No description available.

No description available.

It is widely known that fossil fuels are limited; consequently, the generation of new sources of energy in a clean and environmentally friendly manner is a research priority. Bioethanol appears to be one potential solution, especially second-generation production from renewable biomass. In order to use lignocellulosic feedstock to produce bioethanol, its polysaccharide components, cellulose and hemicellulose, must be hydrolysed into soluble sugars, which can then be converted into ethanol by fermentative microorganisms such as Geobacillus thermoglucosidasius TM242 used by the company ReBio Technologies Ltd. To date, the cost of commercial enzymes used during the hydrolysis process remains a major economic consideration in the production of second-generation bioethanol as an alternative fuel. The research project presented in this thesis aims to improve this rate-limiting step of microbial bioethanol production through an investigation of the different enzymes associated with hemicellulose hydrolysis. Firstly, the TM242 genome sequence revealed a number of genes encoding glycoside-hydrolases. Six of these genes were cloned and expressed in E. coli and the recombinant enzymes characterised; three of them, two β-xylosidases and an α arabinofuranosidase, are relevant to xylan hydrolysis, and were found to be highly active and thermostable. Crystallisation of one of the β-xylosidases permitted the determination of a high-resolution (1.7 Å) structure of the apo-enzyme along with a lower resolution (2.6 Å) structure of the enzyme-substrate complex, resulting in the first reported structure of a GH52 family member (Espina et al., 2014). Secondly, as the TM242 microorganism lacks xylanase enzymes, four genes encoding xylanases from closely-related Geobacillus strains were cloned and expressed in E. coli, with one of them being also successfully cloned and expressed in G. thermoglucosidasius TM242. This heterologous xylanase was secreted in active form representing an enhanced biomass utilisation by TM242. In conclusion, it is felt that the findings presented here have the potential to make a valuable contribution towards second-generation bioethanol production.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

>Magister Scientiae - MSc / The complete degradation of recalcitrant lignocellulose biomass into value-added products requires the efficient and synergistic action of lignocellulose degrading enzymes. This has resulted in a need for the discovery of new hydrolytic enzymes which are more effective than commonly used ones. β-xylosidases and β-glucosidases are key glycoside hydrolases (GHs) that catalyse the final hydrolytic steps of xylan and cellulose degradation, essential for the complete degradation of lignocellulose. Functional-based metagenomics has been employed successfully for the identification and discovery of novel GH genes from a metagenome library. Therefore, this approach was used in this study to increase the chances of discovering novel glycoside hydrolase genes from a horse manure metagenomic DNA library constructed in a previous study. Three fosmid clones P55E4, P81G1, and P89A4 exhibiting β-xylosidase activity were found to encode putative glycosyl hydrolases designated XylP55, XylP81, and BglP89. Amino acid sequence analysis revealed that XylP55, XylP81, and BglP89 are members of the GH43, GH39, and GH3 glycoside hydrolase families, respectively. Phylogenetic analysis of XylP81 and BglP89 indicated that these showed relatively low sequence similarities to other homologues in the respective GH families. The enzymes were expressed and purified, and only XylP81 and BglP89 were biochemically characterized. XylP81 (~58 kDa) and BglP89 (~84 kDa) both showed optimum activity at pH 6 and 50℃ and retained 100% residual activity at 55℃ after 1-hour indicating that they are moderately thermostable. XylP81 had high specific activity against 4-nitrophenyl-β-D-xylopyranoside (pNPX; 122 U/mg) with a KM value of 5.3 mM, kcat/KM of 20.3 s-1mM-1, and it showed enzyme activity against α-L-arabinofuranosidase, β-galactosidase, and β-glucosidase activity. BglP89 had a high specific activity for 4-nitrophenyl-β-D-glucopyranoside (pNPG; 133.5 U/mg) with a KM value of 8.4 mM, kcat/KM of 22 s-1mM-1 and also showed α-L-arabinofuranosidase, β-galactosidase, β-glucosidase, and low β-xylosidase activity. BglP89 also showed low hydrolytic activity on cellobiose, β-glucan, and lichenan indicating that it is a broad specificity β-glucosidase. XylP81 retained ~40% activity in the presence of 3 M xylose whilst BglP89 showed considerable glucose tolerance at 150 mM glucose and retained ~46% residual activity. This study reveals two metagenomic derived enzymes (β-xylosidase and β-glucosidase) showing characteristics that could make them potential candidates for lignocellulose biomass degradation in biotechnological and industrial applications.

No description available.

The world stocks of fossil fuels are dwindling and may be all but out before the end of the century. Despite this there is increasing demand for them to be used for transport, and the ever increasing green house gases which their use produces. Renewable and less environmentally damaging forms of fuel are needed. Biofuels, particularly bioethanol, are a possibility to subsidise or replace fossil fuels altogether. Ethanol produced from fermentation of starch sugars from corn are already in wide use. As this bioethanol is currently produced from crops such as corn and sugar cane, that puts fuel crops in direct competition for space and resources with food crops. This has led to increases in food prices and the search for more arable land. Hydrolysis of lignocellulosic biomass, a waste by-product of many industries, to produce the sugars necessary for ethanol production would ease many of the problems with current biofuels. Degradation of lignocellulose is not simple and requires expensive chemical pre-treatments and large quantities of enzymes usually from fungal species making it about 10 times more expensive to produce than corn starch bioethanol. The production of a consolidated bioprocessor, an organism able to degrade, metabolise and ferment cellulosic material to produce ethanol or other useful products would greatly reduce the cost currently associated with lignocellulosic biofuel. Cellulomonas fimi ATCC 484 is an actinomycete soil bacterium able to degrade efficiently cellulosic material. The US Department of Energy (DOE) released the genome sequence at the start of 2012. In this thesis the released genome has been searched, for genes annotated as encoding polysaccharide degrading enzymes as well as for metabolic pathways. Over 100 genes predicted to code for polysaccharide hydrolysing enzymes were identified. Fifteen of these genes have been cloned as BioBricks, the standard synthetic biology functional unit, expressed in E. coli and C. freundii and assayed for endo β-1,4-glucanase activity using RBB-CMC, endo β-1,4-xylanase activity using RBB-xylan, β-D-xylosidase activity using ONPX, β-D-cellobiohydrolase activity using ONPC and α-L-arabinofuranosidase activity using PNPA. Eleven enzymes not previously reported from C. fimi were identified as active on a substrate with the strongest activities being for 2 arabinofuranosidases (AfsA+B), 4 β-xylosidases (BxyC, BxyF, CelE and XynH), an endoglucanase (CelA), and 2 multifunctional enzymes CelD and XynF, active as cellobiohydrolases, xylosidases and endoxylanases. Four enzymes were purified from E. coli cell lysates and characterised. It was found that AfsB has an optimum activity at pH 6.5 and 45ºC, BxyF has optimum activity at pH 6.0 and 45ºC and XynH has optimum activity at pH 9.0 and 80ºC. XynF exhibited different optima for the 3 substrates with pH 6.0 and 60ºC for ONPC, pH 4.5 and 50ºC for ONPX and pH 5.5 and 40ºC for RBB-xylan. Searching the genome and screening genes for activities will help genome annotation in the future by increasing the number of positively annotated genes in the databases. The BioBrick format is well suited for rapid cloning and expression of genes to be classified. Searching and screening the genome has also given insights into the complex and large network of enzymes required to fully hydrolyse and metabolise the sugars released from lignocellulose. These enzymes are spread across many different glycosyl hydrolase families conferring different catalytic activities. The characterisation of these novel enzymes points towards a system adapted to not only a broad specificity of substrate but also environmental factors such as high temperature and pH. Genomic analysis revealed gene clusters and traits which could be used in the design of a synthetic cellulolytic network, or for the conversion of C. fimi into a consolidated bioprocessor itself.

O cultivo da cana-de-açúcar é uma das atividades agrícolas mais importantes no Brasil, produzindo após a moagem o caldo, utilizado para a produção de açúcar e etanol, e o bagaço, resíduo lignocelulósico. O bagaço é recalcitrante à hidrólise enzimática, em parte pela baixa porosidade, resultante do recobrimento das fibrilas de celulose com lignina e hemicelulose. Neste estudo, o bagaço foi pré-tratado com sulfito alcalino nas concentrações de 2,5% de NaOH e 5% de Na2SO3 versus 5% de NaOH e 10% de Na2SO3 para produzir substratos para hidrólise enzimática. Ambos pré-tratamentos produziram substratos com teor de hemicelulose, grupos ácidos, grau de retenção de água e área superficial semelhantes. O conteúdo de lignina foi bem diferente nos bagaços pré-tratados com 5% de sulfito (21% de lignina) e 10% de sulfito (13% de lignina). A hidrólise da celulose e hemicelulose do bagaço com alto teor de lignina, utilizando a carga enzimática de 40 FPU/g e 80 U/g de ?-glicosidase foram próximas a 50% em 48 horas e, mesmo no bagaço com pouca lignina a conversão dos polissacarídeos não foi completa (90%). Considerando a importância do tipo de enzimas para a conversão dos polissacarídeos dos bagaços pré-tratados, realizou-se um planejamento experimental 24 com 6 pontos centrais ampliado em estrela, para avaliar uma mistura de enzimas partindo de 5 FPU/g do extrato comercial de Trichoderma reesei (celluclast) combinado com enzimas purificadas comerciais: xilanase de Neocallimastix patriciarum (família 10), xilanase de Thermotoga maritima (família 11), ?-xilosidase de Selelomonas ruminantium e ?-glicosidase de Aspergillus niger. A aplicação da mistura de enzimas otimizada no bagaço pré-tratado com alta carga de sulfito aumentou a conversão de celulose e hemicelulose em 6,6% e 15% respectivamente, comparado com a mistura de referência (5FPU de celluclast e 10UI de Novozyme 188 por grama de bagaço). A suplementação da celluclast com ?-xilosidase e ?- glicosidase foi estatisticamente significativa em 24 horas de hidrólise a um nível de 95% de confiança e a interação da xilanase 10 e 11 foi significativa com um nível de confiança de 90%. Quando foram realizados os mesmos ensaios do planejamento com o substrato com alto teor de lignina, as hidrólises da celulose e hemicelulose com a mistura de enzimas foram inferiores à obtida com a referência. A suplementação com xilanases e ?-xilosidase aumentou a conversão enzimática da hemicelulose apenas do substrato com pouca lignina, entretanto nos hidrolisados de ambos os substratos foi detectada a presença de xilooligossacarídeos, indicando a necessidade de adição de mais ?-xilosidase à mistura enzimática. As velocidades iniciais de hidrólise da celulose e hemicelulose foram pouco alteradas quando a lignina do bagaço reduziu de 21% para 13%, porém a conversão em 48 h de reação foi o dobro. Este estudo mostrou que o acesso das enzimas à hemicelulose foi limitado pelo alto teor de lignina do substrato, e que o benefício do uso de xilanases para a conversão de celulose foi obtido no substrato pré-tratado com alta carga de sulfito. / The cultivation of sugarcane is one of the most important agricultural activities in Brazil. The juice obtained from the crushed stalks of sugarcane is used to produce sugar and ethanol and the dry, fibrous residue remaining is the bagasse. Bagasse is recalcitrant to enzymatic hydrolysis, in part by low porosity due to the partial filling of space between the cellulose microfibrils by lignin and hemicelluloses. In this study, bagasses were pretreated with alkaline sulfite at concentrations of 2.5% NaOH and 5% Na2SO3 NaOH versus 5% and 10% Na2SO3 to produce substrates for enzymatic hydrolysis. Both substrates presented similar hemicellulose content, acid groups, water retention and specific surface area. Lignin content differed between pretreated bagasse with 5% sulfite (21%) and 10% sulfite (13%). The hydrolysis of cellulose and hemicellulose of bagasse with high lignin content, using 40 FPU/g and 80 U/g of ?-glucosidase was aproximately 50% in 48 hours and even on bagasse with low lignin content, the polysaccharides conversion was not complete (90%). Considering the importance of the type of enzymes for the conversion of polysaccharides of pretreated bagasses, a 24 full factorial experimental design with six central points was performed to evaluate a mixture of enzymes. A load of 5 FPU/g of Trichoderma reesei extract (celluclast) was combined with purified commercial enzymes: Neocallimastix patriciarum xylanase (family 10), Thermotoga maritima xylanase (family 11), Selelomonas ruminantium ?-xylosidase and ?-glucosidase from Aspergillus niger. The optimized mixture improved the conversion of cellulose and hemicellulose of the substrate with low lignin content in 6.6% and 15% respectively, when compared to the reference mixture (5FPU of celluclast and 10 IU of novozyme 188 per gram of bagasse). Supplementation with ?-xylosidase and ?-glucosidase was statistically significant at 24 hours of reaction and also the interaction of xylanases 10 and 11. When the same assays were performed with the substrate with low lignin, hydrolysis of the cellulose and hemicellulose with a mixture of purified enzymes was inferior to that obtained by the reference. Supplementation with xylanase and ?-xylosidase improved the enzymatic conversion only for substrate with low lignin content, however in supernatants of both substrates was detected the presence of xylo-oligosaccharides, suggesting the need for further addition of ?-xylosidase to the enzyme mixture. Initial rate of cellulose and hemicellulose hydrolysis changed very little when the lignin in the bagasse was reduced from 21% to 13%, but the conversion at 48 h conversion time was twice higher. This study showed that access of enzymes to the hemicellulose was limited by the high lignin content of the substrate, and that the benefit of using xylanases for the conversion of cellulose was obtained on the substrate pretreated with high sulfite load.

O cultivo da cana-de-açúcar é uma das atividades agrícolas mais importantes no Brasil, produzindo após a moagem o caldo, utilizado para a produção de açúcar e etanol, e o bagaço, resíduo lignocelulósico. O bagaço é recalcitrante à hidrólise enzimática, em parte pela baixa porosidade, resultante do recobrimento das fibrilas de celulose com lignina e hemicelulose. Neste estudo, o bagaço foi pré-tratado com sulfito alcalino nas concentrações de 2,5% de NaOH e 5% de Na2SO3 versus 5% de NaOH e 10% de Na2SO3 para produzir substratos para hidrólise enzimática. Ambos pré-tratamentos produziram substratos com teor de hemicelulose, grupos ácidos, grau de retenção de água e área superficial semelhantes. O conteúdo de lignina foi bem diferente nos bagaços pré-tratados com 5% de sulfito (21% de lignina) e 10% de sulfito (13% de lignina). A hidrólise da celulose e hemicelulose do bagaço com alto teor de lignina, utilizando a carga enzimática de 40 FPU/g e 80 U/g de ?-glicosidase foram próximas a 50% em 48 horas e, mesmo no bagaço com pouca lignina a conversão dos polissacarídeos não foi completa (90%). Considerando a importância do tipo de enzimas para a conversão dos polissacarídeos dos bagaços pré-tratados, realizou-se um planejamento experimental 24 com 6 pontos centrais ampliado em estrela, para avaliar uma mistura de enzimas partindo de 5 FPU/g do extrato comercial de Trichoderma reesei (celluclast) combinado com enzimas purificadas comerciais: xilanase de Neocallimastix patriciarum (família 10), xilanase de Thermotoga maritima (família 11), ?-xilosidase de Selelomonas ruminantium e ?-glicosidase de Aspergillus niger. A aplicação da mistura de enzimas otimizada no bagaço pré-tratado com alta carga de sulfito aumentou a conversão de celulose e hemicelulose em 6,6% e 15% respectivamente, comparado com a mistura de referência (5FPU de celluclast e 10UI de Novozyme 188 por grama de bagaço). A suplementação da celluclast com ?-xilosidase e ?- glicosidase foi estatisticamente significativa em 24 horas de hidrólise a um nível de 95% de confiança e a interação da xilanase 10 e 11 foi significativa com um nível de confiança de 90%. Quando foram realizados os mesmos ensaios do planejamento com o substrato com alto teor de lignina, as hidrólises da celulose e hemicelulose com a mistura de enzimas foram inferiores à obtida com a referência. A suplementação com xilanases e ?-xilosidase aumentou a conversão enzimática da hemicelulose apenas do substrato com pouca lignina, entretanto nos hidrolisados de ambos os substratos foi detectada a presença de xilooligossacarídeos, indicando a necessidade de adição de mais ?-xilosidase à mistura enzimática. As velocidades iniciais de hidrólise da celulose e hemicelulose foram pouco alteradas quando a lignina do bagaço reduziu de 21% para 13%, porém a conversão em 48 h de reação foi o dobro. Este estudo mostrou que o acesso das enzimas à hemicelulose foi limitado pelo alto teor de lignina do substrato, e que o benefício do uso de xilanases para a conversão de celulose foi obtido no substrato pré-tratado com alta carga de sulfito. / The cultivation of sugarcane is one of the most important agricultural activities in Brazil. The juice obtained from the crushed stalks of sugarcane is used to produce sugar and ethanol and the dry, fibrous residue remaining is the bagasse. Bagasse is recalcitrant to enzymatic hydrolysis, in part by low porosity due to the partial filling of space between the cellulose microfibrils by lignin and hemicelluloses. In this study, bagasses were pretreated with alkaline sulfite at concentrations of 2.5% NaOH and 5% Na2SO3 NaOH versus 5% and 10% Na2SO3 to produce substrates for enzymatic hydrolysis. Both substrates presented similar hemicellulose content, acid groups, water retention and specific surface area. Lignin content differed between pretreated bagasse with 5% sulfite (21%) and 10% sulfite (13%). The hydrolysis of cellulose and hemicellulose of bagasse with high lignin content, using 40 FPU/g and 80 U/g of ?-glucosidase was aproximately 50% in 48 hours and even on bagasse with low lignin content, the polysaccharides conversion was not complete (90%). Considering the importance of the type of enzymes for the conversion of polysaccharides of pretreated bagasses, a 24 full factorial experimental design with six central points was performed to evaluate a mixture of enzymes. A load of 5 FPU/g of Trichoderma reesei extract (celluclast) was combined with purified commercial enzymes: Neocallimastix patriciarum xylanase (family 10), Thermotoga maritima xylanase (family 11), Selelomonas ruminantium ?-xylosidase and ?-glucosidase from Aspergillus niger. The optimized mixture improved the conversion of cellulose and hemicellulose of the substrate with low lignin content in 6.6% and 15% respectively, when compared to the reference mixture (5FPU of celluclast and 10 IU of novozyme 188 per gram of bagasse). Supplementation with ?-xylosidase and ?-glucosidase was statistically significant at 24 hours of reaction and also the interaction of xylanases 10 and 11. When the same assays were performed with the substrate with low lignin, hydrolysis of the cellulose and hemicellulose with a mixture of purified enzymes was inferior to that obtained by the reference. Supplementation with xylanase and ?-xylosidase improved the enzymatic conversion only for substrate with low lignin content, however in supernatants of both substrates was detected the presence of xylo-oligosaccharides, suggesting the need for further addition of ?-xylosidase to the enzyme mixture. Initial rate of cellulose and hemicellulose hydrolysis changed very little when the lignin in the bagasse was reduced from 21% to 13%, but the conversion at 48 h conversion time was twice higher. This study showed that access of enzymes to the hemicellulose was limited by the high lignin content of the substrate, and that the benefit of using xylanases for the conversion of cellulose was obtained on the substrate pretreated with high sulfite load.