Orientadora: Maria Benincasa Vidotti / Co-orientadora: Angeles Manresa / Banca: Ana Claudia Barana / Banca: Vanildo Luiz Del Bianchi / Banca: Mariana Carina Frigieri / Banca: Janete Apparecida Desiderio / Resumo: Lipoxigenases são enzimas que catalizam a oxido-redução de ácidos graxos poliinsaturados que contém em sua estrutura um ou mais grupos cis 1,4- pentadieno, produzindo hidroperóxidos através da incorporação de um oxigênio molecular. Durante anos a presença de lipoxigenase foi considerada exclusivamente eucariótica, presente em mamíferos, plantas, pequenos invertebrados marinhos e fungos. A função biológica dessas enzimas tem sido amplamente estudada. Porém, pouco tem sido descrito em organismos procariotos. O presente estudo teve por objetivos clonar e caracterizar bioquimicamente duas lipoxigenases de Shewanella woodyi ATCC 51908, caracterizar os produtos de reação destas enzimas e realizar um estudo filogenético e estrutural, comparando-as com lipoxigenases de eucariotos e procariotos. Parâmetros enzimáticos dessas enzimas foram descritos. A influência do pH e temperatura na atividade catalítica foram estudadas. Também foi determinado a preferência por substrato e o efeito da adição de vários íons divalentes que podem interferir na atividade catalítica. A enzima SWPrecLox de S. woodyi apresentou temperatura e pH ótimos de 31ºC e 8,0, respectivamente. Demonstrou afinidade por ácido linoleico, com uma Km de 0,47 mM e Vmax de 2,78 mmols min-1 mg-1. A enzima SWAracLOX teve ácido araquidônico como substrato preferente, com valores de Km 0,20 mM e Vmax 1,6 mmols min-1 mg-1. Atividade ótima foi alcançada com 27ºC e pH 7,0. A partir das estruturas moleculares das lipoxigenases de Plexaura homomalla e Pseudomonas aeruginosa, foram criados hipotéticos modelos estruturais de SWPrecLOX e SWAracLOX. / Abstract: Lipoxygenases are enzymes that catalyze the oxido-reduction of polyunsaturated fatty acids in their structure that contains one or more groups cis 1,4- pentadiene, producing hydroperoxides from the incorporation of molecular oxygen. For years, the presence of lipoxygenases was considered a eukaryotic feature, present in mammals, plants, small marine invertebrates and fungi. The present study aimed to clone and characterize biochemically two lipoxygenases from Shewanella woodyi ATCC 51908, characterize the reaction products of these enzymes and conduct a phylogenetic and structural analysis, comparing them with eukaryotes and prokaryotes lipoxygenases. The biological function of these lipoxygenases has been widely studied. However, only few have been described in prokaryotes. Enzymatic parameters of these enzymes have been described. The influence of the pH and the temperature on the catalytic activity has been studied. Also has been studied the substrate preference and the effect of the addition of several divalent cations that can enhance or eliminate the catalytic activity. The enzyme SWPrecLox of S. woodyi showed temperature and pH optimum were 31 ºC and 8,0, respectively. Demonstrated substrate affinity for linoleic acid, with Km 0,47 mM and Vmax 2,78 mmols min-1 mg-1. The enzyme SWAracLox has arachidonic acid as preferred substrate, with Km 0,20 mM and Vmax 1,6 mmols min-1 mg-1. Optimum activity was reached at 27 ºC and pH 7,0. From the molecular structures of lipoxygenase Plexaura homomalla and Pseudomonas aeruginosa, were created a hypothetical structural model of the SWPrecLox and SWAracLox. / Doutor

No description available.

Submitted in fulfillment for the requirement of a Degree of Master in Biotechnology, Durban University of Technology, 2017. / Microbial xylanases have attracted considerable research interest because of their various applications in biotechnology including the biobleaching of kraft pulp, to increase the nutritional value of foods and animal feed as well as for their potential use in the production of ethanol and methane. In the paper and pulp industry, the bleaching process involves the use of toxic chemicals and in the interim produces harmful gases that have a negative impact on the environment. The application of enzymes for this process will potentially reduce the environmental pollution by this industry. In addition, using an enzyme that is thermostable and alkali tolerant means that they will remain active under the required processing conditions. The xylanase gene, xynA derived from Thermomyces lanuginosus DSM 5826, was previously evolved to produce a number of xylanase variants, which were further enhanced for increased thermostability and alkalinity. In this study, these variants were cloned in Pichia pastoris using the pBGP1 vector to achieve extracellular production of the recombinant proteins. The xylanase genes were isolated using PCR. Both vector and DNA inserts were linearized with restriction enzymes EcoRI and XbaI and ligated. Electroporation was employed to transform the yeast with the recombinant plasmids. This was followed by the expression of the enzymes in P. pastoris grown in yeast peptone glucose (YPD) medium. Enzyme activity was thereafter assessed and the yeast was found to produce 164, 78, 96 and 142 IU/ml of S325, S340, G41 and G53 xylanase respectively, higher levels than bacterial hosts. The enzymes were then characterized and it was established that the optimum temperatures and pH for maximum xylanase activity were, 60°C, pH 6 for S325; 40°C, pH 5 for S340; 60°C, pH 6 for G41 and 60°C, pH 7 for G53. i The pH and temperature stabilities of the respective enzymes were investigated, the S325 variant was exceptionally stable at a pH between 5 and 7 and temperature range of 40-80°C and retained a minimum of 40% of activity at higher pH and temperature after an incubation period of 90 min. The S340 variant was the least thermostable and alkali stable from all four variants, it however retained 40% of activity when subjected to conditions of pH 9, 80°C after 90 min. The G41 and G53 were highly stable under the pH and temperature conditions that they were subjected to. Thus being suitable for potential application in the pulp and paper industry. The enzymes were able to retain 80% of activity at pH 9, 80°C after 120 min. P. pastoris has been proven to be a more suitable protein expression vector than E. coli for a number of reasons, including; the ability to perform complex post-translational modifications and grow to high densities in minimal media resulting in the production of a high yield of heterologous proteins. / M

Bibliography: pages 215-244. / Clostridium acetobutylicum P262 is an endospore forming Gram-positive obligate anaerobe which has been used for the industrial production of acetone and butanol. Strains of C. acetobutylicum have been reported to exhibit some activity towards cellulosic and hemicellulosic substrates. The aim of this thesis was to establish a genebank of C. acetobutylicum P262 DNA in Escherichia coli and to isolate and characterise genes encoding enzymes which show activity towards hemicellulose and cellulose.

No description available.

<p> Full length P1 eDNA clones have been obtained from human and CHO sources (Jindal et al., 1989; Picketts et al., 1989) which contain sequences that show extensive sequence and structural similarity to the chaperonin family of proteins, including the mitochondrial hsp60 protein. In the studies described here human genomic DNA libraries were screened with human P1 (hsp60) eDNA probes and those clones containing P1 related sequences were isolated. One clone, pGem1b, was found to be completely homologous to the human P1 cDNA in both coding and non-coding regions, devoid of intervening sequences, and terminates at a point 24 base pairs upstream of the translation initiation signal (ATG). The other human clones analyzed were all found to be pseudogenes containing numerous additions, deletions and base substitutions, but no introns. A total of six different classes of pseudogenes were identified. Four of these were sequenced completely across the translated region of the functional P1 gene. Sequence homologues of 86.1, 87.4 89.7 and 90.2% were observed. </p> <p> In addition, rat kidney and mouse JTJ cell eDNA libraries were screened similarly for P1 sequences. The rat P1 eDNA sequence was obtained by combining the sequence information from three different clones. The clones obtained lacked the 5'- leader sequence as well as the mitochondrial targeting sequence. However, the entire coding sequence for a mature P1 protein of 547 amino acids could be deduced. The mouse P1 DNA sequence was also obtained from three different clones. These clones contained a portion of the mitochondrial targeting sequence and the entire sequence for the mature P1 protein. The protein sequences of the rat and mouse P1 clones were highly homologous (98-99%) to those obtained from human and CHO sources. The calculated molecular weights of the mature rat and mouse P1 proteins are 57,916 and 57,940 daltons, respectively, which are in close agreement with those predicted for the human (57, 939 daltons) and CHO (57, 949) proteins (Jindal et al. 1 1989; Picketts et al., 1989). </p> / Thesis / Master of Science (MSc)

In this study a library of EcoRI fragments encompassing the entire TOL region of the Pseudomonas putida TOL plasmid pDK1 was constructed in the Escheria coli cloning vector pBR325.

Superoxide dismutase is an enzyme which scavenges superoxide radicals and is thought to be a longevity determinant, as there exists a positive correlation between superoxide dismutase concentration and maximum life span potential. The cytosolic CuZn superoxide dismutase in D. melanogaster has been purified and sequenced, but the gene has not been cloned. However, when it is available the CuZn SOD gene may be reintroduced into the Drosophila genome via the P-element transformation system so its effects on the life span potential of Drosophila may be studied. This study describes attempts to clone the CuZn SOD gene from D. melanogaster using two mixed sequence oligonucleotide probes. The SI probe corresponds to amino acids 43-48 of the protein sequence and contains 128 different oligonucleotide sequences representing all possible codon combinations predicted from the amino acid sequence. The GT3 probe is targeted to amino acids 90-95 of the protein. In this probe, deoxyguanosine was placed in positions where all four nucleotides may occur to decrease probe heterogeneity. The probes were used to screen D. melanogaster Canton-S and Oregon-R genomic lambda libraries. Three positive clones isolated from the Canton-S library had identical nucleotide sequence in the GT3 probe binding region, and sequencing of the probe binding site revealed that one member of the GT3 probe had formed a 15 bp duplex with the phage DNA. Screening of the Oregon-R library produced four clones which hybridized with both GT3 and S1 probes. When these phage DNA were hybridized to polytene chromosomes by in situ hybridization, none mapped to 68AB on the third chromosome, the location of the CuZn SOD gene. These results suggest that modification of the classical strategy used in this study is necessary, and implications on probe design are discussed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

No abstract available. / Thesis (LL.M.)-University of Natal, Durban, 2002.

Esta pesquisa teve como objetivo central a anÃlise da HistÃria da CiÃncia presente nos livros didÃticos de Biologia do ensino mÃdio, tomando como foco o tema Clonagem. A escolha desse tema se deu em virtude de ter gerado muitas discussÃes cientÃficas, religiosas e Ãticas, especialmente desde o advento da ovelha Dolly, em meados da dÃcada de 1990. Um segundo objetivo da pesquisa à o de fornecer ao professor um material de apoio que aborde historicamente a Clonagem e suas implicaÃÃes sociais para ser utilizado durante as aulas sobre o tema. Esta pesquisa apresentou, quanto ao mÃtodo, a abordagem dedutiva, fundada em princÃpios lÃgicos formais de anÃlise dos materiais bibliogrÃficos selecionados para estudo, perseguindo uma anÃlise fundada na lÃgica racional dos argumentos presentes nos materiais selecionados para a pesquisa. Quanto ao nÃvel da pesquisa, caracteriza-se como uma pesquisa exploratÃria, tendo como objetivo mensurar hipÃteses identificÃveis para subsidiar estudos posteriores. Neste caso, a hipÃtese de que o uso da HistÃria das CiÃncias aplicada ao ensino de Biologia no tema clonagem potencializa a melhoria da compreensÃo do tema por parte dos alunos do ensino mÃdio, promovendo a melhoria de sua aprendizagem. Ademais, delineia-se como uma pesquisa bibliogrÃfica, constituÃda por consultas a livros, teses e dissertaÃÃes relacionados ao estudo da HistÃria da CiÃncia, alÃm dos livros didÃticos do ensino mÃdio. A abordagem da HistÃria da CiÃncia teve como funÃÃo evidenciar que a CiÃncia nÃo à linear, nem as descobertas acontecem sem ligaÃÃo com outras pesquisas e eventos sociais e histÃricos. Buscou-se trabalhar, portanto, mediante uma anÃlise de cunho histÃrico que levou aspectos que foram analisados nas narrativas histÃricas, a saber: a ideia de continuidade, de acumulaÃÃo de conhecimento, de linearidade e da apresentaÃÃo de resultados positivos nas pesquisas. Os resultados obtidos na pesquisa evidenciaram que os livros didÃticos do ensino mÃdio apresentam apenas alguns aspectos da HistÃria da CiÃncia, sendo mais evidente a presenÃa de biografias e as principais realizaÃÃes dos cientistas. / This research had as main purpose the science history analysis present in the secondary school biology textbooks, taking as focus the cloning theme. The choice of this theme happened due to many scientific, religious and ethical discussions, especially since the advent of Dolly the sheep in the mid 1990s. A second purpose of this research is providing to teachers a supporting material that talks historically about cloning and its social implications to be used during the classes about the theme. This research presented a deductive approach as method, founded in formal logical principles of bibliographic materials analysis that were chosen to the study, chasing an analysis founded in the rational logic of the arguments present on the selected material of the research. As to the research level, is characterized as an exploratory research, having as purpose to measure identifiable hypotheses to subsidize further studies. In this case the hypothesis that the use of the history of science applied to biology teaching in the cloning theme enhances the improved understanding of the subject by high school students, promoting the improvement of their learning. Still is outlined as a bibliographic research, constructed by search in books, dissertations and thesis related to the study of the history of science, besides the high school textbooks. The approach of history of science had as function to show that the science is not linear, nor the discoveries happen without the connection with other researches and social and historical events. It was tried to work through a historical analysis which took aspects that were analyzed on the historical narratives, such as: idea of continuity, accumulation of knowledge, linearity and presentation of positive results in researches. The results obtained in the research showed that the high school textbooks present only some aspects of history of science, being more evident the presence of biographies and the most important achievements of the scientists.