Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.

Made available in DSpace on 2016-06-02T19:56:54Z (GMT). No. of bitstreams: 1 6022.pdf: 1662458 bytes, checksum: 4e6f5f48dc72f84e96a68be96f5ddf02 (MD5) Previous issue date: 2014-03-28 / Financiadora de Estudos e Projetos / Penicillin G acylase (PGA) is the key enzyme for the industrial production of β-lactam antibiotics. Major current PGA demand is attended for producer organisms as Escherichia coli and Bacillus megaterium genetically modified. The enzyme produced by Escherichia coli accumulates in the periplasmic space of the bacteria and it requires cell disruption for recovery, whereas the enzyme from Bacillus megaterium is secreted into the production medium. This study aimed to evaluate the adsorption of PGA expressed by these two recombinant microorganisms in cationic and anionic resins for recovery and purification of the enzyme. For E. coli PGA was evaluated ionic adsorption of the enzyme in Streamline SP XL® cation exchanger resin after cell disruption by sonication. In this step, we assessed the influence of pH and buffer, to reduce the loss of enzyme inactivation and adjust the pH to a value suitable for a subsequent selective adsorption of the enzyme. The buffers evaluated were acetate, citrate, phosphate and carbonate, and the values of pH were adjusted between 4,5 and 11,0. No denaturation of the enzyme was observed in buffers and pHs evaluated. However, it was observed enzyme adsorption in cellular debris at pH values equal or smaller than 5,4, this debris adsorption was more apparent in the presence of acetate buffer. Thus, cell disruption was defined at pH 6.0 to prevent loss PGA in the adsorption of debris and adjustment of pH and salt molarity by dilution in the appropriate buffer to prevent denaturation of the enzyme by local reduction of the pH when uses concentrated acid. The STREAMLINE SP XL® resin showed higher adsorption capacity of PGA at pH 5,0 and the temperature range (4 and 25°C) did not influence it. The Langmuir isotherm represented adequately the experimental data of the enzyme adsorption resin at 4 and 25°C (pH 5,0) with similar values of qm and KL 25,4 U/g 185,2 U/g, respectively. Purification of PGA in a fixed-bed column showed overall recovery of activity around 50% and purification factor of about 9 times. The adsorption capacity of cationic resin in this mode of operation was 10,9 UNIPAB/mlresin. The PGA purification secreted by Bacillus megaterium recombinant, produced in a synthetic medium, was studied by ion adsorption resins and the following pH values: Streamline SP XL ® and IMMOBEAD IB-C435 (cation-exchanger resins, pH 5,0, 5,5 and 6,0); manae-agarose activated with 40 and 80mmoles/g amino groups (cation exchanger resin, pH 6,0, 7,0 and 7,5); STREAMLINE DEAE XL® and STREAMLINE Q XL® (anionic exchange resins, pH 7,5 and 8,5). Equilibrium experiments in cationic resins Streamline SP XL ® (4°C, pH 5,0) and IB-C435 IMMOBEAD (4°C, pH 5,5) allowed estimation of the parameters of the Langmuir model qm = 76,6 and 91,5 U/g KL = 294,7 and 412,3 U/g, respectively. Despite the high adsorptive capacity of these resins, they were not suited to purification of PGA, because their adsorbes PGA and the contaminants. Manae-agarose resins (40 and 80 μmoles/g) were not effective in PGA adsorption. The STREAMLINE anionic exchangers resins not adsorbed significant amount of PGA in pH evaluated, however, this resin performed a adsorption of almost of 50% proteins present in the medium, demonstrating selective for removal contaminating proteins. Adsorption assays in fixed-bed column Streamline Q XL ® (22°C, pH 8,0) showed that the resin is effective in adsorption of contaminating proteins, it is possible to recover approximately 70 % PGA with a purification factor of 4 times and high specific activity of about 25 U/mg. Already on STREAMLINE SP XL® (22ºC, pH 5,0) resin, the total enzyme recovery was 70 %, but with a purification factor of only 1,61 times and specific activity of around 11 U/mg. It was concluded that adsorption in anionic mode is more advantageous, because presents a better performance and avoid the enzyme dilution. The cultivation of recombinant B. megaterium in a high cell density bioreactor using complex medium, allowed to reach PGA volumetric activity of 50 U/mL. After concentration of the enzyme extract by ultrafiltration to 100 U/mL, was evaluated purification of the enzyme on gel filtration resin column using Superdex 200 Prep Grad (22 °C, pH 7,5). This technique allowed high enzyme recovery (>93%), however with a purification factor of 3 times and specific activity of 13 U/mg. / Penicilina G acilase (PGA) é a enzima chave para a produção industrial de antibióticos β-lactâmicos. Grande parte da demanda atual pela enzima é atendida pela sua produção por Escherichia coli e Bacillus megaterium geneticamente modificados. A enzima produzida por Escherichia coli acumula-se no espaço periplasmático da bactéria, requerendo rompimento celular para sua recuperação, enquanto que a enzima produzida por Bacillus megaterium é secretada para o meio de produção. Este trabalho teve como objetivo avaliar a adsorção de PGA produzida por esses dois microrganismos recombinantes em resinas catiônicas e aniônicas para recuperação e purificação da enzima. Para PGA de E. coli avaliou-se a adsorção iônica da enzima na resina de troca catiônica STREAMLINE SP XL® após rompimento celular por sonicação. Nesta etapa, avaliou-se a influência do tampão e do pH, visando reduzir a perda de enzima por inativação e ajustar o pH a um valor apropriado para uma posterior adsorção seletiva da enzima. Os tampões avaliados foram acetato, citrato, fosfato e carbonato, ajustados para valores de pH entre 4,5 e 11,0. Não se observou desnaturação da enzima nos tampões e pHs avaliados. Contudo, observou-se adsorção da enzima nos debris celulares a valores de pH iguais ou menores que 5,4, sendo mais acentuada essa adsorção na presença de tampão acetato. Definiu-se, assim, rompimento celular em pH 6,0 para evitar perda de PGA por adsorção nos debris e ajuste do pH e da molaridade do sal por diluição no próprio tampão da adsorção, para evitar desnaturação da enzima por redução local do pH quando se utiliza ácido concentrado. A resina STREAMLINE SP XL® mostrou maior capacidade de adsorção de PGA em pH 5,0, não sendo influenciada nas temperaturas avaliadas (4 e 25ºC). A isoterma de Langmuir representou adequadamente os dados experimentais de adsorção da enzima nessa resina a 4 e 25ºC (pH 5,0), com valores similares de qm e KL, 25,4 U/g e 185,2 U/g, respectivamente. A purificação de PGA em coluna de leito fixo apresentou recuperação global de atividade em torno de 50% e fator de purificação de aproximadamente 9 vezes. A capacidade de adsorção da resina de troca catiônica nesse modo de operação foi de 10,9 UNIPAB/mLresina. A purificação de PGA secretada por Bacillus megaterium recombinante, produzida em meio sintético, foi estudada por adsorção iônica da enzima nas seguintes resinas e valores de pH: STREAMLINE SP XL® e IMMOBEAD IB-C435 (resinas de troca catiônica, pHs 5,0, 5,5 e 6,0); MANAE-agarose ativada com 40 e 80 μmoles de grupos amino/g (resina de troca catiônica, pHs 6,0, 7,0 e 7,5); STREAMLINE DEAE XL® e STREAMLINE Q XL® (resinas de troca aniônica, pHs 7,5 e 8,5). Ensaios de equilíbrio para as resinas de troca catiônica STREAMLINE SP XL® (4ºC, pH 5,0) e IMMOBEAD IB-C435 (4ºC, pH 5,5) permitiram a estimativa dos parâmetros do modelo de Langmuir: qm = 76,6 e 91,5 U/g e KL= 294,7 e 412,3 U/g, respectivamente. Apesar da alta capacidade adsortiva dessas resinas, as mesmas não foram adequadas à purificação de PGA, pois adsorveram tanto PGA quanto proteínas contaminantes. As resinas MANAE-AGAROSE (40 e 80 μmoles/g) não foram eficazes na adsorção de PGA. As resinas STREAMLINE de troca aniônica não adsorveram quantidade significativa de PGA nos valores de pH avaliados, entretanto, adsorveram quase 50% de proteínas presentes no meio, mostrando-se assim seletiva para retirada das proteínas contaminantes. Ensaios de adsorção em coluna de leito fixo com STREAMLINE Q XL® (22ºC, pH 8,0) mostraram que a resina é eficaz na adsorção de proteínas contaminantes, sendo possível a recuperação de aproximadamente 70% de PGA com um fator de purificação de 4 vezes e alta atividade específica, em torno de 25 U/mg. Já na resina STREAMLINE SP XL® (22ºC, pH 5,0) a recuperação total da enzima foi de 70%, mas com um fator de purificação de apenas 1,61 vezes e atividade específica em torno de 11 U/mg. Assim, conclui-se que a adsorção no modo aniônico é mais vantajosa, pois além de maior purificação, não dilui a enzima. O cultivo de B. megaterium recombinante em biorreator com alta densidade celular, utilizando meio complexo, permitiu atingir atividade volumétrica de PGA de 50 U/mL. Após concentração do extrato enzimático por ultrafiltração até 100 U/mL, avaliou-se a purificação da enzima em coluna de gel de filtração utilizando a resina Superdex 200 prep grad (22ºC, pH 7,5). Essa técnica permitiu alta recuperação da enzima (>93%), entretanto com um fator de purificação de 3 vezes e atividade específica de 13 U/mg.