Submitted by Biblioteca Central (biblioteca@unicap.br) on 2018-02-19T17:29:12Z No. of bitstreams: 1 maria_cleudenora_cassia_mororo.pdf: 880158 bytes, checksum: ea675b48ea15f324c1021f82c3bd3508 (MD5) / Made available in DSpace on 2018-02-19T17:29:12Z (GMT). No. of bitstreams: 1 maria_cleudenora_cassia_mororo.pdf: 880158 bytes, checksum: ea675b48ea15f324c1021f82c3bd3508 (MD5) Previous issue date: 2017-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES# / #2075167498588264571# / #600 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq# / #-2555911436985713659# / #600 / Cellulases (E.C. 3.2.1.4) are enzymes responsible for the degradation of cellulose, are molecules capable of accelerating chemical reactions and breaking the chemical bonds between glucose units. Cellulases correspond to the complex consisting of three enzymes endoglucanases, exoglucanases and beta-glucosidases, with diverse applications, being the microbial biotechnological processes responsible for a great part of the world economy, yet the costs of production are still very high. In this context, 25 strains of filamentous fungi (Ascomycetes) isolated from mangrove sediments of Rio Formoso, PE, Brazil, were investigated to investigate the production potential of the enzymes of the cellulolytic complex. The initial studies were carried out by selecting the fungi with the highest enzymatic activity, through the detection of cellulolytic activity in solid synthetic medium, with carboxymethylcellulose (CMC) as the substrate. The results indicated the presence of the cellulase enzyme through the formation of halo in 3 strains of the genus Trichoderma, 3 strains of the genus Aspergillus and 1 strain of the genus Penicillium. The most representative enzymatic indices were those of Penicillium sp. UCP 0279 with Index of 2,2, followed by Aspergillus flavus UCP 1413 with enzymatic index of 1,7. Submerged fermentations were carried out to evaluate the endoglucanase activity, exoglucanase and β-glycosidase, using agroindustrial residues, tangerine peel, pineapple peel, pineapple crown, wheat bran and corn bran as substrate. The results indicated a CMCase activity of 20.2 IU / mL for Penicillium sp. UCP 0279, with wheat bran as substrate in 72 h of fermentation and an activity of 18.3 IU / mL in 24 h with the pineapple crown. For the Aspergillus flavus UCP 1413, the yield was 14.9 IU / mL and 14.5 IU / mL with the residues of corn bran and pineapple peel respectively, and both results were obtained with 24 h of fermentation. The FPase activity for Penicillium sp. UCP 0279, using pineapple peel as substrate had 45.5 IU / mL and the tangerine peel 42.8 IU / mL, both in fermentation at 48 h. For A. flavus UCP 1413 the pineapple crown presented 25.0 IU / mL enzymatic activity in 24 h and the pineapple peel 14.4 U / mL at the same time. In the activity of the enzyme β-glycosidase, Penicillium sp. UCP 0279 showed a production of 18.2 IU / mL in 24 h, with the pineapple crown residue and the pineapple peel had 9.1 IU / mL in 48 h. The A. flavus UCP 1413 presented with 96 h of fermentation an activity of 16.9 U / mL and 14.5 U / mL, with wheat bran and corn bran, respectively. / As celulases (E.C. 3.2.1.4) são enzimas responsáveis pela degradação da celulose, são moléculas capazes de acelerar reações químicas e realizar a quebra das ligações químicas existentes entre as unidades de glicose. As celulases correspondem ao complexo constituído por três enzimas endoglucanases, exoglucanases e beta-glicosidases, com diversas aplicações, sendo os processos biotecnológicos microbianos responsáveis por uma grande parcela da economia mundial, contudo os custos de produção ainda são muito elevados. Neste contexto, foi realizada a bioprospecção de 25 linhagens de fungos filamentosos (Ascomycetes) isolados de sedimentos de mangue do município Rio Formoso, PE, Brasil, investigando o potencial de produção das enzimas do complexo celulolítico. Os estudos iniciais foram realizados selecionando os fungos com maior atividade enzimática, através da detecção da atividade celulolítica em meio sintético sólido, tendo como substrato a carboximetilcelulose (CMC). Os resultados indicaram a presença da enzima celulase através da formação do halo em 3 linhagens do gênero Trichoderma, 3 linhagens do gênero Aspergillus e 1 linhagem do gênero Penicillium. Os índices enzimáticos mais representativos foram os de Penicillium sp. UCP 0279 com Índice de 2,2, seguido de Aspergillus flavus UCP 1413 com índice enzimático de 1,7. Em seguida, foram realizadas fermentações submersas para avaliação da atividade endoglucanase, exoglucanase e β-glicosidase, utilizando resíduos agroindustriais, casca de tangerina, casca de abacaxi, coroa de abacaxi, farelo de trigo e farelo de milho como substrato. Os resultados indicaram uma atividade para CMCase de 20,2 UI/mL para Penicillium sp. UCP 0279, com farelo de trigo como substrato em 72 h de fermentação e com a coroa de abacaxi observou-se uma atividade de 18,3 UI/mL em 24 h. Para o Aspergillus flavus UCP 1413, a produção foi de 14,9 UI/mL e 14,5 UI/mL com os resíduos de farelo de milho e de casca de abacaxi respectivamente, e ambos os resultados foram obtidos com 24 h de fermentação. A atividade FPase para Penicillium sp. UCP 0279, usando casca de abacaxi como substrato apresentou 45,5 UI/mL e a casca de tangerina 42,8 UI/mL, ambos em fermentação a 48 h. Para A. flavus UCP 1413 a coroa de abacaxi apresentou 25,0 UI/mL de atividade enzimática em 24 h e a casca de abacaxi 14,4 U/mL no mesmo tempo. Na atividade da enzima β-glicosidase o Penicillium sp. UCP 0279 apresentou uma produção de 18,2 UI/mL em 24 h, com o resíduo da coroa de abacaxi e com a casca de abacaxi apresentou 9,1 UI/mL em 48 h. O A. flavus UCP 1413 apresentou com 96 h de fermentação uma atividade de 16,9 U/mL e 14,5 U/mL, com farelo de trigo e farelo de milho respectivamente.

>Magister Scientiae - MSc / Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method. We first utilized the CRISPR systems for targeting the T.r.eg2 gene to single locus chromosomal sites for single copy integration. Subsequently, we then targeted the same gene to repeated sequences in the genome, namely the delta sites, for multi-copy integration. The procedure was repeated with a different gene, T.e.cbh1, integrated into the same sites to ascertain reporter gene specific effects. High integration efficiency was achieved, since all the strains successfully integrated the genes. However, we discovered significant differences in enzyme activities between the two genes when targeted to different loci, as well as varying copy numbers as determined by qPCR. The T.e.cbh1 gene was highly expressed by yeast transformants targeted at the repeated delta sequences used for multi-copy integration, reaching maximum levels of 248 mU/gDCW. The T.r.eg2 gene was highly expressed in yeast transformants targeted to the single locus site on chromosome 12, reaching a maximum of 160U/gDCW, though it was shown that off-target integration likely occurred. We then used the information from these observations to construct a CBP yeast strain containing three cellulase genes: T.r.eg2, T.e.cbh1, and S.f.BGL1. Significant differences in enzyme activities were observed between the three genes, and it was shown that the S.f.BGL1 gene was poorly expressed by the CBP yeast strain, whereas the T.r.eg2 gene was highly expressed. Notably, due to the fact that marker containing plasmids could be cured from these strains, many additional genetic changes can still be made. Overall, our two CRISPR-Cas9 systems were efficient at engineering strains that produce recombinant proteins and can be used in future studies for a variety of applications, including metabolic engineering in S. cerevisiae

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Made available in DSpace on 2016-06-02T19:02:43Z (GMT). No. of bitstreams: 1 5706.pdf: 7869131 bytes, checksum: 3a35e736b3418ca357ef4fc2e657c0af (MD5) Previous issue date: 2013-10-16 / Universidade Federal de Minas Gerais / Enzymes are potent catalysts, but operationally fragile, expensive and soluble. Industrial applications of enzymes, often, are possible only using immobilized enzyme. Nowadays, various studies have been performed aiming to immobilize enzymes onto magnetic carriers, which allow the selective recovery of the derivative by applying an external magnetic field even in complex reaction media containing other suspended solids. There are many studies using magnetic carriers in enzymes immobilization procedures, however there are no commercially available enzymes immobilized onto magnetic materials. In these studies usually are used carriers with not ideal characteristics for applications in industrial processes. The present study aimed to develop new magnetic carriers and methods for immobilization of enzymes in these carriers, penicillin G acylase (PGA) and cellulases have been used as model enzymes. The thesis was divided into five parts, in the first part (Chapter 1) the state-of-art is presented. The second part (Chapter 2) describes the synthesis of magnetic carriers robust, cheap and with good characteristics for applications in bioprocesses. For this purpose were tested the synthesis of silica magnetic microparticles (SMMps) in water-in-oil micro-emulsion using sodium silicate as silica source and superparamagnetic iron oxide nanoparticles as magnetic core. Materials with good magnetic properties, high surface area and mesoporous structure were obtained. SMMps structure was characterized, it was possible to control the final structure of the material according to the synthesis conditions. In the third part of this study (Chapter 3) was evaluated a new concept in enzymes immobilization using magnetic materials. Magnetic tags were co-aggregated with PGA and cross-linked with glutaraldehyde, producing magnetic cross-linked enzymes aggregates (M-CLEAs). Several reaction conditions were tested producing M-CLEAs with different characteristics and strong response to external magnetic fields. Derivatives with good recovered activity and increased thermal and methanol 50% (v/v) stabilities were obtained. M-CLEAs presented superior performance, in comparison with the free enzyme, in penicillin G hydrolysis experiments, being reused for three reaction cycles without loss of activity. In the fourth part of this study (Chapter 4) the immobilization of the Trichoderma reesei cellulolytic complex onto 17 carriers using 60 different immobilization conditions was evaluated. Covalent methods to cellulases immobilization resulted in total loss of the enzymatic activity. The immobilization by adsorption allowed preserving a portion of the enzymatic activity, however, the enzyme was desorbed from the carrier with the increase in the ionic strength. The best results were achieved for adsorption in MANAE-agarose followed by cross-linking with glutaraldehyde. Hydrolysis experiments using insoluble substrates showed that it is possible to hydrolyze such substrates even using immobilized enzyme onto porous carriers. The derivative was reused for ten reaction cycles (hydrolysis of filter paper) saving more than 90% of its activity. Finally, in Chapter 5, the T. reesei cellulolytic complex was immobilized by adsorption onto SMMp activated with amino groups followed by glutaraldehyde cross-linking achieving good results in terms of recovered activity. / Enzimas são potentes catalisadores, porém frágeis operacionalmente, caras e solúveis. Aplicações industriais desses catalisadores, muitas vezes, são possíveis apenas com o uso de enzima imobilizada. Estudos indicam que o uso de suportes magnéticos para imobilizar enzimas pode permitir a recuperação seletiva do derivado através da aplicação de um campo magnético externo mesmo em meios complexos contendo outros sólidos em suspensão. Apesar de existirem muitos estudos empregando suportes magnéticos para imobilização de enzimas, não existem enzimas imobilizadas em materiais magnéticos disponíveis comercialmente. Nestes estudos geralmente são utilizados suportes magnéticos com características não ideais para aplicações em bioprocessos. O presente estudo teve como principal objetivo o desenvolvimento de novos suportes magnéticos e métodos para imobilização de enzimas nestes suportes, a enzima penicilina G acilase (PGA) e celulases foram utilizadas como modelo. O estudo foi dividido em cinco partes, no Capítulo 1 é apresentada uma introdução indicando o estado da arte. O Capítulo 2 apresenta o preparo de novos suportes magnéticos robustos, baratos e com características ótimas para aplicações em bioprocessos. Nesta etapa foi testada a síntese de micro-partículas magnéticas de sílica (SMMps) em micro-emulsão água-em-óleo, empregando silicato de sódio como fonte de sílica e nanopartículas superparamagnéticas de óxido de ferro como núcleo magnético. Os materiais obtidos apresentaram excelentes propriedades magnéticas, alta área de superfície e estrutura mesoporosa. A partir da caracterização físico-química e morfológica das SMMps foi possível controlar a estrutura final do material de acordo com as condições de síntese. No Capítulo 3 foi avaliado um novo conceito em imobilização de enzimas empregando materiais magnéticos. Neste estudo etiquetas magnéticas foram co-agregadas com PGA e entrecruzadas com glutaraldeído, gerando agregados enzimáticos entrecruzados com propriedades magnéticas (M-CLEAs). Várias condições reacionais foram testadas rendendo M-CLEAs com diferentes características e com resposta robusta a campos magnéticos externos. Derivados imobilizados com boa atividade recuperada e incremento na estabilidade térmica e frente a metanol 50% (v/v) foram obtidos. M-CLEAs apresentaram desempenho superior ao observado para a enzima livre em experimentos de hidrólise de penicilina G, sendo reutilizados por três ciclos reacionais sem perda de atividade. No Capítulo 4 foi avaliada a imobilização do complexo celulolítico de Trichoderma reesei em 17 suportes, empregando 60 diferentes condições de imobilização. Os experimentos de imobilização realizados empregando técnicas de imobilização por união covalente ocasionaram perda total de atividade enquanto métodos de imobilização por adsorção permitiram conservar boa atividade enzimática, porém a enzima dessorveu do suporte com o aumento na força iônica do meio. Os melhores resultados foram alcançados para adsorção em MANAE-agarose seguido de entrecruzamento com glutaraldeído. Experimentos de hidrólise de substratos insolúveis mostraram que é possível hidrolisar este tipo de substrato mesmo com enzima imobilizada em suportes porosos. O derivado foi reutilizado por dez ciclos (hidrólise de papel filtro) conservando mais de 90% de sua atividade. Por fim, no Capítulo 5, o complexo celulolítico de T. reesei foi imobilizado por adsorção em SMMp ativado com grupos amino seguido de entrecruzamento com glutaraldeído apresentando bons resultados em termos de atividade recuperada.

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The theoretical part of work is focused on the issue of biomass which can be used for energy purposes, inparticular agricultural waste, as well as can serve as a substrate for biogas station. It also deals with proteomics, its goals and approaches, separation methods. The aim of this work was to measure each sample of enzyme activity of biomass, which are used as a raw materials for biogas plants and their proteomic identification.