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Under cellulolytic conditions, the white-rot fungus Trametes versicolor produces cellobiose dehydrogenase (CDH), an enzyme with a number of biochemical properties that are potentially relevant to the degradation of lignin and cellulose. To clarify its biochemical properties, CDH was purified from cultures of T. versicolor. Two isoforms of CDH were found: a 97 kDa isoform with both heme and flavin cofactors, and an 81 kDa isoform with a flavin cofactor. Both isoforms of CDH were found to be quite non-specific in their reductive half reactions. The flavin enzyme catalyzed many of the same reactions as the heme/flavin enzyme, but less efficiently. The flavin isoform reduced Fe(III) and Cu(II) only at concentrations well above those found physiologically. Thus the heme/flavin enzyme, but not the flavin enzyme, could be involved in promoting and sustaining the generation of hydroxyl radicals (·OH) by Fenton's chemistry. / To characterize further the structural features of CDH, a genomic clone was isolated and sequenced. CDH was found to consist of 748 amino acids, without its predicted 19 amino acid signal peptide. Consistent with the domain structure of other CDHs, T. versicolor CDH appeared to be divided into an amino terminal heme domain and a carboxy terminal flavin domain, connected by a hydroxyamino acid-rich linker. Within the flavin domain, a putative cellulose-binding domain (CBD) was found by alignment to the hypothesized CBD of P. chrysosporium CDH. The CBD of CDH appeared to be structurally unrelated to other CBDs which have been reported. / A cDNA clone encoding T. versicolor CDH was isolated by RT-PCR. Using this clone, three vectors for the heterologous expression in Aspergillus oryzae of CDH were prepared. These vectors were built by performing in-frame fusions of the cDNA to control sequences from the highly expressed A. oryzae amylase gene. These vectors were transformed into A. oryzae and one strain was isolated which contained the expression construct DNA. / A rapid method for cloning cdh-like genes was developed. Using short stretches of amino acids completely conserved within T. versicolor and P. chrysosporium CDH, PCR primers were designed to amplify a homologous gene from other fungi. The primers were tested using genomic DNA of Pycnoporus cinnabarinus. A 1.8-kb fragment of P. cinnabarinus cdh was thereby amplified and cloned, and its sequence was determined. The three CDHs displayed very high homology at the amino acid level. / Finally, to probe the role of CDH in lignocellulose degradation by T. versicolor, a "knockout" vector was constructed consisting of a phleomycin-resistance cassette inserted into the protein coding sequence of cloned T. versicolor cdh. T. versicolor was transformed with the knockout vector and the transformants were analyzed for their CDH-producing phenotype. Three isolates were found that produced no detectable CDH. Biobleaching and delignification by the CDH(-) strains appeared to be unaffected, suggesting that CDH does not play an important role in these processes.

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An experiment was designed to test the hypothesis that each softwood pulp is unique and requires a specific, well defined mechanical treatment to derive its maximum strength potential. Three bleached softwood Kraft pulps and respective wood samples were sourced from both the Northern and Southern Hemispheres. The raw fibre characteristics of P. patula (Southern Hemisphere), P. menziesii (Northern Hemisphere) and P. mariana (Northern Hemisphere) were measured and compared. The raw pulp sheets were refined at different energies and intensities under controlled laboratory conditions using a 12” single disc pilot refiner. Results were assessed to determine the raw fibre characteristics, optimum refining conditions and the relative refined strength potential for each of the three samples. Results from anatomy measurements on the three wood samples differed significantly. P. patula exhibited a relatively high proportion of springwood growth in the early growing years. As the P. patula aged and formed mature wood there was a significant increase in the frequency of latewood formation. This was characterized by an abrupt and significant increase in the wall thickness, beyond that of the two Northern softwood samples. When the cell wall thickness increased, the lumen width and fibre diameter of the P. patula decreased significantly, yielding extremely coarse, stiff fibres. The Northern P .mariana and P. menziesii samples were characterized by a relatively consistent transition between high and low densities from the pith to the bark of the tree. The Southern P. patula had a unique density trend with an increasing frequency of high density peaks indicative of an increased latewood formation from the pith to the bark. The slower growing Northern P. menziesii and P. mariana samples did not have as clear a differentiation in fibre characteristics between juvenile and mature wood formation. The Northern samples did however contain a significantly higher proportion of juvenile latewood growth than the P. patula. However, the difference in fibre characteristics between earlywood and latewood formation was not as significant as that noted with the Southern P. patula Fibre morphology measurements on the unrefined bleached Kraft pulps also revealed significant differences between the three samples. The average MORFI LAB01 results on the P. patula defined fibres with a high coarseness and relatively low number of fibres per gram of pulp. The extremely coarse latewood fibres formed during mature wood growth being the most likely source. However, P. patula was also characterized with a high fibre flexibility and large lumen, characteristics consistent with earlywood fibres. The Pulmac Z-Span 3000 was used to define the individual fibre strength, when due consideration was given to the number of fibres per gram, the corrected Pulmac results suggested P. patula had the strongest fibres. When refined, using a standard disc refining programme, P. patula exhibited a fast freeness development. Conventional thinking would suggest that this was an indication of a weaker fibre. However, this species had a robust morphology compared to the Northern Hemisphere woods. The theory developed in this dissertation suggests that the effect of coarseness and the concomitant number of fibres per gram plays a significant role. These two parameters are not included in the “traditional” refining calculations. The applied refining load and intensity was calculated on the flow of the pulp passing through the refiner. The calculation did not consider the actual number of fibres present in that specific volume. The implication is that when a fixed refining load is applied to a pulp with coarse fibres there may be a higher effective load on those fewer fibres (resulting in fibre cutting and fines generation). In this case, the Northern samples have a comparatively low coarseness and more fibres per gram with each receiving a smaller portion of the total load and intensity. In terms of refined pulp properties, P. patula developed a relatively high bulk and tear index consistent with coarse, rigid fibres. The Northern P. mariana and P. menziesii samples produced a pulp with good tensile properties, consistent with a greater number of finer, collapsible fibres with a higher relative bonding area. P. patula fibres were extremely heterogeneous in nature containing the smallest relative lumen width during latewood formation and the largest lumen width during earlywood growth. As a result, P. patula contains extremes of both fine and coarse fibres in the same blend. It may be more beneficial for this species than the others to improve both the tear and tensile properties through fibre fractionation with appropriate development of the separate accepts and rejects streams. In terms of fibre development, low intensity refining parameters maximized the tensile strength of the Southern P. patula. The Northern P. mariana and P. menziesii samples had a greater number of fibres per gram of pulp requiring both a higher refining energy and intensity to develop the pulp to its maximum potential. To develop optimum tear results, high intensity refining, with a relatively low specific energy provided optimum results for all 3 samples. Results confirmed that there were significant differences in the fibre morphology both between the three different species and between the two Hemispheres. There was strong evidence that the fibre characteristics dictate the manner in which a fibre responds to refining which in turn determines the relative contribution to specific refined pulp properties. It may be possible to use fibre characteristics to determine the appropriate refining parameters for optimal fibre development which will enhance the value of the end product. To derive the maximum strength potential from P. patula pulp samples, it is recommended that further studies investigate Hydracyclone fractionation and the concomitant benefits of refining the separate streams. Furthermore, a separate study on fibre morphology and refining characteristics of the same species grown in both the Northern and Southern Hemisphere would provide valuable insight. / Thesis (M.Sc.Eng)-University of KwaZulu-Natal, Durban, 2009.

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