Percorrer por autor "Fonseca, L. P."
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- Effect of tween-80 on stability and secretion of hydrophobic tagged-cutinasesPublication . Calado, Cecília; Brandão, M.; Biscaia, J.; Cabral, J. M. S.; Fonseca, L. P.To significantly enhance the downstream processing, two cutinase variants were constructed by genetic fusion small hydrophobic peptides (WP)2 and (WP)4, respectively. However, the fusion of these peptides impairs cutinase secretion by the host cell Saccharomyces cerevisiae and increases cutinase inactivation in the culture broth due to cutinase aggregation, resulting in cutinase activities per biomass of 56 % of cutinase-(WP)2 and of 7 % of cutinase-(WP)4, in relation to cutinase without the hydrophobic tags. It was observed that the addition of non-ionic surfactant Tween-80 into the culture broth could minimise the cutinase inactivation. The addition of Tween-80 also results in the enhancement of cutinase secretion by the yeast cell, leading to 1.25 and 2.51 fold-higher extracellular cutinase-(WP)2 and cutinase-(WP)4, respectively, in relation to cultivations performed in the absence of surfactant. Therefore, the addition of Tween-80 on the culture broth partly minimises the effect of fusion of the hydrophobic tags on the inactivation of the enzymatic activity and on the reduction of the protein secretion. By this way, the use of Tween-80 on the S. cerevisiae cultivation may contribute to the efficiency enhancement of the downstream processing of tagged cutinases.
- Prediction of dynamic plasmid production by recombinant escherichia coli fed-batch cultivations with a generalized regression neural networkPublication . Silva, T.; Lima, P.; Roxo-Rosa, M.; Hageman, S.; Fonseca, L. P.; Calado, CecíliaA generalized regression neural network with external feedback was used to predict plasmid production in a fed-batch cultivation of recombinant Escherichia coli. The neural network was built out of the experimental data obtained on a few cultivations, of which the general strategy was based on an initial batch phase followed by an exponential feeding phase. The different cultivation conditions used resulted in significant differences in bacterial growth and plasmid production. The obtained model allows estimation of the experimental outputs (biomass, glucose, acetate and plasmid) based on the bioreactor starting conditions and the following on-line inputs: feeding rate, dissolved oxygen concentration and bioreactor stirring speed. Therefore, the proposed methodology presents a quick, simple and reliable way to perform on-line feedback prediction of the dynamic behaviour of the complex plasmid production process, based on simple on-line input data obtained directly from the bioreactor control unit and with few cultivation experiments for neural network learning.
- Prediction of retention time of cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatographyPublication . Lienqueo, M. E.; Salazar, O.; Henriquez, K.; Calado, Cecília; Fonseca, L. P.; Cabral, J. M. S.Hydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan–proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Φtagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE < 0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE < 0.022) for the two tagged cutinases at three different stationary phases, except for the case of cut_(wp)2 in octyl sepharose–2 M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of cutinases tagged with hydrophobic peptides in HIC.