Orientador: Jose Augusto Rosario Rodrigues / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-07T19:55:40Z (GMT). No. of bitstreams: 1 Zampieri_LuizArthur_M.pdf: 1219190 bytes, checksum: 50f1c90b246b806bd16bb30df4ecbbaf (MD5) Previous issue date: 2006 / Resumo: Neste trabalho estudou-se a bioxidação do sesquiterpeno valenceno (C15H24), produzindo o bioflavorizante nootkatona (C15H22O), buscando as condições ótimas para obtenção do máximo rendimento. Esta reação foi realizada utilizando dois sistemas enzimáticos distintos: o sistema lacase/mediador (LMS) de Trametes versicolor com mediador HBT (hidroxibenzotriazol) ou TEMPO (tetrametilpiperidin-N-oxil) e o sistema utilizando o complexo enzimático do citocromo P-450 de Chaetomium globosum. Outros microorganismos testados foram Botrytis cinerea, Mortierella isabellina e Mortierella ramaniana. As diversas variáveis (pH, tempo de reação, concentrações de enzima, mediadores e indutores, condições de aeração, entre outras) envolvidas nas respectivas reações foram estudadas através de planejamento fatorial e modelagem de superfície de resposta. A utilização do sistema LMS de Trametes versicolor mostrou ser uma ferramenta viável para obtenção de nootkatona a partir de valenceno, embora tenhamos obtido rendimento inferior (17%, agitador orbital em pequena escala e 15 % em escala preparativa, sob aeração externa) descrito na literatura (25%, sob aeração, escala preparativa), o que torna nosso procedimento pouco viável para utilização em maior escala, apesar disso, os resultados foram condizentes com os obtidos em reações semelhantes descritos na literatura científica, onde o rendimento de sistemas LMS dificilmente ultrapassa os 15%. O sistema enzimático CYP-450 apresentou rendimento inferior ao sistema lacase/mediador e, este último sistema, HBT mostrou ser um mediador mais eficiente que TEMPO. / Abstract: In this work, we study the sesquiterpene valencene bioxidation (C15H24), which produces the nootkatona biological flavor substance (C15H22O), in an attempt to achieve the best conditions for optimum yield. This reaction was carried out using two different enzymatic systems: the Trametes versicolor laccase-mediator system with HBT mediators (hydroxybenzotriazol), or TEMPO (tetramethyl-piperidine-N-oxide), and the system using the Chaetomium globosum cythocrome P-450 enzymatic complex. Other microorganisms were tested, such as Botrytis cinerea, Mortierella isabellina and Mortierella ramaniana. The different variables involved in the respective reactions were studied by means of factorial planning and modeling the response system. The use of the Trametes versicolor LMS system proved to be a viable tool to obtain nootkatone from valencene, although we have obtained an inferior yield (17% with orbital agitator in small scale and 15% in preparatory scale, under external aeration) in comparison with the highest value described in literature (25% under aeration). Thus, our procedure presents little viability for large scale use, although results were in agreement with those obtained in similar reactions described in scientific literature, in which the yield produced by LMS systems rarely exceeds 15%. The CYP-450 enzymatic system presented lower yield in comparison with the laccase-mediator system and in the latter, HBT turned out to be more efficient than TEMPO. / Mestrado / Quimica Organica / Mestre em Química

Today’s most pressing problems would greatly benefit from an integrated production method for food, water, and energy. Biological fuel cells can offer such a production method, but current designs cannot be scaled to meet global demand. The ability of five different fungal strains to secrete laccase was evaluated under optimized culture conditions using three inducers. A specialized electrode was developed to increase the loading of laccase on the cathode. Trametes versicolor was then immobilized at the modified cathode and shown to secrete electrochemically active laccase. This hybrid design combines the power density of an enzymatic catalyst with the robustness of a microbial catalyst by facilitating biological renewal of the enzymatic catalyst laccase.

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

A vinhaça e o bagaço de cana são os principais subprodutos oriundos do processamento da cana-de-açúcar nas indústrias sucroalcooleiras, sendo geradas grandes quantidades dos mesmos. O fungo basidiomiceto Pleurotus ostreatus tem a capacidade de degradar materiais lignocelulolíticos e produzir enzimas lignolíticas de interesse para as indústrias. Com o objetivo de avaliar a produção das enzimas lacases e peroxidase, o fungo Pleurotus ostreatus, foi cultivado em meio contendo bagaço pré-tratado e vinhaça, ou em meio contendo apenas vinhaça, em sistema de fermentação semissólido ou submerso; as enzimas extracelulares foram avaliadas após 7, 10 e 12 dias de cultivo. O bagaço peneirado foi considerado pré-tratado fisicamente (T1); para o pré-tratamento T2 o bagaço umedecido foi submetido a autoclave (121°C e 1 atm por 15 min); nos pré-tratamentos químicos, T3 e T4, o bagaço foi tratado com peróxido de hidrogênio e hidróxido de sódio nas seguintes concentrações: 0,75% H2O2 + 0,75% NaOH (T3) e 0,75% H2O2 + 1% NaOH (T4) na proporção 1:10 (p/v) e, em seguida foram submetidos à autoclave (121°C e 1 atm por 15min). A vinhaça utilizada foi proveniente de uma indústria sucroalcooleira (V1) e outra de destilaria (V2); a composição físico-química mostrou que a primeira possuía os índices de matéria orgânica e fósforo mais elevados que na vinhaça V2, enquanto que a relação C:N foi menor na vinhaça V1. Os extratos enzimáticos foram obtidos após filtração do meio submerso; para o meio semissólido foi necessário a adição de tampão citrato (1:5 p/v) antes da filtração. A atividade de lacasse e peroxidase em meio submerso, nos tratamentos com a vinhaça V1, foi superior ao observado em meio semissólido. A produção das enzimas em fermentação submersa, utilizando a vinhaça V1, apresentou valores de atividade de lacase, no tratamento TL1 e TL2, de 784,9 e 707,5 U.L-1, com atividade específica de 3,04 e 2,86 U.mg-1, respectivamente, e a amostra VL1, contendo apenas vinhaça, de 1,91 U.mg-1, no 12º dia de fermentação. Os valores mais altos de atividade de peroxidase foram obtidos nos tratamentos TL1, TL2, VL1, com 133,1; 131,2 e 126,1 U.L-1, respectivamente, após 12 dias de cultivo. A maior atividade específica obtida foi na VL1 (0,86 U.mg-1) no 7º dia de cultivo. O pré-tratamento físico do bagaço mostrou melhores condições para a produção das enzimas. Para a produção da lacase e da peroxidase é fundamental a composição da vinhaça. / The vinasse and bagasse are the principal by-products derived from the processing of sugarcane in the sugarcane industry, which generated large amounts of them. The basidiomycete fungus Pleurotus ostreatus, has the ability to degrade lignocellulolytic materials and produce lignolíticas enzymes of interest to industry. In order to evaluate the production of laccase and peroxidase enzymes, fungus P. ostreatus was grown in medium containing pre-treated bagasse and vinasse, or in medium containing only vinasse in semi-solid or submerged fermentation system; extracellular enzymes were evaluated after 7, 10 and 12 days of cultivation. The screened bagasse was considered pretreated physically (T1); for the pretreatment T2 moistened residue was subjected to autoclaving (121°C and 1 atm for 15 min). The chemical pretreatments, T3 and T4, the residue was treated with hydrogen peroxide and sodium hydroxide solution in the following concentrations 0,75% H2O2 + 0,75% NaOH (T3) and 0,75% H2O2 + 1% NaOH (T4) 1:10 (w/v) and then underwent autoclaving (121°C and 1 atm for 15 min). Vinasse used was coming from a sugar and alcohol industry (V1) and a distillery (V2); the physico-chemical composition showed that the former had the rates of organic matter and phosphorus higher than in V2 vinasse, whereas the C: N ratio was lower in V1 vinasse. The enzymatic extracts were obtained after filtration medium the submerged; to semisolid medium was necessary the addition of citrate buffer (1:5 w/v) prior to filtration. The activity of peroxidase and lacasse in submerged medium, in the treatments with the V1 vinasse, was higher than observed in semi-solid medium. The production of enzymes by submerged fermentation using the vinasse V1, presented laccase activity values, in the treatment TL1 and TL2, of 784,9 and 707,5 UI.L-1, with specific activity of 3,04 e 2,86 U.mg-1, on the 12th day of fermentation. Higher values peroxidase activity were obtained in the treatments TL1, TL2, VL1, with 133,1; 131,2 and 126,1 UI.L-1, respectively, after 12 days of culture. The highest specific activity was obtained at VL1 (0,86 U.mg-1) on the 7th day of culture. Physical bagasse pretreatment showed better conditions for the production of enzymes. For the production of the laccase and peroxidase is fundamental composition of vinasse.

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

Modern manufacturing processes assume efficient utilization and recycling of natural resources whenever possible. Over the past decade paper recycling has progressed from 33.5% in 1990 to just above 48% in 2002.1 Indeed, for certain select grades, (newspaper and old corrugated containers) greater than 70% is currently being recycled. In contrast, mixed office waste and colored directory papers are often underutilized. A major difficulty in recycling these grades of paper is the problems associated with decolorizing the dyes present in the paper.2 Of the commonly used paper dyes, the stilbene dye Direct Yellow 113 and methine dye Basazol 46L are notorious4 for poor bleachability with the commonly used chemical bleaching agents including chlorine dioxide, oxygen, hydrogen peroxide and sodium dithionite. The ability of white-rot fungi to decolorize colored effluents containing textile dyes is currently the subject of intensive research efforts. The secreted enzymes involved in dye decolorization include manganese peroxidase, lignin peroxidase and laccase. Laccase, a lignolytic enzyme, has also been studied for many years for the biobleaching of wood pulps. The ability of laccase to delignify pulp is greatly enhanced by the addition of small molecule mediators such as 2-2´ azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). This research project focused on applying laccase combined with a mediator to decolorize C.I. Direct Yellow 11 and Basazol 46L. Three mediators were tested: ABTS, HBT and violuric acid. Laccase/ABTS was most effective with 60% of the color being removed. The level of color removal was maintained at 60% even when ABTS concentration was lowered from 5 mM to 0.01 mM. When laccase/1 mM ABTS was applied to Direct Yellow 11 in solution, the majority of color loss occurred within 60 minutes. The ability of soybean (SBP) and horseradish (HRP) peroxidases and laccase to decolorize Direct Yellow 11 and Basazol 46L in solution was also examined. The results demonstrated that these two recalcitrant dyes could be effectively decolorized by enzymatic treatments by horseradish peroxidase, soybean peroxidase, and laccase with ABTS as mediator. SBP is effective from pH 4.5 to 8.5. The stilbene dye Direct Yellow 11 responded to both SBP and laccase/ABTS. For the methine dye Basazol 46L, SBP was a more effective treatment than HRP or laccase/ABTS. Basazol 46L responded quickly to SBP treatment with 74% reduction in signal intensity within 5 minutes. To evaluate the effectiveness of laccase/ABTS treatment, pulp dyed with Direct Yellow 11 and three commercial colored pulps were subjected to seven different bleaching treatments. These treatments consisted of 1)laccase/ABTS; 2)laccase/ABTS followed by alkaline extraction; 3)laccase/ABTS followed by bleaching with sodium dithionite; 4)oxygen bleaching; 5)oxygen bleaching followed by dithionite treatment; 6)alkaline hydrogen peroxide bleaching; and 7)alkaline peroxide bleaching followed by dithionite treatment. The best results were obtained by including reductive bleaching with sodium dithionite. For Direct Yellow 11 dyed pulp, laccase/ABTS followed by dithionite yield comparable reduction in color to oxygen or peroxide followed by dithionite.

Infection of fully expanded leaves of tobacco with INF1 causes the appearance of HR lesions within 12 h and progressive to all infection sites after 48 h treatment. Among the POD isozymes, the increase of cationic PODs and anionic PODs is correlated with the rise of lignin contents in INF1-treated leaves, especially cationic PODs (pI 9.5, pI 8.7, pI 8.3, pI 7.8, pI 7.4). It was suggested that the induction of POD activity resulted in part of H2O2 reduction. The increase of cationic (pI 9.5) and anionic (pI 4.4) POD transcripts was correlated with the increased cationic and anionic PODs activity in INF1-treated leaves. Therefore, the increased POD activity is due to the de novo synthesis of the cationic (pI 9.5) and anionic (pI 4.4) PODs in INF1-treated leaves. The increase in cationic pI 9.6 laccase transcript was also correlated with the increased cationic laccase activity in INF1-treated leaves. Our results suggest that laccase might play a major role on lignin biosynthesis at the early stage (6 h), and as the inoculation time was prolonged, peroxidases (especially cationic POD) and laccases will work together on lignin biosynthesis.

The significant root inhibition of growth in Cd-treated soybean (Glycine max) seedling correlated with the increase of H2O2 levels, PODs and laccases activity. The increase of the activities of PODs (pI 8.8, pI 7.7, pI 5.2, pI 4.5, pI 4.4 and pI 3.7) and laccases (pI 9.2, pI 8.9 and pI 8.3, pI 5.4, pI 4.2 and pI 3.7) are accompanied by a rise of lignin contents in Cd-treated tissues. Our results suggested that laccases work during the early stage of Cd treatment. Laccases and peroxidases work cooperatively in lignin synthesis when the time of Cd treatment was prolonged.