Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-07-13T14:30:26Z No. of bitstreams: 1 texto completo.PDF: 392257 bytes, checksum: cedbde0495c381e0793f30277cca03c4 (MD5) / Made available in DSpace on 2017-07-13T14:30:26Z (GMT). No. of bitstreams: 1 texto completo.PDF: 392257 bytes, checksum: cedbde0495c381e0793f30277cca03c4 (MD5) Previous issue date: 2001-09-06 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os genes Penicillium região pgg1 e pgg2, griseoroseum, codificadora do que codificam poligalacturonases foram clonados e gene pgg1 possui caracterizados. 1251 pares de em A bases (pb) e é interrompida por íntrons de 55 e 67 pb, posicionados por comparação com a seqüência do cDNA correspondente. Uma proteína de 376 aminoácidos (aas) foi obtida da tradução deste gene com e apresenta massa um molecular potencial e pI sítio estimados de em glicosilação 38,4 KDa (Asn 307 ), e 5,31, respectivamente. O gene pgg2, de 1165 pb, também é interrompido por dois íntrons de 58 pb. Os íntrons de pgg1 e pgg2 apresentam posições conservadas, embora o mesmo não tenha sido verificado com as suas seqüências. A proteína deduzida de pgg2 possui 369 aas, massa molecular 38,3 KDa, pI 8,31 e dois possíveis sítios de (Asn 300 glicosilação Asn 338 ). e A identidade entre as proteínas PGG1 e PGG2 foi de 57,5%. Análise mostrou por que hibridização do genes e os únicas no genoma gênero Penicillium pgg1 do fungo. foram DNA total de pgg2 estão Diferentes investigadas P. griseoroseum presentes espécies quanto em de à cópias fungos do presença de genes de PG em seu genoma. Os resultados revelaram que a essas espécies contêm PGs codificadas por poucos genes, ao contrário do observado possuem em Aspergillus famílias de genes niger de PG e Botrytis constituídas cinerea, por no que mínimo cinco membros. A expressão dos genes pgg1 e pgg2 foi analisada por hibridização do RNA total e pela técnica de RT-PCR. Ambos os genes são transcricionalmente regulados, porém com expressão diferenciada entre si. A expressão do gene pgg1 foi verificada apenas em 76 horas de cultivo do micélio em meio com pectina suplementado com extrato de levedura, enquanto o transcrito do gene pgg2 expressão fontes não foi de de com detectado ambos os carbono, em todos genes como também sacarose extrato de levedura. detectados apenas na presença adição extrato de levedura. pgg2 de os e tempos foi de analisada glicose, de pectina, Contudo, a em pgg1 ou foram independente expressão A outras suplementadas Transcritos de cultivo. do da gene foi reprimida somente em meio contendo glicose. A adição de extrato de levedura ao meio de cultivo contendo glicose teve um efeito positivo sobre a expressão de pgg2, embora um nível maior de expressão tenha sido verificado na presença de sacarose e pectina. As regiões 5’ terminais dos genes pgg1 e pgg2 foram sequenciadas para caracterizar cis-elementos e analisar a interação com proteínas reguladoras. Ensaios de migração retardada em gel foram realizadas para comprovar a funcionalidade de alguns cis-elementos. Extratos a a protéicos nucleares foram diferentes proteína preparados fontes foi reguladora de carbono. visualizada do gene de quando pgg2 micélio A crescido em meio formação de complexos fragmentos de DNA contendo os cis-elementos da com DNA- região CCAAT e TATAATTAA foram utilizados. Um possível elemento de resposta ao cAMP foi localizado na posição -757. A região reguladora do gene pgg1 possui um potencial sítio de ligação para o regulador geral CREA sobreposto ao TATA “box”, o que pode ser uma explicação para a ausência de expressão deste gene na presença de glicose. / The polygalacturonase-encoding Penicillium griseoroseum were genes cloned pgg1 and and pgg2 from characterized. The 1251-bp coding region of the pgg1 gene is interrupted by two introns of 55 and 67 bp deduced by comparison with the cDNA sequence. A potencial glycosilation nucleotide mass polypeptide sequence was 38.4 KDa of with 376 site at pgg1. and consists of 1165 bp introns. The positions amino The with a acids residues with a was predicted from protein estimated molecular Asn 307 pI of and is also of the introns 5.31. The pgg2 gene by two 58-bp interrupted were the conserved in both genes however no conservation was seen in their sequences. The pgg2 encodes a protein of 369 residues with estimated mass and pI of 38.3 glycosilation KDa sites and were 8.31, respectively. identified at Asn 300 Two and putative Asn . 338 Based on the deduced amino acid sequence, PGG1 shows 57.5% identity with PGG2. The genome pgg1 of analysis. and P. The pgg2 genes griseoroseum presence of exist as as single copies in the revealed by total DNA blot corresponding PG genes in otherPenicillium species was investigated and the results show that these enzymes are encoded by few genes. A different pattern is reported for Aspergillus niger and Botrytis cinerea where the PG gene family consists of at least five members. RT-PCR and was employed to investigate the expression of pgg1 pgg2 genes. Both genes are regulated at the transcription level, seen. although The pgg1 cultivation while in pgg2 analyzed. some differences gene was pectin Total RNA detected extracted expression after supplemented was was their expressed medium transcript in 76 with in from hours yeast all were extract, time mycelia of points grown on sucrose or glucose, with or without yeast extract. Pectin was the only condition tested that induced the expression of pgg1, even in the expressed absence under of almost yeast all pgg2-specific mRNA could medium the addition while extract. conditions be The studied. observed on yeast extract of pgg2 gene was However, no glucose-containing abolished this repressive effect. The 5’ regions of the pgg1 and pgg2 genes were sequenced in order to interaction of characterize with regulatory electrophoresis extracts were sources. cis-acting proteins mobility prepared from DNA-protein elements was shift mycelia complexes investigated assays. grown were and on their by means Crude nuclear different carbon visualized when DNA fragments containing CCAAT and TATAATTAA were used. A putative cAMP response element translation initiation pgg1 has gene a was site. potential detected The 5’ at –766 upstream binding site from sequence for the the of the global repressor CREA which overlaps the TATA box. This could explain the repressive effect of glucose on pgg1 expression. Only few cis-acting elements are shared by the and this could justify the pgg1 and pgg2 promoters differential expression of the polygalacturonase-encoding genes from P. griseoroseum.

Submitted by Biblioteca Central (biblioteca@unicap.br) on 2018-06-06T18:35:42Z No. of bitstreams: 2 claudia_maria_rocha_moura.pdf: 1212429 bytes, checksum: 64bbf58e7633e49f0648b0ef6ed9e0ab (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-06T18:35:42Z (GMT). No. of bitstreams: 2 claudia_maria_rocha_moura.pdf: 1212429 bytes, checksum: 64bbf58e7633e49f0648b0ef6ed9e0ab (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-06 / Microbial enzymes are one of the bioactive products have increased their production in recent decades, due to its widespread use in many existing industrial and environmental sectors. There are numerous applications of microbial enzymes in various sectors of biotechnology. Due to its widespread use, several studies have been undertaken to isolate and identify new producers microorganisms, especially those isolated from poorly studied environments and with an immense microbial biodiversity as the northeastern Caatinga. Studies have been conducted with filamentous Penicillium isolated from soil samples of the fungi of Pernambuco Caatinga to produce tannase. Initially enzyme production was performed using selection on solid medium 4 samples. The results showed that the samples called SIS 24 and 25 show the formation of the characteristic halos higher production of enzyme at 37°C, with values of 4.0 and 4.5 cm, respectively. Then molecular and morphological tests for identification of the samples were made which had the largest halos production of tannase and the sample identified as Talaromyces (SIS 24) and Penicillium chrysogenum (SIS 25). The tannase production assays were performed with samples SIS 24 and 25 by submerged fermentation using 4 production media with different compositions at 37 °C, 150 rpm, 168 hours. The results show that the SIS 25 showed the highest enzyme production at 96 hours of production 0.1555 U / mL obtained in the medium 1. Continuing to production studies were performed new production using a 23 full factorial design for alternative media formulation containing organic residues (coffee, grape and orange wastes) under the same conditions used in the previous test. The results showed that the assay 3, which showed a pH 4.8, containing coffee 5g, 12g of grape and orange 5g, presented a lipase activity of 0.210 U / mL. The use of agro-industrial waste emerges as a viable alternative for the development of biotechnology products bioactive media of production. / As enzimas microbianas são um dos produtos bioativos que têm aumentado sua produção nas últimas décadas, devido a sua grande utilização em diversos setores industriais e ambientais existentes. São inúmeras as aplicações das enzimas microbianas em diversos setores da biotecnologia. Devido a sua grande utilização, vários estudos têm sido realizados no sentido de isolar e identificar novos micro-organismos produtores, principalmente aqueles isolados de ambientes pouco estudados e com uma imensa biodiversidade microbiana como a Caatinga nordestina. Foram realizados estudos com fungos filamentosos do gênero Penicillium isolados de solo da Caatinga de Pernambuco para produção de tanase. Inicialmente foi realizada uma seleção de produção da enzima utilizando 4 isolados em meio sólido. Os resultados revelaram que os isolados denominadas de SIS 24 e 25, apresentaram a formação dos maiores halos característicos de produção da enzimas à 37oC, com valores de 4,0 e 4,5 cms, respectivamente. Em seguida, foram realizados ensaios moleculares e morfológicos para identificação das amostras selecionadas (SIS 24) identificada como Talaromyces e a (SIS 25) como Penicillium chrysogenum. Em seguida, foram realizados ensaios de produção da tanase com os isolados SIS 24 e SIS 25 através de fermentação submersa, utilizando 4 meios de produção com diferentes composições, à 37oC, 150 rpm, 168 horas. Os resultados revelaram que a amostra SIS 25 apresentou a maior produção enzimática com 96 h de produção de 0,1555 U/mL obtida no meio 1. Ensaios de produção utilizando um planejamento fatorial de 23 completo para formulação de meios alternativos contendo resíduos agroindustriais (casca de café, casca de uva e resíduo de laranja) nas mesmas condições utilizadas no ensaio anterior. Os resultados obtidos evidenciaram que o ensaio denominado 3, que apresentou um valor de pH 4,8, contendo resíduos de café 5g, de uva 12g e de laranja 5g, apresentou uma atividade tanalitica de 0,210 U/mL. A utilização de resíduos agroindustriais surge como uma alternativa viável na elaboração de meios de produção de produtos biotecnológicos bioativos.

Penicillium species have been studied for over 200 years and the genus was first described by Link in 1809. Initially, morphological identification methods were used however, much diversity within the genus resulted in researchers seeking alternative techniques and approaches to improve accuracy. These methods involved biochemical analysis of secondary metabolites in conjunction with morphological examination. With the emergence of more accurate and rapid molecular identification tools, scientists embraced modem technology to address diversity challenges. In order to provide a more holistic approach towards the taxonomy of complex genera, morphological analysis remains an essential component in Penicillium identification. Penicillium species are omnipresent, dominant and problematic in postharvest environments. They are known to cause major losses in export markets due to fruit decay. The aim of this study was to identify species within the South African litchi export chain and develop a rapid method for Penicillium identification. This study used morphological as well as molecular identification methods in order to develop PCR-RFLP restriction maps for a number of dominant Penicillium species. Seventeen species of Penicillium were identified using conventional morphological methodology and DNA sequencing, both of which are laborious and time-consuming. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism provided reliability and repeatability as well as being a cost-effective and rapid identification alternative. A combined phylogenetic study indicated that the taxonomic position of several species may need to be reconsidered. Fourteen species were differentiated from one another through digestion of the â-tubulin gene region with five restriction enzymes. Banding patterns correlated well with phylogenetic and biochemical data of related studies, indicating that this method holds promise as a rapid identification procedure for Penicillium species. / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

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La présente étude vise d'une part, à fournir et définir les paramètres de base nécessaires au développement de la lixiviation fongique des métaux et d'autre part, à améliorer sa rentabilité économique par l'utilisation d'une source de substrat économique. Pour ce faire, différents essais en flacons ont été réalisés en laboratoire. En premier lieu, ces essais ont permis de mettre au point une procédure de lixiviation, notamment le choix d'une méthode de lixiviation adaptée à la lixiviation de deux résidus miniers d'origines différentes. En second lieu, le perméat de lactosérum a été sélectionné parmi 7 résidus agroalimentaires comme une nouvelle source de substrat pour remplacer le sucrose habituellement utilisé au laboratoire lors des essais de lixiviation fongique. Par la suite, l'influence de divers paramètres de production des acides organiques (qui sont les agents de lixiviation) et de lixiviation des métaux sur la solubilisation de 6 métaux lourds (Cu, Fe, Mn, Ni, Pb et Zn) a été évaluée. L'influence de la distribution géochimique des métaux a été étudiée en particulier. Les résultats obtenus montrent que la lixiviation fongique offre la possibilité de récupérer à des concentrations commercialement attractives les métaux lourds retenus dans les importantes quantités de résidus miniers rejetés par l'industrie minière. Elle se révèle ainsi comme une biotechnologie prometteuse qui s'intègre au développement durable non seulement en préservant l'environnement et la santé publique des conséquences néfastes de la présence des métaux lourds, mais aussi en permettant la valorisation de ces derniers comme une source secondaire de matières premières, ainsi que celle du perméat de lactosérum comme une source de substrat pour la biosynthèse des acides organiques.

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