A produção e o consumo de cogumelos comestíveis têm gradualmente aumentado nos últimos anos, em função de sua composição nutricional aliada à qualidade sensorial dos frutos. A radiação ionizante vem sendo estudada em cogumelos frescos, visando manter as características do produto e aumentar sua vida útil. A presente pesquisa teve como objetivo avaliar o efeito de diferentes doses de radiação gama aplicada em cogumelos Pleurotus sajor-caju in natura, por meio de parâmetros físicos, químicos, microbiológicos e sensoriais. O acondicionamento foi realizado em bandejas de poliestireno com 250 g de amostra, envoltos em filme de cloreto de polivinila (PVC). Os cogumelos foram irradiados com doses de 125, 250, 500, 750 Gy e armazenados a 4±1ºC e 90% UR durante 10 dias. Foi determinada a composição centesimal (umidade, fibra bruta, proteína bruta, extrato etéreo e cinzas), sólidos solúveis (SS), pH, textura, cor (L, a*, b*, Croma e ho), atividade enzimática (peroxidase e polifenoloxidase), análise microbiológica (coliformes totais, Escherichia coli e psicrotróficos) e sensorial (cor, aroma e aparência geral) no 1o, 5o e 10o dias de armazenamento. Para a determinação da atividade respiratória, 30 g de amostra foram acondicionadas em frascos de vidro e armazenadas a 4±1ºC e 90% UR durante 8 dias, com leituras realizadas em cromatógrafo gasoso a cada 24 horas durante o período de armazenamento. Os resultados foram submetidos à análise de variância e teste de média, utilizando-se o pacote estatístico SAS. Os valores de proteína e extrato etéreo não diferiram significativamente entre os tratamentos ou períodos de armazenamento, enquanto que a umidade, fibra e cinzas dos cogumelos apresentaram variações estatísticas em função da dose aplicada ou do período de avaliação. As amostras irradiadas com 750 Gy apresentaram escurecimento significativo em relação aos demais tratamentos, e a textura foi mantida no controle durante o experimento. Ocorreu um aumento significativo nos valores de SS, b* e atividade enzimática em todos os tratamentos no final do período de armazenamento. A dose de 250 Gy promoveu o aumento da atividade da polifenoloxidase e peroxidase no último dia de avaliação. Este fato pode ter ocorrido em função do processo de radiólise da água, uma vez que os cogumelos apresentam elevada umidade. A taxa de respiração das amostras irradiadas com 125 Gy foi superior até o 5o dia de armazenamento em comparação aos demais tratamentos, causando redução na vida útil do produto. As amostras irradiadas com 250, 500 e 750 Gy tiveram sua atividade respiratória reduzida em relação ao controle, contribuindo para a manutenção da qualidade pós-colheita durante o armazenamento. As amostras que receberam a dose de 750 Gy obtiveram os melhores resultados nas análises microbiológicas, com a redução de coliformes totais e psicrotróficos durante o período de armazenamento. O controle apresentou os melhores resultados para os atributos aroma, cor e aparência geral, obtendo notas acima do limite de aceitabilidade até o último dia de armazenamento. De maneira geral, as amostras irradiadas foram aceitas para os atributos avaliados até o 5o dia de armazenamento, podendo-se estabelecer, desta forma, a vida útil dos cogumelos irradiados / The production and consumption of edible mushrooms has been increasing in the last years due to its nutritional composition and sensory quality. The irradiation of mushrooms has been used with the purpose of maintaining the fresh product characteristics during shelf-life. The present study evaluated the effect of different radiation doses on the conservation of mushrooms Pleurotus sajor-caju, through by chemical, physical, microbiological and sensorial parameters. The packaging consisted on polystyrene trays with 250 g of sample, wrapped in polyvinyl chloride (PVC). The mushrooms were irradiated with doses of 125, 250, 500 and 750 Gy in a Gammacell 220 type irradiator, and stored at 4±1ºC and 90% UR for 10 days. The proximate composition (moisture, crude fibre, total protein, total fat and ash), total soluble solids, pH, texture, color (L, a*, b*, Chroma and ho), enzymatic activity (polyphenoloxidase and peroxidase), microbiological (total coliform, Escherichia coli and total psychotropic bacteria) and sensory evaluation (color, taste and appearance) were determined in the 1st, 5th and 10th storage days. For the respiratory rate analysis, 30 g of sample were placed in jars and stored at 4±1ºC and 90% UR for 8 days. CO2 was analyzed every day using a gaseous chromatographer. The results were submitted to variance analysis and average test using the SAS statistical package. The total protein and total fat values did not differ significantly between treatments or storage periods, while the moisture, crude fibre and ash values differ between treatments and periods. The dose of 750 Gy darkness the mushroom in the last evaluation, and texture was better in control during the storage period. The color (L, a*), texture and proximate composition values did not differ significantly between treatments or storage periods. A significant increase was observed for soluble solids, b* and enzymatic activity values in all treatments at the end of the storage period. Values of polyphenoloxidase and peroxidase activities increased in the last day of evaluation in samples irradiated with 250 Gy. This fact may be a result of the water radiolysis process, since mushrooms have high water content. The dose of 125 Gy increased the respiratory rate of the samples until the 5th storage day in comparison to the other treatments, causing reduction in the product shelf-life. The samples irradiated with 250, 500 and 750 Gy had a reduction on the respiratory rate if compared with the control, so contributing to the maintenance of the postharvest quality during the storage. The samples which received 750 Gy obtained the best results in the microbiological analyses, with reduction of total coliform and psychotropic bacteria during the storage period. Sensory analyses showed that the control had higher scores for color, aroma and appearance attributes; they were above the acceptability limit until the last storage day. In general, the irradiated samples were accepted for the evaluated attributes until the 5th storage day; thus, establishing the shelf-life for irradiated mushrooms

O lodo proveniente de estações de tratamento de água (ETA) é formado pela decantação do material particulado presente na água e sua composição varia conforme as atividades do entorno do manancial e dos produtos acrescentados durante o processo de potabilização da água. Seu destino é nos cursos de água ou armazenado na ETA, gerando um passivo ambiental. Esse, na forma in natura não apresenta características suficientes para sua aplicação em solos, no entanto, agregado ao bagaço e vinhaça de cana-de-açúcar e tratado com os fungos Pleurotus spp., constituirá um produto com melhor estruturação, devido a capacidade do fungo em degradar, mineralizar e transformar substâncias poluentes. O objetivo desse estudo foi caracterizar o lodo gerado em ETA quanto a sua toxicidade utilizando plântulas de milho e avaliar sua utilização na agricultura como material orgânico após o tratamento com Pleurotus, para uma destinação final adequada do lodo. Foram realizados dois experimentos em laboratório, o primeiro para definir a proporção adequada da composição da mistura em cinco composições (0:1; 1:1; 1:2; 1:3 e 2:1 de lodo/bagaço), inoculadas com cada uma das linhagens dos fungos (P. sajor-caju e P. ostreatus) incubados por 12 dias. Após, avaliou-se o pH; condutividade elétrica, umidade, análises químicas de fertilidade e a atividade de três enzimas (lacase, peroxidase e manganês peroxidase). O segundo experimento foi realizado após a definição da composição da mistura e a linhagem de fungo que proporcionou os melhores resultados. A mistura de 1:2 (lodo/bagaço) com P. sajor-caju foi incubada por 60 dias. Avaliações antes da inoculação e após 60 dias de incubação foram realizadas da mesma maneira que no primeiro experimento, acrescentados das análises dos elementos químicos potencialmente tóxicos. O potencial de utilização agronômico da mistura tratada foi realizado incorporando o material ao solo nas concentrações de 0; 1; 10 e 100 de mistura/solo (g/kg). Dez sementes de milho (Zea mays) foram semeadas, após cinco dias de desenvolvimento realizou-se o desbaste, deixando cinco plântulas por vaso. As plântulas permaneceram em condições de casa de vegetação por mais 14 dias. Foram avaliadas: porcentagem de germinação, comprimento de raiz e parte aérea das plântulas e a produção de matéria seca e, os teores e acúmulo de elementos químicos potencialmente tóxicos nas raízes e parte aérea de milho. A proporção de 1: 2 da mistura tratada com P. sajor-caju foi escolhida com base nas características químicas avaliadas, produção de enzimas, redução de odor e descoloração da mistura. O material tratado não apresentou efeitos de toxicidade em Z. mays até a concentração de 10 g/kg (mistura/solo). Acima desta, na concentração de 100 g/kg observou-se a redução de parte aérea e aumento do crescimento das raízes e, elevadas concentrações de Al, Fe K e P. O teste de mistura e ecotoxicológico utilizando o lodo de ETA evidenciou sua utilização como material componente de um produto orgânico associado ao bagaço e vinhaça, na proporção de 1:2 (lodo/bagaço) inoculado com P. sajor-caju incubado por 60 dias em condições de laboratório / The sludge generation from water treatment plants (WTP) is formed by the setting of the particulate matter. The chemical composition of the sludge varies according to the activities surrounding the watershed and products added during the water purifying process. The normal destination of the sludge are the watercourses or the storage in the area of WTP. The sludge in nature form does not have enough features to their application to land. However, added to bagasse and vinasse, after going through a biological treatment using the fungi of genus Pleurotus, will be transformed in a product with better structuring due to the capacity of degradation, mineralization and conversion of pollutants. Based on this, the objective of this study is to characterize the sludge produced in WTP on its toxicity and evaluate the viability of this utilization in agriculture as an organic material after Pleurotus treatment. There were two experiments in the laboratory, the first to set the proper proportion in the composition of the mixture testing in five compositions 1:0; 1:1; 1:2; 1:3 and 2:1 (sludge/bagasse), inoculated with each strain of the P. sajor-caju and P. ostreatus. After 12 days of incubation were evaluated of pH, electrical conductivity, moisture and the activity of three enzymes (laccase, peroxidase and manganese peroxidase). The second experiment was carried out after setting the composition of the mixture and the fungus strain that provided the best results. The mixture of 1:2 (sludge/bagasse) inoculated with P. sajor-caju with evaluations before and after 60 days of incubation. The treated material was evaluated in the same way as the first experiment, with further analyses of organic carbon, total nitrogen and chemical nutrients or potentially toxic elements. The ecotoxicological test was carried out by incorporating the mixture into the soil in pots at concentrations of 0; 1; 10 and 100 of mixture/soil (g/kg). After that the maize (Zea mays) was sown and grown in conditions of green house for 14 days. Then were evaluated: percentage of germination, plant height, root length, dry matter production, and the content and accumulation of nutrient and/or potentially toxic chemicals elements. The ratio of 1:2 (sludge/bagasse) inoculated with P. sajor-caju was chosen based on the results of chemical characteristics, production of enzymes, reducing odor and discoloration of the treated mixture. This material applied in soils showed no toxic effects in Zea mays up to a concentration of 10 g/kg (mixture/soil). Above this, in the concentration of 100 g/kg, was observed the shoot reduction, root growth and, high concentrations of Al, Fe, K and P. The mixture composition and ecotoxicological tests, revealed the potential of use sludge of WTS in material organic formulation together with bagasse and vinasse in the ratio 1:2 (sludge/bagasse) inoculated with P. sajor-caju incubated for 60 days under laboratory conditions

Orientador: Meire Cristina Nogueira de Andrade / Banca: Geisian Maria de Queiroz Fernandes / Banca: Tadeu Antonio Fernandes da Silva Júnior / Banca: Eustáquio Souza Dias / Banca: José Raimundo de Souza Passos / Resumo: Pleurotus ostreatus (shimeji) está entre os três cogumelos comestíveis mais consumidos no Brasil e no mundo. Dentre os fatores relacionados à sua produtividade elevada, a mais relevante é a produção do spawn. Spawn é a primeira etapa no cultivo de cogumelos e inicia-se com o crescimento micelial in vitro em meios de cultura, chamado de matriz primária, posteriormente transferida para substrato sólido nomeada como matriz secundária, e então utilizado como inóculo para produção de cogumelos. O objetivo desse estudo foi avaliar a influência dos diferentes processos de preparo do spawn sob efeito na eficiência biológica do fungo Pleurotus ostreatus. Na matriz primária avaliou-se número de repicagem, velocidade do crescimento micelial, concentração de nutrientes, concentração de dextrose e fontes diversas de nutrientes no meio de cultura em função da eficiência biológica do fungo. Na matriz secundária: tempo de armazenamento, atividade enzimática de lacase (LAC) e manganês peroxidase (MnP) do spawn em função da eficiência biológica de P. ostreatus. Para isto, a pesquisa foi subdividida em dois capítulos. No primeiro avaliou-se a velocidade do crescimento micelial em doze diferentes combinações de meio de cultura utilizando diferentes concentrações de batata, quirera de milho, composto à base de serragem e dextrose, sob efeitos na interferência da eficiência biológica de P. ostreatus. Posteriormente avaliou-se a atividade enzimática LAC e MnP nos spawns e foi correlacionadas à efic... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pleurotus ostreatus (shimeji) is among the three most consumed edible mushrooms in Brazil and worldwide. Among the factors related to its high productivity, the most relevant is spawn production. Spawn is the first step in the cultivation of mushrooms and begins with mycelial growth in vitro in culture media, called the primary matrix, later transferred to a solid substrate named as secondary matrix, and then used as an inoculum for the production of mushrooms. The objective of this study was to evaluate the influence of different spawn preparation processes under effect on the biological efficiency of the fungus Pleurotus ostreatus. In the primary matrix, the number of grains, mycelial growth velocity, nutrient concentration, dextrose concentration and various nutrient sources in the culture medium were evaluated according to the biological efficiency of the fungus. In the secondary matrix: storage time, enzymatic activity of laccase (LAC) and manganese peroxidase (MnP) of spawn as a function of the biological efficiency of P. ostreatus. For this, the research was subdivided into two chapters. In the first, the mycelial growth rate was evaluated in twelve different combinations of culture medium using different concentrations of potato, maize cherry, sawdust and dextrose based compounds, on effects on the biological efficiency of P. ostreatus. The LAC and MnP enzymatic activity in the spawns was then evaluated and correlated to the biological efficiency of the first and seco... (Complete abstract click electronic access below) / Doutor

All these results advance our understanding on how stimulatory agents can be used to increase the production of useful metabolites by submerged fermentation of mushroom mycelium and indicate its potential biotechnological applications. / By use of one- and two-dimensional gels in proteomic analysis, some functional mycelial proteins that were differentially expressed by the addition of Tween 80 were identified. The up-regulation of heat shock proteins might help to maintain cellular viability under environmental stress. A down-regulation of YALI0E34793p and an up-regulation of ATP citrate lyase isoform 2 might suppress the activity of TCA cycle and subsequently stimulated the EPS production. Up-regulation of fatty acid synthase alpha subunit FasA might promote the synthesis of long-chain fatty acids and their incorporation into the mycelial cell membranes. Up-regulation of mitogen-activated protein kinase might facilitate the signal transduction in these processes. / The BPS, a highly branched glucomannan produced by the addition of Tween 80 in the fermentation broth of PTR mycelium had similar carbohydrate and protein content, monosaccharide composition and glycosidic linkages except by having a significantly lower molecular weight when compared to those of the control. Both BPS, with and without addition of Tween 80, could significantly inhibit (p < 0.05) the in vitro growth of a chronic myelogenous leukemia cells K562 in a dose dependent manner, with an estimated IC50 value of 43.7 and 47.6 microg/mL, respectively. / The effects of different kinds of stimulatory agents including fatty acids, surfactants and organic solvents were compared. The optimum results were achieved when 3.0 g/L Tween 80 was added to the fermentation broth on the 5th day of the fermentation, to give a maximum increase of 51.3 and 41.8% (p < 0.05) in the yield of mycelial biomass and BPS production, respectively. / The underlying mechanisms by which Tween 80 could increase the mycelial growth and EPS production in PTR were investigated by three novel approaches including changes in the nutrient uptake by mycelium, the morphology of mycelial pellets, and the fatty acid composition in the mycelial cell membrane. Firstly, the addition of Tween 80 significantly increased the glucose consumption rate by the mycelium, implying that the efficiency of nutrient uptake from the fermentation broth was enhanced. Secondly the addition of Tween 80 could extend the growth period of the mycelium possibly by maintaining the intact structure of the mycelial pellets and preventing its disintegration caused by shear stress in the fermentation system. Thirdly, the addition of Tween 80 could increase the incorporation of oleic acid which was a constituent of Tween 80 itself into the mycelial cell membrane of PTR, altering its fatty acid composition and increase the cell membrane permeability. The first two results explained the enhancement in the mycelial growth and EPS production while the last one was related to the extracellular transport of EPS to the fermentation broth. / This study aimed at comparing the effectiveness in the use of stimulatory agents with different chemical structures for enhancing the production of mycelial biomass and exopolysaccharide (BPS) by submerged fermentation of an edible mushroom Pleurotus tuber-regium (PTR). The chemical characteristics and antitumor activity of the BPS produced with and without the addition of the most effective stimulatory agent (Tween 80 which is a permitted food additive) were also compared. The underlying mechanisms by which Tween 80 could exert its effect on the mushroom mycelium were investigated by using chemical methods and microscopic techniques as well as proteomic analysis. / Zhang, Bobo. / Adviser: Chi Keung Peter Cheung. / Source: Dissertation Abstracts International, Volume: 73-08, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 132-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lo Sze Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 100-113). / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.iv / List of Figures and Tables --- p.vii / Abbreviations --- p.xii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Edible mushrooms --- p.1 / Chapter 1.1.1 --- Pleurotus sajor-caju --- p.1 / Chapter 1.1.2 --- Flammulina velutipes --- p.2 / Chapter 1.2 --- Lignocellulose and phenolic monomers --- p.4 / Chapter 1.2.1 --- Sources of phenolic monomers --- p.4 / Chapter 1.2.2 --- Toxicity of phenolic monomers --- p.10 / Chapter 1.3 --- Fungal laccases --- p.13 / Chapter 1.3.1 --- Occurrence --- p.13 / Chapter 1.3.2 --- Laccase reaction --- p.14 / Chapter 1.3.3 --- Physiological functions --- p.18 / Morphogenesis --- p.18 / Pathogenicity --- p.19 / Lignin degradation --- p.20 / Chapter 1.4 --- Purpose of study --- p.22 / Chapter 2. --- Materials and Methods --- p.24 / Chapter 2.1 --- General --- p.24 / Chapter 2.1.1 --- Organisms --- p.24 / Chapter 2.1.2 --- Culture medium --- p.24 / Chapter 2.1.3 --- Addition of phenolic compounds --- p.24 / Chapter 2.2 --- Effect of phenolic monomers on the growth of mushroom mycelium on agar plates --- p.25 / Chapter 2.3 --- Effect of phenolic monomers on the production of fungal biomass in liquid culture --- p.25 / Chapter 2.4 --- Effect of phenolic monomers on the extracellular laccase produced by P. sajor-caju and F. velutipes --- p.26 / Chapter 2.5 --- Assay of laccase activity --- p.26 / Chapter 2.6 --- Electrophoresis patterns of laccase proteins --- p.27 / Chapter 2.6.1 --- Non-denaturing polyacrylamide gel electrophoresis --- p.27 / Chapter 2.6.2 --- Localization of laccase activity --- p.27 / Chapter 2.7 --- Purification of extracellular laccases from P. sajor-caju --- p.28 / Chapter 2.7.1 --- Inoculum preparation --- p.28 / Chapter 2.7.2 --- Culture conditions --- p.28 / Chapter 2.7.3 --- Concentration of culture supernatant --- p.29 / Chapter 2.7.4 --- Ammonium sulphate fractionation --- p.29 / Chapter 2.7.5 --- Anion exchange chromatography --- p.29 / Chapter 2.7.6 --- Preparative polyacrylamide gel electrophoresis --- p.30 / Chapter 2.7.7 --- Protein detection and quantification --- p.30 / Chapter 2.8 --- Characterization of Laccase Protein --- p.31 / Chapter 2.8.1 --- "Effect of pH, temperature and substrate concentration" --- p.31 / Chapter 2.8.2 --- Effect of inhibitors --- p.32 / Chapter 2.8.3 --- Determination of isoelectric point --- p.32 / Chapter 2.8.4 --- Determination of molecular weight --- p.33 / Chapter 3. --- Results --- p.34 / Chapter 3.1 --- Effect of phenolic monomers on the growth of P. sajor-caju and F. velutipes --- p.34 / Chapter 3.1.1 --- P. sajor-caju --- p.34 / Chapter 3.1.2 --- F. velutipes --- p.38 / Chapter 3.2 --- Effect of phenolic monomers on laccase production by P. sajor-caju and F. velutipes --- p.41 / Chapter 3.2.1 --- P. sajor-caju --- p.45 / Chapter 3.2.2 --- F. velutipes --- p.49 / Chapter 3.3 --- Electrophoretic patterns of extracellular laccase --- p.53 / Chapter 3.3.1 --- P. sajor-caju --- p.53 / Chapter 3.3.2 --- F. velutipes --- p.56 / Chapter 3.4 --- Purification of laccase protein from P. sajor-caju --- p.58 / Chapter 3.4.1 --- Separation of laccase proteins --- p.58 / Chapter 3.4.2 --- Purification of laccase IV --- p.59 / Chapter 3.5 --- Characterization of laccase IV from P. sajor-caju --- p.64 / Chapter 3.5.1 --- Optimum temperature and thermostability --- p.64 / Chapter 3.5.2 --- Optimum pH and pH stability --- p.67 / Chapter 3.5.3 --- Substrate concentration --- p.70 / Chapter 3.5.4 --- Effect of inhibitors --- p.74 / Chapter 3.5.5 --- Isoelectric point --- p.74 / Chapter 3.5.6 --- Molecular weight --- p.74 / Chapter 4. --- Discussion --- p.78 / Chapter 4.1 --- Phenolic monomers and the growth of P. sajor-caju and F. velutipes --- p.78 / Chapter 4.2 --- Phenolic monomers and laccase production by P. sajor- caju and F. velutipes --- p.81 / Chapter 4.3 --- Electrophoretic patterns of laccase proteins --- p.83 / Chapter 4.4 --- Physiological functions of laccase --- p.85 / Chapter 4.5 --- Purification of selected laccase protein from P. sajor-caju --- p.88 / Chapter 4.6 --- Properties of laccase IV from P. sajor-caju --- p.89 / Chapter 4.6.1 --- Optimum temperature and thermostability --- p.89 / Chapter 4.6.2 --- Optimum pH and pH stability --- p.90 / Chapter 4.6.3 --- Effect of inhibitors --- p.92 / Chapter 4.6.4 --- Km --- p.93 / Chapter 4.6.5 --- Isoelectric point --- p.94 / Chapter 4.6.6 --- Molecular weight --- p.94 / Chapter 4.7 --- Future works --- p.96 / Chapter 5. --- Conclusion --- p.98 / Chapter 6. --- References --- p.100

by Wang Pui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 115-128). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / List of Figures --- p.vi / List of Tables --- p.viii / Abbreviations --- p.ix / Chapter 1. --- Introduction Pg no / Chapter 1.1 --- Ligninolytic enzyme systems --- p.1 / Chapter 1.2 --- Three main ligninolytic enzymes --- p.3 / Chapter 1.2.1 --- Lignin peroxidases (LiP) --- p.3 / Chapter 1.2.2 --- Gene structure and Amino acid sequence structure --- p.7 / Chapter 1.2.3 --- Regulation of expression --- p.8 / Chapter 1.3. --- MnP --- p.8 / Chapter 1.3.1 --- General properties --- p.8 / Chapter 1.3.2 --- Gene structure and Amino acid sequence --- p.9 / Chapter 1.3.3 --- Regulation of Expression --- p.12 / Chapter 1.4 --- Laccase --- p.12 / Chapter 1.4.1 --- General Properties --- p.12 / Chapter 1.4.2 --- Gene structure and Amino acid sequence --- p.14 / Chapter 1.5 --- Pentachlorophenol (PCP) --- p.16 / Chapter 1.5.1 --- Production --- p.16 / Chapter 1.5.2 --- Toxicity --- p.15 / Chapter 1.5.3 --- Persistence --- p.19 / Chapter 1.6 --- Oyster mushroom --- p.22 / Chapter 1.7 --- Application of ligninolytic enzymes in bioremediation --- p.23 / Chapter 1.7.1 --- Genetic modification --- p.23 / Chapter 1.7.2 --- Characterization of enzymes properties --- p.25 / Chapter 1.7.3 --- Ligninolytic enzymes Purification and extraction --- p.26 / Chapter 1.7.4 --- Immobilization of ligninolytic enzymes --- p.26 / Chapter 1.8 --- Fermentation --- p.29 / Chapter 1.8.1 --- Different types of fermentation --- p.29 / Chapter 1.8.1.1 --- Submerged fermentation (SF) --- p.29 / Chapter 1.8.1.2 --- Solid State Fermentation (SSF) --- p.30 / Chapter 1.9 --- Proposal and experimental plan of the project --- p.33 / Chapter 1.9.1 --- Objectives --- p.34 / Chapter 2. --- Methods --- p.36 / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Culture maintenance --- p.36 / Chapter 2.1.2 --- Preparation of Pentachlorophenol (PCP) stock solution --- p.36 / Chapter 2.2 --- Optimization of production of ligninolytic enzymes by effective PCP concentration --- p.37 / Chapter 2.2.1 --- Preparation of mycelial homogenate --- p.37 / Chapter 2.2.2 --- Incubation --- p.37 / Chapter 2.2.3 --- Specific enzyme assays --- p.38 / Chapter 2.2.3.1 --- Laccase --- p.38 / Chapter 2.2.3.2 --- Manganese peroxidase (MnP) --- p.39 / Chapter 2.2.3.3 --- Lignin peroxidase (LiP) --- p.39 / Chapter 2.2.3.4 --- Protein --- p.39 / Chapter 2.3 --- Cloning of specific PCP-degradative laccase cDNA --- p.40 / Chapter 2.3.1 --- Isolation of total RNA --- p.41 / Chapter 2.3.2 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.41 / Chapter 2.3.3 --- First strand cDNA synthesis --- p.42 / Chapter 2.3.4 --- Amplification of laccase cDNA --- p.43 / Chapter 2.3.4.1 --- Design of primers for PCR reaction --- p.43 / Chapter 2.3.4.2 --- Polymerase chain reaction --- p.44 / Chapter 2.3.5 --- Agarose gel electrophoresis of DNA --- p.44 / Chapter 2.3.6 --- Purification of PCR products --- p.45 / Chapter 2.3.7 --- TA cloning of PCR products --- p.46 / Chapter 2.3.8 --- Preparation of Escherichia coli competent cells --- p.46 / Chapter 2.3.9 --- Bacterial transformation by heat shock --- p.47 / Chapter 2.3.10 --- Colony screening --- p.48 / Chapter 2.3.11 --- Mini-preparation of plasmid DNA --- p.48 / Chapter 2.3.12 --- Sequencing --- p.49 / Chapter 2.3.13 --- Identification of sequence --- p.51 / Chapter 2.4 --- Study of regulation temporal expression of laccase genes by PCP --- p.51 / Chapter 2.4.1 --- Semi-quantitative PCR --- p.51 / Chapter 2.4.1.1 --- Design of gene-specific primers --- p.51 / Chapter 2.4.1.2 --- Determination of suitable PCR cycles --- p.54 / Chapter 2.4.1.3 --- Normalization of the amount of RNA of each sample --- p.54 / Chapter 2.5 --- Quantification of residual PCP concentration --- p.55 / Chapter 2.5.1 --- Extraction of PCP --- p.55 / Chapter 2.5.2 --- High performance liquid chromatography --- p.55 / Chapter 2.5.3 --- Assessment criteria --- p.56 / Chapter 2.6 --- Effect of other componds on laccase activity and laccase expression --- p.56 / Chapter 2.6.1 --- Study of different isoform of laccase --- p.57 / Chapter 2.6.2 --- SDS-PAGE analysis of proteins --- p.58 / Chapter 2.7 --- Study of laccase expression and laccase activity in fruiting process of oyster mushroom --- p.59 / Chapter 2.8 --- Statistical analysis --- p.60 / Chapter 3. --- Results --- p.61 / Chapter 3.1 --- Production of Ligninolytic Enzymes by oyster mushroom / Chapter 3.1.1 --- Optimization of laccase production --- p.62 / Chapter 3.1.2 --- Optimization of MnP production --- p.64 / Chapter 3.1.3 --- Change of Protein content at different PCP concentration and time --- p.64 / Chapter 3.1.4 --- Change of specific activity at different PCP concentration and time --- p.64 / Chapter 3.1.5 --- Toxicity of PCP towards mycelial growth --- p.67 / Chapter 3.1.6 --- Enzyme productivities of laccase and MnP --- p.67 / Chapter 3.1.7 --- Change of % of residual PCP concentrations during 14 days --- p.70 / Chapter 3.2. --- Cloning of PCP-degradative laccase genes --- p.70 / Chapter 3.3 --- Regulation of expression of the laccase genes by PCP --- p.74 / Chapter 3.3.1 --- Determination of suitable PCR cycles --- p.74 / Chapter 3.3.2 --- Normalization of total RNA amount of different samples --- p.74 / Chapter 3.3.3 --- Regulation of temporal expression of the laccase genes by PCP --- p.74 / Chapter 3.4 --- Effect of other compounds and physiological status on laccase activity and expression --- p.81 / Chapter 3.5 --- Study of different forms of laccase --- p.86 / Chapter 4. --- Discussion --- p.93 / Chapter 4.1 --- Production of Ligninolytic enzymes by Pleurotus pulmonarius / Chapter 4.1.1 --- Optimization of laccase and MnP production by PCP --- p.95 / Chapter 4.2 --- Cloning of laccase genes --- p.97 / Chapter 4.2.1 --- Cloning strategy --- p.97 / Chapter 4.2.2 --- Analysis of Nucleotide sequence of Lac1 - Lac3 --- p.99 / Chapter 4.2.3 --- Characterization and comparison of deduced amino acid sequences of Lacl-Lac3 --- p.99 / Chapter 4.3 --- Regulation of expression of the laccase genes by PCP --- p.100 / Chapter 4.3.1 --- Regulation of temporal expression by PCP --- p.100 / Chapter 4.4 --- Effect of the potential inducers on laccase activity and expression --- p.103 / Chapter 4.5 --- Effect of the physiological status on laccase activity and expression --- p.105 / Chapter 4.5.1 --- Production of PCP-degradative laccase by Solid-state fermentation --- p.107 / Chapter 4.5.2 --- Uses of molecular probe in bioremediation --- p.107 / Chapter 4.6 --- Different isoforms of laccase --- p.109 / Chapter 4.7 --- Conclusion --- p.112 / Chapter 4.8 --- Further studies / Chapter 4.8.1 --- Confirmation of PCP-degradation by gene product of Lac1 and Lac2 --- p.114 / Chapter 4.8.2 --- Optimization of PCP-degradative laccases production by solid-state fermentation --- p.114 / Chapter 5. --- References --- p.115

Chan Kam Che. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 199-219). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstracts --- p.iii / 摘要 --- p.v / Contents --- p.vii / List of figures --- p.xiv / List of tables --- p.xix / Abbreviations --- p.xxii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Persistent organic pollutants --- p.1 / Chapter 1.2 --- DDT and DDE --- p.2 / Chapter 1.2.1 --- Background --- p.2 / Chapter 1.2.2 --- Health effects --- p.4 / Chapter 1.2.3 --- Environmental exposure of DDE --- p.4 / Chapter 1.2.4 --- Level of DDE in human --- p.9 / Chapter 1.2.5 --- Biodegradation of DDE --- p.10 / Chapter 1.3 --- Remediation methods --- p.11 / Chapter 1.3.1 --- Physical/ chemical treatment --- p.11 / Chapter 1.3.2 --- Bioremediation --- p.13 / Chapter 1.4 --- Fungal Bioremediation --- p.14 / Chapter 1.5 --- Ligninolytic enzymes --- p.15 / Chapter 1.5.1 --- Laccase --- p.15 / Chapter 1.5.2 --- Peroxidases --- p.20 / Chapter 1.5.2.1 --- Manganese Peroxidase (MnP) --- p.20 / Chapter 1.5.2.1 --- Lignin Peroxidase (LiP) --- p.24 / Chapter 1.6 --- Cultivation of Pleurotus pulmonarius --- p.27 / Chapter 1.7 --- Enzyme technology on environmental cleanup and its limitation --- p.28 / Chapter 1.8 --- Aims and objectives of this study --- p.29 / Chapter Chapter II --- Materials and Methods --- p.30 / Chapter 2.1 --- Organism and growth conditions --- p.30 / Chapter 2.2 --- Cultivation and the expression of the ligninolytic enzyme-coding genes during solid-state-fermentation of edible mushroom Pleurotus pulmonarius --- p.30 / Chapter 2.3 --- Treatment of DDE by living P. pulmonarius --- p.31 / Chapter 2.3.1 --- Optimization of DDE removal in broth system --- p.31 / Chapter 2.3.1.1 --- Effects of initial DDE concentration on the removal of DDE --- p.32 / Chapter 2.3.1.2 --- Effects of inoculum size on the removal of DDE --- p.33 / Chapter 2.3.1.3 --- Effects of incubation time on the removal of DDE and transcriptional profiles of the ligninolytic enzyme-coding genes --- p.33 / Chapter 2.3.2 --- Optimization of DDE removal in soil system --- p.34 / Chapter 2.3.2.1 --- Effects of initial DDE concentration on the removal of DDE --- p.34 / Chapter 2.3.2.2 --- Effects of inoculum size on the removal of DDE --- p.35 / Chapter 2.3.2.3 --- Effects of incubation time on the removal of DDE --- p.35 / Chapter 2.3.2.4 --- Transcription of the ligninolytic enzyme-coding genes --- p.35 / Chapter 2.4 --- Treatment of DDE by 1st SMC of p. pulmonarius grown on straw-based compost --- p.36 / Chapter 2.4.1 --- Optimization of DDE removal in soil system --- p.36 / Chapter 2.5 --- Treatment of DDE by crude enzyme preparations of P. pulmonarius grown on straw-based compost --- p.36 / Chapter 2.5.1 --- Optimization of DDE removal in broth system --- p.36 / Chapter 2.5.1.1 --- Effects of initial DDE concentration on the removal of DDE --- p.37 / Chapter 2.5.1.2 --- Effects of amounts of crude enzyme preparations on the removal of DDE --- p.37 / Chapter 2.5.1.3 --- Effects of incubation time on the removal of DDE --- p.37 / Chapter 2.5.2 --- Optimization of DDE removal in soil system --- p.37 / Chapter 2.5.2.1 --- Effects of initial DDE concentration on the removal of DDE --- p.38 / Chapter 2.5.2.2 --- Effects of amount of crude enzyme preparations on the removal of DDE --- p.38 / Chapter 2.5.2.3 --- Effects of incubation time on the removal of DDE --- p.38 / Chapter 2.6 --- Soil characterization --- p.39 / Chapter 2.6.1 --- Identification of organic contaminants in soil sample from Gene Garden using Gas Chromatography/Mass Spectrometry (GC/MS) --- p.39 / Chapter 2.6.2 --- Determination of soil texture --- p.42 / Chapter 2.6.3 --- Fresh soil/air-dried sample moisture --- p.44 / Chapter 2.6.4 --- "Soil pH, electrical conductivity & salinity" --- p.44 / Chapter 2.6.5 --- Total organic carbon contents --- p.44 / Chapter 2.6.6 --- Total nitrogen and total phosphorus --- p.44 / Chapter 2.6.7 --- Available nitrogen --- p.45 / Chapter 2.6.8 --- Available phosphorus --- p.45 / Chapter 2.6.9 --- Potassium value --- p.46 / Chapter 2.7 --- Quantification of residual DDE level --- p.47 / Chapter 2.7.1 --- Preparation of DDE stock solution --- p.47 / Chapter 2.7.2 --- Extraction and quantification of DDE using Gas Chromatography with Electron Capture Detector (GC/μECD) --- p.47 / Chapter 2.7.3 --- Identification of DDE breakdown products by GC/MS --- p.50 / Chapter 2.8 --- Extraction of protein and ligninolytic enzymes --- p.53 / Chapter 2.8.1 --- Protein assay --- p.53 / Chapter 2.8.2 --- Laccase assay --- p.53 / Chapter 2.8.3 --- Manganese peroxidase assay --- p.54 / Chapter 2.8.4 --- Calculation of activity and specific activity of laccase and manganese peroxidase --- p.54 / Chapter 2.9 --- Estimation of fungal biomass --- p.55 / Chapter 2.9.1 --- Preparation of ergosterol standard solution --- p.56 / Chapter 2.9.2 --- Analysis of ergosterol content --- p.56 / Chapter 2.10 --- Expression of the ligninolytic enzyme-coding genes --- p.58 / Chapter 2.10.1 --- Preparation of ribonuclease free reagents and apparatus --- p.58 / Chapter 2.10.2 --- RNA isolation and purification --- p.58 / Chapter 2.10.3 --- cDNA synthesis --- p.59 / Chapter 2.10.4 --- Semi-quantification of ligninolytic enzyme-coding gene expression by RT-PCR --- p.59 / Chapter 2.11 --- Preparation of crude enzyme preparations from P. pulmonarius compost --- p.63 / Chapter 2.12 --- "Assessment criteria: removal efficiency, RE, and removal capacity, RC" --- p.63 / Chapter 2.13 --- Statistical analysis “ --- p.64 / Chapter Chapter III --- Results --- p.65 / Chapter 3.1 --- Soil characterization --- p.65 / Chapter 3.2 --- Cultivation and the expression of the ligninolytic enzyme-coding genes during solid-state-fermentation of edible mushroom Pleurotus pulmonarius --- p.66 / Chapter 3.2.1 --- Mushroom yield --- p.66 / Chapter 3.2.2 --- Protein content --- p.66 / Chapter 3.2.3 --- Specific ligninolytic enzymes activities --- p.66 / Chapter 3.2.4 --- Ergosterol content --- p.69 / Chapter 3.2.5 --- Ligninolytic enzymes productivities --- p.69 / Chapter 3.2.6 --- Expression of the ligninolytic enzyme-coding genes during solid-state-fermentation --- p.72 / Chapter 3.3 --- Treatment of DDE by living P. pulmonaruis --- p.78 / Chapter 3.3.1 --- Optimization of DDE removal in broth system --- p.78 / Chapter 3.3.1.1 --- Effects of initial DDE concentration on the removal of DDE --- p.78 / Chapter 3.3.1.1.1 --- Effects of DDE on biomass development --- p.78 / Chapter 3.3.1.1.2 --- Protein content --- p.78 / Chapter 3.3.1.1.3 --- Specific ligninolytic enzyme activities --- p.78 / Chapter 3.3.1.1.4 --- Ligninolytic enzyme productivities --- p.79 / Chapter 3.3.1.1.5 --- DDE removal and removal capacity --- p.79 / Chapter 3.3.1.2 --- Effects of inoculum sizes on the removal of DDE --- p.84 / Chapter 3.3.1.2.1 --- Effects of DDE on biomass development --- p.84 / Chapter 3.3.1.2.2 --- Protein content --- p.84 / Chapter 3.3.1.2.3 --- Specific ligninolytic enzyme activities --- p.85 / Chapter 3.3.1.2.4 --- Ligninolytic enzyme productivities --- p.85 / Chapter 3.3.1.2.5 --- DDE removal and removal capacity --- p.85 / Chapter 3.3.1.3 --- Effects of incubation time on the removal of 4.0 mM DDE/g biomass --- p.89 / Chapter 3.3.1.3.1 --- Effects of DDE on biomass development --- p.89 / Chapter 3.3.1.3.2 --- Protein content --- p.89 / Chapter 3.3.1.3.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.89 / Chapter 3.3.1.3.4 --- DDE removal and removal capacity --- p.90 / Chapter 3.3.1.3.5 --- Putative degradation derivatives --- p.90 / Chapter 3.3.1.3.6 --- Expression of the ligninolytic enzyme-coding genes during the removal of 4.0 mM DDE/g biomass --- p.94 / Chapter 3.3.1.4 --- Effects of incubation time on the removal of 10.0 mM DDE/g biomass --- p.100 / Chapter 3.3.1.4.1 --- Effects of DDE on biomass development --- p.100 / Chapter 3.3.1.4.2 --- Protein content --- p.100 / Chapter 3.3.1.4.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.100 / Chapter 3.3.1.4.4 --- Expression of the ligninolytic enzyme-coding genes during the removal of 10.0 mM DDE/g biomass --- p.102 / Chapter 3.3.2 --- Optimization of DDE removal in soil system --- p.107 / Chapter 3.3.2.1 --- Effects of initial DDE concentration on the removal of DDE --- p.107 / Chapter 3.3.2.1.1 --- Ergosterol content --- p.107 / Chapter 3.3.2.1.2 --- Protein content --- p.107 / Chapter 3.3.2.1.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.107 / Chapter 3.3.2.1.4 --- DDE removal and removal capacity --- p.108 / Chapter 3.3.2.2 --- Effects of inoculum sizes on the removal of DDE --- p.111 / Chapter 3.3.2.2.1 --- Ergosterol content --- p.111 / Chapter 3.3.2.2.2 --- Protein content --- p.111 / Chapter 3.3.2.2.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.111 / Chapter 3.3.2.2.4 --- DDE removal and removal capacity --- p.112 / Chapter 3.3.2.3 --- Effects of incubation time on the removal of DDE --- p.115 / Chapter 3.3.2.3.1 --- Ergosterol content --- p.115 / Chapter 3.3.2.3.2 --- Protein content --- p.115 / Chapter 3.3.2.3.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.115 / Chapter 3.3.2.3.4 --- DDE removal and removal capacity --- p.116 / Chapter 3.3.2.3.5 --- Putative degradation derivatives --- p.116 / Chapter 3.3.2.4 --- Transcription of the ligninolytic enzyme-coding genes --- p.121 / Chapter 3.4 --- Treatment of DDE by 1st SMC of p. pulmonarius grown on straw-based compost --- p.127 / Chapter 3.4.1 --- Optimization of DDE removal in soil system --- p.127 / Chapter 3.4.1.1 --- Effects of initial DDE concentration on the removal of DDE --- p.127 / Chapter 3.4.1.1.1 --- Ergosterol content --- p.127 / Chapter 3.4.1.1.2 --- Protein content --- p.127 / Chapter 3.4.1.1.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.127 / Chapter 3.4.1.1.4 --- DDE removal and removal capacity --- p.128 / Chapter 3.4.1.2 --- Effects of inoculum sizes on the removal of DDE --- p.132 / Chapter 3.4.1.2.1 --- Ergosterol content --- p.132 / Chapter 3.4.1.2.2 --- Protein content --- p.132 / Chapter 3.4.1.2.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.132 / Chapter 3.4.1.2.4 --- DDE removal and removal capacity --- p.133 / Chapter 3.4.1.3 --- Effects of incubation time on the removal of DDE --- p.136 / Chapter 3.4.1.3.1 --- Ergosterol content --- p.136 / Chapter 3.4.1.3.2 --- Protein content --- p.136 / Chapter 3.4.1.3.3 --- Specific ligninolytic enzyme activities and ligninolytic enzyme productivities --- p.136 / Chapter 3.4.1.3.4 --- DDE removal and removal capacity --- p.137 / Chapter 3.4.1.3.5 --- Putative degradation derivatives --- p.137 / Chapter 3.5 --- Treatment of DDE by crude enzyme preparations of P. pulmonarius grown on straw-based compost --- p.142 / Chapter 3.5.1 --- The crude enzyme preparations of P. pulmonarius grown on straw-based compost --- p.142 / Chapter 3.5.2 --- Optimization of DDE removal in broth system --- p.143 / Chapter 3.5.2.1 --- Effects of initial DDE concentration on the removal of DDE --- p.143 / Chapter 3.5.2.2 --- Effects of amounts of crude enzyme preparations on the removal of DDE --- p.145 / Chapter 3.5.2.3 --- Effects of incubation time on the removal of DDE --- p.147 / Chapter 3.5.2.4 --- Putative degradation derivatives --- p.147 / Chapter 3.5.3 --- Optimization of DDE removal in soil system --- p.151 / Chapter 3.5.3.1 --- Effects of initial DDE concentration on the removal of DDE --- p.151 / Chapter 3.5.3.2 --- Effects of amounts of crude enzyme preparations on the removal of DDE --- p.151 / Chapter 3.5.3.3 --- Effects of incubation time on the removal of DDE --- p.154 / Chapter 3.5.3.4 --- Putative degradation derivatives --- p.154 / Chapter Chapter IV --- Discussions --- p.158 / Chapter 4.1 --- Quantification of the expression of the ligninolytic enzyme-coding genes --- p.158 / Chapter 4.2 --- Artificial cultivation and the expression of the ligninolytic enzyme-coding genes during solid-state-fermentation of edible mushroom Pleurotus pulmonarius --- p.164 / Chapter 4.3 --- Treatment of DDE by living P. pulmonarius --- p.166 / Chapter 4.3.1 --- Optimization of DDE removal in broth system --- p.166 / Chapter 4.3.2 --- Optimization of DDE removal in soil system --- p.169 / Chapter 4.3.3 --- Phylogeny of the ligninolytic enzyme-coding genes --- p.170 / Chapter 4.3.3.1 --- Laccase coding genes --- p.170 / Chapter 4.3.3.2 --- MnP coding genes --- p.175 / Chapter 4.3.4 --- Transcription of the ligninolytic enzyme-coding genes --- p.178 / Chapter 4.4 --- Treatment of DDE by 1st SMC of P. pulmonarius grown on straw-based compost --- p.183 / Chapter 4.4.1 --- Optimization of DDE removal in soil system --- p.183 / Chapter 4.5 --- Treatment of DDE by crude enzyme preparations of P. pulmonarius grown on straw-based compost --- p.184 / Chapter 4.6 --- Cost-effectiveness of the bioremediation method --- p.185 / Chapter 4.7 --- Further investigations --- p.194 / Chapter Chapter V --- Conclusions --- p.197 / References --- p.199

Yau Sze-nga. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 155-177). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.v / Table of Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiv / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Organopollutants and environment --- p.1 / Chapter 1.2 --- Pentachlorophenol --- p.3 / Chapter 1.2.1 --- Application of pentachlorophenol --- p.3 / Chapter 1.2.2 --- Characteristics of PCP --- p.4 / Chapter 1.2.3 --- Toxicity of PCP --- p.5 / Chapter 1.2.4 --- Environmental exposure of PCP --- p.6 / Chapter 1.3 --- Wastewater treatments of organopollutants --- p.9 / Chapter 1.3.1 --- Physical treatment --- p.10 / Chapter 1.3.2 --- Chemical treatment --- p.10 / Chapter 1.3.3 --- Bioremediation --- p.11 / Chapter 1.4 --- Biodegradation of PCP --- p.13 / Chapter 1.4.1 --- Biodegradation of PCP by bacteria --- p.13 / Chapter 1.4.2 --- Biodegradation of PCP by fungi --- p.14 / Chapter 1.5 --- Ligninolytic enzyme --- p.16 / Chapter 1.5.1 --- Lignin peroxidase --- p.16 / Chapter 1.5.2 --- Manganese peroxidase --- p.19 / Chapter 1.5.3 --- Laccase --- p.21 / Chapter 1.5.4 --- Biodegradation of PCP and other organopollutants by ligninolytic enzymes --- p.25 / Chapter 1.6 --- Structure and gene regulation --- p.27 / Chapter 1.6.1 --- MnP gene and structure --- p.27 / Chapter 1.6.1.1 --- Structure of MnP --- p.27 / Chapter 1.6.1.2 --- MnP gene regulation --- p.30 / Chapter 1.6.2 --- Laccase gene and structure --- p.31 / Chapter 1.6.2.1 --- Structure of laccase --- p.31 / Chapter 1.6.2.2 --- Laccase gene regulation --- p.32 / Chapter 1.7 --- Pleurotus pulmonarius --- p.36 / Chapter 1.8 --- Aims of study --- p.37 / Chapter 2 --- MATERIALS & METHOD --- p.39 / Chapter 2.1 --- Optimization of PCP induction in broth system --- p.39 / Chapter 2.1.1 --- Specific enzyme assays --- p.41 / Chapter 2.1.1.1 --- Assay for laccase activity --- p.41 / Chapter 2.1.1.2 --- Assay for manganese peroxidase (MnP) activity --- p.41 / Chapter 2.1.1.3 --- Assay for protein assay --- p.41 / Chapter 2.1.2 --- PCP effect on biomass gain --- p.42 / Chapter 2.1.3 --- Extraction of PCP --- p.42 / Chapter 2.1.3.1 --- Preparation of PCP stock solution --- p.43 / Chapter 2.1.3.2 --- Extraction efficiency of PCP --- p.43 / Chapter 2.1.3.3 --- Quantification of PCP by HPLC --- p.43 / Chapter 2.1.3.4 --- Study of PCP degradation pathway using GC-MS --- p.44 / Chapter 2.2 --- Isolation of laccase and manganese peroxidase coding genes --- p.46 / Chapter 2.2.1 --- Preparation of ribonuclease free reagents and apparatus --- p.46 / Chapter 2.2.2 --- Isolation of RNA --- p.46 / Chapter 2.2.3 --- Quantification of total RNA --- p.47 / Chapter 2.2.4 --- First strand cDNA synthesis --- p.47 / Chapter 2.2.5 --- Polymerase Chain Reaction (PCR) --- p.48 / Chapter 2.2.6 --- Gel electrophoresis --- p.50 / Chapter 2.2.7 --- Purification of PCR products --- p.50 / Chapter 2.2.8 --- Preparation of Escherichia coli competent cells --- p.51 / Chapter 2.2.9 --- Ligation and E. coli transformation --- p.51 / Chapter 2.2.10 --- PCR screening of E. coli transformation --- p.52 / Chapter 2.2.11 --- Isolation of recombinant plasmid --- p.52 / Chapter 2.2.12 --- Sequence analysis --- p.53 / Chapter 2.2.13 --- Construction of dendrogram for Pleurotus sp. laccase and manganese peroxidase dendrogram --- p.54 / Chapter 2.2.13.1 --- Dendrogram of laccase genes --- p.55 / Chapter 2.2.13.2 --- Dendrogram of manganese genes --- p.55 / Chapter 2.3 --- Differential regulation profiles of laccase and manganese peroxidase genes --- p.57 / Chapter 2.3.1 --- Time course of the effects of PCP on levels of laccase and manganese peroxidase mRNAs --- p.57 / Chapter 2.3.1.1 --- Isolation of RNA --- p.57 / Chapter 2.3.1.2 --- RT-PCR --- p.57 / Chapter 2.3.2 --- The effect of different stresses --- p.65 / Chapter 2.3.2.1 --- Pollutant removal analysis --- p.66 / Chapter 2.3.2.2 --- Differential gene expression under different stresses --- p.69 / Chapter 2.4 --- Construction of full-length cDNA --- p.69 / Chapter 2.4.1 --- Primer design --- p.69 / Chapter 2.4.2 --- First-strand cDNA synthesis --- p.71 / Chapter 2.4.3 --- RACE PCR reactions --- p.71 / Chapter 2.5 --- Statistical analysis --- p.73 / Chapter 3 --- RESULT --- p.74 / Chapter 3.1 --- Optimization of PCP induction in broth system --- p.74 / Chapter 3.1.1 --- Enzyme Assay --- p.74 / Chapter 3.1.1.1 --- Protein content --- p.74 / Chapter 3.1.1.2 --- Specific laccase activity --- p.74 / Chapter 3.1.1.3 --- Specific MnP activity --- p.76 / Chapter 3.1.1.4 --- Laccase productivity --- p.78 / Chapter 3.1.1.5 --- MnP productivity --- p.78 / Chapter 3.1.2 --- PCP effect on biomass development --- p.80 / Chapter 3.1.3 --- PCP removal --- p.80 / Chapter 3.2 --- isolation of laccase and manganese peroxidase coding genes --- p.83 / Chapter 3.2.1 --- Dendrogram construction for heterologous MnP and laccase coding genes --- p.83 / Chapter 3.2.2 --- Phylogeny of ligninolytic enzyme coding genes of P. pulmonarius --- p.85 / Chapter 3.2.2.1 --- Phylogeny of MnP coding genes --- p.88 / Chapter 3.2.2.2 --- Phylogeny of laccase coding genes --- p.88 / Chapter 3.3 --- differential regulation profiles of laccase and MnP genes --- p.91 / Chapter 3.3.1 --- Time course of the effects of PCP on levels of MnP and laccase mRNAs --- p.91 / Chapter 3.3.1.1 --- Time course of the effects of PCP on levels of MnP mRNAs --- p.91 / Chapter 3.3.1.2 --- Time course of the effects of PCP on levels of laccase mRNAs --- p.97 / Chapter 3.3.2 --- The effects of different stresses and two lignocellulosic substrates --- p.99 / Chapter 3.3.2.1 --- The effect on laccase and MnP enzyme activities --- p.99 / Chapter 3.3.2.1.1 --- Protein content --- p.99 / Chapter 3.3.2.1.2 --- Specific laccase activity --- p.100 / Chapter 3.3.2.1.3 --- Specific MnP activity --- p.102 / Chapter 3.3.2.1.4 --- Dry weight of P. pulmonarius --- p.102 / Chapter 3.3.2.1.5 --- Laccase productivity --- p.105 / Chapter 3.3.2.1.6 --- MnP productivity --- p.105 / Chapter 3.3.2.2 --- Organopollutant removal --- p.107 / Chapter 3.3.2.3 --- Differential gene expression under different stresses --- p.107 / Chapter 3.3.2.3.1 --- The effect on MnP mRNAs --- p.107 / Chapter 3.3.2.3.2 --- The effect on laccase mRNAs --- p.115 / Chapter 3.4 --- Construction of full-length cDNA --- p.116 / Chapter 3.4.1 --- PPMnP5 --- p.117 / Chapter 3.4.2 --- PPlac2 --- p.120 / Chapter 3.4.3 --- PPlac6 --- p.120 / Chapter 4 --- DISCUSSION --- p.123 / Chapter 4.1 --- Optimization of PCP induction in broth system --- p.123 / Chapter 4.2 --- Isolation of MnP and laccase coding genes --- p.126 / Chapter 4.3 --- Differential regulation profiles of MnP and laccase genes --- p.128 / Chapter 4.3.1 --- The effects incubation time and PCP on levels of MnP and laccase mRNAs --- p.128 / Chapter 4.3.1.1 --- MnP --- p.129 / Chapter 4.3.1.2 --- Laccase --- p.129 / Chapter 4.3.2 --- Regulation of MnP and laccase by different substrates --- p.130 / Chapter 4.3.2.1 --- Regulation of MnP and laccase activities --- p.131 / Chapter 4.3.2.2 --- Organopollutant removal --- p.132 / Chapter 4.3.2.3 --- Regulation of MnP coding genes --- p.136 / Chapter 4.3.2.4 --- Regulation of laccase coding genes --- p.137 / Chapter 4.4 --- "Characterization of full length cDNAs of PPMnP5, PPlac2 and PPLAC6" --- p.140 / Chapter 4.4.1 --- PPMnP5 --- p.140 / Chapter 4.4.2 --- PPlac2 and PPlac6 --- p.144 / Chapter 4.4.3 --- Real-time PCR --- p.146 / Chapter 4.4.3.1 --- Methodology for SYBR-Green real-time PCR --- p.146 / Chapter 4.4.3.2 --- Comparison of conventional PCR and real-time PCR --- p.148 / Chapter 4.5 --- APPLICATION AND FURTHER INVESTIGATION --- p.150 / Chapter 5 --- CONCLUSION --- p.152 / Chapter 6 --- REFERENCES --- p.155

Thesis (M.S.)--Youngstown State University, 2009. / Includes bibliographical references (leaves 63-68). Also available via the World Wide Web in PDF format.

Thesis (M.S.)--Youngstown State University, 2009. / Includes bibliographical references (leaves 38-42). Also available via the World Wide Web in PDF format.