Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-10-01T16:43:18Z No. of bitstreams: 1 Tese Carla Eliana Todero Ritter.pdf: 192233 bytes, checksum: 7751f00ffe27681835ffe3c324f029fa (MD5) / Made available in DSpace on 2015-10-01T16:43:18Z (GMT). No. of bitstreams: 1 Tese Carla Eliana Todero Ritter.pdf: 192233 bytes, checksum: 7751f00ffe27681835ffe3c324f029fa (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES.

As celulases e as xilanases são enzimas que hidrolisam a celulose e a xilana, respectivamente, contidas nos resíduos lignocelulósicos. A possibilidade de aplicar estas enzimas na produção de etanol vem sendo objeto de diversos estudos. No entanto, ainda não há uma tecnologia economicamente viável para a produção deste biocombustível a partir da biomassa lignocelulósica. Entre os microrganismos que apresentam altos títulos para estas enzimas, incluem-se linhagens de Penicillium echinulatum; porém, ainda faltam dados de sua fisiologia e estudos da produção de enzimas em biorreator. Neste trabalho, empregou-se a linhagem mutante celulolítica desreprimida S1M29 de P. echinulatum e o meio de cultivo foi composto por celulose, sacarose, solução de sais, Tween 80, farelo trigo e farelo de soja. Avaliou-se o efeito de diferentes temperaturas e pHs na produção das enzimas. O efeito da concentração da celulose sobre as atividades enzimáticas foi avaliada em regime descontínuo (RD) e regime descontínuo alimentado (RDA). Verificou-se que a temperatura mais apropriada para a produção de celulases e xilanases é de 28ºC e dentre os valores de pHs avaliados, o pH 6,0 apresentou a maior produção das enzimas. O aumento da concentração da celulose no RD proporcionou maiores atividades para endoglicanases, porém o mesmo não foi obtido para xilanases. Para FPA (Filter Paper Activity), aumentos proporcionais nas atividades foram obtidos somente com concentrações de até 3% de celulose em RD, condição que também proporcionou as maiores atividades de - glicosidases. O RDA incrementou as atividades de FPA, endoglicanases e xilanases, mas não de -glicosidases. Estes resultados contribuem para a otimização de processos e para a produção econômica de enzimas por P. echinulatum, visando o desenvolvimento de tecnologias economicamente viáveis para produção de etanol a partir de materiais lignocelulósicos. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cellulases and xylanases are enzymes that hydrolyze cellulose and xylan respectively, which are found in lignocellulosic residues. Although the applicability of these enzymes in the ethanol production has been the subject of several studies, an economically viable technology for the production of biofuel from lignocellulosic biomass is currently not available. Strains of Penicillium echinulatum are among the microorganisms that have high titers of these enzymes. However, data related to physiology and enzyme production in bioreactor for such strains are still missing. A cellulolytic mutant strain of P. echinulatum S1M29 and a culture medium composed of cellulose, sucrose, salt solution, Tween 80, wheat bran and soybean meal were used in this study. The effect of different temperatures and pHs during the enzymes production was evaluated. The effect of cellulose concentration in the enzymatic activity was evaluated in batch cultivation (BC) and fed-batch cultivation (FBC). It was found that the appropriate temperature for the production of cellulases and xylanases is 28°C, while the higher enzyme production occurred at pH 6.0. The high cellulose concentration in BC provided the greatest activities for endoglicanases, but the same result was not obtained for xylanases. For Filter Paper Activity (FPA), proportional increases in activity were obtained only with concentrations up to 3% of cellulose in BC, which is also linked to the highest activities for -glucosidases. FBC increased the activities of FPA, endoglucanases and xylanases, but it did not increase the -glucosidases activities. Such results contribute towards the optimization of enzyme production using P. echinulatum and the development of economically viable technologies for the production of ethanol from lignocellulosic materials.

A produção de enzimas do complexo celulases e de xilanases se destaca no cenário mundial, pois estas são importantes para a geração de biocombustíveis, resinas, álcoois, xilitol, sorbitol, e para a indústria de detergentes e ração animal e processos de biobranqueamento, extração de óleos, pigmentos e essências a partir de biomassa lignocelulósica. A secreção e produção destas enzimas são afetadas pela repressão catabólica, devido à presença da glicose liberada. O objetivo do presente estudo foi verificar o efeito sobre a produção enzimática de Penicillium echinulatum de metabólitos e açúcares durante o cultivo submerso. As estratégias utilizadas para a remoção de substâncias do meio de cultivo incluíram filtração e uso de pellets com adsorventes e uso de filmes adsorventes. O preparo de membranas de suporte cerâmico e polimérico, impregnadas com os polímeros poli(fluoreto de vinilideno) (PVDF) e poliamida (PA), foi realizado por inversão de fases, sendo que a membrana cerâmica impregnada com solução 10% (m/v) de PVDF (C1) reteve 88% da proteínas totais da solução enzimática. A remoção de 10 ou 20% do permeado durante a filtração com membranas planas, em períodos de tempo entre 8h e 24h, resultou em aumento da atividade de β- glicosidase e endoglicanase em frascos Erlenmeyer. Com relação à atividade de xilanases, os títulos observados no 4º dia foram entre 40% e 30% superiores quando houve a filtração em 42 e 44 h, respectivamente. Quando adsorventes foram utilizados em biorreator, a atividade de endoglicanase, CBH e xilanase apresentaram um aumento de 50%, 43% e 12%, respectivamente, em meio concentrado utilizando carvão ativado quando comparado ao padrão. Os ensaios com membranas de nanofiltração permitiram retenção de 95% das enzimas do complexo e, quando utilizadas na remoção de 10% e 20% da fase líquida em frascos, resultaram em aumento na atividade enzimática. Os ensaios 18/20, 24/10, 24/20, 44/10 e 44/20 (tempo de processo, em horas, em que foi feita a filtração / volume percentual de permeado removido) apresentaram, em média, em 72 h, um incremento de 33% na atividade de endoglicanase, enquanto o ensaio 44/20, em 96h, apresentou atividade superior a 40% do padrão. Para os cultivos com Trichoderma reesei Rut C30, os ensaios com filtração de 10 % do volume dos frascos, em 12 h e 20 h apresentaram aumento da atividade de FPA, em 60 h de cultivo, de 15% e 11%, respectivamente. Nos mesmos ensaios, a atividade de endoglicanase apresentou incremento de 40,7% e 52,2%, respectivamente, demonstrando o efeito positivo da remoção de 10% de permeado sobre a produção enzimática. Já para FPA, o aumento representou 15% e 11 % para as mesmas condições / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Cellulases and xylanases enzymes complex production stands out in the world because these enzymes are important for the generation of biofuels, resins, alcohols, xylitol, sorbitol, and for the detergents industry, animal feed, pulp bleaching, and in the extraction of oils, pigments and essences from lignocellulose biomass. Secretion and production of cellulases and xylanases are affected under catabolic repression conditions due to glucose release. The purpose of the present study was to verify the effect on enzymatic production of Penicillium echinulatum compared to metabolites and sugars during submerged cultivation. The strategies used for the substances removal from the medium included filtration application, the use of pellets with adsorbents, and the use of adsorbents films. Membranes preparation from both ceramic and polymeric supports impregnated with polymers PVDF and PA were performed by phase inversion method, and eventually the ceramic membrane impregnated with solution 10% (w/v) of PVDF (C1) held 88% of the total proteins from the enzymatic solution. The removal consisting in 10% or 20% from the permeate during filtration with flat membranes, in periods between 8h and 24h resulted in a rise in the activity regarding β-glucosidase and endoglucanase in Erlenmeyer flasks. In relation to xylanases activity, the titles observed on the fourth day were between 40% and 30% superiors when filtration was made in 42 and 44h, respectively. Subsequently, when adsorbents were used in the bioreactor, activity involving endoglucanase, CBH and xylonase increased 50%, 43% and 12% respectively, in concentration applying activated charcoal comparable to standard source. Moreover, tests performed with nanofiltration membranes allowed 95% retention of the enzymes from the complex, and when they were used in the removal consisted of 10% and 20% in liquid phase in flasks, results showed a rise on the enzymatic activity. The tests 18/20, 24/10, 24/20, 44/10 and 44/20 ( in relation to filtration day and volume permeated) presented, on average, in 72 hours, a 33% increment in the endoglucanase activity and, in 96 hours, the test 44/20 presented activity superior to 40% from standard value. Therefore, for tests with Trichoderma reesei Rut C30, filtration experiments with 10% of the volume of the flasks in 12h and 20 h presented an increase in FPA activity, and in 60 h, the amount found was 15% and 11%, respectively. Subsequently, for endoglucanase enzyme, the activity presented increment levels evaluated in 40,7% and 52, 2% respectively for the mentioned tests, demonstrating the positive factor of the permeate 10% removal from the enzymatic production. Lastly, for FPA the rise represented 15% and 11% in the same test conditions.

Orientador: Anete Pereira de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T01:20:36Z (GMT). No. of bitstreams: 1 Schneider_DilaineRoseSilva_D.pdf: 11263694 bytes, checksum: 808a3a965f6f47fb9c2108a4c971b122 (MD5) Previous issue date: 2010 / Resumo: A brusone do arroz (rice blast disease) causada pelo ascomiceto fitopatógeno Magnaporthe grisea continua a ter um enorme impacto nas culturas de arroz (Oryza sativa) no Brasil e no mundo. PWL2, uma proteína efetora, é um conhecido produto de um gene AVR (avirulência). O gene PWL2 impede que o fungo infecte weeping lovegrass (Eragrostis curvula). Neste trabalho nós identificamos em uma linhagem de M. grisea um gene que produz uma proteína diferente de PWL2, denominada PWL2D. A seqüência do gene PWL2D tem duas bases que diferem do gene PWL2, as quais produzem alterações nos resíduos de 90 e 142 da proteína. A alteração do resíduo 90 (de D90 para N90) é fundamental para a avirulência. Neste trabalho foram efetuadas a clonagem do gene PWL2D no vetor pET32-Xa/LIC, a expressão em Escherichia coli e a avaliação da estrutura de PWL2D por técnicas espectroscópicas. A proteína PWL2D fusionada à cauda TRX é propensa a agregação, e sua solubilidade é melhorada quando super-expressa sem o seu peptídeo-sinal original. Os resultados estruturais obtidos indicam que a proteína PWL2D possivelmente é intrinsecamente desordenada. Foi elaborado um modelo para a resistência/susceptibilidade do hospedeiro à M. grisea considerando a atuação de PWL2D como uma proteína intrinsecamente desordenada. Os resultados obtidos deverão facilitar a análise estrutural de PWL2D e podem contribuir para a compreensão da função do gene nas interações fungo / planta. Oito diferentes genes de M. grisea, além de PWL2D, foram também estudados neste trabalho. Dentre estes, destacam-se o gene que produz a xilanase XYL5 e seu domínio catalítico, o gene que codifica a chaperona ABC1 e seus dois domínios funcionais, e o gene que codifica a trealase PTH9, sendo todos estes relacionados à patogenicidade do fungo M. grisea. A xilanase XYL5 (EC 3.2.1.8) e seu domínio catalítico conservado (XYL5/DOM) foram fusionados à Maltose Binding Protein (MBP) ou à tiorredoxina (TRX) e expressas em E. coli. A produção de proteína solúvel e ativa foi influenciada pelo tipo de fusão. Os extratos solúveis contendo as proteínas de fusão MBP-XYL5 e MBP-XYL5/DOM apresentaram atividade xilanolítica em relação ao controle. Entretanto, durante o processo de purificação, a atividade foi perdida. Assim, obteve-se pela primeira vez o gene de patogenicidade XYL5 de M. grisea expresso com sucesso em E. coli e sua atividade enzimática xilanolítica foi demonstrada. Não foi possível expressar a chaperona ABC1 na forma solúvel nos sistemas de expressão utilizados, e a sequência gênica referente à trealase PTH9 - por mostrar a presença de introns após o seqüenciamento do gene amplificado na linhagem de M. grisea em estudo, mostrou-se inadequado para a sua expressão protéica no sistema de expressão procariótico utilizado durante a realização deste trabalho / Abstract: The rice blast disease caused by the ascomycete phytopathogen Magnaporthe grisea continues to have a huge impact on crops of rice (Oryza sativa) in Brazil and worldwide. PWL2, an effector protein, is a product of an AVR (avirulence) gene . The gene PWL2 prevents fungus from infecting weeping lovegrass (Eragrostis curvula). In this work we identified in a strain of M. grisea a gene that produces a protein different from PWL2, called PWL2D. The gene sequence PWL2D has two bases that differ from PWL2 gene, which produce changes in residues 90 and 142 of the protein. The change of residue 90 (from D90 to N90) is critical to avirulence. In this work it was realized the cloning of the gene in the vector PWL2D pET32-Xa/LIC, the expression in Escherichia coli and the assessment of PWL2D structure by spectroscopic techniques. The protein fused to the tag PWL2D TRX is prone to aggregation, and its solubility is improved when overexpressed without its original signal peptide. The structural results obtained indicate that possibly the protein PWL2D is intrinsically disordered. A model for the resistance/susceptibility of the host to M. grisea was developed considering the performance of PWL2D as an intrinsically disordered protein. The results should facilitate structural analysis of PWL2D and may contribute to the understanding of gene function in the interactions fungus/plant. Eight different genes of M. grisea, besides PWL2D, were also studied in this work. Among these, stands out the gene that produces xylanase XYL5 and its catalytic domain, the gene that codify the chaperone ABC1 and its two functional domains, and the gene that codify the trehalase PTH9, all them being related to the pathogenicity of the fungus M. grisea. The xylanase XYL5 (EC 3.2.1.8) and its retained catalytic domain (XYL5/DOM) were fused to the solubilizing proteins (MBP) or thioredoxin (TRX) and expressed into E. coli. The production of soluble and active protein was influenced by the type of fusion. The soluble extracts containing the fusion proteins MBP- XYL5 and MBP-XYL5/DOM showed xylanolytic activity compared to the control. However, during the purification process, the activity was lost. Thus, we obtained for the first time the gene pathogenicity XYL5 M. grisea expressed successfully in E. coli and its enzymatic xylanolytic activity was demonstrated. It was not possible to express the chaperone ABC1 in soluble form in the expression systems used, and the gene sequence related to trehalase PTH9 - by showing the presence of introns after the sequencing of the gene amplified in the strain of M. grisea under study, rendered inadequate for its protein expression in the prokaryotic system used during the realization of this work / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Orientador: Francisco Maugeri Filho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T18:37:14Z (GMT). No. of bitstreams: 1 Motta_FernandaLopes_M.pdf: 741375 bytes, checksum: 2dd5dcca312294d6118957b0bf359271 (MD5) Previous issue date: 2010 / Resumo: O interesse no estudo dos sistemas das xilanases vem sendo estimulado pela sua utilidade em uma variedade de processos biotecnológicos. Estas enzimas podem ser aplicadas em indústrias de panificação, melhorando a textura, o volume da massa e o tempo de prateleira dos produtos; em vinícolas e cervejarias, favorecendo a etapa de filtração e a clarificação, além de ser adicionada à ração animal de aves e suínos para aumentar a digestibilidade. Diante da importância da aplicação das xilanases e da necessidade desenvolver condições que otimizem a produção dessas enzimas a fim de tornar sua utilização comercial menos restrita, estudos têm sido realizados visando obter maior produtividade e substratos de baixo custo. O objetivo deste trabalho foi otimizar a produção de xilanase a partir de uma cepa de levedura silvestre isolada da Mata Atlântica utilizada em estudo anterior, indicando potencial para produção de uma enzima estável. Duas cepas foram testadas inicialmente a fim de selecionar a mais adequada para este estudo. Uma vez selecionada, a cepa foi utilizada para testar a influência do tampão fosfato sobre a produção de xilanase e os resultados mostraram que a adição de tampão fosfato ao meio de cultura exerce um efeito negativo tanto em relação à biomassa da levedura quanto à produção e atividade da enzima. A partir desses resultados optou-se pela exclusão do tampão do meio e foi aplicado um planejamento experimental variando as condições de fermentação visando a otimização da produção da xilanase. Primeiramente, realizou-se um delineamento Plackett & Burman (PB) para determinar os efeitos principais das sete variáveis estudadas em relação à atividade enzimática, onde a temperatura e a concentração de xilanase foram consideradas estatisticamente significativas. Em seguida, realizou-se um Delineamento Composto Central Rotacional (DCCR) de 11 ensaios onde as variáveis independentes foram aquelas consideradas significativas pelo PB. Os resultados obtidos pelos DCCR foram analisados por ferramentas estatísticas que confirmaram a confiabilidade do modelo preditivo para a atividade da enzima xilanase. De acordo com este modelo, a máxima atividade enzimática (67,45 UI) foi encontrada para os pontos centrais do planejamento, onde a concentração de xilana foi de 20 g.L-1 e a temperatura de 30°C / Abstract: The interest in the study of systems of xylanases has been stimulated by its usefulness in a variety of biotechnological processes. These enzymes could be applied to bakery, improving texture, volume and mass of the shelf life of products, wineries and breweries, supporting the step of filtration and clarification; also it could be added in poultry and pigs feed to increase digestibility. Due to this importance of application of xylanases and the necessity to develop conditions that optimize the production of these enzymes in order to make its commercial use less restricted, studies have been done to achieve higher productivity with low cost substrates. The objective of this work was to optimize the production of xylanase from a wild yeast strain isolated from Mata Atlântica. Two strains were tested initially in order to select the most appropriated one for this study. Once selected, it was tested the influence of phosphate buffer on the production of xylanase and the results showed that the addition of phosphate buffer to the culture medium has a negative effect on yeast biomass and activity enzyme produced. From these results it was decided to exclude the buffer used in medium and it was applied an experimental design aimed to optimize the conditions for xylanase production. At first, a design Plackett & Burman (PB) was done to determine the main effects of each variable studied in relation to enzymatic activity, where the temperature and the concentration of xylanase were considered statistically significant. Then, a central composite rotational design (DCCR) of 11 trials was carried out, where the independent variables were those considered significant by the PB. The results obtained by DCCR were analyzed by statistical tools that confirmed the reliability of the predict model to the xylanse activity enzyme. According to this model, the maximum enzyme activity (67,45 UI) was found at the central points of planning, where the concentration of xylan was 20 gL-1 and temperature of 30 ° C / Mestrado / Mestre em Engenharia de Alimentos

As xilanases (EC 3.2.1.8), enzimas produzidas por diversos organismos, são capazes de hidrolisar as ligações -1,4 da cadeia principal da xilana, o mais abundante polissacarídeo hemicelulósico da natureza. O grande potencial biotecnológico das xilanases consiste na sua aplicação nas etapas de branqueamento do papel, nas quais a xilana é hidrolisada sob condição de temperatura elevada para facilitar a remoção da lignina (substância responsável pela coloração), diminuindo a quantidade de compostos clorados utilizados nestas etapas. A termoestabilidade e a especificidade pela xilana são as propriedades responsáveis pelo grande interesse biotecnológico e comercial que as xilanases têm atraído. As xilanases mesofílica, XBC, de temperatura ótima 55ºC (produzida pela bactéria Bacillus circulans) e termofílica, XTL, de temperatura ótima 70ºC (produxida pelo fungo Thermomyces lanuginosus) foram estudadas comparativamente por simulação de dinâmica. Os sistemas foram modelados pelo campo de força GROMOS-96(43A1) e as simulações realizadas pelo programa GROMACS 3.2. O objetivo do trabalho é relacionar as diferenças estruturais, energéticas e dinâmicas com as diferentes termostabilidades exibidas por estas enzimas. Os estudos por simulação sugerem claramente a existência de dois grandes tipos de regiões nas enzimas xilanases XBC e XTL: uma conservada e de grande estabilidade, que é o domínio palma, e a outra que pode sofrer grande movimentação, no caso o domínio polegar. Uma movimentação do tipo abre-fecha de dobradiça foi identificada. O monitoramento das ligações de hidrogênio inter/intramoleculares e pontes salinas ao longo do tempo e em função da temperatura permitem explicar clara e detalhadamente as diferentes termoestabilidades exibidas por duas proteínas da mesma família que compartilham de uma estrutura tridimensional altamente semelhante. Foi possível identificar 14 resíduos carregados que estão presentes na XTL e ausentes na XBC, tais resíduos devem ser considerados sítios potenciais de mutação na XBC. De uma maneira geral, tanto na XBC quanto na XTL, a presença do substrato não altera as características de cada domínio/região mas confere estabilidade para o domínio polegar. Nenhuma diferença clara na afinidade pelo substrato foi detectada pelas interações intermoleculares proteína-substrato. / The enzymes xylanases (EC 3.2.1.8) are produced by several microorganisms and used to hydrolyze the -1,4 bonds of the xylan main chain, the most abundant hemicellulose in nature. The great biotechnological potential of the xylanases is due to its application in the pulp-bleaching processes when the xylan is hydrolyzed under high temperature condition to optimize the lignin removal. This procedure presents the advantage to reduce the amount of chlorine chemicals used in the pulp-bleaching process. The required properties of a biotechnologically useful xylanase include thermostability and high affinity for xylan. The mesophilic, XBC, (from Bacillus circulans) and thermophilic, XTL, (from Thermomyces lanuginosus) xylanases were studied by molecular dynamics simulations. The primary structures of these enzymes are almost completely different while the tertiary structures are identical. The objective of the study is to get some insight on the factors that are responsible for the xylanase thermostability. The systems were modeled by the GROMOS96-(43A1) force field and the molecular dynamic simulations were performed by the GROMACS 3.2 package in the temperature range from 25 to 80ºC. The results obtained with both xylanases were compared. The existence of two kinds of regions was identified in XBC and XTL: the first one conserved and highly stable is formed by the so-called palm and fingers domains. The second region exhibits large movements: this is the thumb domain. A kind of open-close motion was identified that maybe can facilitate the access of the xylane to the active center. The inter/intramolecular hydrogen bonds and salt bridges allow to explain at great length the thermostability differences between the two enzymes. It was possible to identify 14 charged residues present in the XTL with no similar in the XBC: such residues must be considered outstanding mutation sites in XBC. In the presence of the substrate, the characteristics of each domain/region are not modified but the stability of the thumb domain is increased. No difference in the affinity for the substrate was detected between the xylanases and it can be suggested that the activation energies are similar. Two water molecules were found in the active site supporting the hydrolysis mechanisms proposed in the literature.

Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2000 / M

No description available.

Submitted in complete fulfillment of the requirements for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, Durban, South Africa. 2014. / The global demand for green technology has created a need to search for microbes that can play an active role in advancing a greener and cleaner future. Microbial enzymes are nature’s keys to life and their efficiency, specificity and environmental-friendliness has lead to their increased use in industrial processes. Thermomyces lanuginosus is a thermophilic fungus that can degrade plant biomass and produces a variety of enzymes that have industrial application. The fungus T. lanuginosus SSBP has been reported in literature to produce the highest level of xylanase among other Thermomyces strains and some of its enzyme s viz., amylase and lipase are already being used. Because of this ability, it has been identified as one of the organisms that can have various industrial applications. Although a few proteins from this fungus have been cloned and used commercially, the vast majority are still unknown. In order to identify new protein candidates and understand their biochemical interactions, the T. lanuginosus genome (DNA) and the transcriptome (mRNA) were sequenced using 454 Roche and Solexa sequencing platforms. Genome and transcriptome data was assembled using Newbler software forming a genome size of 23.3 Mb contained 30 scaffolds. Protein prediction identified 5105 candidates as protein-coding genes and these gene models were supported by expressed sequence tag and transcriptomic data. The annotated data was assembled into metabolic pathways in order to identify functional pathways and validate the accuracy of the annotation process. T. lanuginosus is usually found in composting plant material thus protein related to plant hydrolysis were analysed. The total number of plant biomass-degrading and related proteins that fall into the carbohydrate-active enzyme (CAZy) family was 224. Most of these proteins were similar to proteins found in other filamentous fungi. Surprisingly, T. lanuginosus contained a single gene coding for xylanase which hydrolyses xylan although this organism is well known for being among the highest producers of this enzyme. An important subset of the above group of proteins is the cellulose degrading-proteins as this can be used in biofuel production. Eight candidates belonging to this group were identified, making this fungus significant in the biofuels. Among the eight cellulase candidates, phylogenetic analysis revealed that three of them were closely related to Trichoderma reesei, a well known industrial cellulase-producer. Utilization of cellulase-related compounds was validated by phenotypic microarray experiments, with cellobiose having inducing biomass in T. lanuginosus. Proteins that are involved in high temperature survival are vital for the survival. of this thermophilic fungus. Interestingly, T. lanuginosus contains 19 heat shocking proteins which are responsible for thermostability. Another adaptation identified in this fungus is the accumulation of trehalose to combat heat stress. Furthermore, T. lanuginosus contains the highest reported number methyltransferases, which have been linked to producing thermostable proteins and higher energy production. Also because of this organism’s ability to grow on composting environments, the assimilation and ability to produce biomass on different carbon sources were analysed using phenotypic microarray technique. The results showed that xylose was the best compound to induce biomass followed by trehalose, maltose and maltotriose. The genomic sequencing of this fungus has provided valuable information that can be used for various biotechnological applications, as well as providing greater insights into its thermostability. Understanding the metabolic pathways involved may allow for manipulation to increase production of these enzymes or cloning into other hosts. This can have an impact in the field of biofuel production and other plant biomass-related processes.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.