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 2 2 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% of bromelain activity was recovered. Ultra-filtration retained 100% 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.

ABSTRACT

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.

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