Cloning and expression of protease ClpP from Streptococcus pneumoniae in Escherichia coli: study of the influence of kanamycin and IPTG concentration on cell growth, recombinant protein production and plasmid stability.

Infections caused by Streptococcus pneumoniae are one of the main causes of death around the world. In order to address this problem, investigations are being made into the development of a protein-based vaccine. The aims of this study were to clone and express ClpP, a protein from S. pneumoniae serotype 14 in Escherichia coli, to optimize protein expression by using experimental design and to study plasmid segregation in the system. ClpP was cloned into the pET28b vector and expressed in E. coli BL21 Star (DE3). Protein expression was optimized by using central composite design, varying the inducer (IPTG) and kanamycin concentration, with a subsequent analysis being made of the concentration of heterologous protein, cell growth and the fraction of plasmid-bearing cells. In all the experiments, approximately the same concentration of ClpP was expressed in its soluble form, with a mean of 240.4mg/L at the center point. Neither the IPTG concentration nor the kanamycin concentration was found to have any statistically significant influence on protein expression. Also, higher IPTG concentrations were found to have a negative effect on cell growth and plasmid stability. Plasmid segregation was identified in the system under all the concentrations studied. Using statistical analysis, it was possible to ascertain that the procedures for determining plasmid stability (serial dilution and colony counting) were reproducible. It was concluded that the inducer concentration could be reduced tenfold and the antibiotic eliminated from the system without significantly affecting expression levels and with the positive effect of reducing costs.

Concentration by membrane separation processes of a medicinal product obtained from pineapple pulp

The concentration of pineapple juice is needed to retain the bromelain activity and to standardize the composition and proteolytic activity. Thus, this work aimed to obtain a pure bromelain extract from the Ananas comosus L. Merril juice by membrane separation process. A 2² experimental planning was used to study the influence of pH and transmembrane pressure on the activity recovery by micro-filtration using a plain membrane. In second step, this enzyme was purified by the ultra-filtration using a 10 kDa millipore kit. The best operation condition to bromelain concentration using the plain membrane was at pH 7.5 and transmembrane pressure of 0.05 bar, while 85 percent of bromelain activity was recovered. Ultra-filtration retained 100 percent of proteolytic activity and concentrated in 10 fold the bromelain extract. SDS-PAGE electrophoresis showed that the ultra-filtrated had high purity and the bromelain from A. comosus pulp had a molecular weight of 24.5 kDa.

A concentração do suco de abacaxi é necessária para manter a atividade da bromelina e padronizar a composição e atividade proteolítica. Assim, este trabalho objetivou a obter um extrato de bromelina pura do suco do Ananas comosus L. Merril por processos de separação por membranas. Um planejamento experimental do tipo 2² foi feito para estudar a influência do pH a da pressão transmembranar sobre a recuperação da atividade por micro-filtração usando uma membrana plana. Em uma segunda etapa, purificou-se a enzima alvo por ultra-filtração usando um "kit millipore" de 10 kDa. A melhor condição para a concentração da bromelina foi a pH 7,5 e pressão transmembranar de 0,05 bar, onde 85 por cento da atividade da bromelina foi recuperado. A ultra-filtração manteve 100 por cento da atividade proteolítica e concentrou em 10 vezes o extrato de bromelina. A eletroforese via SDS-PAGE mostrou que o ultra-filtrado teve alta pureza e a bromelina da polpa do Ananas comosus tem um peso molecular de 24.5 kDa.

Response surface methodology to evaluation the recovery of amylases by hollow fiber membrane

This work aimed to study the pH and the transmembrane pressure effects during the recovery of alpha and beta amylases enzymes from corn malt (Zea mays) by hollow fiber membrane. The optimal condition was obtained for a statistical model, established by response surface methodology (RSM). The response surface analysis showed that the best operation condition for amylolitics enzymes recovery by hollow fiber membrane was 0.05 bar and pH 5.00, while the enzymes were purified about of 26 times.

Este trabalho objetivou estudar o efeito do pH e da pressão trans-membrana durante a recuperação das enzimas alfa e beta amilases do malte de milho (Zea mays) por membranas de fibras ocas, a obtenção das condições ótimas foi feita por um modelo estatístico, estabelecido pela metodologia de superfície de resposta (RSM). A análise da superfície de resposta mostrou que as melhores condições operacionais para a recuperação das enzimas amiloliticas por membranas de fibras ocas foi 0,05 bar e pH 5,00; onde as enzimas foram purificadas cerca de 26 vezes.

Partitioning optimization of proteins from Zea mays malt in ATPS PEG 6000/CaCl2

This work aimed to establish the relationship between the compositions and pH of ATPS PEG 6000/CaCl2 and the proteins partition from maize malt and also to simplify the process optimization in ATPS for a statistical model, established by response surface methodology (RSM). Results showed that these were no influence of pH on the phase diagrams and on the composition of tie line length of PEG 6000/CaCl2 ATPS. SRM analyses showed that elevated pH and larger tie line length were the best conditions for recovering of maize malt proteins. The maximum partition coefficient by PEG 6000/CaCl2 ATPS was about 4.2 and was achieved in ATPS in a single purification step. The theoretical maximum partition coefficient was between 4.1-4.3. The process was very suitable for continuous aqueous two-phase purification due to the stability of proteins (e.g. and -amylases) and could increase their content into middle.

Este trabalho objetivou encontrar uma relação entre a composição e o pH do sistema bifásico aquoso (SBA) PEG 6000/CaCl2 e a partição de proteínas do malte de milho, e assim simplificando a otimização do processo por um modelo estatístico, estabelecido por metodologia de superfície de resposta (RSM). Os resultados mostraram que não houve influência do pH sobre os diagramas de fases e sobre a composição das linhas de amarração do SBA PEG/CaCl2. As analises RSM mostraram que em pH elevado e nas maiores linha de amarração encontra-se a melhor condição para a recuperação das proteínas do malte de milho. O coeficiente de partição máximo foi cerca de 4,2 para uma única etapa de purificação no SBA 6000/CaCl2. O coeficiente de partição máximo encontrado teoricamente esteve entre 4,1-4,3. O processo é adequado para a purificação contínua via sistemas bifásicos aquosos, já que as proteínas do malte (ex: e -amilases) são estáveis e podendo elevar sua concentração no meio.