO were submitted to GenBank (NCBI), accession number JQ410355. Both PsVAO and FmVAO were cloned into the pET28b(+) vector adding N-terminal His-tags and expressed in E. coli BL21(DE3)pRARE2. Using this strain to compensate for rare codons improved the expression of PsVAO but it was still not possible to detect discernable VAO bands of either PsVAO or FmVAO on SDS-PAGE gels. Comparison of substrate specificity of PsVAO and FmVAO in assays done with cell free extracts and whole cell biotransformations revealed that FmVAO preferred vanillyl alcohol as substrate and can thus be regarded as a "true" vanillyl-alcohol oxidase - possibly the first. Vanillyl-alcohol oxidase activities of PsVAO and FmVAO in cell-free extracts were respectively 0.028 and 0.018 U/mg protein, while eugenol oxidase activities were 0.030 and 0.005 U/mg protein. In whole cell biotransformations of vanillyl alcohol, specific activities of PsVAO and FmVAO were respectively 6.1 and 5.7 U/g dry weight, while with eugenol as substrate activities were 11.0 and 2.2 U/g dry weight. In whole cell biotransformations FmVAO showed higher activity with ethylphenol, again indicating its different substrate specificity. PsVAO was also cloned and expressed in the yeasts Kluyveromyces marxianus and Arxula adeninivorans while FmVAO was also cloned and expressed in A. adeninivorans. The K. marxianus vector pKM63 which gave excellent but unstable expression in K. marxianus contains 18S rDNA fragments from K. marxianus for genomic integration, a geneticin resistance marker and the native inulinase promoter of K. marxianus to drive expression of the cloned gene. The wide range vector pKM118 used for cloning into A. adeninivorans only differs from pKM63 in that it contains a hygromycin resistance marker and uses the Yarrowia lipolytica TEF promoter to drive expression of the cloned gene. Comparison of the specific activities in cell free extracts of both FmVAO and PsVAO expressed in A. adeninivorans and E. coli revealed that expression in the yeast increased the activity in cell-free extracts, with FmVAO benefiting more from expression in A. adeninivorans. The vanillyl-alcohol oxidase activity of FmVAO in A. adeninivorans was 0.045 U/mg protein and the eugenol oxidase activity, 0.015 U/mg protein. Both the vanillyl-alcohol oxidase and eugenol oxidase activities of PsVAO in A. adeninivorans were 0.04 U/mg protein. Differential centrifugation of cell free extracts showed that both PsVAO and FmVAO activity could only be detected in the soluble fraction.en_ZA
dc.description.abstractAfrikaans: Daar is tans slegs twee bevestige vanilliel-alkohol oxidases (VAOs) afkomstig van swamme, een van Penicillium simplicissimum (genoem PsVAO) en een van Byssochlamys fulva. Slegs die geen volgorde van PsVAO is beskikbaar. Fusarium spp. is geteiken as 'n bron van meer VAOs, want hulle is plantpatogene wat bekend is vir die produksie van lignien afbreekbare ensieme en die produksie van aromatiese verbindings. Die BLAST navrae wat gerig was aan die databasisse van die Swam Genoom Inisiatief van die Broad Institute met PsVAO as navraag ondersteun hierdie keuse. ‘n Voorspelde proteïen (genoem FvVAO), geen aantal FVEG 03424 van Fusarium verticillioides,wat so verkry is het ‘n 63% aminosuur identiteit gedeel met PsVAO en was gegroepeer saam met PsVAO in 'n filogenetiese analise. Sewe Fusarium stamme van drie spesies. F. verticilliodes (sinoniem Fusarium moniliforme), Fusarium graminearum en Fusarium oxysporum is vir VAO aktiwiteit ondersoek. F. moniliforme MRC 6155 het konsekwent VAO aktiwiteit, vanaf 0.036 U/mg proteïen in selvrye fraksies getoon nadat veratryl alkohol bygevoeg is vir induksie. Teiken DNA fragmente wat gebaseer is op die FvVAO geen, is gebruik om die VAO geen (genoem FmVAO) van F. moniliforme MRC 6155 van beide DNA en mRNA te amplifiseer. ‘n Vergelyking van die genomiese DNA van FvVAO en FmVAO het dit aan die lig laat kom dat albei dieselfde vier introns het. Daar was 'n totaal van 42 nukleotied verskille, terwyl die afgeleide aminosuurvergelyking met sewe aminosure verskil het. Die genoomkode van die nuwe FmVAO was ingedien by GenBank (NCBI), Aanwysingsnommer: JQ410355. Beide PsVAO en FmVAO is gekloon in die pET28b(+) vektor en n-terminale His-etikette is bygevoeg vir uitgedrukking in E. coli BL21(DE3)pRARE2. Die spesifieke subspesie is gebruik om te vergoed vir seldsame kodons om dus so uitdrukking van PsVAO te verbeter, maar dit was nog nie moontlik om waarneembare VAO bande van, of PsVAO of FmVAO op SDS PAGE gels waar te neem nie. ‘n Vergelyking van die substraat spesifisiteit van PsVAO en FmVAO in toetse wat gedoen is met 'n selvrye fraksies sowel as met heelsel biotransformasies het aan die lig gebring dat FmVAO voorkeur aan vanilliel alkohol as substraat gee en kan dus beskou kan word as 'n "ware" vanilliel-alkohol oxidase - moontlik die eerste. Vanilliel-alkohol oxidase aktiwiteite van PsVAO en FmVAO in selvrye fraksies was onderskeidelik 0,028 en 0,018 U/mg proteïen, terwyl eugenol oksidase aktiwiteit 0,030 en 0,005 U/mg proteïen onderskeidelik was. Heelsel-biotransformasies van vanilliel alkohol het onderskeidelik 6,1 en 5,7 U/g droë gewig gegee vir PsVAO en FmVAO gegee, terwyl die eugenol substraat aktiwiteite, 11,0 en 2,2 U/g droë gewig onderskeidelik was. In heelsel-biotransformasies het FmVAO hoër aktiwiteit met ethylphenol getoon, wat weereens klem lë op die verskil in substraat spesifisiteit. PsVAO is ook gekloon en uitgedruk in die-giste Kluyveromyces marxianus en Arxula adeninivorans, terwyl FmVAO gekloon en uitgedruk is in A. adeninivorans. Die K. marxianus vektor pKM63 het uitstekende, maar onstabiele uitdrukking in K. marxianus veroorsaak. Dit bevat 18s rDNA fragmente afkomstig vanaf K. marxianus vir genomiese integrasie, 'n geneticin weerstands merker en die inheemse inulinase promotor afkomstig van K. marxianus vir uitdrukking van die gekloonde geen. Die wye verskeidenheids vektor, pKM118 wat gebruik is vir kloning in A. adeninivorans verskil slegs van pKM63 in dat dit 'n hygromycin weerstands merker bevat sowel as die Yarrowia lipolytica TEF promotor vir uitdrukking van die gekloonde geen . Vergelyking van die spesifieke aktiwiteite in 'n sel vrye ekstrakte van beide FmVAO en PsVAO uitgedruk in A. adeninivorans en E. coli het die lig gebring dat die uitdrukking in die gis die aktiwiteit in die selvrye ekstrak met FmVAO bevoordeel het. Die vanilliel-alkohol oksidase aktiwiteit van FmVAO in A. adeninivorans was 0,045 U/mg proteïen en die eugenol oksidase aktiwiteit, 0,015 U/mg proteïen. Beide die vanilliel-alkohol oxidase en eugenol oksidase aktiwiteite van PsVAO in A. adeninivorans was 0,04 U / mg proteïen. Differensiële sentrifugering van 'n selvrye ekstrakte het gewys dat beide PsVAO en FmVAO aktiwiteit slegs waargeneem kan word in die oplosbare oplosbare fraksie.af
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectThesis (Ph.D. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2012en_ZA
dc.subjectOxidasesen_ZA
dc.subjectFungal enzymesen_ZA
dc.subjectFusarium -- Genetic engineeringen_ZA
dc.subjectMolecular cloningen_ZA
dc.subjectFungien_ZA
dc.subjectVanillyl-alcohol oxidaseen_ZA
dc.subjectPhylogenetic analysisen_ZA
dc.subjectGeneen_ZA
dc.subjectFusariumen_ZA
dc.subjectSpecific activityen_ZA
dc.subjectHeterologous expressionen_ZA
dc.subjectRare codonen_ZA
dc.titleIdentification, cloning and heterologous expression of fungal vanillyl-alcohol oxidasesen_ZA
dc.typeThesisen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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