Orientador : Glaucia Maria Pastore / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-28T00:46:26Z (GMT). No. of bitstreams: 1 Santos_CristineFiorClementeDos_D.pdf: 43427689 bytes, checksum: 6cdb1d14cef42b14acdca1f05afc3e01 (MD5) Previous issue date: 2001 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Doutorado / Doutor em Ciência de Alimentos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2016. / Made available in DSpace on 2016-09-20T04:19:36Z (GMT). No. of bitstreams: 1 339941.pdf: 1997654 bytes, checksum: fe62e4da921f4d0a81e1c7d2733bd6a1 (MD5) Previous issue date: 2016 / Os biossurfactantes são compostos anfifílicos produzidos por microrganismos e que são capazes de diminuir as tensões superficiais e interfaciais. A surfactina, produzida por Bacillus subtilis, apresenta-se como um dos biossurfactantes mais poderosos em termos de atividade superficial. Contudo, o alto custo e o baixo rendimento associados à sua produção acaba limitando sua aplicação extensiva na indústria. Assim, faz-se necessário o desenvolvimento de estratégias que visem aumentar o rendimento e diminuir os custos envolvidos na produção desse biossurfactante. O fornecimento de oxigênio dissolvido suficiente, através da agitação mecânica e da aeração, é essencial para garantir a eficiência do processo de produção da surfactina. Entretanto, quando se conduz um processo em biorreator em aerobiose, é necessário avaliar a capacidade de transferência de oxigênio do sistema através do coeficiente volumétrico de transferência de oxigênio (kLa). Diante do que foi exposto, este estudo propõe a caracterização da agitação e aeração do biorreator a ser empregado, através da determinação do coeficiente volumétrico de transferência de oxigênio (kLa) em condições de baixas vazões de aeração, além da execução de ensaios para a produção de surfactina por Bacillus subitlis ATCC 21332 em determinados valores de kLa. Os ensaios foram conduzidos em biorreator descontínuo, em meio salino composto por 10 g.L-1 de glicose e suplementado com extrato de levedura. Os resultados mostraram que as maiores produtividades em surfactina foram obtidas quando houve uma transferência de oxigênio mais eficiente, ou seja, quando se utilizaram os maiores valores de kLa, Para os ensaios realizados com aeração em profundidade, a concentração de surfactina foi muito maior no líquido efluente do que dentro do biorreator, e, assim, a maior parte do biossurfactante produzido foi recuperada através da espuma. Além disso, na maioria dos ensaios houve uma redução da concentração celular na espuma, bem como da concentração de substrato, em relação ao que foi encontrado no reator, o que pode ser de grande importância, dependendo de como se pretende operar o biorreator.<br> / Abstract: Biosurfactants are amphiphilic compounds that are able to lower the surface and interfacial tension. Surfactin appears as one of the most powerful biosurfactants due its high superficial activity. However, the high cost and low yield associated with surfactin production end up limiting its widespread implementation in industry. Thus, it is necessary to develop strategies that allow higher yields and lower costs in the production of this biosurfactant. The supply of sufficient dissolved oxygen through mechanical agitation and aeration appears to play important roles in the efficiency of surfactin production. Nevertheless, when we operates a reactor under aerobic conditions, it is necessary to evaluate the oxygen transfer capacity of the system through the volumetric oxygen transfer coefficient (kLa). Based on the above considerations, the aim of this study is to characterize the aeration and agitation of the bioreactor, which will be used, by determining the volumetric oxygen transfer coefficient under low rates of aeration and agitation, as well as carrying on experiments for surfactin production by Bacillus subtilis ATCC 21332 according to the certain kLa values. The fermentations were carried on in a stirred tank batch bioreactor in a mineral salts medium composed by 10 g.L-1 of glucose and supplemented with yeast extract. The results showed that the highest surfactin productivities were achieved when there was an efficient oxygen transfer, in other words, when larger kLa values were employed. For the experiments conducted under a series of combination of aeration and agitation rates, surfactin concentration was higher in the effluent liquid than into the bioreactor, and thus, most of the biosurfactant was recovered in the foam. In addition, in most of the fermentations there was a biomass concentration decrease in the foam, as well as a substrate concentration decrease, which may be valuable information for process design.

No description available.

CwlD has sequence similarities to N-acetyl muramoyl-L-alanine amidases, a class of enzymes known to cleave the bond between the peptide side chain and the N-acetyl muramic acid residue in cortex peptidoglycan formation during sporulation. A major difference between vegetative peptidoglycan and spore peptidoglycan is the presence of muramic-<FONT FACE="Symbol">d</FONT> -lactam (MAL) in spore peptidoglycan. It was previously determined that a <I>cwlD</I> null mutant does not contain muramic-<FONT FACE="Symbol">d</FONT> -lactam in the spore cortex peptidoglycan and the mutant spores were unable to complete germination. Therefore, it is believed that CwlD plays a role in MAL formation during sporulation. However, the specific role of the protein had not been demonstrated. It was also previously found that <I>cwlD</I> is in a two-gene operon with <I>orf1</I>. Orf1 is produced within the forespore with CwlD. The hypothesized role of Orf1 is to inhibit CwlD activity from within the forespore. Muramoyl-L-alanine amidase activity was demonstrated by CwlD <I>in vivo</I>. Therefore, CwlD is carrying out the first step of MAL synthesis, cleaving the peptide side chain while other enzymes are needed to complete MAL formation. Two different forms of CwlD were over-expressed, with and without the protein's signal peptide sequence. Both forms of the protein were purified and in both cases activity was undetectable. Antibodies specific for CwlD were obtained which can be used in future research as a tool to further characterize CwlD activity. A series of <I>B. subtilis</I> <I>cwlD</I> operon mutants were constructed altering the expression patterns of Orf1 and CwlD within the mother cell and forespore compartments. Various resistance properties and the germination ability of the mutant dormant spores were analyzed. It was determined that the absence of just Orf1 or Orf1 and CwlD from within the forespore has no effect on the phenotypes tested. Peptidoglycan from developing mutant forespores was extracted and analyzed throughout sporulation. Evidence was obtained demonstrating that the role of Orf1 is not to inhibit CwlD from within the forespore as hypothesized. / Master of Science

Call number: LD2668 .T4 1984 L33 / Master of Science

No description available.

No description available.

Master of Science / Graduate Biochemistry Group / Michael R. Kanost / Prophenoloxidase-activating proteinase (proPAP) activates prophenoloxidase when bacteria or fungi invade Manduca sexta. Upon activation, phenoloxidase initiates synthesis of melaninin, which can encapsulate the invaders and kill them. M. sexta contains three proteases that can activate prophenoloxidase, proPAP1, proPAP2, and proPAP3. The study of proPAP function has been slowed by the difficulty of expressing the proteins in recombinant systems. ProPAP1 contains one clip domain and one serine proteinase domain, a simpler structure than proPAP2 and proPAP3, which have two clip domains. For this reason, proPAP1 was selected for this investigation, to develop an improved system for expression of recombinant proPAP zymogens. In past experiments proPAP1 had a low expression level in insect cells using a baculovirus vector. In Escherichia coli, proPAP1 was expressed as an insoluble protein that could not be refolded successfully. The Bacillus subtilis expression system offers a potential improvement for expression of recombinant clip domain proteases because it can secrete recombinant proteins into the medium, it is a Biosafety Level 1 organism that is easy to handle, and it is less expensive to culture than insect cells. Four constructs for expression of proPAP1 and proPAP1 mutants were produced in the plasmid shuttle vector pHT43, which is compatible with both E. coli and B. subtilis. Experiments were carried out to test and optimize expression and purification of proPAP1 in B. subtilis. Conditions were optimized for IPTG (isopropyl β-D-1-thiogalactopyranoside) concentration, IPTG induction time, growth medium and induction temperature. Results showed that 0.5mM IPTG with 20 hours induction at 37°C in 2xYT medium was the optimum condition for proPAP1 production in the B. subtilis system. The recombinant proPAP1 was precipitated from the medium in 50% saturated ammonium sulfate and partially purified by nickel affinity chromatography. In addition to the full length proPAP1 protein, degradation of proPAP1 was also observed. Further experiments should be done to try to solve this problem. With purified protein, future work can be aimed at study of the structure and function of proPAP1.

Bacteria can quickly adapt to changing environmental conditions by activating alternative sigma factors. It has been shown previously that single cell approaches can reveal hidden dynamics in sigma factor activation. Here, we investigate the single cell response dynamics of the B. subtilis extracytoplasmic function sigma factors, which are an important part of the cell envelope stress response, under their specific stresses. To do this we use transcriptional reporters of sigma factors, quantitative single cell snapshots, time-lapse microscopy, and microfluidics. By developing an improved microfluidics setup for single cell time-lapse microscopy, as well as improved single cell analysis code, we are able to observe new sigma factor dynamics. First, we observe heterogeneous entry into a higher $\sigma^{V}$ activity state in response to lysozyme, which displays a memory, as the heterogeneity is lost on removal and reapplication of the stress. Next, we observe a pulse amplitude and duration modulated sigma factor response of $\sigma^{M}$ to bacitracin. Finally, for $\sigma^{M}$ under ethanol and acidic stress, and for $\sigma^{Y}$ under ethanol stress, we observe a noisy increase in activity to a new steady state level, where the degree of variability between cells depends on the stress condition. This thesis also discusses efforts on building a single cell microfluidic device based on the ”mother machine” design, for the rod-shaped cyanobacterium, S. elongatus, which forces the cells to grow in a straight line. Growing this organism in a traditional mother machine device has, so far, proved challenging. To adapt the mother machine for cyanobacteria we modify the channel geometry using electron beam lithography, and improve the loading protocol. The research presented here reveals the range of regulatory dynamics possible for ECF sigma factors in B. subtilis, and provides improved microfluidics and analysis code that will enable easier quantification of bacterial gene circuits at the single cell level in the future.

No description available.