Made available in DSpace on 2014-11-10T11:09:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-05-29Bitstream added on 2014-11-10T11:58:01Z : No. of bitstreams: 1 000789630_20141229.pdf: 409322 bytes, checksum: ffbf7ab412d7ca57518c2014df4f7a52 (MD5) Bitstreams deleted on 2015-01-05T11:00:54Z: 000789630_20141229.pdf,Bitstream added on 2015-01-05T11:01:50Z : No. of bitstreams: 1 000789630.pdf: 2048959 bytes, checksum: 453ad5bd7d63b78e68c4bf76a6845403 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Este trabalho visou dar tratamento tecnológico a extratos enzimáticos ricos em endoglucanases obtidos por cultivo em estado sólido (CES) do fungo termofílico Myceliophtora thermophila M77, através da secagem por spray-drying. A fim de garantir a estabilidade das enzimas durante um período de armazenamento, o extrato enzimático bruto (EEB) foi seco em spray-dryer sendo investigados os parâmetros operacionais e os adjuvantes de secagem para garantir a maior retenção da atividade enzimática após a secagem. Os ensaios foram divididos entre discriminação do adjuvante que proporcionasse maior proteção às enzimas, otimização das condições de secagem, armazenamento do EEB e dos pós secos, avaliação do efeito da secagem sobre as propriedades físico-químicas das endoglucanases e morfologia dos pós. Utilizou-se de planejamentos estatísticos para analisar os efeitos das variáveis temperatura de saída do ar de secagem, vazão de alimentação de solução, proporção entre o EEB e adjuvante, e teor de sólidos na formulação, sendo as respostas retenção de atividade enzimática e umidade dos pós. Nos ensaios de discriminação foi selecionado o adjuvante goma arábica. Em seguida, nos ensaios para otimização das condições de secagem, foi identificado menor vazão de alimentação e menores temperaturas de saída do ar de secagem para maiores retenções de atividade enzimática e maiores temperaturas e menores vazões para menores teores de umidade dos pós, o teor de sólidos revelou-se não significativo. O pó obtido nas condições mais favorável e menos favorável para a retenção da atividade enzimática foi armazenado à temperatura ambiente e sob refrigeração e a atividade enzimática determinada quinzenalmente. A análise estatística revelou não haver diferença na retenção da atividade enzimática após longos períodos para ambas as condições de secagem selecionadas e alternativas de armazenamento... / This work aimed to provide technological treatment to enzymatic extract rich in endoglucanase, obtained by solid state culture using the fungus Myceliophtora thermophila M77, through spray-drying. Operational parameters and adjuvants were varied in order to obtain stable enzymes with high activities during storage. The experiments were divided into adjuvant discrimination, optimization of the drying conditions, storage of the raw enzymatic extract (REE) and powders, evaluation of drying on the physical-chemical characteristics of the endoglucanase, and powder morphology. Statistical experimental designs were used to evaluate the effect of the variables exit air temperature, solution flow rate, proportion REE/adjuvant, and total solid content on the enzyme activity retention and powder moisture content. The adjuvant Arabic gum was selected from the discrimination assays. From the optimization experiments, it was determined that the lower flow rate and air exit temperature resulted in the highest enzyme activity retention, while low temperature and solution flow rate resulted in the lowest retention. The total solid concentration was not statistically significant. The powders produced in the Best and in the worst drying conditions were stored at room temperature and under refrigeration and the enzymatic activity was measured at 15 days intervals. The statistical analysis did not show any difference in the enzymatic activity retention after long periods of storage for both drying conditions and both storage alternatives. The influence of temperature and pH on the enzyme activity was also investigated before and after the drying process, and it was noticed that these physical-chemical properties were not affected by the adjuvants and by the drying process. The morphology of the powders, obtained using the inert gas adsorption method, indicated that the powder obtained under the worst drying condition presented specific superficial area twice ...

Orientador: João Cláudio Thoméo / Banca: Gustavo Orlando Bonilla Rodriguez / Banca: Izabela Dutra Alvin / Resumo: Este trabalho visou dar tratamento tecnológico a extratos enzimáticos ricos em endoglucanases obtidos por cultivo em estado sólido (CES) do fungo termofílico Myceliophtora thermophila M77, através da secagem por spray-drying. A fim de garantir a estabilidade das enzimas durante um período de armazenamento, o extrato enzimático bruto (EEB) foi seco em spray-dryer sendo investigados os parâmetros operacionais e os adjuvantes de secagem para garantir a maior retenção da atividade enzimática após a secagem. Os ensaios foram divididos entre discriminação do adjuvante que proporcionasse maior proteção às enzimas, otimização das condições de secagem, armazenamento do EEB e dos pós secos, avaliação do efeito da secagem sobre as propriedades físico-químicas das endoglucanases e morfologia dos pós. Utilizou-se de planejamentos estatísticos para analisar os efeitos das variáveis temperatura de saída do ar de secagem, vazão de alimentação de solução, proporção entre o EEB e adjuvante, e teor de sólidos na formulação, sendo as respostas retenção de atividade enzimática e umidade dos pós. Nos ensaios de discriminação foi selecionado o adjuvante goma arábica. Em seguida, nos ensaios para otimização das condições de secagem, foi identificado menor vazão de alimentação e menores temperaturas de saída do ar de secagem para maiores retenções de atividade enzimática e maiores temperaturas e menores vazões para menores teores de umidade dos pós, o teor de sólidos revelou-se não significativo. O pó obtido nas condições mais favorável e menos favorável para a retenção da atividade enzimática foi armazenado à temperatura ambiente e sob refrigeração e a atividade enzimática determinada quinzenalmente. A análise estatística revelou não haver diferença na retenção da atividade enzimática após longos períodos para ambas as condições de secagem selecionadas e alternativas de armazenamento... / Abstract: This work aimed to provide technological treatment to enzymatic extract rich in endoglucanase, obtained by solid state culture using the fungus Myceliophtora thermophila M77, through spray-drying. Operational parameters and adjuvants were varied in order to obtain stable enzymes with high activities during storage. The experiments were divided into adjuvant discrimination, optimization of the drying conditions, storage of the raw enzymatic extract (REE) and powders, evaluation of drying on the physical-chemical characteristics of the endoglucanase, and powder morphology. Statistical experimental designs were used to evaluate the effect of the variables exit air temperature, solution flow rate, proportion REE/adjuvant, and total solid content on the enzyme activity retention and powder moisture content. The adjuvant Arabic gum was selected from the discrimination assays. From the optimization experiments, it was determined that the lower flow rate and air exit temperature resulted in the highest enzyme activity retention, while low temperature and solution flow rate resulted in the lowest retention. The total solid concentration was not statistically significant. The powders produced in the Best and in the worst drying conditions were stored at room temperature and under refrigeration and the enzymatic activity was measured at 15 days intervals. The statistical analysis did not show any difference in the enzymatic activity retention after long periods of storage for both drying conditions and both storage alternatives. The influence of temperature and pH on the enzyme activity was also investigated before and after the drying process, and it was noticed that these physical-chemical properties were not affected by the adjuvants and by the drying process. The morphology of the powders, obtained using the inert gas adsorption method, indicated that the powder obtained under the worst drying condition presented specific superficial area twice ... / Mestre

Der biologische Aufschluss lignocellulosehaltiger Biomasse mit Hilfe von Mikroorganismen oder ihrer Enzyme ist im Vergleich zu bekannten physikochemischen Verfahren umwelt- und ressourcenschonend. Der Einsatz geeigneter bakterieller oder pilzlicher Hydrolasen und Oxidoreduktasen in isolierten Form bedarf jedoch, aufgrund der noch zu geringen katalytischen Effizienzen und der nach wie vor zu hohen Herstellungskosten der Enzyme, weiterer Optimierung. Vor diesem Hinter-grund besteht, neben dem Ansatz einer gentechnischen Verbesserung der En-zym-Eigenschaften mittels protein oder metabolic engineering, die Möglichkeit einer prozesstechnischen Optimierung der Enzym-Präparate und ihrer Einsatzbe-dingungen. Dem letzteren Ansatz widmete sich die vorliegende Arbeit, in der ein bisher kommerziell nicht erhältliches Glycosidase-Gemisch aus dem ascomycetalen Schimmelpilz Penicillium janthinellum, der für seine hohen β-Glycosidase-Aktivitäten bekannt ist, im Zusammenhang mit dem enzymatischen Aufschluss faserreicher Substrate (Lignocellulosen) untersucht wurde. Ein Schwerpunkt lag dabei auf der kombinierten Anwendung des Glycosidase-Präparats mit zwei pilzlichen Peroxidasen (Mangan-Peroxidase, MnP und Dye-decolorizing-Peroxidase, DyP). Darüber hinaus wurden vergleichende Untersuchungen zu biologischen Aufschlussverfahren unter Einsatz pilzlicher Glycosidasen und/oder mikrobieller Vorkulturen durchgeführt. Die untersuchten Lignocellulose-Substrate (Hölzer, strohähnliche Materialien) stammten aus der gemäßigten und tropischen Klimazone (Europa bzw. Kambodscha), und wurden in den Experimenten in zerkleinerter Form, allerdings ohne weitere Vorbehandlung, eingesetzt. Die Konzentration niedermolekularer Zucker (insbesondere Monosaccharide), die während des enzymatischen Aufschlusses aus den Substraten freigesetzt wurden sowie der Biogasertrag, der mittels anaerober Fermentation aus den enzymatisch und/oder mikrobiell vorbehandelten Substraten erzielt wurde, dienten zur Beurteilung der Effektivität der jeweiligen Vorbehandlung. Außerdem wurde die in den Experimenten verwendete mikrobielle Vorkultur soweit molekularbiologisch untersucht, dass die Bakterienart identifiziert werden konnte, die maßgeblich am Aufschluss der lignocellulosehaltigen Biomasse beteiligt war. Die enzymatische Umsetzung der ausgewählten lignocellulosehaltigen Substrate mit Hilfe des Glycosidase-Gemisches aus P. janthinellum verlief erfolgreich und ist vergleichbar mit Ergebnissen, die laut Literatur unter Zuhilfenahme der effektivsten industriellen Cellulase-Präparate erzielt worden sind. Es wurden vorrangig Glucose und Xylose aus den verschiedenen Zellwand-Polysacchariden freigesetzt, wobei die Umsetzung von Cellulose und Hemicellulosen im Holz tropischer Laubbäume effizienter verlief als im Holz europäischer Laubbäume. Der Gehalt an Lignin und organischen Extraktiven beeinflusste – abgesehen von einigen artenspezifischen Inhibitoren – nur geringfügig den enzymatischen Aufschluss der Polysaccharid-Komponenten. Die Vorbehandlung mit dem Glycosidase-Präparat aus P. janthinellum führte zu einer Verbesserung der Biogasbildung und zum Ausbleiben der für faserreiche Substrate typischen Lag-Phase während der ers-ten Tage der anaeroben Vergärung der Lignocellulose aus Triticum sp. (Weizen-stroh) und Pinus sylvestris (Kiefernspäne). Dabei erhöhte sich der finale Biogasertrag innerhalb des Untersuchungszeitraums signifikant. Die genannten positiven Effekte einer enzymatischen Vorbehandlung könnten sich in kontinuierlich betriebenen großtechnischen Biogasanlagen als nützlich erweisen: Zum einen ließen sich die Gaserträge deutlich erhöhen und zum anderen könnte die erforderliche Verweilzeit des Substrates im Bioreaktor (Fermenter, Faulturm) und somit das benötigte Anlagenvolumen reduziert werden. Eine vorausgehende Oxidation des im Substrat enthaltenen Lignins mit Hilfe der MnP erwies sich in der nachfolgenden Behandlung mit Glycosidasen als förderlich hinsichtlich der Freisetzung von Zuckern aus dem Holz von Fagus sylvatica (Rotbuche). Verglichen mit der häufig verwendeten Malonsäure war die Citronensäure, ein pilzlicher Metabolit des Intermediär-Stoffwechsels (Zitronensäurezyklus), ein wirksamerer Mangan-Chelator für diese Voroxidation mittels MnP. Dies hing möglicherweise mit der höheren chemischen Reaktivität der Citronensäure zu-sammen, was eine verstärkte Bildung chemischer Radikale zur Folge hatte. Eine enzymatische Vorbehandlung mittels DyP und dem Glycosidase-Gemisch in einer Reaktionskaskade wirkte sich ebenfalls positiv auf die Biogasbildung, in diesem Fall aus Bagasse von Saccharum officinarum (Zuckerrohr), aus. Dabei kam es wahrscheinlich auch zu einer partiellen Oxidation und Zerstörung des Lignins und damit zu einer Verbesserung der Zugänglichkeit der Zellwand-Polysaccharide. Im Ergebnis konnten Cellulose und Hemicellulosen in späteren Phasen der anaeroben Vergärung von den entsprechenden Mikroorganismen (Bakterien, Archaeen) besser verwertet werden. Der Voraufschluss mit Glycosidasen führte hingegen in der initialen Phase der anaeroben Vergärung zu positiven Effekten bezüglich der Biogasbildung, indem die bereitgestellten Einfachzucker (z.B. Glucose, Xylose) rasch in Methan umgewandelt wurden. Beim Vergleich verschiedener biologischer Aufschlussverfahren erwies sich eine kombinierte Vorbehandlung des Substrates („Stroh“ von Miscanthus × giganteus), bestehend aus einer Vorhydrolyse durch das Glycosidase-Gemisch und einer Vorfermentation mit einer Mischkultur gärender Mikroorganismen, als der effektivste Weg. Durch die kombinierte biologische Vorbehandlung konnte ein ähnlich hoher Methanertrag wie für Maissilage (das derzeit optimale Substrat in Biogasanlagen) erreicht werden. In der entsprechenden mikrobiellen Vorkultur wurde ein Bacillus-Vertreter aus dem so genannten Bacillus-subtilis-Artkomplex (Bacillus subtilis species-complex) mittels klassischer mikrobiologischer und molekularbiologischer Analysen als möglicher „abbaurelevanter Organismus“ identifiziert. / The biological disintegration of lignocellulosic biomass by microorganisms and their enzymes is – in comparison to established physical and chemical approaches –environmentally friendly and sustainable. The broad use of isolated bacterial or fungal hydrolases and oxidoreductases requires, however, still substantial optimization because of too low catalytic performance and too high production costs for the enzymes. Against this background, there is the possibility, besides genetic improvement of enzyme properties by protein and metabolic engineering, to optimize the process performance of enzymes as well as the reaction conditions. The latter approach has been subject of the present dissertation, in the course of which a non-commercial preparation of glycosidases from the ascomycetous mold Penicillium janthinellum, which is well-known for its high β-glycosidase activities, was used for the enzymatic disintegration of fiber-rich substrates (lignocelluloses). Experimental work focused on the combined action of the glycosidase mixture with two fungal peroxidases (manganese peroxidase, MnP and dye-decolorizing peroxidase, DyP). Furthermore, comparing studies were carried out regarding enzymatic/biological lignocellulose disintegration by isolated fungal glycosidases and/or microbial precultures. Lignocellulose substrates studied (wood, straw-like materials) originated from temperate and tropic climate zones (Europe and Cambodia, respectively) and were used after chopping in all experiments without further pretreatment. The concentration of low-molecular mass sugars (in first place monosaccharides), which were being released from the substrates during enzyme action as well as the biogas yield that was achieved via fermentation of enzymatically or microbiologically pretreated samples, were taken into consideration to evaluate the efficacy of respective treatments. Moreover, the microbial preculture used in the above experiments was analyzed on the molecular level to an extent that it was possible to identify a bacterial key species that was involved in the degradation of lignocellulosic biomass. The enzymatic treatment of selected lignocellulosic substrates with the glycosidase mixture of P. janthinellum was successful and the results are – according to literature data – comparable to results reported for the best industrial cellulase preparations. In first place, glucose und xylose were released from different cell-wall polysaccharides, and the conversion of cellulose und hemicelluloses in the wood of tropical broad-leaved trees was more efficient than in wood of respective European trees. The content of lignin and organic extractives only slightly affected the enzymatic disintegration of polysaccharide components (apart from a few species-specific inhibitors). Substrate pretreatment with the glycosidase preparation of P. janthinellum resulted in an enhancement of biogas formation and in the disappearance of the lag-phase being characteristic for the conversion of fiber-rich substrates during the first days of anaerobic treatment of lignocelluloses from Triticum sp. (wheat straw) und Pinus sylvestris (wood shavings). In this context, the final biogas yields significantly increased in the course of the experiments. The observed positive effects of enzymatic pretreatment may be beneficially ap-plied in continuously working biogas plants. That way, on one hand, the gas yields could be considerably enhanced and on the other hand, the required retention time of the substrates in the bioreactor (fermenter, digestion tower) and hence the required reactor volume could be reduced. The preceding oxidation of substrate-bound lignin with MnP turned out to be beneficial for the subsequent glycosidase treatment with respect to the release of sugars from beech wood (Fagus sylvatica). In comparison to widely used malonic acid, citric acid – a ubiquitous fungal metabolite of the intermediary metabolism (tricarboxylic acid cycle) –proved to be the more effective manganese chelator for the pre-oxidation of lignin by MnP. Probably this corresponds to the higher chemical reactivity of citric acid, which entails a forced formation of chemical radicals. Enzymatic substrate pretreatment with DyP and the glycosidase mixture within a reaction cascade had also a positive effect on the formation of biogas, in this case, from bagasse of Saccharum officinarum (sugar cane). During the respective treatment, the lignin might partially be oxidized as well and thereby, the availability of cell-wall polysaccharides was improved for hydrolase action. As the result, microorganisms (bacteria, archaea) consumed cellulose and hemicelluloses more efficiently during later phases of anaerobic fermentation. On the other hand, glycosidase pretreatments had positive effects in the initial phase of fermentation, regarding biogas formation from the ‘made-available’ monosaccharides (e.g. glucose, xylose) that were immediately converted into methane. When comparing different biological methods to disintegrate lignocellulose, pre-hydrolysis with a glycosidase mixture combined with fermentative pretreatment proved to be the most effective option (demonstrated by the example of ‘straw’ from Miscanthus × giganteus). That way, a similarly high methane yield could be achieved as with maize silage (for the time being, the most suitable substrate used in biogas facilities). In the respective microbial preculture, a Bacillus species from the Bacillus subtilis species-complex was identified as a relevant potential degrader microbe by classic microbiological and molecular analyses.

"This thesis is based on the following articles, referred to in the text by the Roman numerals given below. In addition some unpublished results are presented. I. Caiyan Wu ... [et al] Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I (amyI) gene from Ophiostoma floccosum [pub. in ] Gene 384: 96-103 -- II. Caiyan Wu ... [et al.] Activity-based identification of secreted serine proteases of the filamentous fungus Ophiostoma. Accepted by Biotechnology letters DOI 10.1007/s10529-007-9333-6 -- III. Caiyan Wu ...[et al.] Expression of a thermostable bacterial xylanase in the filamentous fungus Ophiostoma floccosum. Submitted to Letters in applied microbiology in July 2007." - leaf 9. / Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2007. / Bibliography: leaves 100-123. / Introduction -- Materials and methods -- Results and discussion -- Conclusion and future aspects -- References -- Publications I, II and III. / Ophiostoma spp. belong to the Ophiostomataceae family, a large group of ascomycetes, which are the most frequent blue stain fungi isolated from stained wood. Most Ophiostoma species do not compromise the strength properties of wood, but do reduce the aesthetic quality of timber and therefore decrease the economic value of lumber. Some albino variants of O. floccosum and O. piliferum have been used as biological control agents to prevent blue staining. This successful whole organism approach plus the added capability of extracellular protein secretion makes Ophiostoma spp. attractive for industrial application. In addition, Ophiostoma produces only a small range of abundantly secreted proteins in liquid culture, which can facilitate downstream purification of any recombinant gene product introduced into the system. Genes encoding efficiently secreted proteins provide a potential souce for strong promoters for high-level gene expression. These characteristics provide an excellent starting point for the development of a novel expression system. / In this study, UV-mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein secretion after repeated rounds of mutagenic treatment. Several mutants of O. floccosum derived by UV mutagenesis were isolated and the total amount of secreted protein was increased by 4 to 6 times. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the amount of total secreted protein was about six times greater to that of the parental strain. Proteinase profiles in the culture supernatants of several key mutants were characterized for the future matching of an expression host with a particular gene product. N-terminal sequencing of the five dominant proteins separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an_-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the _-amylase amino acid sequence. The amplified fragment contained the entire gene encoding_-amylase (amyl) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single _-amylase gene locus. / A series of expression vectors containg the _-amylase regulatory sequences and partial amyl gene were constructed. Several selection markers were screened and the hph gene conferring hygromycin resistance under the regulation of the Aspergillus nidulans gpd promoter was chosen and inserted into the amyl expression vectors. The gene encoding a red fluorescent protein DsRed-E5 was used as a reporter gene to test the expression system using mutant MQ.5.1 as host. However, no transformants were obtained by either biolistic transformation or protoplast transformation. Subsequently, an alternative strategy was developed using a thermostable xylanase B as a reporter. Thermostable xylanase activity was detected in the culture supernatants of several transformants. Production of xylanase by transformant SS41 which exhibited high secreted xylanase activity was investigated. Xylanase activity in the culture supernatant of SS41 was visualized by a zymogram gel assay. Two active proteins with molecular masses of around 27 and 30 kDA, which were larger than the predicted Mr of 25 kDA were detected. This is the first report describing successful expression of a recombinant thermostable bacterial enzyme in Ophiostoma. / Mode of access: World Wide Web. / 158 leaves col. ill