ISEL - Engenharia Química e Biológica
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- Diffuse reflectance studies of β-phenylpropiophenone and benzophenone inclusion complexes with calix[4], [6] and [8]arenesPublication . Ferreira, Luís F. Vieira; Ferreira, Margarida R. Vieira; Oliveira, Anabela S.; Branco, T. J. F.; Prata, José V.; Moreira, J. C.The formation of inclusion complexes of β-phenylpropiophenone and benzophenone with p-tert-butylcalix[4], [6] and [8]arenes (powdered solid samples) was studied with the use of diffuse reflectance techniques. Experimental evidence was obtained for inclusion in all cases. The benzophenone n → π* absorption band exhibits hypsochromic shifts with an increase of the calixarene ring aperture, providing evidence for an increase in cavity polarity. β-Phenylpropiophenone exhibits strong room temperature phosphorescence in contrast to its solution behaviour, and this is a clear evidence for the inclusion of this probe within the calixarene molecules. Calix[4]arene and calix[8]arene provide a more rigid environment to β-phenylpropiophenone whereas calix[6]arene appears to be more flexible. Transient absorption spectra of inclusion complexes with benzophenone (1 : 1 mol : mol, excited at 355 nm) show major triplet formation in the calix[4]arene case, while the ketyl radical of benzophenone is formed in the calix[6]arene case and is predominant in the calix[8]arene case. Phenoxyl radical transient absorption also becomes evident in the last two cases. For β-phenylpropiophenone (1 : 1 mol : mol) phenoxyl radicals are formed but no evidence was found for triplet or ketyl radical formation of this ketone in the calix[4]arene case. However, strong support for the ketyl radical of the aryl alkyl ketone was found for calix[6]arene and calix[8]arene inclusion complexes.
- Intramolecular addition of acyldiazenecarboxylates onto double bonds in the synthesis of heterocyclesPublication . Prata, José V.; Clemente, Dina-Telma S.; Prabhakar, Sundaresan; Lobo, Ana M.; Mourato, Isabel; Branco, Paula SérioAppropriate aryl-substituted unsymmetrical azodicarbonyl compounds, generated from bishydrazides by oxidation, undergo intramolecular cyclisations to furnish a variety of useful heterocycles such as N-substituted oxindoles, carbostyrils, benzazepinones, benzazocinones, benzimidazolones, benzoxazinones and pyrazolones in varying degrees of efficiency. Methods are described to remove the N-acyl groups from the heteroaromatic compounds. Under mildly acidic conditions where equal opportunities are available for an ipso or a normal cyclisation it is the former process that occurs preferentially. Evidence is presented in favour of a C-to-C migration in the ipso product for the formation of a methoxy-substituted carbostyril derivative. One of the spiro substances is shown to participate in dienone–phenol rearrangement to provide the corresponding quinolone–phenol in high yield.
- Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiaePublication . Calado, Cecília; Monteiro, Sandra M. S.; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The importance of controlling the expression of heterologous cutinase in a recombinant Saccharomyces cerevisiae SU50 strain was investigated. Maximum specific growth rate and the biomass yield increased 1.91 and 1.16 fold, respectively, when cutinase production was induced by galactose in a pre-fermentation step. However, only 19% of specific cell activity was obtained in comparison to other fermentations following a pre-fermentation step without induction of cutinase expression. Thus, the pre-fermentation step was performed using a selective medium not containing galactose, and the fermentation was performed with a cheaper and complex non-selective medium containing galactose. Under these conditions, and with the aim of maximising the specific cutinase activity, a pre-fermentation with low volume and high density of viable cells must be used. However, due to the low pre-fermentation volume, low yeast cell concentrations and low specific cell activities were obtained after 96 h of fermentation. Otherwise, when the aim was to maximise cutinase yield and productivity, a pre-fermentation volume of 10% (v/v) in relation to fermentation and in the exponential growth phase with a cell concentration between 1.1 and 1.8 g dcw/l should be used. A higher pre-fermentation volume, such as 20% (v/v), would still be economical in the case of a pre-fermentation with low cell density or low cell viability.
- Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strainsPublication . Calado, Cecília; Mannesse, Maurice; Egmond, Maarten; Cabral, Joaquim M.S.; Fonseca, Luís P. P.This study focused on the growth of Saccha‐romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild‐type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)2 and (WP)4, respectively. Different cultivation and strategies were tested in a 2‐L shake flask and a 5‐L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed‐batch culture, where wild‐type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg−1 with activity per protein broth (specific activity) of 266 Umg−1, whereas cutinase‐(WP)2 was secreted with a specific cell activity of 2.1 Umg−1 with a specific activity of 200 Umg−1, and cutinase‐(WP)4 with a specific cell activity of 0.7 Umg−1 with a specific activity of 15 Umg−1. The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed‐batch culture).
- Optimisation of culture conditions and characterisation of cutinase produced by recombinant Saccharomyces cerevisiaePublication . Calado, Cecília; Taipa, M. Ângela; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The optimum culture dissolved oxygen concentration and culture pH for the production of cutinase from Fusarium solani pisi by the recombinant Saccharomyces cerevisiae SU50 strain was investigated. Dissolved oxygen concentrations in the fermentation culture of 5, 30 and 60% of air saturation were evaluated. A high cutinase production, specific cutinase activity and the highest cutinase yield on biomass and the highest specific cutinase production rate were obtained with a 5% of air saturation, which could have impact on process economics. Furthermore, at a low dissolved oxygen concentration, the specific growth rate, specific cutinase production rate, cutinase yield on biomass, cutinase activity and specific cutinase activity were increased when the pH control was changed from 5.25 to 6.25. The cutinase, accumulated in the yeast culture presents two glycosilated isoforms with molecular weights of 22.8 and 24.9 kDa measured by SDS-PAGE. Furthermore, cutinase in clarified culture samples presents a linear relationship between estereolytic and lypolitic activity and a high stability at room temperature.
- Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinasePublication . Calado, Cecília; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The effect of culture media composition, and fermentation conditions and strategy on the growth and cutinase production of recombinant Saccharomyces cerevisiae and subsequent cutinase purification by expanded bed adsorption (EBA) was studied. The reduction in the amount of yeast extract used as nitrogen source from 20 g dm−3 to 10 g dm−3 in batch cultures led to a 29% decrease in the heterologous cutinase production, while the 5% cutinase dynamic adsorption capacity (q5%) on the cation Streamline SP XL was increased 6.7‐fold. By dilution of the whole fermentation broth, performed with the lowest yeast extract content, which reduces conductivity, the q5% was additionally increased by 1.9‐fold. After implementation of a fed‐batch strategy the cutinase concentration, cutinase yield on carbon source, cutinase yield on nitrogen source and productivity were increased by 10.8‐, 2.9‐, 5.3‐ and 6.4‐fold, respectively, in relation to the previously‐mentioned batch fermentation. However, the increased cutinase production was compromised by heterologous protein loss during the EBA recovery operation and the cutinase dynamic adsorption capacity and purification productivity decreased by 90% and 75%, respectively. Thus, target protein production by S cerevisiae fermentation and a downstream process with EBA cannot be considered as separate entities, where the understanding of the factors that affect the interactions among them are crucial towards optimization of the overall production process of heterologous proteins.
- Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strainPublication . Calado, Cecília; Almeida, Claúdio; Cabral, Joaquim M.S.; Fonseca, Luís P. P.Saccharomyces cerevisiae SU50 strain was cultivated with different concentrations of glucose and galactose with the aim of increasing cutinase activity, cutinase yield on the carbon source, and bioreactor productivity. Cultivations in shake flasks with galactose as the sole carbon source, with sugar concentrations between 10 and 40 g/l, exhibited growth-associated cutinase production and a constant specificity of cutinase secretion. Furthermore, as the galactose concentration increased to values higher than 15 g/l, a progressively higher maximum specific galactose consumption rate and a consequent higher alcoholic fermentation occurred, resulting in progressively lower biomass yields on the carbon source and cutinase yields on biomass. Using high glucose and galactose concentrations in a well-aerated bioreactor resulted in a high biomass productivity (0.5 g dcw/l/h), a high cutinase yield on biomass (21.5 U/mg dew), a final high cutinase secretion efficiency (97%), and plasmid stability (99%). Based on these studies, a two phase fed-batch cultivation strategy was developed. A batch phase with high glucose and galactose concentrations, followed by a fedbatch with a constant feed rate with galactose as the sole carbon source in order to minimize the repression of the GAL 7 promoter, were established. The feed rate was estimated to maintain a pre-determined concentration of galactose (20 g/l) on the culture medium in order to maximize the efficiency of cutinase secretion and minimize the galactose alcoholic fermentation. By this cultivation strategy, enhancements of 3.6-fold in cutinase activity, 1.2-fold in cutinase yield on the carbon source, and 8.7-fold culture productivity were obtained in relation to a batch cultivation performed in shake flasks with 20 g/l of galactose.
- Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspectsPublication . Ferreira, Bruno S.; Calado, Cecília; Keulen, Frederik van; Fonseca, Luís P. P.; Cabral, Joaquim M.S.; R., Da Fonseca M. M.Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell—required for the induction of cutinase production—and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.
- Novel laser-induced luminescence resulting from benzophenone/O-propylated p-tert-butylcalix[4]arene complexes: a diffuse reflectance studyPublication . Ferreira, Luis F. Vieira; Ferreira, Margarida R. Vieira; Silva, José P. da; Machado, Isabel Ferreira; Oliveira, Anabela S.; Prata, José V.Laser-induced room temperature luminescence of air-equilibrated benzophenone/O-propylated p-tert-butylcalix[4]arene solid powdered samples revealed the existence of a novel emission, in contrast with benzophenone/p-tertbutylcalix[4]arene complexes, where only benzophenone emits. This novel emission was identified as phosphorescence of 1-phenyl-1,2-propanedione, which is formed as the result of an hydrogen atom abstraction reaction of the triplet excited benzophenone from the propoxy substituents of the calixarene. Room temperature phosphorescence was obtained in air-equilibrated samples in all propylated hosts. The decay times of the benzophenone emission vary greatly with the degree of propylation, the shortest lifetimes being obtained in the tri- and tetrapropylated calixarenes. Triplet–triplet absorption of benzophenone was detected in all cases, and is the predominant absorption in the p-tert-butylcalix[4]arene case, where an endo-calix complex is formed. Benzophenone ketyl radical formation occurs with the O-propylated p-tert-butylcalix[4]arenes hosts, suggesting a different type of host/guest molecular arrangement. Diffuse reflectance laser flash photolysis and gas chromatography–mass spectrometry techniques provided complementary information, the former about transient species and the latter regarding the final products formed after light absorption. Product analysis and identification clearly show that the two main degradation photoproducts following laser excitation in the propylated substrates are 1-phenyl1,2-propanedione and 2-hydroxybenzophenone, although several other minor photodegradation products were identified. A detailed mechanistic analysis is proposed. While the solution photochemistry of benzophenone is dominated by the hydrogen abstraction reaction from suitable hydrogen donors, in these solid powdered samples, the α-cleavage reaction also plays an important role. This finding occurs even with one single laser pulse which lasts only a few nanoseconds, and is apparently related to the fact that scattered radiation exists, due to multiple internal reflections possibly trapping light within non-absorbing microcrystals in the sample, and is detected until at least 20 µs after the laser pulse. This could explain how photoproducts thus formed could also be excited with only one laser pulse.
- Syntheses and properties of hydride–cyanamide and derived hydrogen-cyanamide complexes of molybdenum(IV). Crystal structure of [MoH2(NCNH2)2(Ph2PCH2CH2PPh2)2][BF4]2Publication . Martins, Luisa; Alegria, Elisabete; Hughes, David L.; Fraústo Da Silva, João; Pombeiro, ArmandoThe first hydride–cyanamide (or –cyanoguanidine) complexes of molybdenum, [MoH2(NCR)2(dppe)2][BF4]2 (R = NH2 1a, NMe2 1b, NEt2 1c or NC(NH2)2 1d; dppe = Ph2PCH2CH2PPh2), have been prepared by treatment of [MoH4(dppe)2] in THF with the appropriate cyanamide (or cyanoguanidine) in the presence of HBF4. Reaction of 1a with a base leads to the bis(hydrogen-cyanamide) [or bis(hydrogen-cyananoimide)] complex trans-[Mo(NCNH)2(dppe)2][BF4]2 2 or to the bis(cyanoimide) complex trans-[Mo(NCN)2(dppe)2] 3, via basecatalysed or base-promoted dehydrogenation, whereas cathodically-induced dehydrogenation appears to form [MoH2(NCNH)(NCNH2)(dppe)2] 4. The spectroscopic properties of the complexes are also reported along with their electrochemical behaviours and the molecular structure of 1a as established by X-ray crystallography which indicates the presence of the NCNH2 ligands involved in two hydrogen bonds connecting the ions in dimeric units.