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Diferentes meios de cultivo e condições operacionais na produção de penicilina G acilase por Bacillus megaterium
(Universidade Federal de São Carlos, 2007-08-16) Souza, Vanessa Ribeiro de; Giordano, Raquel de Lima Camargo; https://lattes.cnpq.br/3368639921194981
Penicillin G acylase (PGA) is an important enzyme for industry, used to produce aminopenicillanic acid, a key intermediate in the synthesis of ampicillin, among other betalatam antibiotics. The production of this enzyme by Bacillus megaterium has been studied by the research group for several years. This work advances this research, aiming not only at the enhancement of the enzyme production, but also at a better understanding of B. megaterium regulatory expression mechanisms for PGA. A systematic study of the inoculum, including conservation strategies for the microorganism, was performed. Different cultivation media and operational conditions for the production of PGA were investigated, especially temperature and dissolved oxygen concentration. Enzyme recovery was also studied, including methodologies for minimizing protein contents in whey. Cheese whey is an important nutrient for enzyme production, but its use implies the presence of contaminant proteins in the culture broth. Finally, the enzyme was kinetically characterized. During the inoculum study, PGA yields of microorganisms conserved using different techniques were compared: endospores in 20%-glycerol, -50oC (cryotubes), endospores in solid medium, in refrigerator ( slants ) and vegetative cells in 5%-glycerol, -50oC ( eppendorffs ). It was observed that cryotubes (frozen spores) preserve the enzyme production levels for 12 months, 521 IU/L ±20 IU/L with great reproducibility. Conservation in slants shows a marked fall of production after one month, with a low reproducibility along months. Freezing vegetative cells leads to the highest patterns of enzyme production, up to 900 IU/L, but preservation is sustained for only five months. Maximum specific growth rates changed according to the conservation method, with µmax eppendofor > cryotube > slant . A study of the effect of the inoculum growth period on enzyme yield has shown that, for the three conservation methods, harvesting between 8 and 12 h leaded to similar cell mass and enzyme concentrations after 24 h of cultivation. The procedure of harvesting the inoculum after 12 h, with 10%-bioreactor inoculum volume, showed to be a good method for inoculum standardization. The effect of temperature on the cultivation of B. megaterium for production of PGA was assessed in the range 24-40oC. Maximum cell concentration and maximum enzyme yield were achieved at 30oC. The effect of the concentration of dissolved oxygen on the production of PGA was studied, using air to feed the bioreactor (culture medium volume: 1.2-2.0 L). A second B. megaterium strain showed a lower growth rate than the original one, demanding 24 h for germination/propagation, while the original one requires 12 h. Thus, different oxygen (air) feeding strategies were tested for both strains. Along the years, enzyme yields in agitated flasks have been higher than in bioreactor, for almost all assays. For the second strain, sustaining the dissolved oxygen at 10% of saturation leaded to the highest enzyme productivity among all tested conditions, with a bioreactor productivity similar to the agitated flasks. For the original strain a similar production was only achieved with an increasing stirring profile, implying a very low dissolved oxygen concentration, equal to zero during long periods of the cultivation. The two strains presented different dissolved oxygen requirements. Changes in the cultivation medium also implied different dissolved oxygen requirements for a maximum enzyme yield. Cheese whey has a still no-identified substance that is an essential nutrient for enzyme production. The use of enzymatically hydrolyzed cheese whey improves the downstream process. Assays in agitated flasks, with the original strain, using hydrolyzed whey, leaded to PGA levels similar to the ones obtained using integral whey. However, in bioreactor the yield of enzyme was always lower than in agitated flasks, no matter the aeration strategy that was used. Three possible explanations for this fact were investigated: 1) microorganism preservation method; 2) changes in the time necessary for attaining and sustaining the metabolic state where enzyme expression occurs, what could be related to the ratio between vegetative cells and spores along time, in flasks and in the bioreactor; 3) increase in the production of proteases in the bioreactor, compared to the flasks. The lower yield using hydrolyzed whey does not seem to be related to none of these hypotheses, and up to now it was not possible to generalize the study of the effect of this variable on the PGA production by B. megaterium. Enzyme purification and concentration, via ultra-diafiltration, in the presence of integral and hydrolyzed whey was another subject for research. The effect of pH and of the number of washing cycles on enzyme recovery was assessed. Results showed that this technique is efficient, provided it is run at low temperatures. Hydrolyzed whey indeed eases the purification of the enzyme using membranes, and great part of the contaminant whey proteins can be removed. However, an expressive loss of PGA occurs during the diafiltration stages, which must be minimized. pH did not influence enzyme recovery. The kinetic characterization of the produced enzyme, using the hydrolysis of penicillin G as standard reaction, has showed that maximum PGA activity is at pH 8 and 37oC. Estimated Michelis-Menten parameters were Vmax= 0.0344 mMPenG/min and Km=1.83 mM, with activation energy equal to 27.12 KJ/mol.
Efeito do ultra-som pulsado de baixa intensidade sobre fêmur de ratas ovariectomizadas
(Universidade Federal de São Carlos, 2007-08-16) Albertin, Lucélia Maria; Nonaka, Keico Okino; https://lattes.cnpq.br/4551036775738296; https://lattes.cnpq.br/8533605259639231
Osteoporosis is a metabolic disease characterized by the damage of the bone strength due to the integration of bone density, expressed by the quantity of mineral material by bone area and volume, and the bone quality related to bone architecture, remodeling and mineralization. It is a disease that induces bone fragility and fracture spinal column and in the femur, where trabecular bone are predominant. The osteoporotic fractures are responsible by great morbidity and mortality, therefore representing a great public health problem. Studies with alternative non pharmacological treatments have been proposed and one of them is the pulsed ultrasound with low intensity (US). It is well known its osteogenic effect as a mechanical stimulus in isolates cells and bone fractures. However, these effects are not completely explained in the osteoporosis. Also, studies with aim to avoid the osteopenia with mechanical stimulus as US are rare. Therefore, the present study had the objective to analyze the effect of US (30 mw/cm2 SATA, 200µs pulse width, 1 KHz of frequency of repetition and frequency of 1.5 MHz) in femur of rats. The ovariectomized Wistar rats were used as animal model of experimental osteoporosis. Biomechanical, physical and histomorphometric parameters were analyzed. The treatment began 24h after ovariectomy, 5 days/week, during 8 weeks. Female rats with 4 months of age were divides in 6 experimental groups: US 30 and US 20 - OVX rats US stimulated during 30 and 20 minutes; Sham 30 and Sham 20 OVX rats US sham stimulated; Intact 30 and Intact20 intact rats US sham stimulated. The efficacy of the osteopenia model induced by ovariectomy was demonstrated by the physical, biomechanical and histomorphometric parameters. These parameters also showed that US was not efficiency to avoid the bone microarchiteture deterioration induced by castration.
Semicontinuidade superior de atratores para semigrupos multívocos
(Universidade Federal de São Carlos, 2007-08-16) Simsen, Jacson; Gentile, Cláudia Buttarello; https://lattes.cnpq.br/1295174684198407
In this work we developed an abstract theory for existence and characterization of attractors for multivalued semigroups defined by generalized semi ows and we apply the abstract results to systems of p-laplacian differential inclusions and we obtain upper semicontinuity of the attractors as the parameter of diffusion goes to infinity.