This particular work is proposed as a test of the expedience of using the fluorescent-antibody technique as a method for enumeration and identification of certain strains of B. cereus that have been found to be effective in preventing taste and odor in water supplies resulting from certain Actinomycete blooms.

A agricultura moderna exige que as operações de manejo das plantas daninhas sejam economicamente viáveis, e principalmente seguras em termos de minimização da contaminação ambiental. A preocupação sobre a utilização intensiva de herbicidas sintéticos tem sido debatida constantemente, de tal forma que pesquisas têm sido desenvolvidas estrategicamente para a descoberta de novas moléculas herbicidas, baseadas em produtos naturais, para aplicação direta como agente de controle ou utilização indireta como aleloquímico. O solo é habitado por uma grande variedade de microrganismos, sendo que a maioria deles não foi estudada e identificada até o momento, conseqüentemente muitas pesquisas ainda podem ser desenvolvidas com o objetivo de explorar metabólitos secundários produzidos por estes microrganismos. Sendo assim, foi desenvolvida a presente pesquisa com o objetivo de testar e desenvolver uma fase inicial na seleção e descoberta de microrganismos do solo (actinomicetos) com potencial de produção de compostos fitoinibitórios. O método geral de seleção constituiu-se na coleta de amostras de solo de 0-20 cm de profundidade, a partir de áreas com diferentes sistemas de manejo e/ou vegetação. Estas amostras de solo foram então submetidas ao isolamento de actinomicetos (10 g de solo de cada amostra) através da diluição em série e plaqueamento em meio seletivo. Foram isoladas e cultivadas em meio líquido glicerina-caseína 103 colônias, sendo que, a solução de metabólitos acelular foi obtida por centrifugação e filtragem. A partir das soluções contendo os metabólitos foi conduzido um teste de germinação (screening primário) através de bioensaios de laboratório, utilizando como plantas teste pepino (Cucumis sativa) e sorgo (Sorghum bicolor). Com o objetivo de verificar a concentração do meio de cultura que exerceria o mínimo de efeito na germinação e crescimento/desenvolvimento das plântulas, foi realizado um teste de germinação com o meio de cultura sem os metabólitos microbiológicos, sendo avaliado através de análise de regressão dos resultados obtidos. O screening secundário foi realizado em condições de câmara-de-crescimento e consistiu na aplicação do meio de cultura acelular em condições de pré e pós-emergência das plantas de pepino e sorgo. Os screenings primário e secundário resultaram na seleção de sete microrganismos produtores de composto fitoinibitórios, estes utilizados para condução do experimento em condições de casa-de-vegetação, sendo aplicadas as soluções de metabólitos em condições de pré e pós-emergência das plantas de pepino, sorgo, picão-preto (Bidens pilosa) e capim colchão (Digitaria ciliaris). A conclusão principal desta pesquisa foi de que o método para seleção de isolados de actinomicetos, com potencial de produção de fitotoxicinas é adequado, e pode ser utilizado em um programa de descoberta de novos compostos com potencial herbicida, no entanto, os resultados obtidos não permitiram isolar actinomicetos com suficiente potencial fitoinibitório, para ser utilizado de forma direta em um programa de manejo de plantas daninhas na agricultura. / The modern agriculture requires that the weed management practices are economically feasible, and mainly safe for the minimization of the environmental contamination. The concern on the intensive use of synthetic herbicides has been debated constantly, in such way that researches have been developed strategically for the discovery of new herbicides molecules, based on natural products, either for direct application as control agent, or for indirect use as allelochemical. The soil is colonized by a great variety of microorganisms, however most of them were not studied and identified at the moment, consequently many researches still can be done, with the objective of exploring secondary metabolites produced by these microorganisms. Therefore, it was developed this research with the objective of testing and developing a initial process in the selection and discovery of soil microorganisms (actinomycetes) with potential of producing phytoinhibitory compounds. The general method used in the research consisted of soil sampling at 0-20 cm depth, from areas that had been cultivated with different cropping systems and/or vegetation. These soil samples were submitted to actinomycets isolation (10 g of soil per sample) through series dilution in selective medium. It was isolated and cultivated in liquid casein-glycerin medium 103 colonies, being the no cellular metabolite solution obtained by centrifugation and filtration. From the solutions containing the metabolites it was conducted a germination test (primary screening) through a laboratory bioassay with test plants of cucumber (Cucumis sativus) and sorghum (Sorghum bicolor). Objectifying to verify each of the cultivation medium concentrations that would cause the minimum effect on germination and growth/development of seedlings, it was also conducted a germination test with medium without the microbial metabolites, being evaluated through regression analysis results. The secondary screening was done in the growth chamber conditions, and consisted of application of no cellular medium in pre and post emergence conditions of the plants of sorghum and cucumber. The primary and secondary screening resulted in the selection of seven microorganisms producers of phytoinhibitory compounds, these used to conduct an experiment in the greenhouse, being sprayed the metabolic solutions in pre and post emergence conditions of cucumber, sorghum, Bidens pilosa and Digitaria ciliaris. The main conclusion of the research was that the method used for actinomycets selection and isolation, with potential of phytotoxins production is adequate and can be used in a new compounds discovery program with potential herbicide effects; however the results obtained did not allow isolating actinomycets with enough phytoinhibitory potential to be directly used in a program of weed management in agriculture.

A agricultura moderna exige que as operações de manejo das plantas daninhas sejam economicamente viáveis, e principalmente seguras em termos de minimização da contaminação ambiental. A preocupação sobre a utilização intensiva de herbicidas sintéticos tem sido debatida constantemente, de tal forma que pesquisas têm sido desenvolvidas estrategicamente para a descoberta de novas moléculas herbicidas, baseadas em produtos naturais, para aplicação direta como agente de controle ou utilização indireta como aleloquímico. O solo é habitado por uma grande variedade de microrganismos, sendo que a maioria deles não foi estudada e identificada até o momento, conseqüentemente muitas pesquisas ainda podem ser desenvolvidas com o objetivo de explorar metabólitos secundários produzidos por estes microrganismos. Sendo assim, foi desenvolvida a presente pesquisa com o objetivo de testar e desenvolver uma fase inicial na seleção e descoberta de microrganismos do solo (actinomicetos) com potencial de produção de compostos fitoinibitórios. O método geral de seleção constituiu-se na coleta de amostras de solo de 0-20 cm de profundidade, a partir de áreas com diferentes sistemas de manejo e/ou vegetação. Estas amostras de solo foram então submetidas ao isolamento de actinomicetos (10 g de solo de cada amostra) através da diluição em série e plaqueamento em meio seletivo. Foram isoladas e cultivadas em meio líquido glicerina-caseína 103 colônias, sendo que, a solução de metabólitos acelular foi obtida por centrifugação e filtragem. A partir das soluções contendo os metabólitos foi conduzido um teste de germinação (screening primário) através de bioensaios de laboratório, utilizando como plantas teste pepino (Cucumis sativa) e sorgo (Sorghum bicolor). Com o objetivo de verificar a concentração do meio de cultura que exerceria o mínimo de efeito na germinação e crescimento/desenvolvimento das plântulas, foi realizado um teste de germinação com o meio de cultura sem os metabólitos microbiológicos, sendo avaliado através de análise de regressão dos resultados obtidos. O screening secundário foi realizado em condições de câmara-de-crescimento e consistiu na aplicação do meio de cultura acelular em condições de pré e pós-emergência das plantas de pepino e sorgo. Os screenings primário e secundário resultaram na seleção de sete microrganismos produtores de composto fitoinibitórios, estes utilizados para condução do experimento em condições de casa-de-vegetação, sendo aplicadas as soluções de metabólitos em condições de pré e pós-emergência das plantas de pepino, sorgo, picão-preto (Bidens pilosa) e capim colchão (Digitaria ciliaris). A conclusão principal desta pesquisa foi de que o método para seleção de isolados de actinomicetos, com potencial de produção de fitotoxicinas é adequado, e pode ser utilizado em um programa de descoberta de novos compostos com potencial herbicida, no entanto, os resultados obtidos não permitiram isolar actinomicetos com suficiente potencial fitoinibitório, para ser utilizado de forma direta em um programa de manejo de plantas daninhas na agricultura. / The modern agriculture requires that the weed management practices are economically feasible, and mainly safe for the minimization of the environmental contamination. The concern on the intensive use of synthetic herbicides has been debated constantly, in such way that researches have been developed strategically for the discovery of new herbicides molecules, based on natural products, either for direct application as control agent, or for indirect use as allelochemical. The soil is colonized by a great variety of microorganisms, however most of them were not studied and identified at the moment, consequently many researches still can be done, with the objective of exploring secondary metabolites produced by these microorganisms. Therefore, it was developed this research with the objective of testing and developing a initial process in the selection and discovery of soil microorganisms (actinomycetes) with potential of producing phytoinhibitory compounds. The general method used in the research consisted of soil sampling at 0-20 cm depth, from areas that had been cultivated with different cropping systems and/or vegetation. These soil samples were submitted to actinomycets isolation (10 g of soil per sample) through series dilution in selective medium. It was isolated and cultivated in liquid casein-glycerin medium 103 colonies, being the no cellular metabolite solution obtained by centrifugation and filtration. From the solutions containing the metabolites it was conducted a germination test (primary screening) through a laboratory bioassay with test plants of cucumber (Cucumis sativus) and sorghum (Sorghum bicolor). Objectifying to verify each of the cultivation medium concentrations that would cause the minimum effect on germination and growth/development of seedlings, it was also conducted a germination test with medium without the microbial metabolites, being evaluated through regression analysis results. The secondary screening was done in the growth chamber conditions, and consisted of application of no cellular medium in pre and post emergence conditions of the plants of sorghum and cucumber. The primary and secondary screening resulted in the selection of seven microorganisms producers of phytoinhibitory compounds, these used to conduct an experiment in the greenhouse, being sprayed the metabolic solutions in pre and post emergence conditions of cucumber, sorghum, Bidens pilosa and Digitaria ciliaris. The main conclusion of the research was that the method used for actinomycets selection and isolation, with potential of phytotoxins production is adequate and can be used in a new compounds discovery program with potential herbicide effects; however the results obtained did not allow isolating actinomycets with enough phytoinhibitory potential to be directly used in a program of weed management in agriculture.

The purpose of this paper is to present a comparative study of marine bacteria, molds and actinomycetes in regard to their ability to degrade certain pure and mixed complex compounds possibly occurring in the lagoon waste traps of the Texas Gulf Coast. This comparison was made using a differential oxygen uptake as the index of specific compound utilization.

Les actinomycètes sont des bactéries ubiquitaires et certains sont reconnus comme potentiellement pathogènes pour l’Homme, dans l’air de certains lieux de travail. C’est notamment le cas dans l’air des plates-formes de compostage où les concentrations peuvent atteindre des valeurs relativement élevées. L’exposition des salariés à ce type de bioaérosols peut être la cause de pathologies diverses (notamment des pneumopathies d’hypersensibilité). Bien que le problème soit reconnu, la bibliographie démontre un manque de connaissances à propos de l’évaluation du risque : aucune méthode globale de prélèvement et d’analyse n’est, à l’heure actuelle, standardisée pour l’étude de ces bioaérosols, si bien qu’il n’existe aucune relation dose-effets pour la plupart de ces agents ni même de valeur limite d’exposition professionnelle. Les méthodes traditionnellement utilisées ne sont pas sans inconvénient (sous-estimation de la concentration réelle notamment) et le plus souvent non-spécificiques. C’est pourquoi l’objectif de la thèse, ici décrite, est le développement et l’évaluation de la technique de biologie moléculaire qu’est la PCR temps réel pour la quantification de bactéries dans ces bioaérosols. La méthode a tout d’abord été développée et optimisée notamment par le dessin d’oligonucléotides, par la comparaison de protocoles d’extraction d’ADN et par la réalisation de gammes étalons. Elle a ensuite été comparée aux techniques plus traditionnelles, encore largement utilisées, que sont le dénombrement du bacteries cultivable par mise en culture et l’épifluorescence, à la fois sur des cultures de cellules et sur des bioaérosols expérimentaux. Ce n’est qu’après l’avoir caractérisé qu’elle a été appliquée sur des bioaérosols prélevés en conditions réelles d’exposition, sur des plates-formes de compostage.La méthode développée, basée sur une extraction d’ADN et une PCR temps réel, permet la quantification de l’ADN de Thermoactinomyces vulgaris (basée sur l’amplification du gène GyrB), de Thermobifida fusca et T. alba (gène ecf) et des streptomycètes mésophiles (ARNr 23S). La PCR permet l’obtention de résultats fortement corrélés à ceux issus du dénombrement sur milieux gélosés mais offre de réels avantages par rapport à la culture. Comme ces quantifications prennent en compte n’importe quelle forme de la bactérie (cellules végétatives et spores), la PCR dépasse les inconvénients de sous-estimation liés aux méthodes traditionnelles. La technique a un réel avantage de spécificité, elle est répétable et sensible. Les campagnes de prélèvements effectuées sur 5 plates-formes de compostage en France ont permis de mesurer les concentrations en bactéries mésophiles et thermophiles par culture et d’établir celles en Thermoactinomyces vulgaris, Thermobifida sp. et Streptomyces sp. par PCR. L’étude confirme que les activités de compostage sont génératrices de bioaérosols avec parfois des valeurs relativement élevées selon les points échantillonnés. Elle met également en exergue des informations comme la distribution granulométrique du bioaérosol ou l’adéquation entre le type de prélèvement effectué et l’analyse par PCR. Les travaux menés, du développement de la méthode qPCR appliquée au groupe des actinomycètes à son application sur des échantillons environnementaux, apportent de nombreuses données pour la quantification des actinomycètes aéroportés. Ils ont permis d’acquérir des éléments de validation concernant la méthode mise en place et ont livré les seules mesures de concentrations disponibles à l’heure actuelle, pour T. vulgaris, Thermobifida sp., et les streptomycètes mésophiles dans l’air des plates-formes de compostage / Actinomycetes are ubiquitous bacteria and some can be potentially pathogen for Humans in the air of some working areas. It’s notably the case in composting plants where bacteria concentrations can reach high values. Workers exposure to these inhalable bioaerosols can be source of various diseases (hypersensitivity pneumonitis notably). Although this problem is admitted, bibliography reveals a lack of knowledge about risk assessment: currently, none global method for bioaerosols sampling and analysis is standardized. So much that neither dose-effects relationship for most of these bacteria, nor Threshold Limit Value exists. Traditional methods, that are used, have some drawbacks (concentrations underestimation notably) and most often, aren’t specific.It’s the reason why the aim of the thesis, here described, is the development and the evaluation of the biomolecular technique of real time PCR for the quantification of bacteria in these bioaerosols. First, this method was developed and improved by oligonucleotides design, by comparison of many DNA extraction protocols and by the construction of standard ranges. Then, the method was compared to traditional widely used methods such as cultivable bacteria counting by cultures and epifluorescence microscopy, both on cells culture samples and experimental bioaerosols. After this characterization, the analytic method was applied on environmental bioaerosols sampled on real exposure conditions (composting plants).The method that we have developed, based on DNA extraction and real-time PCR, allows the quantification of Thermoactinomyces vulgaris DNA (based on gyrB gene amplification), of Thermobifida fusca and T. alba (ecf gene) and of mesophilic streptomycetes (rDNA 23S). The results obtained by PCR are strongly correlated with those obtained by counting on agar but PCR method offers more advantages than cultures. As PCR quantifies any form of the bacteria (vegetative cells and spores), the method goes over the drawbacks of traditional methods, like underestimation. The method has a real advantage of specificity, it’s also repeatable and sensitive. Sampling campaigns realized on 5 composting plants implanted in France have permitted measuring mesophilic and thermophilic bacteria concentrations by culture and establishing Thermoactinomyces vulgaris, Thermobifida sp. and Streptomyces sp. ones by PCR. The study confirms that composting activities release bioaerosols. And according to the localization of the sampling, the values could be rather high. It also underlines some informations as particles size distribution of the bioaerosol or the adequacy between sampling apparatus and PCR analysis. The works carried out, from qPCR method development for actinomycetes group to its application on environmental samples, give a lot of datas concerning airborne actinomycetes quantification. It permit to validate the developed method and give the only currently available measures for T. vulgaris, Thermobifida sp., and mesophilic streptomycetes in the air of composting plants

Natural products are bioactive secondary metabolites produced by living organisms and are prevalently utilized as pharmaceutical drugs. Marine adapted organisms are a promising source of new natural products possessing unique chemical structures and biological activities. By studying the biosynthetic pathways employed by living organisms to produce natural products, insights into new strategies to generate molecules to combat disease and overcome drug resistance may be gained. This thesis study aimed to uncover the biosynthetic pathway employed by a marine actinomycete, Nocardiopsis sp. CMB-M0232, to catalyze the assembly of the nocardioazines. These molecules are a group of 2,5-diketopiperazine natural products that feature structurally unique functional groups. Nocardioazine A, the hypothesized end product of the nocardioazine biosynthetic pathway, exhibits anticancer activity. Bioinformatics analyses revealed three biosynthetic gene clusters from Nocardiopsis encoding proteins with hypothesized roles in nocardioazine A biosynthesis. Two cyclodipeptide synthases (CDPSs), NozA and NcdA, were biochemically characterized in vivo and in vitro to reveal that both are substrate specific enzymes that utilize tryptophan-charged tRNA substrates to catalyze assembly of cyclo(L-Trp-L-Trp), a proposed precursor of nocardioazines. Fidelity is uncommon amongst characterized CDPSs, making NozA and NcdA important CDPS family additions. This study also aimed to characterize NozD and NozE, two cytochrome P450 homologs with predicted roles as diketopiperazine-tailoring enzymes. Heterologous expression of these enzymes in Streptomyces strains was not able to confirm the functions of NozD and NozE but set the stage for future studies to optimize conditions for probing their roles in nocardioazine A biosynthesis. The results gathered from this study, along with future work to better understand the engineering of unique functional groups from Nocardiopsis may provide opportunities to produce new bioactive molecules.

No description available.

Der Actinomycet T. fusca KW3, isoliert aus Kompost, bildete während der Kultivierung im Mineralsalz-Spurenelement-Vitamin-Minimalmedium nach Zusatz von PET-Fasern eine 52 kDa Carboxylesterase (TfCa), welche effizient zyklische PET Trimere (CTR) hydrolysiert. Die TfCa besitzt einen pI von 4,8, eine Substratspezifität gegenüber kurzkettigen p-Nitrophenyl-Estern und wird durch Phenylmethylsulfonylfluorid (PMSF) und Tosyl-L-Phenylalanin-Chloromethylketon (TPCK) in der Aktivität gehemmt. Die Carboxylesterase hydrolysiert kein Cutin oder Poly-ε-caprolacton (PCL). CTR hingegen wurden durch die TfCa mit einem Km von 0,5 mM und einer Vmax von 9,3 μmol/min/mg bei optimalen Bedingungen (60°C, pH 6) hydrolysiert. Das aktive Zentrum der Carboxylesterase besteht aus den Aminosäuren Ser185, Glu319 und His415, wobei das Serin in das katalytische Motiv G-E-S-A-G eingebettet ist. Während der Reaktion setzte die TfCa auch Hydrolyseprodukte aus PET-Fasern und -Filmen frei. Der Nachweis der Hydrolyse erfolgte durch Umkehrphasen-Hochleistungsflüssigkeitschromatographie der Abbauprodukte und bei den PET-Filmen zusätzlich mittels Rasterelektronenmikroskopie. Dabei zeigte die Carboxylesterase verglichen mit anderen PET-Hydrolasen eine geringere Effizienz, was durch die Lage des aktiven Zentrums in einer Bindungstasche und der daraus folgenden schlechten Zugänglichkeit für polymere Substrate begründet werden kann. Bei der Hydrolyse der viel kleineren CTR war die TfCa deutlich effektiver, was auf eine höhere Spezifität gegenüber kurzkettigen PET Substraten hinweist.

Der Actinomycet T. fusca KW3, isoliert aus Kompost, bildete während der Kultivierung im Mineralsalz-Spurenelement-Vitamin-Minimalmedium nach Zusatz von PET-Fasern eine 52 kDa Carboxylesterase (TfCa), welche effizient zyklische PET Trimere (CTR) hydrolysiert. Die TfCa besitzt einen pI von 4,8, eine Substratspezifität gegenüber kurzkettigen p-Nitrophenyl-Estern und wird durch Phenylmethylsulfonylfluorid (PMSF) und Tosyl-L-Phenylalanin-Chloromethylketon (TPCK) in der Aktivität gehemmt. Die Carboxylesterase hydrolysiert kein Cutin oder Poly-ε-caprolacton (PCL). CTR hingegen wurden durch die TfCa mit einem Km von 0,5 mM und einer Vmax von 9,3 μmol/min/mg bei optimalen Bedingungen (60°C, pH 6) hydrolysiert. Das aktive Zentrum der Carboxylesterase besteht aus den Aminosäuren Ser185, Glu319 und His415, wobei das Serin in das katalytische Motiv G-E-S-A-G eingebettet ist. Während der Reaktion setzte die TfCa auch Hydrolyseprodukte aus PET-Fasern und -Filmen frei. Der Nachweis der Hydrolyse erfolgte durch Umkehrphasen-Hochleistungsflüssigkeitschromatographie der Abbauprodukte und bei den PET-Filmen zusätzlich mittels Rasterelektronenmikroskopie. Dabei zeigte die Carboxylesterase verglichen mit anderen PET-Hydrolasen eine geringere Effizienz, was durch die Lage des aktiven Zentrums in einer Bindungstasche und der daraus folgenden schlechten Zugänglichkeit für polymere Substrate begründet werden kann. Bei der Hydrolyse der viel kleineren CTR war die TfCa deutlich effektiver, was auf eine höhere Spezifität gegenüber kurzkettigen PET Substraten hinweist.:Inhaltsverzeichnis 1 Einführung 1 1.1 Actinomyceten 1 1.1.1 Klassifizierung 1 1.1.2 Thermobifida (Thermomonospora) fusca 2 1.2 Biopolyester Cutin und Suberin 4 1.2.1 Bestandteile des Cutins 4 1.2.2 Bestandteile des Suberins 5 1.2.3 Struktur der Biopolymere 7 1.3 Hydrolasen 12 1.3.1 Struktur und katalytischer Mechanismus 12 1.3.2 Carboxylesterasen 16 1.3.3 Lipasen 19 1.3.4 Cutinasen 22 1.4 Polyethylenterephthalat 23 1.4.1 Konventionelle PET-Faserbehandlung 24 1.4.2 Charakterisierung von PET 26 1.4.3 Biofunktionalisierung von PET 30 1.4.4 PET-Hydrolasen 38 1.5 Zielsetzung 49 2 Material und Methoden 50 2.1 Materialien 50 2.1.1 Verwendete Mikroorganismen 50 2.1.2 Verwendete Enzyme 50 2.1.3 Größenstandards 51 2.1.3.1 Low Molecular Weight Marker (GE Healthcare) 51 2.1.3.2 Roti®-Mark Standard (Fa. Carl Roth GmbH) 51 2.1.3.3 SpectraTM Multicolor Broad range Protein Ladder (Fermentas) 51 2.1.3.4 PageRulerTM plus prestained protein ladder (Fermentas) 51 2.1.3.5 Kalibrierungskit für pI Bestimmung (pH 3-10, GE Healthcare) 52 2.1.3.6 Kalibrierungskit für die Größenausschlusschromatographie (LMW, GE Healthcare) 52 2.1.4 Chemikalien 53 2.1.5 Geräte und Materialien 55 VI 2.1.6 Software 58 2.1.7 Nährmedien 58 2.1.8 Suberin- und Cutin-Präparationen 60 2.1.9 PET-Substrate für die Abbauuntersuchungen 60 2.1.10 Puffer und Lösungen 60 2.2 Mikrobiologische Methoden 65 2.2.1 Stammhaltung und Kultivierung 65 2.2.2 Mikroskopische Untersuchungen 65 2.2.3 Trockengewichtsbestimmung 65 2.3 Proteinchemische Methoden 66 2.3.1 Proteinaufreinigung der Wildtyp TfCa 66 2.3.2 Proteinaufreinigung der rekombinanten TfCa, TfCut1 und TfCut2 67 2.4 Analytische Methoden 67 2.4.1 Esterase-Aktivitätsbestimmung 67 2.4.1.1 Bestimmung mittels Spektrophotometer 68 2.4.1.2 Bestimmung mittels Plattenleser 68 2.4.2 Cutinase-Aktivitätsbestimmung 68 2.4.3 PCL-Abbauuntersuchung 69 2.4.3.1 Bestimmung mittels Hofbildung 69 2.4.3.2 Bestimmung mittels Plattenleser 69 2.4.4 Quantitative Protein-Bestimmung nach Bradford (1976) 69 2.4.5 SDS Polyacrylamidgelelektrophorese (SDS-PAGE) 70 2.4.5.1 Esteraseaktivitäts-Färbung 70 2.4.5.2 Coomassie-Färbung 71 2.4.5.3 Silberfärbung der Proteine 71 2.4.6 Bestimmung des pI 71 2.4.7 Bestimmung der Molaren Masse 72 2.4.8 Bestimmung der Temperatur und pH-Wert Stabilität 72 2.4.9 Bestimmung der Stabilität gegenüber Inhibitoren 72 2.4.10 Bestimmung der kinetischen Konstanten für die Hydrolyse von p-NP Ester 72 2.4.11 Bestimmung der kinetische Konstanten für die Hydrolyse von CTR 73 2.4.12 Bestimmung optimaler Temperatur und pH-Wert für die Hydrolyse von CTR 73 2.4.13 N-terminale Sequenzierung 73 2.4.14 MALDI-TOF Sequenzierung 74 2.4.15 Abbaustudien 74 VII 2.4.16 Analytik der PET Abbauprodukte 75 2.4.17 Konzentrationabhängige CTR-Abbaustudien 75 2.5 Homologie-Modelling der TfCa und weitere PET-Hydrolasen 76 3 Ergebnisse und Diskussion 77 3.1 Screening nach PET-Hydrolasen aus T. fusca 77 3.1.1 Wachstum von T. fusca KW3 im Czapek-Medium 77 3.1.2 Wachstum von T. fusca KW3 im MSV-Medium 78 3.1.2.1 Esterasebildung mit verschiedenen synthetischen und natürlichen Polyestern 78 3.1.2.2 Esterasebildung mit einer Suberinpräparation 81 3.1.2.3 Esterasebildung mit PET-Fasern 83 3.1.3 Esterasebildung bei T. fusca KW3 DSM 6013, T. fusca DSM 43792 und DSM 43793 mit PET-Fasern und Diethylterephthalat 85 3.2 Charakterisierung der PET-Hydrolasen aus T. fusca KW3 95 3.2.1 Aufreinigung 95 3.2.1.1 Aufreinigung der TfCa 95 3.2.1.2 Aufreinigung der rekombinanten PET-Hydrolasen 105 3.2.2 pI der TfCa 113 3.2.3 Molare Masse der TfCa 113 3.2.4 Temperatur- und pH-Stabilität der TfCa 114 3.2.5 Wirkung von Inhibitoren auf die TfCa 117 3.2.6 Kinetik der Hydrolyse von verschiedenen Esterasesubstraten durch die TfCa 118 3.2.7 Optimale Temperatur und optimaler pH-Wert der CTR-Hydrolyse durch die TfCa 119 3.2.8 Cutinaseaktivität der TfCa 120 3.2.9 PCL-Abbau durch die TfCa 121 3.2.10 N-terminale Sequenz der TfCa 122 3.2.11 MALDI-TOF Sequenzierung der TfCa 123 3.2.12 Homologie-Modeling der TfCa und Vergleich der Struktur mit anderen Hydrolasen 126 3.2.13 Vergleich der TfCa mit bekannten PET-Hydrolasen 128 3.3 Hydrolyse von PET Substraten durch prokaryotische und eukaryotische Hydrolasen 138 3.3.1 Partielle Hydrolyse von APET-Filmen durch die PET-Hydrolasen 140 3.3.2 Partielle Hydrolyse von PET-Fasern durch die PET-Hydrolasen 148 3.3.3 Hydrolyse von PET-Trimeren durch die PET-Hydrolasen 151 4 Zusammenfassung 165 VIII 5 Literaturverzeichnis 166 6 Publikationen 181 7 Poster und Vorträge 182 8 Lebenslauf 183