A biomassa lignicelulósica tem sua estrutura composta por celulose, hemicelulose e lignina. Dentre essas, a lignina tem se mostrado interessante por ser uma fonte precursora sustentável de fragmentos aromáticos antes obtidos apenas de combustíveis fósseis. Sua estrutura é composta por resíduos de fenilpropanóides p-hidroxibenzeno (H), guaiacil (G) e siringil (S) unidas por ligações C–C e C–O–C em que a ligação β–O–4 é a predominante (mais de 50%). Devido à sua complexidade estrutural e conformacional, a clivagem de suas ligações é pouco seletiva e a caracterização dos fragmentos resultantes é complexa. Uma estratégia comumente empregada para evitar esses desafios é o uso de modelos mais simples. Entretanto, poucas metodologias são reportadas na literatura para a sua síntese e a maioria delas envolve o emprego de halocetonas. O presente trabalho desenvolveu duas novas metodologias promissoras para síntese desses oligômeros, contendo ligação β–O–4 por meio da química de diazo: (a) reação de inserção O–H entre fenol e α–aril diazocetonas, e (b) compostos α–diazo β-cetoéster. Ademais, a utilização de monômeros contendo a função fenol e diazocetona no mesmo anel permitiria a síntese de cadeias de diversos tamanhos em uma única etapa. Como ponto de partida para o estudo, limitou-se à síntese de dímeros, visando entender a reação de inserção O–H. Os produtos desejados foram obtidos em rendimentos de 27–51% após catálise com Cu(hfac)2. Por fim, os modelos de lignina propriamente ditos foram sintetizados após simples adição aldólica e redução em rendimentos globais de 51–78%. Os estudos envolvendo a inserção de fenol em α–diazo β-cetoéster mostraram resultados promissores, corroborando para uma nova estratégia sintética para a obtenção de modelos de lignina. Novos estudos em nosso laboratório estão sendo desenvolvidos para se obter resultados mais conclusivos. / Lignocellulosic biomass is composed of cellulose, hemicellulose and lignin. Among these, the macromolecule lignin has widely received attention as an interesting source of aromatic building blocks to replace those currently obtained from fossil–based fuels. Lignin is composed by units of phenylpropanoids p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) linked by C–C and C–O bonds which β–O–4 linkages are dominant by more than 50%. Unfortunately, due to the complex structure and 3D arrangement of lignin, it is difficult to control selectivity during its degradation as well as characterize the resulting fragment. A common strategy to avoid these challenges is the use of model substrates; however, few methods exist for the synthesis of these models oligomers. The present work developed two promising routes towards the synthesis of these oligomers containing β–O–4 linkages through use of diazo chemistry: (a) O–H insertion with a phenol and α–aryldiazoketones, and (b) α–diazo β–ketoether compounds. Additionally, monomers containing a phenol and diazoketone unit on the same aromatic ring could allow for the synthesis of a variety of side chains in a single step. We began our studies on the synthesis of dimers via O–H insertion and obtained 27–51% yield of the desired products using catalytic Cu(hfac)2. The mimetic lignin models were synthesized after simple Aldol addition and reduction in 51–78% overall yield. Our work involving this O–H insertion into α–diazo β–ketoether is a promising strategy to rapidly synthesize lignin model oligomers and is an active area of research within our lab.

Os métodos analíticos empregados atualmente para a caracterização química de materiais lignocelulósicos foram desenvolvidos para a madeira e são empregados com pequenas modificações ao bagaço e à palha da cana-de-açúcar. A não existência de metodologia específica para estes materiais leva a obtenção de resultados inadequados e dificultam tanto o planejamento quanto a interpretação de resultados. Sendo assim, o objetivo principal deste trabalho é desenvolver metodologias analíticas específicas para a caracterização química da palha e do bagaço de cana-de-açúcar. A determinação de lignina foi estudada a partir da hidrólise e solubilização da celulose e hemiceluloses em solução de ácido sulfúrico. A fração insolúvel foi analisada por análise elementar, espectroscopia no infravermelho, espectrometria de ressonância magnética nuclear de C13 no estado sólido e análise termogravimétrica (TGA). Os açúcares hidrolisados e produtos derivados destes foram analisados por meio de cromatografia líquida de alta eficiência (CLAE). Foi realizada uma análise de holocelulose que é complementar à determinação de lignina. O procedimento consiste no tratamento do material lignocelulósico (pré-extraído com cicloexano/etanol) com solução de clorito de sódio em meio ácido. Os resultados obtidos não apresentaram similaridade nas correlações entre os métodos analíticos, entretanto mostraram que o bagaço integral e suas frações separadamente apresentam comportamentos diferentes, frente ao tratamento ácido, o que ressalta a necessidade de métodos analíticos específicos. / The analytical methods currently used for chemical characterization of lignocellulosic materials were developed for wood and are applied with minor modifications for sugar cane bagasse and straw analysis. The lack of specific methodology for these materials leads to inadequate results and hamper both the planning and the interpretation of results. Thus, the main aim of this work is to develop specific analytical methodologies to the chemical characterization of sugar cane bagasse and straw. The determination of lignin was studied by the hydrolysis and dissolution of the polysaccharide fraction in sulfuric acid solution. The insoluble fraction was analyzed by elemental analysis, Fourier Transform Infrared (FT-IR), Carbon-13 nuclear magnetic resonance spectroscopy (13C NMR), Gel permeation chromatography (GPC) and Thermogravimetric analysis (TGA). The sugars and derivatives of these hydrolysates were analyzed by High performance liquid chromatography (HPLC). It was also performed a complementary analysis from the holocellulose content determinations in order to check the values obtained by the klason procedure. The results showed the dependence of sulfuric acid concentration on lignin content determinations and the role of condensation reactions in the lignin characteristics. Despite the similarities in chemical composition, klason lignins obtained from straw exhibited very low molar masses. Preliminary results obtained from holocellulose determinations showed also the need for optimized oxidation procedures in order to be successful applied to sugar cane bagasse analysis.

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

O objetivo deste trabalho foi avaliar a disponibilidade de Fe, Cu e Zn em presença de mesocarpo de babaçu, pois o mesocarpo contem compostos antinutricionais (fitato e lignina) que podem diminuir a disponibilidade de nutrientes minerais. As concentrações de Fe, Cu e Zn encontradas no mesocarpo de babaçu foram 17, 7,0 e 2,6 µg g-1, respectivamente. Para um melhor entendimento das interações dos elementos com o mesocarpo, foi realizada a extração de substâncias complexantes com soluções de NaOH (pH 7 e 12). Análises por espectrometria de absorção molecular indicaram que o principal complexante presente no extrato era o fitato. Soluções de Fe, Cu e Zn (10 a 300 mg L-1) foram adicionadas aos extratos, para que, após agitação e centrifugação, fossem determinadas as recuperações desses elementos. Os resultados mostraram que os elementos formam complexos com o fitato e a solubilidade desses complexos é dependente da razão elemento/fitato, quanto maior essa razão menor é a solubilidade. Também foi avaliada a interação dos elementos com o sólido remanescente da extração. Fe, Cu ou Zn foram adicionados ao mesocarpo lavado. Não foi possível afirmar que houve interação do mesocarpo lavado com Fe e Cu devido à precipitação decorrente da hidrólise. Porém, o Zn não sofreu hidrólise e teve baixas recuperações, indicando que houve interação com o sólido. Além disso, pode ser observado que ao adicionar maiores concentrações de Zn obtiveram-se melhores recuperações, provavelmente devido à saturação dos sítios de ligação. Para os estudos de disponibilidade, foi utilizado o procedimento de digestão simulada in vitro da US Pharmacopeia. Fe e Zn não foram extraídos durante a digestão do mesocarpo, portanto esses elementos não estariam disponíveis para absorção pelo organismo. Entretanto, cerca de 120 µg L-1 de Cu foi extraído do mesocarpo. Foram realizadas digestões do mesocarpo com adição de Fe, Cu e Zn e apenas 48% do Fe, 65% do Cu e 75% do Zn foram recuperados. Digestões contendo essa mesma concentração dos elementos foram realizadas com adições de Ca e Mg. A presença do Ca diminuiu a recuperação de Fe e Zn. Para simular uma situação próxima ao real, foram realizadas digestões gastrointestinais de leite, mistura de leite e mesocarpo e mistura de leite e lignina. Apesar do ferro presente no leite apresentar baixa disponibilidade, quando o mesmo foi misturado ao mesocarpo observou-se um aumento da concentração de Fe solúvel. Compostos presentes no leite ou no mesocarpo podem aumentar a solubilidade dos elementos. Não foi possível observar se havia alguma alteração da disponibilidade do Cu presente no leite na presença do mesocarpo, pois a concentração de Cu no leite é muito baixa. A fração de Zn solúvel na mistura de leite e mesocarpo permaneceu a mesma, porém a fração dialisável foi praticamente nula. A adição de lignina ao leite aumentou a extração dos elementos. Entretanto, esses elementos continuaram não sendo dialisados / The aim of this study was to evaluate the availability of Fe, Cu and Zn in the presence of mesocarp of babassu because it contains antinutritional compounds (phytate and lignin) that may decrease the availability of mineral nutrients. The concentrations of Fe, Cu and Zn found in the mesocarp of babassu were 17, 7.0 and 2.6 mg g-1, respectively. For a better understanding of elements interactions with mesocarp, it was performed the extraction of complexing substances with NaOH solutions (pH 7 and 12). Analysis by molecular absorption spectrometry indicated that the main complexing present in the extract was the phytate. Solutions of Fe, Cu and Zn (10 to 300 mg L-1) were added to the extracts for determining the recoveries of these elements, after agitation and centrifugation. The results showed that the elements form complexes with phytate and the solubility of these complexes is dependent on the ratio element / phytate, the greater this ratio the lower the solubility. The elements interaction with the remaining solid from extraction was also evaluated. Fe, Cu or Zn were added to the washed mesocarp. It was not possible to affirm that there was interaction of washed mesocarp with Fe and Cu due to precipitation derived from hydrolysis. However, Zn did not suffer hydrolysis and had low recoveries, indicating that there was an interaction with the solid. Furthermore, it can be seen that adding higher concentrations of Zn, better recoveries were obtained, probably due to saturation of the binding sites. For the studies of availability, it was used an in vitro simulated digestion procedure from U.S. Pharmacopeia. Fe and Zn were not extracted during digestion of the mesocarp, therefore these elements would not be available for absorption by the organism. However, about 120 mg L-1 of Cu was extracted from the mesocarp. Mesocarp digestions were performed with addition of Fe, Cu and Zn and only 48% of Fe, 65% of Cu and 75% of Zn were recovered. Digestions containing the same concentration of the elements were performed with additions of Ca and Mg. The presence of Ca decreased the recovery of Fe and Zn. For simulating a real situation, gastrointestinal digestion of milk, milk and mesocarp mixture and milk and lignin mixture were performed. Although the iron present in milk had low availability, when it was mixed with mesocarp it was observed an increase in the concentration of soluble Fe. Compounds present in milk or in mesocarp can increase the solubility of the elements. It was not possible to observe if there was any change in the availability of Cu present in milk in the presence of mesocarp, because the Cu concentration in the milk is very low. The fraction of soluble Zn in milk and mesocarp mixture remained the same, but the dialysable fraction was practically null. Lignin addition increases the milk elements extraction. However, these elements remain not dialyzed

"Cellulose nanocrystals, one of the most abundant materials in nature, have attracted great attention in the biomedical community due to qualities such as supreme mechanical properties, biodegradability, biocompatibility and low density. In this research, we are interested in developing a bio-inspired material-by-design approach for cellulose-based composites with tailored interfaces and programmed microstructures that could provide an outstanding strength-to-weight ratio. After a preliminary study on some of the existing biomaterials, we have focused our research on studying the nanostructure and nanomechanics of the bamboo fiber, a cellulose-based biocomposite, designed by nature with remarkable strength-to-weight ratio (higher than steel and concrete). We have utilized atomistic simulations to investigate the mechanical properties and mechanisms of interactions between cellulose nanofibrils and the bamboo fiber matrix which is an intertwined hemicellulose and lignin called lignin-carbohydrate complex (LCC). Our results suggest that the molecular origin of the rigidity of bamboo fibers comes from the carbon-carbon or carbon-oxygen covalent bonds in the main chain of cellulose. In the matrix of bamboo fiber, hemicellulose exhibits larger elastic modulus and glass transition temperature than lignin whereas lignin shows greater tendency to adhere to cellulose nanofibrils. Consequently, the role of hemicellulose is found to enhance the thermodynamic properties and transverse rigidity of the matrix by forming dense hydrogen bond networks, and lignin is found to provide the strength of bamboo fibers by creating strong van der Waals forces between nanofibrils and the matrix. Our results show that the amorphous region of cellulose nanofibrils is the weakest interface in bamboo microfibrils. We also found out that water molecules enhance the mechanical properties of lignin (up to 10%) by filling voids in the system and creating hydrogen bond bridges between polymer chains. For hemicellulose, however, the effect is always regressive due to the destructive effect of water molecules on the hydrogen bond in hemicellulose dense structure. Therefore, the porous structure of lignin supports the matrix to have higher rigidity in the presence of water molecules. "

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 145-148. / by Theodore Robert Nunn. / M.S.

O objetivo deste trabalho foi avaliar a disponibilidade de Fe, Cu e Zn em presença de mesocarpo de babaçu, pois o mesocarpo contem compostos antinutricionais (fitato e lignina) que podem diminuir a disponibilidade de nutrientes minerais. As concentrações de Fe, Cu e Zn encontradas no mesocarpo de babaçu foram 17, 7,0 e 2,6 µg g-1, respectivamente. Para um melhor entendimento das interações dos elementos com o mesocarpo, foi realizada a extração de substâncias complexantes com soluções de NaOH (pH 7 e 12). Análises por espectrometria de absorção molecular indicaram que o principal complexante presente no extrato era o fitato. Soluções de Fe, Cu e Zn (10 a 300 mg L-1) foram adicionadas aos extratos, para que, após agitação e centrifugação, fossem determinadas as recuperações desses elementos. Os resultados mostraram que os elementos formam complexos com o fitato e a solubilidade desses complexos é dependente da razão elemento/fitato, quanto maior essa razão menor é a solubilidade. Também foi avaliada a interação dos elementos com o sólido remanescente da extração. Fe, Cu ou Zn foram adicionados ao mesocarpo lavado. Não foi possível afirmar que houve interação do mesocarpo lavado com Fe e Cu devido à precipitação decorrente da hidrólise. Porém, o Zn não sofreu hidrólise e teve baixas recuperações, indicando que houve interação com o sólido. Além disso, pode ser observado que ao adicionar maiores concentrações de Zn obtiveram-se melhores recuperações, provavelmente devido à saturação dos sítios de ligação. Para os estudos de disponibilidade, foi utilizado o procedimento de digestão simulada in vitro da US Pharmacopeia. Fe e Zn não foram extraídos durante a digestão do mesocarpo, portanto esses elementos não estariam disponíveis para absorção pelo organismo. Entretanto, cerca de 120 µg L-1 de Cu foi extraído do mesocarpo. Foram realizadas digestões do mesocarpo com adição de Fe, Cu e Zn e apenas 48% do Fe, 65% do Cu e 75% do Zn foram recuperados. Digestões contendo essa mesma concentração dos elementos foram realizadas com adições de Ca e Mg. A presença do Ca diminuiu a recuperação de Fe e Zn. Para simular uma situação próxima ao real, foram realizadas digestões gastrointestinais de leite, mistura de leite e mesocarpo e mistura de leite e lignina. Apesar do ferro presente no leite apresentar baixa disponibilidade, quando o mesmo foi misturado ao mesocarpo observou-se um aumento da concentração de Fe solúvel. Compostos presentes no leite ou no mesocarpo podem aumentar a solubilidade dos elementos. Não foi possível observar se havia alguma alteração da disponibilidade do Cu presente no leite na presença do mesocarpo, pois a concentração de Cu no leite é muito baixa. A fração de Zn solúvel na mistura de leite e mesocarpo permaneceu a mesma, porém a fração dialisável foi praticamente nula. A adição de lignina ao leite aumentou a extração dos elementos. Entretanto, esses elementos continuaram não sendo dialisados / The aim of this study was to evaluate the availability of Fe, Cu and Zn in the presence of mesocarp of babassu because it contains antinutritional compounds (phytate and lignin) that may decrease the availability of mineral nutrients. The concentrations of Fe, Cu and Zn found in the mesocarp of babassu were 17, 7.0 and 2.6 mg g-1, respectively. For a better understanding of elements interactions with mesocarp, it was performed the extraction of complexing substances with NaOH solutions (pH 7 and 12). Analysis by molecular absorption spectrometry indicated that the main complexing present in the extract was the phytate. Solutions of Fe, Cu and Zn (10 to 300 mg L-1) were added to the extracts for determining the recoveries of these elements, after agitation and centrifugation. The results showed that the elements form complexes with phytate and the solubility of these complexes is dependent on the ratio element / phytate, the greater this ratio the lower the solubility. The elements interaction with the remaining solid from extraction was also evaluated. Fe, Cu or Zn were added to the washed mesocarp. It was not possible to affirm that there was interaction of washed mesocarp with Fe and Cu due to precipitation derived from hydrolysis. However, Zn did not suffer hydrolysis and had low recoveries, indicating that there was an interaction with the solid. Furthermore, it can be seen that adding higher concentrations of Zn, better recoveries were obtained, probably due to saturation of the binding sites. For the studies of availability, it was used an in vitro simulated digestion procedure from U.S. Pharmacopeia. Fe and Zn were not extracted during digestion of the mesocarp, therefore these elements would not be available for absorption by the organism. However, about 120 mg L-1 of Cu was extracted from the mesocarp. Mesocarp digestions were performed with addition of Fe, Cu and Zn and only 48% of Fe, 65% of Cu and 75% of Zn were recovered. Digestions containing the same concentration of the elements were performed with additions of Ca and Mg. The presence of Ca decreased the recovery of Fe and Zn. For simulating a real situation, gastrointestinal digestion of milk, milk and mesocarp mixture and milk and lignin mixture were performed. Although the iron present in milk had low availability, when it was mixed with mesocarp it was observed an increase in the concentration of soluble Fe. Compounds present in milk or in mesocarp can increase the solubility of the elements. It was not possible to observe if there was any change in the availability of Cu present in milk in the presence of mesocarp, because the Cu concentration in the milk is very low. The fraction of soluble Zn in milk and mesocarp mixture remained the same, but the dialysable fraction was practically null. Lignin addition increases the milk elements extraction. However, these elements remain not dialyzed

<p>Plant biomass is an abundant source of renewable energy, but the efficiency of its conversion into liquid fuels is low. One reason for this inefficiency is the recalcitrance of biomass to extraction and saccharification of cell wall polysaccharides. This recalcitrance is due to the complex and rigid structure of the plant cell wall. A better understanding of the genes effecting cell wall composition in bioenergy crops could improve feedstock quality and increase conversion efficiency. To identify genetic loci associated with biomass quality traits, we utilized genome-wide association studies (GWAS) in an 840-line <i>Sorghum</i> diversity panel. We identified several QTL from these GWAS including some for lignin composition and saccharification. Linkage disequilibrium (LD) analysis suggested that multiple polymorphisms are driving the association of SNPs within these QTL. Sequencing and further analysis led to the identification of a SNP within the coding region of a gene encoding phenylalanine ammonia-lyase (PAL) that creates a premature stop codon and co-segregates with an increase in the ratio of syringyl (S) to guaiacyl (G) lignin. A comparison of net PAL activity between lines with and without the mutation revealed that this mutation results in decreased PAL activity. </p>

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

La première partie de ce projet à consisté à isoler la lignine à partir de liqueurs industrielles et à la purifier pour s’affranchir des sucres, des minéraux et autres constituants. Pour cela, un protocole efficace en trois étapes a été proposé pour obtenir des échantillons de lignine avec une pureté satisfaisante (95%) et pour récupérer 68% de la lignine initialement présente dans la liqueur alcaline de départ. La lignine alcaline purifiée a ensuite été caractérisée d’un point de vue moléculaire et par analyses thermiques. L’élucidation de la structure de la lignine alcaline a permis d’appréhender sa fonctionnalisation par oxydation. Les analyses par chromatographie d’exclusion stérique de la lignine après oxydation ont montré une diminution de la masse molaire confirmant ainsi le clivage. Trois fractions différentes ont été isolées après l’oxydation de la lignine selon le solvant d’extraction ; d’une part des oligomères (plus ou moins fonctionnalisés) et d’autre part des molécules aromatiques (dont15% de vanilline). Ce travail de thèse aura abouti à la synthèse de composés aromatiques à haute valeur ajoutée (vanilline) et d’oligomères de lignine fonctionnalisés par des fonctions acide carboxylique. Les applications envisageables de ces « polyacides » issus de lignine sont nombreuses pour la formation de nouveaux polymères bio-sourcés tels que des polyesters, polyamides ou encore polyuréthanes. / A valorization of alkaline lignin from an industrial pulping liquor has been proposed for this project. Before considering any chemical modification or potential applications, the lignin structure has been elucidated. An efficient three-steps protocol for extraction and purification of lignin from industrial liquor has been established. This protocol leads to high purity sample of lignin (95%) and allows the recovery (68%) of the lignin initially present in the alkaline liquor. Alkaline lignin has been characterized utilizing analytical methods and thermogravimetric analysis. This precise structure elucidation was critical for proceeding to chemical modification of alkaline lignin. Chemical modification of alkaline lignin has been done by oxidation in alkaline media. Three major oxidized products have been isolated depending on the extraction solvent: oligomers bearing carboxylic groups and aromatic molecules. This thesis work led to the synthesis of value-added bio-sourced chemicals and functionalized oligomers. The polyacids from lignin obtained should be studied to form new biobased polymers such as polyesters, polyamids or polyurethanes.