Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Author "De Koker, T. H. (Theodorus Hermanus), 1965-"

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    Genetic and enzymatic characterisation of wood degrading strains of Phanerochaete species
    (Stellenbosch : Stellenbosch University, 2000-03) De Koker, T. H. (Theodorus Hermanus), 1965-; Janse, B. J. H.; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: White rot fungi are of interest in the paper and pulp industry because of their removal of lignin from wood. In this study over 600 Basidiomycete fungi were isolated from indigenous forests as well as from commercial Eucalyptus spp. and Pinus spp. plantations in South Africa. One hundred isolates were identified to genus level. Biochemical tests were done to screen the fungal cultures for characteristics that are favourable for biopulping, e.g. low cellulase activity with concomitant high activity of ligninolytic enzymes. Various Phanerochaete isolates with potentially high ligninolytic activity were identified. Although Phanerochaete chrysosporium Burds. has previously been isolated from the indigenous forest at Knysna in South Africa, this study showed that P. chrysosporium was a natural coloniser of wood chip piles in South Africa, indicating potential for application in industry. A possible new species of Phanerochaete, viz. Phanerochaete pseudomagnoliae nom. provo (strain PP25) from decayed wood collected in Stellenbosch, South Africa, was described and illustrated. It differs from previously described Phanerochaete species in having smaller basidiospares, and in the formation of few chlamydospores on malt extract agar but more on xylose containing media. The potential of using internal transcribed spacer DNA sequences (ITS) to infer phylogenetic relationships among species of the genus Phanerochaete was investigated. Consensus phylogenetic trees could be presented, but the presence of ambiguous aligning sequences within the ITS made inferring of phylogenetic relationships within the whole genus difficult. Fifty-five South African strains of P. chrysosporium were screened for lignin peroxidase (liP), manganese peroxidase (MnP) and glyoxal oxidase (GLOX) enzymes. Manganese peroxidase activity was quantified on agar media. The liP and GLOX activities of 13 selected strains, including control strains and P. pseudomagnoliae (PP25), were also quantified on agar media. Differences in MnP and GLOX activities existed among the strains. Preliminary biochemical characterisation of strain PP25 indicated that the most important difference was the apparent unique regulation of ligninolytic enzymes. Under low nitrogen, liP activity of the selected strains showed no significant variation, whereas strain PP25 had significantly increased liP levels under high nitrogen conditions. Restriction fragment length polymorph isms of the lignin and manganese peroxidase gene DNA fragments showed variability among strains, whereas there was probably only allelic variation for the glox gene DNA fragments. Previous research has indicated xylose oxidation activity within P. chrysosporium. To investigate whether GLOX can oxidise xylose, a purified recombinant GLOX (rGLOX) from P. chrysosporium BKM-F-1767 Burds. was used in this study. This rGLOX oxidised D-xylose and D-glucose (D-xylose > D-glucose) to produce H202. Xylose was oxidised to xylono-1 ,4-lactone with a 1:1 stoichiometric relationship between H202 produced and xylose used. Xylono-1,4-lactone was converted non-enzymatically to xylonate. This suggested that the furanose form of xylose, rather than the pyranose form, is a substrate of GLOX. The production of H202 and the removal of inhibitory compounds by GLOX could enhance ligninolytic activity. . To conclude, unique strains of P. chrysosporium have been isolated from South Africa with potential biotechnological use in paper manufacturing. The relationship of P. pseudomagnoliae nom. provo to other Phanerochaete species was evaluated and light was shed on the possible role of GLOX in lignin degradation.