Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1983. / Extra t.p. with thesis statement inserted. Includes the author's four published papers. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1983. / Extra t.p. with thesis statement inserted. Includes the author's four published papers. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Due to the high resistance of the spores, the bacterium Paenibacillus larvae is the most dangerous bacterial pathogen of the honey bee (Apis mellifera). Thanks to its biological properties and restricted pathogenicity, this bacterium can be used as a model organism to study gram positive sporulating aerobic rods. This work is focused on completing information about secreted proteases of this bacterium and in a study of proteases bound in a spore structure. MYPGP medium was used for the cultivation of P. larvae. In this medium, lysis of the culture was shown after 40 hours of cultivation. The pH of the medium decreased below 6.4 by lysis. The induction of temperate bacteriophage BLA was detected as a causative agent of this lysis. A new sporulation medium called HCBB agar was proposed for the sporulation of P. larvae. In comparison with HCBB agar with MYPGP agar by 31 strains of P. larvae stored in our collection, HCBB agar was evaluated as an appropriate sporulation medium with a median of sporulatin 4.2 ' 106 spores per cm2 in aerobic conditions and 5.65 ' 106 spores per cm2 in aerobic conditions with 10 % CO2. For purification of the secreted proteases, a one-day culture incubated at room temperature was used. Optimal purification of 87/74 kDa and 42/40 kDa proteases was observed after application of this...

The study of rapid cleavage of organophosphate esters by metal ions is of great interest as it is the most important reaction in both biological and environmental sciences. A good understanding of organophosphate hydrolysis by metal ions is important as it can be exploited in formulation of useful detoxifying agents for organophosphate contaminants in the environment. The knowledge can also help in developing effective artificial enzymes. The hydrolysis of 4-NPP in the presence of Ferrous and Ferric ions in o/w microemulsion media was investigated. The reaction was monitored by measuring the absorbance of the 4-nitrophenolate ion produced in the reaction aliquots with time. The order of effectiveness of the ME media towards the hydrolysis of 4-NPP was found to be CME > AME > aqueous in the presence of Fe(II), Fe(III) and Prussian blue at neutral pH. In comparison with individual metal ions used in the investigation, it was found that polymetallic Prussian blue showed enhanced rate of hydrolysis. The degree of effectiveness is as follows; Prussian blue (insoluble) > Prussian blue (soluble) > Fe(III) > Fe(II). The result of the present investigation enriches our understanding of the possible roles polymetallic ions play in hydrolysis reactions and the effect of different reaction media. The reactions mimic the roles of purple acid phosphatases in the hydrolysis of phosphate esters. The application of the above systems for environmental decontamination of organophosphates is also envisaged. / Chemistry / M.Sc.(Chemistry)

Orientador: Fabio Marcio Squina / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T12:41:11Z (GMT). No. of bitstreams: 1 Alvarez_ThabataMaria_D.pdf: 22982311 bytes, checksum: 1bc2a260df2bfab4948ffb299f13a63f (MD5) Previous issue date: 2013 / Resumo: Devido à necessidade do desenvolvimento de fontes de energias renováveis é de grande interesse a descoberta de novas enzimas envolvidas na desconstrução da parede celular vegetal para a produção de bicombustíveis. A metagenômica é uma poderosa ferramenta para a descoberta de novos genes em comunidades microbianas que não são passíveis de cultivo pelas técnicas tradicionais. Neste contexto, o objetivo desta tese foi o desenvolvimento de estratégias metagenômicas para prospecção de novas enzimas atuantes na degradação da biomassa vegetal no metagenoma de solo de canavial bem como a caracterização funcional das mesmas. A biblioteca metagenômica construída com DNA extraído de um consórcio microbiano especializado na degradação de bagaço de cana-de-açúcar explodido a vapor e deslignificado foi empregada nos experimentos de triagem funcional de alto desempenho. Como resultado, foram identificados três clones positivos com atividade celulolítica e dois clones com atividade xilanolítica. A análise dos insertos de cada um dos clones resultou na localização de ORFs cujas sequências de aminoácidos apresentaram identidade com domínios conservados de glicosil hidrolases da família 5 (celulases E-1 e E-2), família 6 (celulase E-3), família 10 (xilanase X-1) e família 16 (glicosil hidrolase X-2). A celulase E-1 apresentou em sua estrutura além do domínio catalítico, E-1 Cat, um domínio de ligação a carboidratos, denominado E-1 CBM, que não apresentou identidade de sequência com domínios conservados conhecidos. A análise funcional do E-1 CBM revelou tratar-se de um CBM específico para cadeias de glucano com grau de polimerização mínimo de cinco unidades de glicose. Ensaios de atividade enzimática em diferentes substratos mostraram que E-1 Cat atuou especificamente na hidrólise das ligações glicosídicas do tipo ß(1,4) entre resíduos de glicose. Os maiores valores de atividade enzimática foram obtidos em pH 7,0 e temperatura de 50ºC. Os parâmetros cinéticos calculados em CMC foram Km igual a 6,05 ± 0,37 mg/mL, Vmax de 42,51 ± 1,2 ?mol/min/mg e eficiência catalítica kcat/Km de 4,06 mL/mg/s. A enzima apresentou termoestabilidade a 40ºC por cinco horas. A atividade enzimática de E-1 Cat em celulose cristalina e bagaço de cana-de-açúcar explodido a vapor resultou na liberação de açúcares solúveis, evidenciando sua potencial aplicação em processos de conversão da biomassa vegetal. Ensaios de atividade em diferentes substratos mostraram que X-1 apresentou maior atividade enzimática em xilana não ramificada, nas condições de pH e temperatura de 6,0 e 45ºC, respectivamente. Os parâmetros cinéticos calculados utilizando como substrato xilana de madeira de faia foram Km de 2,18 ± 0,13 mg/mL, Vmax de 1.435 ± 30,4 ?mol/min/mg e kcat/Km de 496,32 mL/mg/s. Em relação à termoestabilidade, a enzima se manteve estável a 40ºC e 50ºC por seis horas. A hidrólise de substratos complexos com X-1 resultou na liberação de xilooligossacarídeos, xilobiose e xilose, que são compostos que apresentam potencial aplicação nas indústrias alimentícias e de biocombustíveis. Os resultados obtidos neste estudo validaram a abordagem metagenômica desenvolvida para a descoberta de novos genes codificantes para glicosil hidrolases. Além disso, a estratégia descrita nesta tese pode ser estendida para a descoberta de uma miríade de bioprodutos de interesse biotecnológico / Abstract: Due to the necessity of development of renewable sources of energy, it is of great interest the discovery of novel enzymes involved in plant cell wall deconstruction for biofuels production. Metagenomics is a powerful tool for the discovery of novel genes in microbial communities that are not liable to cultivation by traditional techniques. In this context, the aim of this thesis was the development of metagenomic strategies for prospection of novel enzymes involved in plant biomass degradation in sugarcane field soil metagenome and functional characterization of the identified enzymes. The metagenomic library constructed with DNA extracted from a microbial consortium specialized in degradation of steam exploded delignified sugarcane bagasse was used in the experiments of high-performance functional screening. As a result, we identified three positive clones with cellulolytic activity and two clones with xylanolytic activity. The analysis of the inserts from each clone resulted in the location of ORFs whose amino acid sequences showed identity to conserved domains of glycoside hydrolase family 5 (cellulases E-1 and E-2), family 6 (cellulase E-3), family 10 (xylanase X-1) and family 16 (glycoside hydrolase X-2). Cellulase E-1 exhibited in addition to the catalytic domain, E-1 Cat, a carbohydrate binding module, called E-1 CBM, which showed no sequence identity with known conserved domains. Functional analysis of E-1 CBM showed that it is a CBM specific for glucan chains with a degree of polymerization of at least five units of glucose. Assays with a set of different substrates revealed that E-1 Cat hydrolyzed specifically ß(1,4) glycoside bonds between glucose residues. The highest value of enzymatic activity was obtained at pH 7.0 and temperature of 50°C. The kinetic parameters Km, Vmax and catalytic efficiency kcat/Km calculated using CMC were 6.05 ± 0.37 mg/mL, 42.51 ± 1.2 ?mol/min/mg and 4.06 mL/mg/s, respectively. The enzyme showed thermal stability at 40°C for five hours. The enzymatic activity of E-1 Cat in crystalline cellulose and steam exploded sugarcane bagasse resulted in the release of soluble sugars, demonstrating its potential application in processes of biomass conversion. The xylanase X-1 showed higher enzyme activity in debranched xylan, in reactions conducted in pH 6.0 and temperature of 45°C. The kinetic parameters Km, Vmax and catalytic efficiency kcat/Km calculated using beechwood xylan were 2.18 ± 0.13 mg/mL, 1,435 ± 30.4 ?mol/min/mg and 496.32 mL/mg/s, respectively. In relation to thermal stability, the enzyme was stable at 40°C and 50°C for six hours. The hydrolysis of complex substrates resulted in the release of xylo-oligosaccharides, xylobiose and xylose, which are compounds that have potential application in food and biofuels industries. The results of this study validated the metagenomic approach developed for the discovery of novel genes coding for glycoside hydrolases. Moreover, the strategy described in this work can be extended to the discovery of a myriad of byproducts of biotechnological interest / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

M.Sc. (Biochemistry) / Pseudomonas, as originally defined by Migula (1894), was a genus encompassing an assembly of rod-shaped, Gram-negative, aerobic gammaproteobacteria, possessing one or more polar flagella for motility. This vague description resulted in the misclassification of polarly flagellated proteobacteria that had not been completely characterized. Re-evaluation of the taxonomy of Pseudomonads was set into motion by advancements in molecular biology. Following this revision, Pseudomonads were clustered based on the homogeneity in their 16S rRNA sequence (Anzai et al., 2000). Research on Pseudomonas species has gained momentum owing to their significant prospects in biotechnological processes. Their exploitation for use as cleaning, biocontrol and bioremediation agents dates back to the 1980s (Sebastian and Kolattukudy, 1988; Weller, 1988)...

Using proteolytic probes, the pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was analyzed for its native topography in the cytoplasmic membrane. Before analyses could be performed, the isolation of transhydrogenase-enriched ISO (inside-out) cytoplasmic membrane vesicles was accomplished by modification of the procedure followed by Clarke (Clarke, D. M. and Bragg, P. D. (1985) Eur. J. Biochem. 149, 517-523) in purifying the enzyme from overexpressing E.coli JM83pDC21 cells. Two major changes were made. One was that the solubilization of the bacterial membrane and subsequent purification steps were omitted. The other was the separation of outer membranes from the cytoplasmic membrane preparation by sucrose gradient density centrifugation. This was essential owing to the contaminating presence of a 30 kD protein in the outer membrane of the original preparation. Transhydrogenase-enriched RSO (right-side-out) membrane vesicles were isolated by a different procedure using lysozyme-mediated breakage of E.coli spheroplasts and subsequent vesicular reformation. To identify possible transhydrogenase fragments arising from proteolytic cleavage, anti-E.coli transhydrogenase polyclonal antibodies were generated in rabbits. Two sets of polyclonal antibodies were produced. One set cross-reacted with both the α (52 kD) and β (48 kD) subunits of the transhydrogenase. The other reacted with the α subunit only. Trypsin and proteinase K were the main proteolytic probes used against both ISO and RSO cytoplasmic membrane vesicles, although chymotrypsin was also used in preliminary experiments with ISO membrane vesicles. Identification of fragments resulting from proteolytic cleavage of the enzyme was obtained using anti-transhydrogenase antibodies and by N-terminal sequencing and/or C-terminal sequencing. In some of these experiments, isolation of the proteolytic fragments was necessary prior to analysis. This was done using a number of different methods. The particular methods applied, which included column chromatography strategies and elution procedures from SDS-Polyacrylamide gels, depended on the type of analysis carried out. The analyses indicated that the α subunit has at least a 41 kD sequence extending from its N-terminus which is exposed to the cytoplasmic side of the membrane. This sequence may contain an active site of the enzyme. This is suggested by the binding of this fragment to a NAD-affinity column. The membrane-imbedded region of the α subunit anchoring the 41 kD region predicted by hydropathy plotting (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) could not be detected by our methods. Susceptible tryptic cleavage sites along the 41 kD region were identified by partial proteolysis and may reflect areas in the subunit's tertiary or quaternary structure that are exposed to the surrounding medium. Major cleavage sites were at arg₁₅, Iys₂₂₇, Iys₂₆₄, arg₂₆₈, Iys₂₇₅, arg₃₅₅, and arg₃₆₁. There do not appear to be significant portions of the subunit protruding into the periplasm as neither trypsin nor proteinase K had any effect on the subunit in RSO-oriented membrane vesicles. Proteinase K experiments with ISO and RSO membrane vesicles suggest that a 20 kD portion of the β subunit is protected from cleavage and is imbedded in the membrane. The identity of this fragment could not be confirmed. Hydropathy analysis of the transhydrogenase gene-derived amino acid sequence (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) suggests that this could be a sequence extending from the N-terminus of the β subunit. This is a hydrophobic sequence containing 7 possible transmembranous helices and having a theoretical molecular weight in the range of 20 kD. The proteinase K results also indicate that the rest of the β subunit is exposed to the cytoplasmic side of themembrane rather than the periplasmic side. The results obtained here are consistent with hydropathy predictions made with regard to this subunit. In addition, two different experiments indicate that an α-α subunit interaction may be present in the oligomeric structure of the membrane-bound enzyme (Hou, C, Potier, M. and Bragg, P. D. (1990), Biochim. Biophys. Acta 1018, 61-66). Substrates of the enzyme did not appear to affect the transhydrogenase's general conformation upon binding as detected by experiments using partial tryptic proteolysis. Partial trypsinolysis also revealed that selective detergent extraction of transhydrogenase-enriched ISO vesicles with Triton X-100 and sodium cholate did not affect the overall conformation of the membrane-bound enzyme despite greatly reducing the enzymatic activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

This study purposes to examine extracellular proteases of the anaerobic rumen bacterium, Bacteroides amylophilus H-18. An enzyme was isolated and purified from 29 litres of 23 hr cell-free culture supernatant using DEAE Sephadex A-50, Sephadex G-200 and isoelectrofocusing techniques. Although proteolytic activity in the supernatant had a peak of activity at pH 6.7, there was activity at pH values from 4.5 to 11.5. Therefore, an attempt was made to purify the pH 6.7 activity and to follow the activity at other pH values as an index of purity. It was found that separation of the activities at different pH values was not achieved, even though the enzyme was purified 1265 times. Gel filtration of this purified material revealed the presence of two proteases, one of 60,000 and the other of 30,000 molecular weight. Since these enzymes were otherwise identical, they could have represented the monomeric and dimeric forms of a single protein. If the protease of 30,000 molecular weight was separated and resubjected to gel filtration, protease activity of molecular weight 60,000 reappeared. Ultracentrifugation of the 30,000 molecular weight protease demonstrated only one component. Therefore, if the two forms were in equilibrium, it appeared that the dimer was the more stable form of the enzyme. The purified protease did not contain cysteine, so that any tertiary structure in the enzyme could not involve disulfide bridges. All attempts to dissociate the dimeric into the monomeric form were unsuccessful. Examination of the inhibition of Nα benzoyl-L-arginine methyl ester esterase and protease activities with Nα tosyl-L-chloromethane revealed a complete inhibition of esterase activity at pH 8.0 but only a 30% inhibition of protease activity at the same pH, suggesting that more than one enzyme was responsible for the proteolytic activity exhibited by the purified enzyme. Because it was not possible to achieve separation of proteolytic activities at different pH values after a 1265 times purification, it must be assumed that if there are actually different proteases present they must have very similar structures. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate