The extracellular culture supernatants of the white rot fungus Trametes versicolor can bleach and delignify unbleached kraft pulps, however the process is too slow for commercial application. Though at least two oxidative enzymes, laccase and manganese peroxidase (MnP) produced by this organism can catalyze a partial delignification of kraft pulp, the effect observed is small relative to that obtained with the complete fungal system. To develop a synthetic (cell-free) delignification system, other protentially important components of the culture supernatant were identified and their contributions to biobleaching and delignification were evaluated. The presence of pulp did not significantly affect the overall carbon balance of the fungus, but a number of non-volatile metabolites (oxalic, fumaric, glyoxylic and phenyllactic acids) induced by the presence of pulp were identified. In T. versicolor 52J, the secretion of manganese peroxidase (MnP), and cellobiose:quinone oxidoreductase (CBQase) enzymes were inducible by pulp whereas cellulase and laccase were not. Several low molecular weight metabolites secreted by T. versicolor functioned as effective Mn(III) complexing agents at their physiological concentrations and promoted MnP activity. / Two distinct CBQase proteins are secreted by T. versicolor 52J, CBQase 4.2 a a 113kDa homodimer containing both heme and flavin cofactors and CBQase 6.4, a 48 kDa monomer with a flavin cofactor only. Superficially, these enzymes appear very similar to the cellobiose oxidase (CBO) and CBQase reported in Phanerochaete chrysosporium. CBQase 4.2 was shown to reduce insoluble manganese dioxide to its soluble Mn(II) and Mn(III) forms with the concommitant oxidation of cellobiose. The sugar acids formed by CBQase could function as effective complexing agents for Mn(III),and complement the Mn(II) to Mn(III) oxidation activity of MnP. / It is proposed that a redox cycling of lignin molecules by certain fungal oxidative and reductive enzymes occurs during delignification and that this cycling ultimately promotes net lignin degradation. A redox cycle wa established between T. versicolor CBQase and laccase which allowed the O$ sb2$ comsumption rate of laccase to remain at a constant level and the total O$ sb2$ consumption by the enzyme was much treater than if the substrate were incubated with laccase alone. A new assay for CBQase based on the ability of this enzyme to reduce the radical intermediates formed during laccase-mediated chlorpromazine oxidation was developed. A redox cycle for these two enzymes was established using both model substrates like CPZ, and with a kraft lignin preparation. CBQase inhibited the formation of polymeric material by laccase; however no evidence was found indicating that cycling with these two enzymes favors depolymerization of kraft lignin. However, the alkali extractability of residual lignin in kraft pulp was increased by a sequential treatment with MnP followed by CBQase.

Although several enzymes presumed to be part of the delignification/kraft pulp biobleaching system of the white-rot basidiomycete Trametes versicolor have been studied, characterized, and even exploited as pulp treatments, the complete system remains poorly understood. Little is known about which enzymes are essential for delignification, how they are regulated, or whether there remain unknown enzymes essential to wood decay in this system. Auxotrophic mutants of T. versicolor 52J were developed and characterized. Plasmid pUC18 was complemented with the T. versicolor 52J genome to create a gene library. Transformation with this plasmid-gene library converted argB and ade2 auxotrophs of T. versicolor 52J to prototrophy. Attempts to rescue the plasmids responsible were unsuccessful. Several different pre-existing plasmid constructs were examined for their potential as selectable markers on the fungus. One of these, pGPhT, worked well at conferring phleomycin resistance. Given that there are now available partial or complete sequences for four T. versicolor laccases, the single cellobiose dehydrogenase, and a lignin peroxidase isozyme, these genetic tools should be very useful in dissecting the mechanisms of white-rot delignification.

Thesis (M.Sc. (Clinical Tropical Medicine))--Mahidol University, 1984.

No description available.

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Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Humirianthera ampla Miers (Icacinaceae), Simarouba verscicolor A. St.-Hil. (Simaroubaceae), Lippia Alba (Mill) N.E. Br. (quimiotipos carvoneifera, citralifera e mirceneifera), Lippia gracillis Schauer e Lippia sidoides Cham., foram avaliadas como provÃveis fontes de compostos bioativos possuidores de atividade anticÃncer (antioxidante e/ou citotÃxica) atravÃs de ensaios in vitro, e os seus constituintes quÃmicos foram identificados e quantificados atravÃs mÃtodos espectroscÃpicos e espectromÃtricos. Em H. ampla foram identificados e quantificados cinco constituintes em suas folhas (esqualeno, α-tocoferol, campesterol, estigmasterol e β-sitosterol) e dez em suas raÃzes (Ãcido vanÃlico, Ãcido 3,4-di-hidroxibenzÃico, Ãcido sirÃngico, Ãcido gÃlico, campesterol, estigmasterol, annonalida, simiarenol, β-sitosterol e lupeol). A annonalida isolada das raÃzes de H. ampla apresentou potente atividade citotÃxica contra vÃrias linhagens de cÃlulas tumorais in vitro. Em S. versicolor foram identificados e quantificados seis constituintes em suas cascas (campesterol, estigmasterol, taraxerona, β-sitosterol, lupenona e sitostenona). A avaliaÃÃo do potencial antioxidante de H. ampla e S. versicolor apresentou resultados desapontadores quando comparados com outras espÃcies. Os Ãleos essenciais das espÃcies de Lippia foram avaliados por CG/EM e os constituintes majoritÃrios foram timol (Lippia sidoides, 65,5 %), carvona (Lippia alba carvoneifera, 48,6%) geraniol (Lippia alba citraleifera, 38,4 %; Lippia alba mircenefera, 31,9 %), nerol (Lippia alba citraleifera, 28,3 %; Lippia alba mirceneifera, 23,2 %), carvacrol (Lippia gracilis, 31,5 %) e limoneno (Lippia alba carvoneifera, 25,7 %). Os Ãleos essenciais e os extratos etanÃlicos das espÃcies de Lippia tiveram suas capacidades antioxidantes avaliadas, e somente o Ãleo essencial e o extrato etanÃlico de Lippia alba carvoneifera apresentaram resultados promissores. / Humirianthera ampla Miers (Icacinaceae), Simarouba verscicolor A. St.-Hil. (Simaroubaceae), Lippia Alba (Mill) N.E. Br. (chemotypes carvoneifera, citralifera and myrceneifera), Lippia gracillis Schauer and Lippia sidoides Cham., were evaluated as potential sources of bioactive compounds possessing anticancer activity (antioxidant or cytotoxic) by in vitro assays, and the chemical compounds were identified and quantified by epectroscopic and spectrometric methods. In H. ampla were identified and quantified five compounds in the leaves (squalene, α-tocoferol, campesterol, stigmasterol e β-sitosterol) and ten in the roots (vanillic acid, 3,4-dihydroxybenzoic acid, syringic acid, galic acid, campesterol, stigmasterol, annonalide, simiarenol, β-sitosterol and lupeol). Annonalide isolated from roots of H. ampla showed very high cytotoxicity against several cancer cell lines in vitro. In S. versicolor were identified and quantified six compounds in the bark (campesterol, stigmasterol, taraxerone, β-sitosterol, lupenone e sitostenone). The evaluation of the antioxidant capacity of H. ampla e S. versicolors showed disappointing results when compared with other species. The essential oils of Lippia species were evaluated by CG/MS and the major compounds were timol timol (Lippia sidoides, 65,5 %), carvone (Lippia alba carvoneifera, 48,6%) geraniol (Lippia alba citraleifera, 38,4 %; Lippia alba myrcenefera, 31,9 %), nerol (Lippia alba citraleifera, 28,3 %; Lippia alba myrceneifera, 23,2 %), carvacrol (Lippia gracilis, 31,5 %) and limonene (Lippia alba carvoneifera, 25,7 %). Antioxidant capacity of the essential oils and ethanolics extracts of Lippia species were evaluated, and only the essential oil and the ethanolic extract of Lippia alba showed promising results.