Browsing by Author "Cabral, J. M. S."
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- 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.
- Micro-analytical GO/HRP bioreactor for glucose determination and bioprocess monitoringPublication . Vojinović, V.; Calado, Cecília; Silva, A. I.; Mateus, M.; Cabral, J. M. S.; Fonseca, Luís P. P.A bi-enzymatic micro-analytical bioreactor integrated in a FIA system for glucose measurements is described. Its robustness and small dimensions (working volume of about 70 μl containing approximately 1.2 mg GO and 0.26 mg HRP) make it easy to operate. The column is based on immobilisation of glucose oxidase (GO) and horseradish peroxidase (HRP) on alkylamine controlled pore glass (CPG) beads. The column has excellent shelf life (no significant loss of activity after 1 year if kept at 4 °C), and a very high operational stability that was demonstrated through extensive usage for glucose determinations over 1 year period during which the column retained almost all of its activity. More importantly, this operational stability allows glucose monitoring in the culture media without a decay of signal over the experiment time and consequently no signal correction or re-calibration is needed. This high operational stability was also confirmed by continuous glucose conversion with 30% activity loss after converting quantity of glucose equivalent to 21600 FIA injections of 20 μl with 1.7 mM glucose. Such good performance is a result of an optimised immobilisation method and moreover of the implementation of in situ enzyme stabilisation strategy which consisted on promoting the instantaneous H2O2 consumption produced by the GO. This strategy has the additional advantage of allowing concomitant assay of the H2O2 based on the HAP catalysed co-oxidation of phenol-4-sulphonic acid (PSA) in the presence of 4-aminoantipyrine (4-AAP). The glucose measurements are reproducible with high precision against the standard HPLC method. Linear range and sensitivity depend on sample injection volume; the upper limit is about 1.1 g/l. Lower detection limit is 10 mg/l. The column performance has been validated for E. coli and S. cerevisiae fermentation monitoring, and glucose measurements in an animal cell culture (rat Langerhans islets).
- 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.
- Supercritical carbon dioxide extraction of bioactive compounds from microalgae and volatile oils from aromatic plantsPublication . Palavra, A. M. F.; Coelho, Jose; Barroso, J. G.; Rauter, A. P.; Fareleira, J. M. N. A.; Mainar, A.; Urieta, J. S.; Nobre, B. P.; Gouveia, L.; Mendes, R. L.; Cabral, J. M. S.; Novais, J. M.A discussion of the most interesting results obtained in our laboratories, during the supercritical CO(2) extraction of bioactive compounds from microalgae and volatile oils from aromatic plants, was carried out. Concerning the microalgae, the studies on Botryococcus braunii and Chlorella vulgaris were selected. Hydrocarbons from the first microalgae, which are mainly linear alkadienes (C(23)-C(31)) with an odd number of carbon atoms, were selectively extracted at 313 K increasing the pressure up to 30.0 MPa. These hydrocarbons are easily extracted at this pressure, since they are located outside the cellular walls. The extraction of carotenoids, mainly canthaxanthin and astaxanthin, from C. vulgaris is more difficult. The extraction yield of these components at 313 K and 35.0 MPa increased with the degree of crushing of the microalga, since they are not extracellular. On the other hand, for the extraction of volatile oils from aromatic plants, studies on Mentha pulegium and Satureja montana L were chosen. For the first aromatic plant, the composition of the volatile and essential oils was similar, the main components being the pulegone and menthone. However, this volatile oil contained small amounts of waxes, which content decreased with decreasing particle size of the plant matrix. For S. montana L it was also observed that both oils have a similar composition, the main components being carvacrol and thymol. The main difference is the relative amount of thymoquinone, which content can be 15 times higher in volatile oil. This oxygenated monoterpene has important biological activities. Moreover, experimental studies on anticholinesterase activity of supercritical extracts of S. montana were also carried out. The supercritical nonvolatile fraction, which presented the highest content of the protocatechuic, vanilic, chlorogenic and (+)-catechin acids, is the most promising inhibitor of the enzyme butyrylcholinesterase. In contrast, the Soxhlet acetone extract did not affect the activity of this enzyme at the concentrations tested. (C) 2011 Elsevier B.V. All rights reserved.