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1
Smith, David John. "A genetic study of penicillin biosynthesis in Penicillium chrysogenum." Thesis, University of Bristol, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292440.
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Martínez, Benítez Eva. "Estudio de especies micotoxígenas del género Penicillium: Penicillium verrucosum Dierckx." Doctoral thesis, Universitat Autònoma de Barcelona, 2003. http://hdl.handle.net/10803/5598.
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Mycotoxins are toxic secondary metabolites produced after growth of fungal species of different genera. This mycotoxin production can be found in different substrata, including food and feed. Nowadays there is an increasing interest in the toxigenic properties of the strains isolated from food, feed and raw materials, and in the factors that can avoid this production. Species of the genus Penicillium, together with Aspergillus and Fusarium spp., are the majority in these substrata and also the main producers of mycotoxins. Ochratoxin A is one of the more studied mycotoxins currently, having a recent legislation in the European Union, mainly due to its high toxicity and wide distribution. Its production is associated with different Aspergillus spp., and uniquely to one species of the genus Penicillium: Penicillium verrucosum. This species is principally isolated from cereals and have a wider distribution in cold climate countries.In the present thesis, it has been evaluated the presence of Penicillium spp. in 178 samples of feed and cereal destined to animal consumption. A total of 152 strains belonging to 34 different species of the genus were isolated, dominating the species of the subgenus Penicillium. The species Penicillium aurantiogriseum has been the only one isolated from every kind of substrata tested. Penicillium verrucosum, the ochratoxin A producer species, has been isolated only from cereals, and mainly from barley.
A series of physiologic (in the culture media CREA, CSN, YES, NSA, Raulin-Thom and in a liquid culture medium with urea) and biochemical (indole) characteristics, that have been proposed during the last years as taxonomic tools for Penicillium spp., have been evaluated in a total of 298 strains. The media CREA and CSN and the biochemical test for detecting indole metabolites have obtained good results for species distinction in the genus Penicillium. In the culture medium YES, the species P. verrucosum have a violet brown colour in the reverse of the colony, but just by around a 50% of the strains. This colour seems to appear more commonly in fresh cultures than in strains subjected to numerous subcultures.
The capacity of the 298 strains of Penicillium spp. to produce different mycotoxins (aflatoxins, citrinin, sterigmatocystin, ochratoxin A, zearalenone, penicillic acid and penitrem A) was also evaluated. A total of 119 strains had the ability of synthesise one or more of these mycotoxins (except for aflatoxins, sterigmatocystin and zearalenone). A percentage of the 58% of strains of P. aurantiogriseum produced penicillic acid, and the 100% of strains of P. citrinum and P. crustosum synthesised citrinin and penitrem A, respectively. An 85% of strains of P. verrucosum were producers of ochratoxin A, in different concentracions, and around the 50% of them produced the mycotoxin citrinin.
The study by the technique RAPD of the genomic DNA of P. verrucosum allowed the distinction in to groups, A and B, between the strains of the species analysed, corresponding with the two recently proposed species, P. verrucosum and P. nordicum. The study of the sequence of the fragment ITS 1-5.8S-ITS 2 of the DNA codifying for the ribosomal DNA was very homogeneous between the strains of P. verrucosum assayed.
The production of the toxins ochratoxin A and citrinin by P. verrucosum was evaluated under different environmental conditions, obtaining the highest concentrations at values of water activity of 0,94 and 0,96, at temperatures between 15 and 25ºC, at a range of pH between 6 and 10 and with saccharose as carbon source (with higher concentrations of ochratoxin A than in media with fructose or glucose, and all those three media with higher concentrations of the toxins than media with wheat, corn, rice and potato starches).
3
Newbert, Roger William. "A genetic analysis of high titre penicillin production strains of Penicillium chrysogenum." Thesis, University of Sheffield, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420327.
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Karhoot, J. M. "Production of penicillin G by Penicillium chrysogenum immobilized in rotating disc fermenters." Thesis, University of Strathclyde, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381320.
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De, Noronha Pissarra Pedro Maria Do Carmo. "Towards a metabolic engineering approach for optimising penicillin production by penicillium chrysogeneum." Thesis, King's College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244795.
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Silva, José Vinícius da. "Estudo de alterações metabólicas nos fungos endofíticos Penicillium brasilianum e Penicillium griseoroseum." Universidade Federal de São Carlos, 2011. https://repositorio.ufscar.br/handle/ufscar/6532.
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Previous issue date: 2011-08-26Universidade Federal de Sao Carlos
Microorganisms, known as being relatively simple, have shown a great adaptation power to different nutritional situations by changing their metabolism with lack or supply of nutrients in the culture medium. This feature allows a wide range of the use of microorganisms to produce metabolites with several interests. This study has been named OSMAC (One Strain Many Compounds) approach, which was applied in this study by using two fungi from Penicillium genus. Through culture medium modification with FeCl3 during growth of P. brasilianum, it was able to produce isoquinolinic alkaloid. On the other hand, the addition of CaCl2, another alkaloid belonging to tremogenic class was isolated and analysed by spectroscopic methods. When P. griseoroseum was cultivated with NH4Br, it was observed the production of a halogenated secondary metabolite, which was not reported before (brominated roquefortine C). Analyses by GC/MS led to detect three phenolic compounds with bromine atoms in its structure. The secondary metabolites of P. griseoroseum with no modification in the culuture medium were studied and three metabolites were isolated. Two of them belong to the tetronic acid class and the other one was isolated as a dimer from clavatol. Analyses by LC/ MS-MS were able to identify other tetronic acids in the ethyl acetate extract from P. griseoroseum.
Os microorganismos, por serem de organização relativamente simples, têm um poder de grande adaptação a variadas situações nutricionais, modificando seu metabolismo com a carência ou com o fornecimento de nutrientes ao meio de cultivo. Isso permite uma ampla flexibilidade em sua utilização, podendo-se induzir microorganismos a produzir determinadas substâncias de interesse. Esta abordagem pode levar a uma variedade de novos metabólitos secundários interessantes a partir de uma única cepa de um microorganismo. Este estudo vem sendo denominado de abordagem OSMAC (One Strain Many Compounds), a qual foi utilizada no presente trabalho com dois fungos do gênero Penicillium. Foi verificado que ao modificar o meio de cultura com FeCl3 durante o crescimento de P. brasilianum houve a produção de um alcalóide isoquinolínico, enquanto com a adição de CaCl2 houve a produção de outro alcalóide pertencente à classe dos alcalóides tremogênicos. Com relação ao fungo P. griseoroseum, foi isolado um metabólito halogenado (roquefortina C contendo um átomo de bromo) quando ao meio de cultura foi adicionado NH4Br. As substâncias isoladas foram analisadas por técnicas espectroscópicas. Também foram detectados por GC/MS mais três compostos fenólicos contendo átomos de bromo em sua estrutura. O metabolismo secundário de P. griseoroseum foi analisado e foram isolados policetídeos pertencentes à classe dos ácidos tetrônicos e um dímero do clavatol, inédito na literatura. Análises por LC/MS-MS detectaram outros ácidos tetrônicos presentes no extrato de acetato de etila.
7
何耀祥 and Yiu-cheung Timothy Ho. "Biotyping in Penicillium marneffei." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31969732.
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White, Stewart. "Autolysis in Penicillium chrysogenum." Thesis, University of Strathclyde, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367044.
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Ho, Yiu-cheung Timothy. "Biotyping in Penicillium marneffei." Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B22050310.
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Adatia, Remy. "The nephrotoxins of Penicillium aurantiogriseum." Thesis, Imperial College London, 1991. http://hdl.handle.net/10044/1/46637.
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Ariyo, Bolatito Taiwo Onigbogi. "Studies on the effects of oligosaccharides on penicillin G production in cultures of Penicillium chrysogenum." Thesis, University of Westminster, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283468.
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Cheung, Yee-lam Elim, and 張以琳. "Cloning and characterization of putative molecular targets of Penicillium marneffei identified by random genome exploration." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B26642608.
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Boualem, Khadidja. "Influence de conditions environnementales sur la conidiation et les propriétés de surface de Penicillium camemberti." Dijon, 2008. http://www.theses.fr/2008DIJOS012.
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La conidiation est un mécanisme de reproduction asexuée universel qui permet aux champignons filamenteux de se reproduire et de se propager dans l’environnement sous forme de conidies. Ce mécanisme est très utilisé pour produire des conidies pour fabriquer des produits fermentés comme les fromages, pour les biotechnologies avec la production d’enzymes et de composés d’intérêt, et pour la lutte biologique. Les conidies sont produites le plus souvent en culture de surface en milieu solide car la conidiation en culture liquide mmergée des champignons filamenteux n’est pas possible ou pas maîtrisée. C’est le cas de Penicillium camemberti, un des champignons les plus utilisés en agroalimentaire, mais très peu étudié au niveau physiologique et génétique, qui a servi de modèle pour cette thèse. L’objectif a été d’étudier le comportement de cette espèce en culture solide de surface et en culture liquide en faisant varier certains paramètres du milieu comme notamment la composition en azote et la température de croissance. Sur la base de mécanismes et données bio-informatiques de champignons modèles comme Aspergillus, des gènes clés impliqués dans la conidiation (brlA, wetA) et propriété de surface (rodA) ont été clonés et caractérisés pour la première fois chez P. Camemberti, et leur expression a été étudiée par RT-PCR quantitative dans différents types de cultures. L’absence de conidiation en culture liquide immergée est corrélée avec la très faible expression du gène rodA codant pour une hydrophobine, un type de protéines hydrophobes essentielles pour la biologie des champignons filamenteux. Des modifications de la composition azotée du milieu ont permis d’obtenir une conidiation en culture liquide immergée avec des modifications de propriétés de surface et un phénotype nouveau pour les conidies ainsi produites. Enfin, ce travail a révélé pour la première fois que certaines températures de croissance étaient capables, dans certaines conditions de composition azotée, d’induire une croissance de cette espèce en milieu liquide sous forme de microcycles de conidiation, avec une production massive de conidies (5. 108. Ml-1 de milieu)Conidiation is an universal asexual reproduction mechanism enabling filamentous fungi to reproduce and propagate in the environment as conidies. This process is used to produce conidies that are employed in the manufacturing of fermented food, like cheese, in biotechnologies with the production of enzymes and compounds of interest and biological control. Conidies are generally produced in surface of solid-state culture because conidiation of submerged filamentous fungi in liquid medium is usually not possible or not controlled. This is particularly true for Penicillium camemberti, a very popular fungi in the food industry but that has not been much studied at the physiological or genetical levels. This microorganism was the subject of our study. The objective was to investigate its behaviour in solid- or liquid-state culture by modifying some medium parameters such as the nitrogen composition and the growth temperature. Based on mechanisms and bio-informatic data from model fungi such as Aspergillus, key genes involved in conidiation (brlA, wetA) and surface properties (rodA) were cloned and characterised for the first time in P. Camemberti, and their expression was studied by quantitative RT-PCR in different types of culture. The absence of conidiation in submerged liquid culture is correlated with the very weak expression of the rodA gene encoding a hydrophobin, a hydrophobic protein essential for the filamentous fungi biology. Modifications of the nitrogen composition of the medium resulted in conidiation in submerged liquid-state culture with a modification of the surface properties and a new phenotype for the conidies obtained in this way. Finally, this work showed for the first time that some growth temperatures, for specific nitrogen compositions, induced growth in liquid medium of conidiation microcycles with a massive production of conidies (5. 108. Ml-1 of medium)
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Böhm, Julia [Verfasser], Ulrich [Gutachter] Kück, and Dominik [Gutachter] Begerow. "Kreuzungstypgene und Sexualzyklus bei dem Penicillin-Produzenten Penicillium chrysogenum / Julia Böhm ; Gutachter: Ulrich Kück, Dominik Begerow." Bochum : Ruhr-Universität Bochum, 2016. http://nbn-resolving.de/urn:nbn:de:hbz:294-50202.
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Böhm, Julia [Verfasser], Ulrich Gutachter] Kück, and Dominik [Gutachter] [Begerow. "Kreuzungstypgene und Sexualzyklus bei dem Penicillin-Produzenten Penicillium chrysogenum / Julia Böhm ; Gutachter: Ulrich Kück, Dominik Begerow." Bochum : Ruhr-Universität Bochum, 2016. http://d-nb.info/1119447143/34.
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Böhm, Julia Verfasser], Ulrich [Gutachter] Kück, and Dominik [Gutachter] [Begerow. "Kreuzungstypgene und Sexualzyklus bei dem Penicillin-Produzenten Penicillium chrysogenum / Julia Böhm ; Gutachter: Ulrich Kück, Dominik Begerow." Bochum : Ruhr-Universität Bochum, 2016. http://d-nb.info/1119447143/34.
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Halidi, Ben Saida Ali. "Fermentation d'acides gras par Penicillium roqueforti." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/mq33669.pdf.
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Launen, Loren A. "Pyrene biodegradation by Penicillium janthinellum SFU403." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0022/NQ51887.pdf.
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Chow, Wang-ngai, and 周弘毅. "Identification of microRNA in penicillium marneffei." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/198803.
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Penicillium marneffei is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, the existence of miRNAs in fungi was less well studied and their potential roles in fungal dimorphism were largely unknown. Based on available genome sequence of P. marneffei, it is hypothesized that miRNA-like small RNAs (milRNAs) may be expressed in the dimorphic fungus and dicer- or argonuate-like proteins may be involved in dimorphism or virulence in P. marneffei.
I attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 (2502 reads) candidates in mycelial and seven (232 reads) in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 and qde-2 of P. marneffei were more closely related to the homologues in the thermal dimorphic pathogenic fungi, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Coccidioides immitis than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 and qde-2 among other thermal dimorphic fungi. Moreover, dcl-2 and qde-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds and 2 folds respectively (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1KO, dcl-2KO, dclDKO and qde-2KO deletion mutants, it was shown that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. While deletion of qde-2, but not the two dcl genes, was found to decrease the virulence level of P. marneffei in mice model, the deaths of the qde-2KO conidia challenged mice were delayed for over 10 days. The qde-2KO conidia have lower recovery rate both in human THP1 and murine J774 macrophage cell lines and also reduced resistance to hydrogen peroxide than the wild type.
This study provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism. This is also the first study to reveal the relationship between argonuate-like QDE-2 protein and virulence in P. marneffei in mice model. This study provides a foundation for the milRNAs study in pathogenic thermal dimorphic fungi.published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy
20
Mellon, F. M. "Aspects of interspecific hybridization in penicillium." Thesis, University of Nottingham, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374813.
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Ferreira, Gislene. "Produção de patulina por Penicillium spp." Universidade Federal de Viçosa, 2000. http://www.locus.ufv.br/handle/123456789/10629.
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Fundação de Amparo a Pesquisa do Estado de Minas Gerais
Seis linhagens de Penicillium foram estudadas quanto à capacidade de produzir a micotoxina patulina. Dentre as seis linhagens de Penicillium testadas, apenas P. expansum GF produziu patulina, em todos os tempos de cultivo, meios testados e em quantidades superiores às permitidas por legislação (50μg/L). A curva de produção da patulina foi determinada para as linhagens de P. expansum isolada de maçã (GF) e de sementes de plantas florestais (VIC), bem como suas capacidades em produzir patulina nas mesmas condições em que há a produção das enzimas poligalacturonase (PG) e xilanase. O isolado de P. expansum (GF) apresentou maior produção de patulina entre o 9 o e o 15 o dia de crescimento, mas apenas em condições de incubação estática. Sob agitação rotacional, concentrações baixas de 0,16 μg/mL de patulina foram detectadas apenas no 9 o dia de crescimento. A linhagem VIC não produziu patulina em nenhuma condição de incubação. Os fungos P. expansum GF e VIC não produziram patulina sob as mesmas condições em que houve a produção de enzimas despolimerizantes da parede celular de plantas, PG e lanase. As características morfológicas e genéticas xidas três linhagens de P. expansum (GF, VIC e CCT-4608) foram testadas por comparação e mostraram que as linhagens de P. expansum GF e VIC possuem grande semelhança entre si, mas se relacionam muito pouco com a terceira linhagem (CCT- 4608), adquirida como linhagem padrão de P. expansum.
Six Penicillium strains were studied in relation to their capacity to produce the mycotoxin patulin. Among them, only P. expansum GF produced patulin in all cultivation times and media tested and in amounts above those allowed by legislation (50 μg/L). Patulin production curves were determined under the same conditions of polygalacturonase (PG) and xylanase production for Penicillium strains isolated from apples (GF) and seeds of forest trees (VIC). The isolate P. expansum GF presented the highest production of patulin between the 9 th and the 15 th day of growth, albeit only under static incubation. Under rotational shaking, concentrations as low as 0.16 μg/L patulin were detected in the 9 th day of growth. The strain VIC did not produce patulin under any incubation condition tested. The strains GF and VIC did not produce patulin under the same conditions under which the production of plant cell wall depolymerizing enzymes, PG, and xylanase was observed. The morphological and genetic characteristics of three strains of P. expansum (GF, VIC, and CCT- 4608) were compared and showed that the strains GF and VIC were highly similar to each other, but were poorly related to CCT-4608, a standard strain of P. expansum.
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Lam, Sze-ki Clare, and 林思琪. "Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification and antifungal susceptibility of penicillium marneffei." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/206605.
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Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in HIV-infected patients in Southeast Asia. However, laboratory diagnosis based on microscopic morphology and mycelial-to-yeast conversion is time-consuming and expertise-dependent. The performance of the Bruker MALDI-TOF MS system for identification of mold and yeast cultures of 59 P. marneffei strains were evaluated by using the direct transfer method. Using the Bruker databases, BDAL v4.0.0.1 and Filamentous Fungi Library 1.0, the 59 P. marneffei strains grown in mold and yeast phase were identified as P. funiculosum (score <1.7) and P. purpurogenum (<1.7) respectively. When the combined database was expanded with inclusion of spectra from 20 P. marneffei strains grown in mold, yeast or both phases, all the remaining 39 P. marneffei strains grown in both mold and yeast phase were correctly identified to the species level with score >2.0. The spectra of P. marneffei exhibited significant difference to those of the closely related species, P. brevi-compactum, P. chrysogenum, Talaromyces aurantiacus and T. stipitatus (one strain included for each species). P. brevi-compactum was identified to the genus level (as P. brevi-compactum but with score <2.0) and P. chrysogenum was unidentified (as P. chrysogenum but with score <1.7) using the combined database with or without spectra from P. marneffei. Both T. aurantiacus and T. stipitatus were unidentified (as wrong species with score <1.7). MALDI-TOF MS is useful for rapid identification of both yeast and mold cultures of P. marneffei, but this requires expansion of the database using P. marneffei strains.
Since the susceptibilities of P. marneffei to the newer antifungal drugs are not well studied, their in vitro antifungal activities against the 59 isolates of P. marneffei were also investigated in accordance with CLSI M27-A3 microdilution method. MICs of itraconazole, voriconazole, posaconazole and anidulafungin for yeast form of P. marneffei were determined. The MICs of itraconazole, voriconazole, posaconazole and anidulafungin were 0.00128-0.00256 μg/ml, 0.01565-0.0625 μg/ml, 0.000978-0.001956 μg/ml, 2-8 μg/ml respectively. The results suggested that the azoles have similarly good activities against P. marneffei, whereas anidulafungin was the least active.published_or_final_version
Microbiology
Master
Master of Medical Sciences
23
Bittencourt, Mona Lisa Sousa de Assis. "Avaliação do perfil de proteases expressas por Penicillium fellutanum e Penicillium restrictum isolados do solo do cerrado brasileiro." reponame:Repositório Institucional da UnB, 2014. http://repositorio.unb.br/handle/10482/23086.
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As proteases se referem a um grupo de enzimas cuja função catalítica é hidrolisar proteínas. Enzimas proteolíticas encontram ampla aplicação em diversas indústrias e preparações farmacêuticas. Os fungos filamentosos são usados em muitos processos industriais para a produção de enzimas e metabólitos. Alguns desses fungos são produtores de uma série de enzimas, como amilases, pectinases e proteases. O presente trabalho teve como objetivo principal caracterizar proteases expressas por fungos filamentosos isolados de diferentes amostras do Cerrado do Centro-Oeste brasileiro, frente à produção de proteases de interesse industrial e farmacêutico em diversas condições de cultivo. Inicialmente, foi realizada uma triagem para avaliar a capacidade de 17 fungos quanto à produção de protease em meio de cultura contendo Ágar-leite. Oito espécies formaram halo no cultivo em placas de Petri contendo 10% de leite desnatado em Ágar indicando serem produtoras de proteases. Em seguida, quando cultivadas em estufa, no meio sabouraud, peptona e leite desnatado, seis espécies de fungos apresentaram altas atividades de protease sendo então cultivadas sob condição de agitação. Uma melhoria nas atividades de protease para as espécies Aspergillus foetidus, Penicillium variotti, Penicillium citrinum e Penicillium fellutanum foi obtida quando utilizado o cultivo em shaker. Um importante aumento na atividade proteolítica foi obtido para a espécie P.restrictum e P. fellutanum quando avaliado meio de cultivo contendo resíduo agroindustrial. No meio contendo farelo de trigo como fonte de carbono, a maior atividade proteolítica foi identificada quando realizado cultivo por P. restrictum (81,1 UI/mL), a as proteases presentes no meio possuem temperatura ótima igual a 45 °C e pH ótimo em uma faixa de 5,0 a 9,0. Sendo termoestáveis por 2 horas em pH e temperatura ótima. Desta forma, estas enzimas podem ser consideradas promissoras para aplicação industrial.
Proteases are a group of enzymes whose catalytic function is the hydrolysis of proteins. Proteolytic enzymes have wide application in various industries and pharmaceutical preparations. Due to technical and economic advantage, microorganisms are the preferred source of industrial application of protease enzymes, although it can be obtained from animals and plants. Filamentous fungi have beeb used in many industrial processes for the production of enzymes and metabolites. Some of these fungi are producers of several enzymes as amylases, proteases and pectinases. This work aimed to characterize proteases expressed by filamentous fungi isolated from different samples of the Cerrado of Central Brazil, focusing the production of these enzymes of industrial and/or pharmaceutical interest in different growing conditions. Initially, a screening was performed to assess the ability of 17 fungi initially isolated for production of proteases in culture media containing 10% skim milk in agar. Eight species formed a clear zone surrounding colonies, indicating production of protease. These species were then cultivated in Sabouraud broth, peptone and skim milk. Six species showed high protease activity and were then incubated under agitation. Protease activity for the species Aspergillus foetidus, Penicillium variotti, Penicillium citrinum and Penicillium fellutanum improved when used in the cultivation in shaker.A significant increase in proteolytic activity was obtained for the species Penicillium restrictum and Penicillium fellutanum when evaluated in culture media containing agro industrial residues. In a media containing wheat bran as carbon source, the major proteolytic activity has been identified as performed by growing Penicillium restrictum (81.1 IU/mL). Proteases present in the medium have an excellent temperature of 45 °C and optimum pH in a range 5.0 to 9.0. We evaluated the physicochemical characterization and the enzymatic profile of the species Penicilliumrestrictum and Penicillium fellutanum for production of protease in different culture media. Thus, these enzymes can be considered promising for industrial application.
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Eugênio, Patrícia de Fátima Menegoci. "Análises de meroterpenos produzidos por Penicillium brasilianum." Universidade Federal de São Carlos, 2008. https://repositorio.ufscar.br/handle/ufscar/6419.
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Previous issue date: 2008-02-14Financiadora de Estudos e Projetos
This work describes the use of the high performance liquid cromatography (HPLC) technic coupled to the mass spectrometry technique for the analysis of fungal extracts, and the main purpose was the detection of meroterpens produced by P. Brasilianum. The fungal species P. Brasilianum was cultivated in a variety of artificial culture media: rice, wheat, maize, rice broth, solid decanted from rice broth and liquid medium Czapeck. In all the culture media tested the fungus presented a very good development, and also, the ions related to the meroterpens in study were detected in all the fungal extracts produced by these cultivations, except in Czapeck medium. The atmospheric pressure chemical ionization (APCI) was compared to the electrospray ionization, for the meroterpens analysis, and the result obtained was a great increase in detection power for this class of compounds, with the use of APCI. The P. brasilianum fungus was cultivated in Czapeck medium in the presence of Pinus taeda resin, alizarin, oleanolic, benzoic, salicylic, 2,5-dihydroxybenzoic, 2,6-dihydroxy-4-methyl-benzoic, 3,5-dinitrosalicylic, 3,5-dinitrobenzoic and ferulic acids, and 2 ,4 ,6 -trihydroxyacetophenone. The presence of these exogenous aditives modified the fungal metabolic profile, because the cromatographic profiles of the extracts produced showed a differentiation, in comparation to the fungal extract without aditive. Only the fungal extract produced in the presence of ferulic acid presented the meroterpens PSP-3 and PSP-8. The fungal extracts produced without any aditive also did not present meroterpens.
Este trabalho descreve o uso da técnica de cromatografia líquida de alta eficiência (HPLC) acoplada com a técnica de espectrometria de massas para análise de extratos fúngicos, visando principalmente a detecção de meroterpenos, produzidos por P. brasilianum. A espécie fúngica P. brasilianum foi cultivada em diversos meios de cultura artificiais: arroz, trigo, milho, caldo de arroz, filtrado do caldo de arroz e meio líquido Czapeck. Em todos os meios de cultivo testados o fungo apresentou um ótimo crescimento, e também, em todos os extratos fúngicos produzidos através destes cultivos foram detectados íons relacionados aos meroterpenos de interesse, exceto no meio Czapeck. A técnica de ionização química a pressão atmosférica (APCI) foi comparada com a de electrospray, para a análise destes meroterpenos, e o resultado obtido foi um aumento significativo no poder de detecção desta classe de compostos com o uso de APCI. O fungo P. brasilianum foi cultivado em meio Czapeck, na presença de resina de Pinus taeda, alizarina, ácidos oleanólico, benzóico, salicílico, 2,5-dihidroxibenzóico, 2,6-dihidroxi-4-metilbenzóico, 3,5-dinitrosalicílico, 3,5-dinitrobenzóico e ferúlico, e a 2 ,4 ,6 -trihidroxiacetofenona. A presença destes aditivos exógenos modificou o perfil metabólico do fungo, pois os perfis cromatográficos dos extratos produzidos se diferenciaram em relação ao extrato fúngico sem nenhum aditivo. Somente o extrato fúngico produzido na presença de ácido ferúlico apresentou os meroterpenos PSP-3 e PSP-8. Os extratos fúngicos produzidos na ausência de aditivos também não apresentaram meroterpenos.
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Tam, Wan-ting, and 譚韻婷. "Characterization of polyketide synthases in penicillium marneffei." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/197137.
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Penicillium marneffei is a thermal dimorphic fungus that causes systemic mycosis in HIV-positive patients. The fungus displays unique phenotypic properties, including the yellow and black pigments on its conidia as well as the secretion of a diffusible red pigment during growth in mycelial phase. However, all these pigments have not been characterized. Investigation into the pigment production of the fungus can provide insights into the functions of the respective pigment to the fungus as well as their roles in fungal pathogenesis.
This study reports the identification and characterization of 23 polyketide synthase (PKS) and 2 polyketide synthase non-ribosomal peptide synthase hybrid (PKS-NRPS) genes in the genome of P. marneffei. Systematic knockdown of the PKS genes showed a loss of black pigment on the conidia of the pks4 (alb1) knockdown mutant, a loss of yellow pigment in the mycelial form of pks11 and pks12 knockdown mutants and a loss of red pigment production in the pks3 knockdown mutant.
PKS4 in P. marneffei is responsible for melanin production. Knockdown of pks4 resulted in the loss of melanin production and reduced ornamentation on the conidial surface. Mice that were challenged with the pks4 knockdown mutant survived significantly better than those challenged with wild type conidia (P<0.005). The sterilizing doses of hydrogen peroxide giving a 50% survival reduction of the fungal conidia were 11 minutes and 6 minutes for wild type and the pks4 knockdown mutant, respectively. These together suggested that melanin in P. marneffei contributed to its pathogenesis by reducing its susceptibility to killing by hydrogen peroxide.
HPLC-MS analysis revealed the identity of the yellow pigment of P. marneffei to be mitorubrinic acid and mitorubrinol. Mice that were challenged with the pks11and pks12 knockdown mutants survived significantly better than those challenged with wild type conidia (P<0.05). The survival of the pks11and pks12 knockdown mutants in J774 and THP1 macrophages were also both significantly lower than the wild type, suggesting mitorubrinic acid and mitorubrinol contribute to fungal pathogenesis by improving its survival in macrophages.
The red pigment secreted by P. marneffei was found to compose of monascorubrin, rubropunctatin, ankaflavin, citrinin and different amino acid conjugated with monascorubrin/rubropunctatin. The biosynthetic pathway of the red pigment involved a polyketide synthase (pks3), a transcription activator (rp1), a fatty acid synthase subunit beta (rp2), a 3-oxoacyl-[acyl-carrier-protein] synthase (rp3) and an oxidoreductase (rp4). RP2, PR3 and RP4 are responsible for fatty acid production. PKS3 is responsible for the biosynthesis of an intermediate polyketide, and RP1 is responsible for the biosynthetic activation. Through esterification, the fatty acid attaches to the intermediate polyketide to form monascorubin, an orange pigment, which is secreted out of the cell. Amino acids in the culture medium were found to conjugate with monascorubrin to form pigments ranging from orange to red in color. Ankaflavin is synthesized by the reduction of monascorubrin. PKS3 and RP1 are also responsible for the biosynthesis of citrinin. In conclusion, the chemical composition, biosynthetic pathways and potential roles in virulence of the black, yellow and red pigments in P. marneffei were characterized.published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy
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Rogers, S. D. "DNA repair and mutagenesis in Penicillium chrysogenum." Thesis, University of Westminster, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233044.
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Smith, T. M. "Molecular and biochemical studies in Penicillium chrysogenum." Thesis, University of Westminster, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371397.
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Zampieri, Denise. "Expressão do complexo celulolítico em Penicillium echinulatum." reponame:Repositório Institucional da UCS, 2011. https://repositorio.ucs.br/handle/11338/1005.
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O Penicillium echinulatwn linhagem 9A02Sl é um fungo filamentoso que apresenta um sistema celulolítico com potencial para aplicaqão em processos de degradação de materiais lignocelulósicos para produção de etanol. O crescente interesse nesse combustível e a abundância de materiais lignocelulósicos que podem ser usados como matéria-prima fez aumentar o interesse no estudo de celulases. Neste estudo, a linhagem 9A02Sl de Penicillium echinulatum foi cultivada em cultivos submersos em frascos mantidos sob agitação recíproca, com variações quanto às fontes de carbono. Além de crescimento, foram avaliadas as produções de celulases, ~-glicosidases e xilanases e a expressão das enzimas através ale zimogramas em géis de poliacrilamida para detemlinação da massa molecular. Observou-se que o crescimento micelial provocou a redução do pH do meio de cultivo, e que não está relacionado a produção de enzimas. A celulose apresentou-se como indutora para todas as enzimas analisadas. A carboximetilcelulose mostrou-se uma eficiente fonte de carbono para a produção de atividade sobre papel filtro, endoglicanases e xilanases, apesar do baixo crescimento micelial. Celobiose, glicerol e glicose estimularam a produção de ~glicosidases. Uma banda de atividade endoglicanêlsica de 74 kDa foi detectada nos zimogramas de todos os caldos enzimáticas obtidos na presença d1e diferentes fontes de carbono, sugerindo esta seja uma enzima constitutiva. A expressão da ~-glicosidase oconeu ao fmal do cultivo (5° e 6° dias), sendo que em todos os cultivos avaliados houve a expressão de uma banda de 220 kDa, indicando tratar-se de uma enzima constitutiva. A expressão de outras bandas com diferentes massas moleculares sugerem que diferentes genes, fotmats multiméricas ou modificações pós-traducionais estão envolvidos no perfil destas enzimas em Peni'cillium echinulatum.Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-09-22T17:54:12Z No. of bitstreams: 1 Dissertacao Denise Zampieri.pdf: 18671785 bytes, checksum: 0c85c4e913bd6377f21bfe2a1fe9e8ff (MD5)
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Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq
The st:rain of Penicillium echinulatum 9A02Sl is a filamentous fungus that presents a cellulolytic system with potential application in processes of degradation of lignocellulosic materiais for ethanol production. The growing interest in fhel and the abundance of lignocellulosic materiais that can be used as raw material has inc:reased the interest in cellulases. In this study, the strain P echinulatum 9A02Sl was grown in submerged cultivation in agitated flasks in presence of different carbon sources. In addition to growth, it was evaluated the production of cellula.ses, Pglucosidases and xylanases, and enzyme expres:sion in activity polyacrylamide gels in order to detemlinate the molecular ma.ss. The mycelial growth decreased pH o f the medium and this fact was not related to enzyme production. The cellulose was an inducer for all the enzymes ana.lyzed. The carboximetilcellulose was found to be an effi[cient carbon source for production o f filter paper a.ctivity, endogluca.nases and xylanases, despite the low mycelial growth. Cellobiose, glycerol and glucose stimulated the production of P-glucosidases. An endoglucanase band of 74 kDa was detected in zymograms o f all enzyme broths obtained in the presence o f different carbon sources, suggesting it is a constitutive enzyme. The expression of P-glucosidase occuned at the end of cultivation (5 and 6 days), and in all medium that was evaluated was observed a 250 kDa band, indica.ting that this is a. constitutive enzyme. The expression of other bands with different molecular mass suggest that different genes, multimeric fonns or post-translational modifications are involved in the expression ofthese enzymes in P echinulatum.
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Chalier, Pascale. "Production de composés d'arôme par "Penicillium roqueforti"." Montpellier 2, 1991. http://www.theses.fr/1991MON20178.
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La bioconversion par penicillium roqueforti d'acides gras a courte, moyenne et longue chaine a ete etudiee. La production d'heptanone-2 a partir d'acide octanoique varie en fonction de la nature de la souche, de l'etat physiologique du champignon, de la concentration en acide gras, de la presence, dans le milieu de culture, de glucose. Elle peut etre fortement augmentee par l'addition maitrisee de tween ou d'huile de soja, dans le milieu. L'action simultanee d'un lipase est necessaire a la bioconversion des acides gras constitutifs, des triglycerides de l'huile de coprah. Mais une lipolyse trop intense est defavorable a la production de methyl-cetones et provoque l'apparition de methyl cetones insaturees. Lorsque le champignon est cultive en presence d'acides gras a longue chaine saturee ou insaturee, d'autres molecules odorantes interessantes sont identifiees par cpg-sm. L'action simultanee d'une lipase exogene et des spores de penicillium roqueforti sur l'huile de soja permet de former des dodecalactones a odeur de peche. Sur milieu zapeck saccharose modifie enrichi en huile de soja, penicillium roqueforti produit des composes a structure terpenique et sesquiterpenique. Des pyrazines, des phenols sont mis en evidence dans les cultures de penicillium roqueforti sur lait de soja
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Walter, Ruth. "Untersuchungen zur Grünfäule (Penicillium spec.) an Weintrauben." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:100-opus-2975.
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Maria, de Barros Rodrigues Priscila. "Produção de protease pelo Penicillium aurantiogriseum URM4622." Universidade Federal de Pernambuco, 2008. https://repositorio.ufpe.br/handle/123456789/2255.
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Previous issue date: 2008Conselho Nacional de Desenvolvimento Científico e Tecnológico
As proteases microbianas representam cerca de 60 % do total mundial de vendas de enzimas. Estas proteases têm sido extensivamente estudadas, devido à necessidade de novas proteases com diferentes características, para atender o rápido crescimento das indústrias baseadas na tecnologia de produção de enzimas. O presente trabalho objetivou a produção e caracterização parcial da protease produzida pelo Penicillium aurantiogriseum URM4622 em biorreator utilizando um planejamento experimental (23), visando sua aplicação em detergentes. A produção da protease ocorreu em bioreator (Fermenter RALF 2,0 L) de tanque agitado com 1,5 L de volume de trabalho, equipado com controles de temperatura, pH e oxigênio dissolvido. Amostras foram coletadas a cada 12 horas, para determinações da biomassa, curva de pH e atividade proteásica. As melhores condições para a produção da enzima, correspondente a maior atividade específica (43,67 ±1,98 U/mg) foram 26 oC; pH 7,0 e 25 % O2. A protease do caldo fermentado mostrou-se estável em uma ampla faixa de pH 5,8 - 9,5 e a temperaturas de 25 40 °C. A atividade proteolítica decresceu cerca de 26 % na presença do íon Zn2+ e aumentou 29 % com o íon Mn2+. Cerca de 96,2 % e 70,8 % da atividade proteolítica foram mantidas após 90 min de incubação com H2O2 a 5 % e 10 % (v/v), respectivamente. A inibição pelo PMSF revelou a presença de proteases do tipo serina; nenhuma inibição ocorreu em presença de Tween 80 e Triton X-100 e mais de 50% de sua atividade foi retida em presença de vários detergentes comerciais. Os resultados obtidos sugerem que o Penicillium aurantiogriseum URM4622 é uma fonte viável de produção de protease alcalina com potencial interesse na indústria de detergentes
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Vivan, Juliana. "Produção da micotoxina citrinina por Penicillium spp." Universidade Federal de Viçosa, 2002. http://www.locus.ufv.br/handle/123456789/11395.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
O fungo Penicillium expansum, produtor de enzimas pectinolíticas e xilanolíticas, as quais possuem aplicação na indústria têxtil, na indústria de bebidas, principalmente na clarificação de sucos e vinhos, e na indústria de alimentos, está incluído entre as espécies toxigênicas capazes de produzir micotoxinas, dentre elas a citrinina. A presença dessa micotoxina inviabiliza a utilização de P. expansum na indústria de alimentos. Com o propósito de analisar a possibilidade desse fungo produzir simultaneamente as enzimas de interesse e a citrinina, foram verificadas seis linhagens de Penicillium , que vêm sendo utilizadas em estudos de produção de pectinases e xilanases na Universidade Federal de Viçosa. Dois métodos de análise, cromatografia em camada delgada (CCD) em sílica-gel e cromatografia líquida de alta eficiência (CLAE) em fase reversa, foram utilizados com o único propósito de detectar citrinina. Dentre as seis linhagens, P. citrinum (principal produtor de citrinina ), P. expansum GF (linhagem toxigênica isolada de maçãs deterioradas), P. expansum VIC, P. griseoroseum , P. roqueforti e P. camemberti , apenas P. citrinum e P. expansum GF produziram citrinina nos três meios de cultivo testados (Timonin, YES e Aveia). P. roqueforti produziu citrinina apenas em caldo Timonin e os demais não a produziram em nenhum dos meios testados. Apenas o fungo P. citrinum foi capaz de produzir citrinina sob condições de incubação estática e em agitação rotacional a 150 rpm, em longos períodos de tempo (40 dias). O fungo P. expansum GF produziu a citrinina somente em condições estáticas, os demais fungos testados, P. expansum VIC, e P. roqueforti , também cultivados em ambas as condições, foram incapazes de produzir citrinina. Deste modo, pode-se concluir que os fungos P. expansum VIC e P. griseoroseum não representam perigo quanto à produção de citrinina, quando utilizados na indústria de alimentos, pois não têm capacidade de produzir a micotoxina nas mesmas condições em que produzem as enzimas de interesse, poligalacturonase e xilanase.
The fungus Penicillium expansum , which produces pectinolytic and xylanolytic enzymes with application in the textile, food and beverage industries, is one of the toxigenic species capable of producing mycotoxins, one of which is citrinin. The presence of this mycotoxin would preclude the use of P. expansum in the food industry. Six lineages of Penicillium that are being used in studies of pectinase and xylanase production at the Universidade Federal de Viçosa were therefore evaluated for possible simultaneous production of the enzymes of interest and citrinin. Both thin layer (TLC) and reverse phase liquid (HPLC) chromatographies were used to detect citrinin. Of the six lineages evaluated, P. citrinum (principal citrinin producer) , P. expansum GF (a toxigenic lineage isolated from rotten apples), P. expansum VIC, P. camemberti , and P. roqueforti , only P. citrinum and P. expansum GF produced citrinin in the three culture media tested (Timonin, YES and oatmeal). P. roqueforti produced citrinin only in Timonin broth while the other lineages did not produce this mycotoxin in any of the media used. Only P. citrinum was able to produce citrinin when cultivated both with (150 rpm) and without agitation for long periods (40 days). The fungus P. expansum GF only produced citrinin under static growth conditions. The other fungi evaluated ( P. expansum VIC, and P. roqueforti ) were also cultivated with and without agitation but did not produce citrinin under either growth condition. It can therefore be concluded that P. expansum VIC and P. griseoroseum represent no threat to the food industry in terms of citrinin production since these fungi are unable to produce this mycotoxin under the conditions used to produce the enzymes of interest polygalacturonase and xylanase.
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Hashim-Mostafa, Abdulkarim. "Recherche de l'activité antitumorale chez les micromycètes : étude particulière de la patuline, métabolite secondaire de Penicillium italicum." Université Joseph Fourier (Grenoble), 1987. http://www.theses.fr/1987GRE18002.
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Pimenta, Eli Fernando. "Investigação das condições de crescimento e produção de metabólitos secundários das linhagens de fungos Penicillium citrinum e Penicillium oxalicum." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/75/75131/tde-03032011-163613/.
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No presente estudo, duas espécies de fungos isoladas do ambiente marinho tiveram seus extratos brutos ativos contra Staphylococcus aureus e Candida albicans para uma espécie de P. citrinum e, atividade citotóxica e contra Mycobacterium tuberculosis para uma espécie de P. oxalicum. Estas foram estudadas com a finalidade de otimizar suas condições de crescimento para produção de metabólitos secundários. Foram realizadas análises multivariadas utilizando planejamento fatorial fracionário. Foram obtidas duas condições ótimas de crescimento para as duas linhagens de Penicillium. O crescimento em maior volume em condições de cultura otimizadas para P. oxalicum permitiu observar a presença de três metabólitos secundários no extrato do meio de cultura. Dois puderam ser isolados e identificados: a meleagrina 52 e a oxalina 26. A metodologia utilizada para se obter uma maior quantidade de metabólitos secundários proporcionou, ainda, o incremento em cerca de 150% na área do pico da oxalina. A partir do crescimento em maior volume em condições de cultura otimizadas para P. citrinum foi possível observar presença de pelo menos onze diferentes metabólitos na análise dos extratos obtidos do meio de cultura. Foi possível identificar e isolar quatro compostos já conhecidos: a (8E)-1-(2,3-diidropirrol-1-il)-2-metildec-8-eno-1,3-diona 56; a 1-(2,3-diidropirrol-1-il)-2-metildecano-1,3-diona 58; a 2-((E)-hept-5-enil)-6,7,8,8a-tetraidro-3-metilpirrolo[2,1-b][1,3]oxazin-4-ona 59 e a citrinina 31. Também foram isolados dois novos alcalóides indólicos contendo um grupo nitro na molécula que foram nomeadas de citrinalinas A 60 e B 62. Foi realizado, também, um estudo com P. citrinum visando a maior produção das citrinalinas, que possibilitou o incremento na produção da citrinalina B.In this study, two species of fungi isolated from the marine environment had their active extracts against Staphylococcus aureus and Candida albicans to a strain of P. citrinum, and activity cytotoxic and against Mycobacterium tuberculosis to a strain of P. oxalicum. This studied aims the optimization their growth conditions for the production of secondary metabolites. Multivariate analysis using a fractional factorial design were used to establish optimal growth conditions for both Penicillium strains. Two optimal growth conditions were obtained for both Penicillium strains. A largest growth volume of P. oxalicum using optimized conditions enabled the detection of three secondary metabolites in the culture media crude extract. Two of such compounds were isolated and identified: meleagrin 52 and oxaline 26. The methodology used to increase the production of secondary metabolites by P. citrinum enabled an increase of 150% in the peak area of oxaline. A largest growth volume of P. citrinum led to the detection of eleven different metabolites in the culture media. Four of these compounds were isolated and identified as the known (8E)-1-(2,3-dihydropyrrol-1-yl)-2-methyldec-8-ene-1,3-dione 56; the 1-(2,3-dihydropyrrol-1-yl)-2-methyldecane-1,3-dione 58; the 2-((E)-hept-5-enyl)-6,7,8,8a-tetrahydro-3-methylpyrrolo[2,1-b][1,3]oxazin-4-one 59 and citrinin 31. Two new indole alkaloids containing a nitro group were also isolated and identified, named citrinalins A 60 and B 62. A further study with P. citrinum aiming an enhanced production of citrinalins allowed a significant increase in the production of citrinalin B.
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Gaspar, Júnior Pascoal José 1971. "Caracterização de holocelulases fúngicas na otimização da biomassa lignocelulósica." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314694.
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Orientadores: Sérgio Marangoni, Saulo Luis da SilvaTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A triagem de fungos produtores de holocelulases é uma estratégia para a obtenção de enzimas capazes de hidrolisar o material lignocelulósico da biomassa vegetal, contribuindo para aumentar a viabilidade da produção de etanol celulósico Este trabalho avaliou o potencial enzimático dos fungos Penicillium corylophilum e Penicillium simplicissimum com relação às enzimas (endoglicanase, exoglicanase, ?-glicosidase, FPase, xilanase, pectinase e mananase) sobre substratos lignocelulósicos e comerciais como fonte de carbono. Além disso, avaliou-se também a influência da adição de diferentes fontes orgânicas e inorgânicas de nitrogênio sobre a atividade enzimática. Os fungos foram cultivados em triplicata em meio líquido suplementar com 1% de substrato lignocelulósico como fonte de carbono, em pH 7,0. A inoculação foi feita por suspensão de esporos (108 esporos/mL). O cultivo foi feito sob agitação a 120 rpm, a 28°C e os filtrados resultantes em 12, 24, 72 e 120 horas foram utilizados como fontes enzimáticas pelo método do DNS (ácido-3,5- dinitrosalicílico) em triplicata. De todas as atividades enzimáticas analisadas nas fontes lignocelulósicas, a atividade de xilanase do P. simplicissimum sobre a linhaça foi a mais expressiva (3,87 e 3,97 UI/ml), cultivados em 72 e 120 horas, respectivamente, e selecionada para os testes de purificação proteica. A atividade xilanásica específica aumentou consideravelmente após os passos cromatográficos de gel filtração e troca iônica (CTI), sendo inicialmente 3x10-3 atv/ µg de proteína no liofilizado e 19,2 atv/ µg de proteína na fração proveniente da CTI. Nos testes de pH, observou-se que a atividade de xilanase foi maior em pH=4,0 e temperatura de 50°C. Com relação à adição de substratos comerciais, a celulose microcristalina e a xilana apresentaram os resultados mais expressivos da indução da produção de xilanase, sendo que a concentração de 0,5% de xilana mostrou a melhor atividade enzimática em ambos os fungos estudados. A xilose apresentou uma concentração indutora mínima de 0,04% que foi suficiente para aumentar a atividade de xilanase do P. simplicissimum. Com relação à suplementação de fontes de nitrogênio no meio de cultivo para a produção de holocelulases, a adição de (NH4)2SO4 e caseína é uma alternativa importante para a potencialização das atividades de pectinase e de endoglicanase respectivamente pois foram fontes de nitrogênio que proporcionaram um aumento da atividade enzimática em ambos os fungos estudados como também nas duas fontes de carbono testadas. As frações contendo xilanases após cromatografia de gel filtração seguida de fase reversa, após análises por espectrometria de massas apresentam relação massa/carga de 18831,26 Da. A utilização do resíduo lignocelulósico da linhaça, como fonte de carbono para o cultivo submerso do Penicillium simplicissimum é uma opção ecologicamente correta, exequível e de baixo custo para a produção de xilanases. Diversas aplicações biotecnológicas como a utilização na ração animal, na indústria do papel e no etanol de segunda geração, dentre outros, possibilitam um acréscimo substancial do valor agregado desse substrato, permitindo uma ampliação da utilização da linhaça, além do aproveitamento do óleo, sem aumentar a área plantada
Abstract: Screening for producing fungi holocelulases is a strategy for obtaining enzymes that hydrolyze the lignocellulosic material from plant biomass, helping to increase the viability of cellulosic ethanol production This study evaluated the enzymatic potential of Penicillium simplicissimum and Penicillium corylophilum regarding enzymes (endoglicanase, exoglicanase, ?-glucosidase, FPase, xylanase, pectinase and mannanase) for commercial and lignocellulosic substrates as a carbon source. Furthermore, it was also evaluated the influence of the addition of different organic and inorganic nitrogen sources on enzyme activity. The fungus was grown in triplicate in a liquid medium supplement with 1% lignocellulosic substrate as carbon source, pH 7.0. The inoculation was done by the spore suspension (108 spores/ml). The cultivation was done with stirring at 120 rpm at 28 °C and the resulting filtered in 12, 24, 72 and 120 hours were used as enzyme sources for DNS (acid-3,5- dinitrosalicilic) method in triplicates. All enzymatic activities analyzed in lignocellulosic sources, the xylanase activity of P. simplicissimum about flaxseed was greater (3.87 and 3.97 IU/ml), grown at 72 and 120 h of cultivation, respectively, and selected for testing for protein purification. The specific xylanase activity increased considerably after the chromatographic steps of gel filtration and ion exchange (CTI), initially 3x10-3 atv/ mg of protein in lyophilized and 19.2 atv/ mg of protein in the fraction from the CTI. In pH testing, it was noted that the xylanase activity was higher at pH 4.0 and 50 °C. With respect to the addition of commercial substrates, microcrystalline cellulose and xylan showed the most significant results of induction of xylanase production, and the concentration of 0.5% xylan showed the best enzyme activity in both fungi studied. The xylose showed a minimal inducing concentration of 0.04% which was sufficient to increase the activity of xylanase from P. simplicissimum. With respect to supplemental nitrogen sources in the culture medium for the production of holocelulases, the addition of (NH4)2SO4 and casein can be an important tool for the enhancement of pectinase and endoglicanase activities respectively as alternative nitrogen sources that were provided an increase in enzyme activity in both fungi studied as well as the two carbon sources tested. The use of lignocellulosic residue of flaxseed as a source of carbon for submerged cultivation of Penicillium simplicissimum is an environmentally friendly, feasible and cost effective for the production of xylanases option. The fractions containing xylanases after gel filtration chromatography followed by reverse fase after analysis by mass spectrometry are related mass/charge of 18831 26 Da. The use of lignocellulosic waste of flaxseed as a source of carbon for submerged cultivation of Penicillium simplicissimum is an environmentally friendly, feasible and cost effective for the production of xylanases option. Various biotechnological applications such as use in animal feed, in the paper industry and in second generation ethanol, among others, allow a substantial increase in the value of this substrate, allowing an expansion of the use of flaxseed, plus the use of oil, without increasing acreage
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular
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Perraud, Xavier. "Characterization of lipoxygenases and associated enzymes from selected microorganisms." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0032/NQ64642.pdf.
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Schneider, Willian Daniel Hahn. "Secretômica e atividades enzimáticas da linhagem selvagem 2HH e do mutante S1M29 de Penicillium echinulatum." reponame:Repositório Institucional da UCS, 2014. https://repositorio.ucs.br/handle/11338/884.
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O emprego de enzimas lignocelulolíticas secretadas por microrganismos para a produção de etanol de segunda geração tem motivado pesquisas na área da engenharia de processos fermentativos, genômica e secretômica. Entre os microrganismos com potencial para a produção de celulases destacam-se variantes genéticos do fungo filamentoso Penicillium echinulatum caracterizados por produzirem altos títulos enzimáticos. Neste trabalho, estudouse a secretômica da linhagem selvagem 2HH e do mutante S1M29 de P. echinulatum, em cultivo submerso, empregando diferentes fontes de carbono: glicose, glicerol, celulose e bagaço de cana-de-açúcar pré-tratado por explosão a vapor. Análises enzimáticas possibilitaram identificar que P. echinulatum produz celulases, hemicelulases, esterases e, em menor proporção, pectinases e amilases. Outrossim, os maiores títulos enzimáticos para a maioria das enzimas foram verificados na linhagem mutante. Nos meios formulados com bagaço de cana-de-açúcar ou celulose verificou-se a indução das maiores produções enzimáticas para ambas as linhagens. A análise do secretoma por 1D-PAGE seguido de LCMS/ MS das amostras de 96 horas de cultivo permitiu identificar que em ambas as linhagens há predominância de enzimas CAZy, sendo celulases, hemicelulases e enzimas degradadoras de parede celular fúngica as mais predominantes. Celobiohidrolases, endoglicanases, β-glicosidases, xilanases, β-xilosidases e mananases foram identificadas e, em quantidades menores, ligninases, pectinases, amilases, esterases e solenina, entre outras proteínas (adesão, chaperonas, oxidoredutases, proteases, peptidases, lipases, glutaminases e hipotéticas). Os meios elaborados com glicose ou glicerol foram utilizados pelo fungo para a produção de amilases, ligninases e enzimas degradadoras da parede celular fúngica. Destaca-se a secreção 2 a 3 vezes maior de celulases pela linhagem mutante, sendo que o meio de cultivo elaborado com bagaço de cana-de-açúcar proporcionou a secreção de maiores quantidades de celulases para o mutante. Nesta condição, o complexo celulolítico da linhagem S1M29 constitui-se de 55% de celobiohidrolases, 38% de endoglicanases e 1% de β-glicosidases. Estes dados sugerem que durante o melhoramento genético do fungo ocorreram mudanças, embora não direcionais, possivelmente em nível da regulação da expressão gênica, modificações póstraducionais e alterações na capacidade para secretar proteínas extracelulares que tornaram a linhagem mutante S1M29 com potencial para ser empregada na hidrólise de lignocelulósicos.Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-02-12T12:51:30Z No. of bitstreams: 1 Dissertacao Willian Daniel Hahn Schneider.pdf: 2455680 bytes, checksum: 6b4ce5e7e2a0bd9fc7714d8979b4ee29 (MD5)
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The use of lignocellulolytic enzymes secreting by microorganisms for the production of second-generation ethanol has motivated research in the field of fermentation processes engineering, genomics and secretomics. Among the microorganisms with the potential for cellulases production are genetic variants of the filamentous fungus Penicillium echinulatum characterized by produce high enzymatic titers. In this work, it was studied the secretome of the wild type 2HH and mutant S1M29 of P. echinulatum in submerged cultivation on different carbon sources: glucose, glycerol, cellulose and sugar cane bagasse pretreated by steam explosion). Enzymatic analysis allowed verifying that P. echinulatum produces cellulases, hemicellulases, esterases, and minor proportion, pectinases and amylases. Furthermore, the major enzymes titers for most enzymes dosed were verified in the mutant strain. It was verified in the media formulated with sugar cane bagasse or cellulose the induction of the highest enzyme production for both strains. The analysis of secretome by 1DPAGE followed by LC-MS/MS, of samples collected at 96 hours of cultivation, showed that in both strains there is a predominance of CAZy enzymes, being cellulases, hemicellulases and fungal cell wall degrading enzymes the most prevalent. Cellobiohydrolases, endoglucanases, β-glucosidase, xylanase, endoxylanase, β-mannanases and xylosidases were identified and, in smaller amounts, ligninases, pectinases, amylases, esterases and swollenin, among other proteins (adhesion, chaperones, oxidoreductases, proteases, peptidases, lipases, glutaminases and hypothetical). The media elaborated with glucose or glycerol were used for producing of amylases, ligninases and fungal cell wall degrading enzymes. Highlights the secretion of 2-3 times more cellulases by the mutant, being the medium prepared with sugar cane bagasse afforded the secretion of large cellulases quantities for the mutant. In this condition, the cellulolytic complex of S1M29 strain consists of 55% cellobiohydrolases, 38% endoglucanases and 1% β-glucosidase. These data suggest that during the genetic improvement of the fungus changes occurred, although not directional, possibly at the level of regulation of gene expression, post-translational modifications and changes in the ability to secrete extracellular proteins, that have made the mutant S1M29 a potential strain to be employed in hydrolysis of lignocellulose.
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Ritter, Carla Eliana Todero. "Efeito da adsorção e filtração na produção de celulases e xilanases." reponame:Repositório Institucional da UCS, 2015. https://repositorio.ucs.br/handle/11338/1020.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES.
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Sautour, Marc. "Physiologie, biochimie et modélisation de la germination et de la croissance de Penicillium chrysogenum et autre micromycètes." Dijon, 2001. http://www.theses.fr/2001DIJOS024.
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La microbiologie prévisionnelle vise à prévoir le comportement de microorganismes à l'aide de modèles mathématiques. Cependant, les travaux de recherche dans ce domaine ne concernent, en majorité, que le règne bactérien et la modélisation de la croissance des micromycètes n'a pas du tout reçu le même niveau d'attention. L'objectif de ce travail a donc été l'élaboration de modèles décrivant la croissance de champignons filamenteux tel que Penicillium chrysogenum, en fonction de paramètres environnementaux. Une première partie est conscrée à la sélection des facteurs environnementaux à prendre en compte pour la construction des modèles mathématiques. Par une approche mettant en œuvre des plans d'expériences, il est mis en évidence les effets prédominants de l'activité de l'eau (aw) et de la température sur la germination des spores de P. Chrysogenum. La deuxième partie concerne l'élaboration de modèles décrivant l'influence de l'aw et/ou de la température sur le développement mycélien de P. Chrysogenum et d'autres micromycètes. Enfin une troisième partie est consacrée à la recherche de marqueurs biochimiques de la germination des spores de P. Chrysogenum. . .
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du, Plooy W., T. Regnier, and S. Combrinck. "Essential oil amended coatings as alternatives to synthetic fungicides in citrus postharvest management." Elsevier, 2009. http://encore.tut.ac.za/iii/cpro/DigitalItemViewPage.external?sp=1001494.
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Abstract
A newapproach to the control of postharvest pathogens, while maintaining fruit quality, has been implemented
by the application of essential oil amended coatings to citrus. This approach eliminates the need
for synthetic fungicides, thereby complying with consumer preferences, organic requirements and reducing
environmental pollution. In vitro studies indicated that the essential oils and some of the terpenoid
components tested were active against Penicillium digitatum. In a series of subsequent semi-commercial
and commercial trials, Mentha spicata and Lippia scaberrima essential oils, as well as pure (d)-limonene
and R-(−)-carvone were incorporated into a variety of commercial citrus coatings. These amended coatingswere
applied postharvest to ‘Tomango’ oranges in the absence of the standard fungicide dip. Excellent
disease control was achieved with the amended coatings, while measured quality parameters indicated
that overall fruit quality was maintained. Moreover, moisture loss was decreased significantly in fruit
treated with essential oil enriched coatings. The efficacy of amended coatings as a viable alternative or
supplement to existing fruit protection strategies was demonstrated in a commercial trial.
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du, Plooy W., S. Combrinck, and T. Regnier. "Essential oil amended coatings as alternatives to synthetic fungicides in citrus postharvest management." Elsevier, 2009. http://encore.tut.ac.za/iii/cpro/DigitalItemViewPage.external?sp=1001711.
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a b s t r a c t
A newapproach to the control of postharvest pathogens, while maintaining fruit quality, has been implemented
by the application of essential oil amended coatings to citrus. This approach eliminates the need
for synthetic fungicides, thereby complying with consumer preferences, organic requirements and reducing
environmental pollution. In vitro studies indicated that the essential oils and some of the terpenoid
components tested were active against Penicillium digitatum. In a series of subsequent semi-commercial
and commercial trials, Mentha spicata and Lippia scaberrima essential oils, as well as pure (d)-limonene
and R-(−)-carvone were incorporated into a variety of commercial citrus coatings. These amended coatingswere
applied postharvest to ‘Tomango’ oranges in the absence of the standard fungicide dip. Excellent
disease control was achieved with the amended coatings, while measured quality parameters indicated
that overall fruit quality was maintained. Moreover, moisture loss was decreased significantly in fruit
treated with essential oil enriched coatings. The efficacy of amended coatings as a viable alternative or
supplement to existing fruit protection strategies was demonstrated in a commercial trial.
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Légier, Valérie. "Réactions d'acylations d'alcools en réacteur semi-continu catalysées par des lipases endogènes périplamiques de nouvelles souches fongiques." Aix-Marseille 3, 1992. http://www.theses.fr/1992AIX30026.
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Dans la premiere partie de ce travail nous nous sommes interesse a deux souches fongiques: fusarium oxysporum et penicillium cyclopium. Ces deux champignons inferieurs ont ete isoles au laboratoire a partir d'un milieu naturel. F. Oxysporum et p. Cyclopium secretent tous deux, outre une lipase exogene, une lipase endogene thermoresistante dont nous avons determine les proprietes physico-chimiques. Par ailleurs nous nous sommes egalement attaches a optimiser la croissance et la production de lipase endogene de ces souches. Le second volet de ce travail a ete la mise en application des lipases myceliennes periplasmiques de ces deux champignons et d'une autre espece. Rhizopus arrhizus, pour la synthese d'esters. Pour realiser ces syntheses nous avons mis au point un reacteur a lit fixe avec recyclage. Les lipases de p. Cyclopium et de r. Arrhizus presentant les meilleures aptitudes biosynthetiques, nous avons optimise les conditions d'obtention du laurate de lauryle en utilisant la methodologie de la recherche experimentale et les reseaux uniformes de doehlert. Enfin nous avons termine notre etude par l'application des lipases de p. Cyclopium et r. Arrhizus a l'esterification d'alcools tertiaires et de diols; nous avons egalement aborde dans ce travail l'interesterification catalysee par la lipase de p. Cyclopium en vue de la synthese de triglycerides modeles pour des etudes de nutrition
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Lafond, Mickaël. "Etude du mécanisme d'action du sécrétome de Penicillium funiculosum sur la digestion des polysaccharides des blés Caphorn et Isengrain par la méthode in vitro TIM-I - Expression, caractérisation et étude de spécificité de l'endo-β(1,4)-xylanase D." Aix-Marseille 3, 2010. http://www.theses.fr/2010AIX30008.
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Le Rovabio™ Excel est un cocktail enzymatique commercialisé par la société Adisseo et utilisé en nutrition animale pour augmenter la digestibilité des céréales comme le blé. Le but de notre étude a été dans un premier temps d'évaluer, de localiser dans l'espace et dans le temps et de comprendre l'effet du Rovabio sur la digestibilité des blés Caphorn et Isengrain à l'aide d'un modèle in vitro de digestion, le TIM-I. La digestion a été suivie à l'aide de la matière organique, des sucres réducteurs, des monosaccharides totaux et de certains produits finaux. La digestibilité de la matière organique pour les deux blés obtenue avec le T1M-1 est la même que celle obtenue chez le poulet. Le Rovabio améliore la digestibilité des deux blés principalement entre 180 et 360 min de digestion avec tous les marqueurs utilisés. L'effet positif du cocktail enzymatique sur le blé Caphom, est majoritairement jéjunal contrairement au blé Isengrain où l'effet est principalement iléal. Dans un deuxième temps et dans le but d'augmenter l'efficacité du Rovabio sur des substrats riches en glucides non digestibles de types arabinoxylanes, nous avons exprimé la xylanase D (GH10) de P. Funiculosum dans le système eucaryote Pichia pastoris. Après l'avoir purifiée, caractérisée biochimiquement et comparée au niveau enzymologique aux autres xylanases du même champignon, nous avons évalué son efficacité hydrolytique sur les deux blés cités précédemment par la méthode TIM-I. Nous avons pu mettre en évidence un intérêt indéniable de cette enzyme qui représente à dose équivalente, environ 80 % de l'efficacité du cocktail enzymatiqueRovabio™ Excel is a food additive marketed by Adisseo and used in animal nutrition to increase the digestibility of cereals like wheat. It is an enzymatic cocktail produced by Penicillium funiculosum filamentous fungi. The goal of our study was initially to evaluate, locate and understand the effect of Rovabio on the digestibilities of wheats Caphorn and Isengrain using TIM-I, an in vitro model for the gastro-intestinal digestive tract. The digestibility markers used were organic matter and the liberated reducing sugars, total monosaccharides and some end products. Whatever the marker used, we highlighted a positive effect of Rovabio on the digestion of both wheat cultivars, especially between 180 and 360 min of digestion. The positive effect of Rovabio on digestion of Caphorn occurred mainly in jejuna] compartment, whereas that on Isengrain occurred on the ileal one. This was linked to the higher content of Caphom soluble polysaccharides as compared to Isengrain. In addition, with an aim of increasing the effectiveness of Rovabio on the substrates rich in non digestible polysaccharides (arabinoxylans), we expressed the xylanase D (GH10) of P. Funiculosum in the eukaryote system Pichia pastoris. After its purification, biochemical characterization and comparison with xylanases B and C (GH11) under kinetic viewpoint, we tested its hydrolytic effectiveness on Caphorn and Isengrain wheat cultivars TIM-I model. We could highlight an undeniable interest of this enzyme since it performed approximately 80% of the enzymatic cocktail effectiveness
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Liu, Qintao. "Conversion of triacylglycerols into monoacylglycerols by penicillium roquefortii." Thesis, Sheffield Hallam University, 1998. http://shura.shu.ac.uk/3098/.
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The synthesis and use of monoacylglycerols in food systems havc been reviewed. The use of monoacylglycerols alone or in combination with free fatty acids as food preservatives has been discussed. Model systems have been devised to produce monoacylglycerols (MAGs) from butter and Shea oils with two strains of Penicillium roquefortii, FRR 2456 (isolated from a spoilt melon) and Wisbey PJ (a commercial dairy strain) A semi-micro method was developed using Preparative Thin Layer Chromatography (PTLC) and Gas Chromatography - Mass Spectrometry (GC-MS) of the MAG trimethylsilyl ether derivatives to determine the identification, fatty acid composition and structural isomers of the individual MAG. The main monoacylglycerols produced by spores and emerging mycelium were 1(3)-monoacylsn-lycerols (in suspension culture). Monopalmitin was the major MAG from butter and Shea oils. Monoacylglycerols produced by fungal mycelium (in solid-state culture) were mainly the 1 (3 )-monoacyl-sn-glycerols although approximately 30% were present as 2-monoacyl-snglycerols. Again the main MAG was monopalmitin. It suggested that P. roquefortii produced two lipases, one during germination with specificity to the sn-2 position in the original triacylglycerols (TAGs) and one L3-specific during growth of the fungal mycelium. In addition, flavour compounds, methyl ketones and y-lactones, were found in solid-state culture. The composition of the MAGs formed by lipolysis using a commercial lipase (E.C.3.1.1.3) with 1,3- specificity gave the expected 2-isomers when butter oil was the substrate but gave 1 (3)monostearin rather than the expected 2-monoolein when Shea oil was the substrate. It suggested that acyl migration occurred due to the reactive nature of the original oleate group at the sn-2 position in the Shea oil TAGs. There were no significant differences with fungal strain or temperature of incubation (10 °C and 25°C) on the composition of the MAGs. The mechanism of formation of MAGs from butter and Shea oils has been discussed. It has been suggested that l(3)-MAGs together with free fatty acids may be part of a natural antimicrobial system in high pH foods such as blue mould-ripened cheese where growth of foodborne pathogens such as Listeria monoGytogcnes can be a problem from time to time
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Renno, Didier. "Molecular genetics and physiological studies of Penicillium chrysogenum." Thesis, University of Westminster, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306433.
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Krasniewski, Isabelle. "De l'induction de la conidiation à la qualité des conidies de Penicillium camemberti en culture de surface : une étude pluridisciplinaire." Dijon, 2005. http://www.theses.fr/2005DIJOS057.
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La conidiation de Penicillium camemberti, un ferment d'affinage, a été étudiée en culture de surface, d'un point de vue physiologique et moléculaire. Le développement d'une technique d'inoculation par trempage de filtre a permis de travailler sur un matériel biologique homogène et de réaliser des transferts de mycélium d'un milieu à un autre. Dans ces conditions de culture, il a été démontré que le nitrate de potassium stimule la conidiation, alors que le sulfate d'ammonium l'inhibe. Le temps nécessaire à l'acquisition de la compétence a été évalué à 51 heures. Différents facteurs nutritionnels ont ensuite été testés pour déterminer leur influence, soit positive, soit négative, sur la conidiation de Penicillium camemberti. L'étude de l'effet du calcium et du rapport carbone sur azote (C/N) a alors été approfondie. D'une point de vue moléculaire, nous avons identifiés les gènes brlA et wetA, potentiellement impliqués dans le contrôle de la conidiation de Penicillium camemberti. L'analyse de l'expression du gène brlA a mis en évidence une corrélation entre le niveau d'expression de ce gène et le taux de conidiation. La qualité des conidies a été étudiée en fonction des conditions de culture, notamment liquide et solide. Le dosage du contenu des conidies en polyols et tréhalose ainsi qu'une analyse ultrastructurale nous a permis d'interpréter les différences de résistance à la lyophilisation et de capacité de couverture d'un milieu fromageThe conidiation of Penicillium camemberti, a ripening fungus, was examined on solid media from a physiological and molecular point of view. The development of a technique of inoculation by soaking filters made possible to work on a homogeneous biological material and to transfer easily mycelium from a medium to an another. Under these conditions of culture, it was shown that potassium nitrate stimulates the conidiation, whereas ammonium sulfate inhibits it. The time necessary to acquire the competence was evaluated at 51 hours. Various nutritional factors were then tested to determine their influence, either positive, or negative, on the conidiation of Penicillium camemberti. The effect of calcium and the carbon to nitrogen ratio (C/N was then thoroughly studied. From a molecular point of view, we identified the brlA and wetA genes, potentially implied in the control of the conidiation of Penicillium camemberti. The analysis of the expression of the brlA gene highlighted a correlation between the level of expression of this gene and the rate of conidiation. The quality of the conidies was studied according to the conditions of culture, in particular liquid and solid. The determination of the polyols and trehalose contents of the conidies as well as an ultrastructural analysis enabled us to interpret differences into resistance to freeze-drying and capacity of covering a cheese medium
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Cardoso, Patrícia Gomes. "Organização do gene de pectina liase em Penicillium expansum e obtenção de linhagens recombinantes de Penicillium griseoroseum com alta produção de pectina liase." Universidade Federal de Viçosa, 2004. http://www.locus.ufv.br/handle/123456789/10691.
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A seqüência de nucleotídeos contendo o gene ple1 que codifica pectina liase em Penicillium expansum foi clonada. Esta seqüência contém 874 nucleotídeos da região promotora, 1342 nucleotídeos da região codificadora e 469 nucleotídeos da região terminadora. A região codificadora do gene ple1 é interrompida por dois íntrons, de 101 e 116 nucleotídeos, confirmados pela seqüência do cDNA. Na seqüência da região promotora de ple1 foi observado cis-elementos envolvidos na regulação da expressão desse gene, como TATA box (TATATAA) na posição -91 do códon de início da tradução, sendo esta seqüência precedida por uma região rica em pirimidinas e CAAT box (CCAATT) a -568 do códon de inicio da tradução. A proteína PLE1, deduzida a partir da seqüência de nucleotídeos possui 374 resíduos de aminoácidos, com massa molecular estimada de 40,1 KDa e pI calculado de 9,46. Análise por hibridização do DNA total de P. expansum e P. griseoroseum com um fragmento de DNA de 2,1 Kb que corresponde ao gene pelA de A. niger, indicou organização semelhante dos genes de PL no genoma destes fungos. A expressão do gene ple1, avaliada pela hibridização do RNA total com um fragmento de DNA que corresponde à região estrutural do gene ple1 mostrou que o transcrito foi detectado durante todo tempo de cultivo em presença de pectina. No entanto, quando cultivado em presença de sacarose, o transcrito somente foi detectado em 12 e 24 horas de cultivo, com adição de extrato de levedura. A caracterização morfológica de P. expansum e P. griseoroseum mostrou diferenças nítidas tanto no diâmetro quanto na coloração do verso das colônias destes fungos. Diferenças na organização da região subtelomérica dos cromossomos de P. expansum e P. griseoroseum, foram observadas quando o plasmídeo pTEL13 foi utilizado como sonda. A região ITS do rDNA de P. expansum tem 600 pb e de P. griseoroseum 594 pb. As linhagens transformantes obtidas com os plasmídeos pPlg1 e pNPG1, apresentaram aumentos na atividade de PL de no máximo 3 vezes. Análise por hibridização do DNA total dos transformantes indicou a ocorrência de integrações homólogas e heterólogas de pelo menos duas cópias do gene plg1. Foi construído um vetor de expressão, denominado pAN52-Plg1 que continha o gene plg1 sob o controle do promotor forte constitutivo do gene (gpdA) de A. nidulans. A transformação da linhagem mutante PG63 utilizando este vetor e o pNPG1, resultou na obtenção de uma linhagem recombinante (105) com aumento de 58 vezes na atividade de PL, quando cultivada em presença de glicose como fonte de carbono. Seis linhagens recombinantes foram avaliadas por hibridização apresentando integração heteróloga de mais de uma cópia do gene plg1 no genoma. Avaliação das proteínas extracelulares por eletroforese (SDS-PAGE) mostrou a presença de uma banda nítida e forte de aproximadamente 40 KDa presente no sobrenadante de cultivo da linhagem recombinante 105 que corresponde a PLG1. A atividade específica aparente de PL sintetizada por esta linhagem foi 44 e 27 vezes maior do que aquela obtida para linhagem mutante PG63 e de uma preparação comercial de pectinases “Citrus Clear”, respectivamente. O cultivo da linhagem recombinante 105 em meio contendo caldo de cana promoveu uma atividade de PL 132 vezes maior do que a atividade obtida pela linhagem PG63 cultivada nesta mesma fonte de carbono. A atividade de PL da linhagem recombinante 105, cultivada em diferentes volumes de meio, aumentou linearmente com o tempo. Pectina cítrica, quando utilizada como substrato, promoveu maior atividade de PL. A enzima mostrou ser estável em ampla faixa de pH e temperatura de armazenamento. Estes resultados mostram que a linhagem recombinante 105 é promissora para produção de PL em escala industrial, principalmente, para aplicação na indústria de sucos e vinhos, onde o emprego apenas da PL apresenta várias vantagens na qualidade do produto final.
The sequence of the pectin lyase-enconding gene ple1, from Penicillium expansum, was cloned. This sequence consists of 874 nucleotides of the promoter region, 1342 nucleotides of the coding region, and 469 nucleotides of the terminator region. The coding region of ple1 is interrupted by two introns of 101 and 116 nucleotides, confirmed by cDNA sequencing. Two cis-elements, TATA box (TATATAA) and CAAT box (CCAATT), were found at the positions 91 and 568 upstream from the translation start code, respectively. The deduced amino acid (aa) sequence (374 aa) of PLE1 has an estimated molecular mass of 40.1 KDa and a calculated pI of 9.46. One putative N- glycosylation site was found at Asn 112 . Southern blot analysis of genomic DNA from P. expansum and P. griseoroseum with a 2.1 kb-DNA fragment of the gene pelA from A. niger indicated that this gene is similarly organized in the genomes of these fungi. The hibridization of total RNA with a fragment of the coding region of ple1 showed that the transcript was detected diring the whole of cultivation in a medium containing pectin. However, when the fungus was cultivated in the presence of sucrose with addition of yeast extract, the transcript was detected only at 12 and 24 hours of cultivation. The morphologic characterization of P. expansum and P. griseoroseum showed clear differences in the xdiameter and in the coloration of the botton of the colonies. Differences in the organization of the subtelomeric region of chromosomes of P. expansum and P. griseoroseum, were observed when the plasmid pTEL13 was used as a probe. The ITS region of the rDNA of P. expansum has 600 bp and that of P. griseoroseum 594 bp. The transformants obtained with the plasmids pPlg1 and pNPG1 presented increases in the activity of PL of at the least 3 times the active of the wild type. The hibridization profile of the genomic DNA of the transformants showed the occurrence of homologous and heterologous integrations of at least two additional copies of the gene plg1 in the genome. It was not possible to define the exact number of copies and correlate it with PL activity. An expression vector was built, designated pAN52-Plg1 that contained the gene plg1 under the control of the strong constitutive promoter gpdA of A. nidulans. The transformation of the mutant strains PG63 using this vector and the pNPG1 resulted in the isolation of a recombinant strain (105) with a PL activity 58 times higher than that of the wild type, when cultivated in glucose as the sole carbon source. Six recombinants were analised by hybridization that presented heterologous integration of more than one copy of plg1. Evaluation of the extracellular proteins by electrophoresis (SDS-PAGE) showed the presence of a clear and strong band of approximately 40 KDa in the cultivation filtrate of the recombinant 105. This band corresponded to PLG1. The apparent specific activity of PL synthesized by this strain was 44 and 27 times higher than that obtained for the strain PG63 and for a commercial preparation of pectinolytic enzymes (Citrus Clear), respectively. The cultivation of the recombinant strain 105 in a medium containing sugar cane juice promoted a PL activity that was 132 times higher than that for the strain PG63 cultivated with the carbon source. PL activity of the recombinant strain 105, cultivated in different volumes of medium, increased linearly with time. Citric Pectin, when used as substrate, supported higher PL activity. The enzyme was stable in a wide range of pH and storage temperature.
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Valentines, i. Escolà M. Carme. "Bases bioquímiques de resistència a Penicillium expansum en poma." Doctoral thesis, Universitat de Lleida, 2005. http://hdl.handle.net/10803/8373.
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L'objectiu principal d'aquesta tesi era l'estudi dels mecanismes bioquímics que determinen la resistència de les pomes contra un atac de Penicillium expansum. Els diferents estudis es van centrar en determinar el paper que el peròxid d'hidrogen juga en la resistència, directament o com a inductor d'altres processos també involucrats en la resistència dels fruits, com és la lignificació. Es van estudiar també les possibles relacions entre la capacitat d'enfosquiment enzimàtic del fruit i la resistència. D'altra banda, i amb els objectius de comprovar els resultats obtinguts en fruits sencers i d'establir un model d'interpretació, es va utilitzar un cultiu cel·lular de poma. Tots aquests estudis tenien com finalitat determinar les mecanismes bioquímics claus involucrats en la resistència a P. expansum i proposar un marcador de susceptibilitat en pomes. Els resultats presentats en aquesta tesi demostren la participació del peròxid d'hidrogen en la resistència, ja que s'observava una acumulació d'aquest compost en fruits en resposta a l'atac fúngic. Aquesta acumulació era present en fruits immadurs (fruits resistents), però no en els fruits madurs (fruits sensibles). En fruits sencers, la generació de peròxid d'hidrogen estava determinada per un increment en l'activitat de l'enzim superòxid dismutasa (SOD) i una disminució dels enzims involucrats en la degradació del peròxid d'hidrogen, especialment de la peroxidasa (POX). Els resultats obtinguts en el model in vitro confirmen la intervenció del peròxid d'hidrogen en el procés de resistència segons un model d'interacció de tipus compatible. En aquest model, l'acumulació del peròxid estava principalment determinada per l'acció de l'enzim NADPH oxidasa, mentre que els altres enzims (SOD, CAT i POX) eren secundaris. Quan el cultiu cel·lular de poma s'inoculava amb suspensió aquosa de P. expansum filtrada, el model es comportava com un model d'interacció de tipus incompatible, fet que mostra l'existència d'un inductor hidrosoluble en el patogen que pot promoure l'acumulació de peròxid d'hidrogen. Els resultats obtinguts en fruit sencer, també demostren que el peròxid d'hidrogen actua principalment promovent l'acumulació de lignines en la zona ferida, i que el procés d'enfosquiment enzimàtic no està vinculat a la resistència dels fruits. Els canvis metabòlics descrits amb anterioritat semblen ésser induïts principalment per l'acció de la ferida i no són constitutius. Com a conseqüència, cap d'aquests paràmetres podria ser utilitzat com a marcador de susceptibilitat del fruit a la collita.El principal objetivo de la presente tesis era el estudio de los mecanismos bioquímicos que determinan la resistencia de las manzanas contra un ataque por Penicillium expansum. Los diversos estudios se centraron en determinar el papel que el peróxido de hidrógeno juega en la resistencia, directamente o como inductor de otros procesos también involucrados en la resistencia de los frutos, como es la lignificación. Se estudiaron también las posibles relaciones entre la capacidad de empardecimiento enzimático del fruto y la resistencia. Por otro lado, y con los objetivos de comprobar los resultados obtenidos en fruto entero y de establecer un modelo de interpretación, se utilizó un cultivo celular de manzana. Todos estos estudios tenían como finalidad determinar los mecanismos bioquímicos clave involucrados en la resistencia a P. expansum y proponer un marcador de susceptibilidad de las manzanas. Los resultados presentados en esta tesis demuestran la participación del peróxido de hidrógeno en la resistencia, ya que se observaba una acumulación de este compuesto en frutos en respuesta al ataque fúngico. Esta acumulación se daba en frutos inmaduros (frutos resistentes), pero no en los frutos maduros (frutos sensibles). En frutos enteros, la generación de peróxido de hidrógeno estaba determinada por un incremento de actividad de la enzima superòxido dismutasa (SOD) y una disminución de las enzimas involucradas en la degradación del peróxido de hidrógeno, especialmente de la peroxidasa (POX). Los resultados obtenidos en el modelo in vitro confirman la intervención del peróxido de hidrógeno en el proceso de resistencia según un modelo de interacción de tipo compatible. En este modelo, la acumulación del peróxido estaba principalmente determinada por la acción de la enzima NADPH oxidasa, mientras que las otras enzimas (SOD, CAT y POX) eran secundarias. Cuando el cultivo celular de manzana se inoculaba con una suspensión acuosa de P. expansum filtrada, el modelo se comportaba como un modelo de interacción de tipo incompatible, hecho que muestra la existencia de un inductor hidrosoluble en el patógeno que puede promover la acumulación de peróxido de hidrógeno. Los resultados obtenidos en fruto entero, también muestran como el peróxido de hidrógeno actúa promoviendo la acumulación de ligninas en la zona dañada y que el proceso de empardecimiento enzimático no está relacionado con la resistencia de los frutos. Los cambios metabólicos descritos anteriormente parecen ser inducidos principalmente por la acción de la herida y no son constitutivos. En consecuencia, ninguno de estos parámetros podría ser utilizado como marcador de susceptibilidad del fruto a la cosecha.
The main objective of this thesis was to study the biochemical mechanisms that determine the resistance of apple in response to an attack by Penicillium expansum. Emphasis was given in determining the role that hydrogen peroxide played in the resistance, directly or in relation to the induction of other processes also involved in fruit resistance, such as lignification. The possible relationship between the enzymatic browning potential of the fruit and resistance was also studied. On the other hand, studies on apple cell suspension were also carried out in order to verify the results obtained in whole fruit and establish an interpretation model. The purposes of all these studies were to determine the key biochemical mechanisms involved in the resistance to P. expansum and to propose a marker of apple susceptibility. As shown in our results, hydrogen peroxide appeared to play an underlying role in resistance, because an accumulation of this compound was observed in response to the fungal attack. This accumulation was detected in immature fruit (resistant fruit) but not in mature fruit (susceptible fruit). In whole fruit, the generation of hydrogen peroxide was mainly determined by the increase of superoxide dismutase enzyme activity (SOD) but also by a decrease of H2O2-scavenging enzymes, especially peroxidase (POX). The results obtained in the in vitro model confirmed the contribution of hydrogen peroxide in the resistance process according to a compatible interaction model. In this model the accumulation of the peroxide was mainly determined by the action of NADPH oxidase enzyme, whereas the other enzymes (SOD, CAT and POX) were secondary. When apple cell suspension was inoculated with the filtered aqueous suspension of P. expansum, the model behaved as an incompatible model, showing the existence of a pathogen-related hydrosoluble elicitor that may also elicits the accumulation of hydrogen peroxide. The results obtained in whole fruit also showed that hydrogen peroxide is mainly involved in defence mechanism through its action on lignin accumulation and that the enzymatic browning process was not linked to fruit resistance. All the parameters involved in apple resistance were not constitutive and appeared to be triggered by the wounding response. In consequence, none of these parameters could be used to predict the fruit susceptibility at harvest.
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Rivera, Karol Geraldina. "Taxonomy, systematics and DNA barcoding of selected Penicillium groups." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28200.
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Cytochrome c oxidase subunit 1 (Cox 1) is the barcode for many animal groups, protists, and macroalgae. Previously in fungi, the efficiency of Cox 1 as a genetic marker was only analysed in Penicillium subgenus Penicillium and Leohumicola spp. In this thesis, two species isolated from the intestinal tracts of caterpillars from Costa Rica, and two potential species complexes, P. sclerotiorum and P. oxalicum belonging to Penicillium subgenus Aspergilloides and Furcatum, were studied using the Genealogical Concordance Concept (Gee) recognition criterion and barcoding methods. Analyses with beta-tubulin (BenA), the nuclear internal transcriber spacer (ITS) region, Cox 1, translation elongation factor 1-alpha (TEF1-alpha), and calmodulin (CaM) revealed that the Penicillium species isolated from Costa Rica are undescribed, and that P. sclerotiorum is a complex of seven phylogenetic species (including the Costa Rican species) that fit the prevailing morphological concept of P. sclerotiorum. The phylogenetic species were compared and newly discovered diagnostic morphological characters were used to create a taxonomic key to the species of the complex. The new species are formally described as P. guanacastense, P. mallochii, P. krugii, P. cainii, P. jacksonii and P. ciebiessum. Analyses of multiple strains of P. oxalicum revealed that it is a single phylogenetic species, despite having a world wide distribution, an unusually high degree of morphological variation, and a diversity of ecological roles.
Cox1 proved to be a good barcode for identifying the selected Penicillium groups, and provided a species level resolution of 83.3%. ITS provided the same resolving ability. BenA (91.7%), TEF1-alpha (100%) and CaM (100%) provided a higher species level resolution than Cox1, but BenA, TEF1-alpha, and CaM were difficult to amplify or sequence for some Penicillium groups. A secondary barcode marker is suggested in addition to Cox1 for Penicillium.
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Nakagi, Vanessa de Souza [UNESP]. "Caracterização da atividade da fosfatase ácida de Penicillium implicatum." Universidade Estadual Paulista (UNESP), 2007. http://hdl.handle.net/11449/94888.
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A produção de fosfatase ácida extracelular pelo fungo Penicillium implicatum foi estudada em diferentes concentrações de fosfato no meio de cultivo e na presença e ausência de agitação. A produção de fosfatase ácida extracelular repressível foi cerca de 15 vezes maior em meio agitado e na presença de 50 ìM de KH2PO4 quando comparada ao tratamento controle. A enzima foi purificada 19 vezes em Fenil Sepharose CL-4B, e apresenta uma atividade específica de 27,3U/mg. A enzima é um monômero de massa molecular (Mr) da ordem de 45KDa. O pH ótimo aparente das atividades p-NFFásica e de pirofosfatase é de 5,5. Os íons Zn, Mg, e Co; EDTA, tartarato e iodoacetamida exerceram pouca ou nenhuma influência sobre a atividade da fosfatase ácida de P. implicatum; enquanto que a enzima foi inibida por molibdato e arsenato. A enzima apresenta atividade pirofosfatásica de 297,78U/mg a 4mM de pirofosfato e pH 5,5. A cinética de hidrólise simultânea dos substratos PNFF e PPi mostrou que ambos os substratos ligaram-se ao mesmo sítio. A aplicação da fosfatase na hidrólise de fitato em ração animal sugere uma forma de disponibilizar fosfato para o organismo e também minimizar o impacto ambiental.
The extracellular production of acid phosphatase by Penicillium implicatum was studied at different concentrations of phosphate and in the presence and absence of agitation growth medium. The production of extracellular repressible acid phosphatase was about 15-fold highest in agitated medium with 50ì M of KH2PO4 when compared to the control treatment. The enzyme was purified 19-fold on Phenyl Sepharose CL-4B and showed specific activity of 27,3U/mg. The enzyme is a monomer with molecular weight (Mr) of ~ 45KDa. The optimum pH of the p-NPP and pyrophosphate activities was 5,5. The Zn, Mg, Ca, Mn and Co ions, EDTA and tartarate had no effects; but it was inhibitored by arsenate and molybdate. The pyrophosphate activity was 298U/mg at 4mM and pH 5,5. The kinetics data of simultaneous p-NPP and pyrophosphate hydrolysis, showed that both substrates had bound the same active site. The phosphatase application on fitate hydrolysis in animal food suggests a mean to dispose phosphate to the organism and also to minimize the environmental impact.