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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-08162012-112452 |
| Date | 16 August 2012 |
| Creators | van Rooyen, Newlandè |
| Contributors | Dr DJ Opperman, Prof MS Smit |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-08162012-112452/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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