Browsing by Author "Barreiros, Susana F."
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- Cutinase activity in supercritical and organic media: water activity, solvation and acid–base effectsPublication . García, Sílvia D.; Vidinha, Pedro; Arvana, Helena; Silva, Marco D.R. Gomes da; Ferreira, Olga; Cabral, Joaquim M.S.; Macedo, Eugénia A.; Harper, Neil; Barreiros, Susana F.We performed a comparative study on the activity of Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY, in n-hexane, acetonitrile, supercritical ethane (sc-ethane) and sc-CO2, at two different water activity (aW) values set by salt hydrate pairs in situ and at acid–base conditions fixed with solid-state buffers of aqueous pKa between 4.3 and 10.6. The reaction studied was the transesterification of vinyl butyrate by (R,S)-2-phenyl-1-propanol. The transesterification activity of cutinase was highest and similar in sc-ethane and in n-hexane,about one order of magnitude lower in acetonitrile and even lower in sc-CO2. Activity coefficients (γ) generated for the two substrates indicated that they were better solvated in acetonitrile and thus less available for binding at the active site than in the other three solvents. γ data also suggested higher reaction rates in sc-ethane than in n-hexane, as observed, and provided evidence for a direct negative effect of sc-CO2 on enzyme activity. Manipulation of the acid–base conditions of the media did not afford any improvement of the initial rates of transesterification relative to the blanks (no added acid–base buffer, only salt hydrate pair), except in the case of cutinase immobilized on zeolite NaA in sc-ethane at aW = 0.7. The poor performance of the blank in this case and the great improvement observed in the presence of a basic buffer suggest a deleterious acidic effect in the medium which, an experiment without additives confirmed, was not due to the known acidic character of the salt hydrate pair used to set aW = 0.7. In acetonitrile, increasing aW was accompanied by a decrease in initial rates of transesterification, unlike in the other solvents. There was considerable hydrolysis in acetonitrile, where initial rates of hydrolysis increased about 20-fold from aW = 0.2 to 0.7. Hydrolysis was less pronounced in sc-ethane and in n-hexane, and only at aW = 0.7, and in sc-CO2 butyric acid was detected only at very long reaction times, in agreement with a generally low catalytic activity. Cutinase enantio-selectivity towards the alcohol substrate was low and unaffected by any manipulation of medium conditions.
- Decyl acetate synthesis by enzyme catalysis in sc-CO2Publication . Rebocho, Sílvia F.; Ribeiro, Adriano S.; Ferreira, Olga; Oliveira, Manuela V.; Vidinha, Pedro; Barreiros, Susana F.; Macedo, Eugénia A.; Loureiro, José M.The synthesis of decyl acetate, from the transesterification of vinyl acetate with decanol, was studied in a high-pressure experimental set-up, equipped with a variable volume batch reactor, operating isothermally at 35 ºC and 100 bar, using CO2 in supercritical conditions as solvent, and Candida antarctica lipase B (CALB), immobilized on the macroporous resin Lewatit B (Novozym 435®), as catalyst. The enzymatic content was determined for each particle size of the catalyst. It was shown that the smallest particles have a larger specific amount of enzyme, and the results indicate that the enzyme is located in an external shell of the particle, following an “egg-shell” model type, with a thickness of ca. 60 μm (assuming a homogeneous distribution), independent of the particle size. Both external and internal mass transfer resistances were evaluated. External resistances were easily eliminated by stirring and internal diffusional limitations can be considered as negligible. The effect of the feed concentration of substrates on the initial reaction rate was also studied. It was observed that, above a certain concentration of decanol in excess relatively to vinyl acetate, the reaction is inhibited by the alcohol. On the contrary, the reaction is favoured when the reactor is fed with excess of vinyl acetate, with a significant enhancement of its initial rate. These results are consistent with a Ping-pong bi-bi type mechanism with competitive inhibition by the alcohol, commonly used in the description of enzymatic reactions of esterification/ transesterification.
- Decylacetate synthesis by enzyme catalysis in SC-CO2Publication . Oliveira, Manuela V.; Rebocho, Sílvia F.; Ribeiro, Adriano S.; Ferreira, Olga; Vidinha, Pedro; Barreiros, Susana F.; Macedo, Eugénia A.; Loureiro, José M.The main purpose of this work was the study of long chain esters production, using decyl acetate as model compound, by enzymatic catalysis in supercritical media, with the aim of developing a sustainable, clean and efficient process as an alternative to the traditional chemical processes. The combination of a sustainable and clean technology, as biocatalysis, with a green/natural solvent, as supercritical CO2, besides allowing the establishment of processes with less environmental costs, leads to products considered as natural, which results in a significant increase of their market value. Decyl acetate is a high added value product. It figures in the “Food and Drug Administration” list of authorized flavoring agents and additives, and it also finds applications in the fragrance industry because of its floral essence. Usually, these kind of esters are obtained by extraction from natural expensive oils and waxes or produced through the esterification of carboxylic acids by acid catalysis. In this work, it was synthesized by the transesterification reaction of vinyl acetate with decanol in a high-pressure experimental set-up, equipped with a variable volume batch reactor, operating isothermally at 35 ºC and 100 bar. Candida antarctica Lipase B (CALB), immobilized in the macroporous resin Lewatit B (Novozym 435®), was used as catalyst and CO2 in supercritical conditions was used as solvent. Results on the enzyme intraparticle distribution, external and internal diffusional limitations, and effect of the feed concentration of substrates in the initial reaction rate are presented. Modeling and simulation results are also addressed in this work. Some important details of the experimental set-up and experimental procedure are described. The enzymatic content was determined for each particle size of the catalyst. The smallest particles have a larger specific amount of enzyme, with an inversely proportional relation between the enzymatic content and the particle size. The results also show that the enzyme is most probably located in an external shell of the particle, following an “egg-shell” model type, with a thickness of ca. 60 µm (assuming a homogeneous distribution in this outer shell), independent of the particle size. The mass transfer resistances, both external and internal, were found to be negligible. The experimental results obtained while varying the concentration of each reactant individually were qualitatively consistent with the Ping-pong bi-bi kinetic model with competitive inhibition by the alcohol: the reaction was enhanced by concentrations of vinyl acetate ≥ 100 mM, and inhibited by concentrations of decanol ≥ 100 mM. The reactor behavior was simulated using a simplified Ping-pong bi-bi reaction rate equation with four parameters. These were determined using an adaptative random search optimization algorithm. Simulation results are in very good agreement with experimental data, within the range of concentrations tested (50 – 200 mM).
- Enzymatic production of decyl acetate: kinetic study in n-hexane and comparison with supercritical CO2Publication . Ribeiro, Adriano S.; Oliveira, Manuela V.; Rebocho, Sílvia F.; Ferreira, Olga; Vidinha, Pedro; Barreiros, Susana F.; Macedo, Eugénia A.; Loureiro, José M.The kinetics of the lipase-catalyzed synthesis of decyl acetate, by the transesterification reaction of vinyl acetate with decanol, was investigated at 30 °C using n-hexane as the solvent. Novozym 435 was found to be the most active catalyst among the immobilized lipases tested. Given the nonideality of the reaction mixture, only a thermodynamic activity-based kinetics was found to be suitable to represent the experimental data in the entire range of compositions tested (0.1-1.4 M). The reaction follows a ping-pong bi-bi mechanism, in which inhibition only by excess of alcohol was identified. Although intraparticle diffusional limitations were detected, intrinsic kinetic parameters were obtained by crushing the catalyst particles. The results were compared to those obtained with supercritical CO2 as the solvent. For the conditions tested, Candida antarctica lipase B showed higher activity in the organic medium.
- Geranyl acetate synthesis in a packed-bed reactor catalyzed by novozym in supercritical carbon dioxide and in supercritical ethanePublication . Couto, Ricardo; Vidinha, Pedro; Peres, Célia Peres; Ribeiro, Adriano S.; Ferreira, Olga; Oliveira, Manuela V.; Macedo, Eugénia A.; Loureiro, José M.; Barreiros, Susana F.The esterification reaction of geraniol with acetic acid (100 mM/100 mM) catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was studied in supercritical carbon dioxide (sc-CO2) and in sc-ethane in a packed-bed reactor(PBR). In sc-CO2 it was easy to adjust the water activity (aw) in the reaction mixture to levels leading to good enzyme performance. Enzyme stability was high and steady-state conversions could be achieved that exceeded the largest conversions measured in batch stirred-tank reactors (BSTRs), which is probably due to the lower aw levels achieved in the PBR. In sc-ethane, where the solubility of water is lower, high steady-state conversions could be attained only by preventing the accumulation on the enzyme bed of the water produced during reaction. The kinetic parameters for the reaction in sc-CO2 were determined using previously published data obtained in a BSTR, and a model was developed for the PBR that included those kinetic parameters. This model was able to predict with reasonable accuracy the behavior of the PBR. Slight differences were observed for some operating regions, probably due to the influence of aw in the activity of the enzyme, which is not included in the model.
- Production of esters by biocatalysed transesterification in supercritical CO2 and HexanePublication . Ribeiro, Adriano S.; Rebocho, Sílvia F.; Ferreira, Olga; Vidinha, Pedro; Barreiros, Susana F.; Macedo, Eugénia A.; Loureiro, José M.The development of sustainable processes that use renewable raw materials and minimize chemical and energetic waste has attracted considerable attention and represents a great challenge to both academic researchers and industrial experts. The use of enzymes to catalyse chemical transformations can constitute a more sustainable alternative to some traditional chemical processes and more than 100 biotransformations are already operated at an industrial scale [1]. Lipases usually operate in mild conditions and can catalyse both hydrolytic and synthetic reactions, depending on the surrounding medium. The hydrolysis of esters can be performed in water, but the reverse production reactions are not favoured in this medium, and are usually performed in organic solvents. Supercritical CO2 can constitute a more sustainable alternative to organic solvents as a reaction medium, provided that it does not have a direct adverse effect on the enzyme's active site or significantly reduce its activity. Decyl acetate was chosen as a model compound and its production by a transesterification reaction catalysed by Novozym 435 (immobilized Candida Antarctica Lipase B) was studied in both hexane and supercritical CO2. A comparative analysis between these two alternatives was performed, focusing on the differences on the enzyme's catalytic activity, solubilities of the substrates and mass transfer rates; which significantly affect the outcome of the reaction process and its productivity and provide information on when such solvents can be used. [1] Straathof, A.J.J., Panke, S., Schmid, A. The production of fine chemicals by biotransformations. Current Opinion in Biotechnology, 2002, 13(6), 548-556.